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TOXIC RESPONSES OF THE
REPRODUCTIVE SYSTEM
Michael J. Thomas and John A. Thomas
Biotransformation of Exogenous Chemicals
Testes
Ovary
DNA Repair
Alkylating Agents
Lead
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INTRODUCTION
History
Endocrine Disruptors
Reproductive Hazards
GENERAL REPRODUCTIVE BIOLOGY
Sexual Differentiation
Gonadal Sex
Genotypic Sex
Phenotypic (Genital) Sex
TARGETS FOR CHEMICAL TOXICITY
CNS
Gonads
Sertoli Cells
Steroidogenesis
GONADAL FUNCTION
Central Modulation
Testicular Function
Apoptosis
Spermatogenesis
Sertoli Cells
Interstitium (Leydig Cells)
Posttesticular Processes
Efferent Ducts
Epididymides
Accessory Sex Organs
Erection and Ejaculation
Ovarian Function
Oogenesis
Ovarian Cycle
Postovarian Processes
Oviducts
Uterus
Cervix
Vagina
Fertilization
Implantation
Placentation
EVALUATING REPRODUCTIVE CAPACITY
TESTING MALE REPRODUCTIVE CAPACITY
General Considerations
Flow Cytometry
Sex Accessory Organs
Semen Analyses
Sperm Counts and Motility
Androgens and Their Receptors
Other Secretory Biomarkers
TESTING FEMALE REPRODUCTIVE CAPACITY
General Considerations
Oogenesis/Folliculogenesis
Estrogens and Their Receptors
Ovulation/Fertilization/Implantation
REPRODUCTIVE TESTS AND REGULATORY
REQUIREMENTS
General Considerations
Guidelines
Endpoints—Females
Endpoints—Males
INTEGRATIVE PROCESSES
HUMAN RISK FACTORS AFFECTING FERTILITY
Hypothalamo-Pituitary-Gonadal Axis
Puberty
General Considerations
Male
Female
SEXUAL BEHAVIOR AND LIBIDO
GENERAL TOXICOLOGIC/PHARMACOLOGIC
PRINCIPLES
EXTRAPOLATION OF ANIMAL DATA TO HUMANS
EPIDEMIOLOGIC STUDIES
Blood–Testis Barrier
673
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INTRODUCTION
History
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The endocrine function of the gonads is primarily concerned with
perpetuation of the species. The survival of any species depends
on the integrity of its reproductive system. Sexual reproduction involves a very complex process for the gonads. Genes located in
the chromosomes of the germ cells transmit genetic information
and modulate cell differentiation and organogenesis. Germ cells
ensure the maintenance of structure and function in the organism
in its own lifetime and from generation to generation.
The twentieth century has undergone an industrial renaissance; through scientific and technical advances, there has been a
significant extension in life expectancy and generally an enhanced
quality of life. Concomitantly with this industrial renaissance, an
estimated 50,000 to 60,000 chemicals have come into common use.
Approximately 600 or more new chemicals enter commerce each
year. The production of synthetic organic chemicals has risen from
less than 1 billion lb in 1920 to over 20 billion lb in 1945, to 75
billion lb in 1960, and to over 200 billion lb in the late 1980s (cf.
Lave and Ennever, 1990). Several endocrine disorders are associated with industrial chemicals (cf. Barsano and Thomas, 1992).
Overall, occupational diseases in the United States may be responsible for about 60,000 deaths per year (Baker and Landrigan,
1991). The impact of new chemicals (or drugs) on the reproductive system was tragically accentuated by the thalidomide disaster
in the 1960s (cf. Fabrio, 1985). This episode led to increased awareness on a worldwide basis and brought forth laws and guidelines
pertaining to reproductive system safety and testing protocols. This
new awareness of reproductive hazards in the workplace has led
to corporate policies and legal considerations (Bond, 1986;
McElveen, 1986). In 1985, the American Medical Association
(AMA) Council on Scientific Affairs of charged its Advisory Panel
on Reproductive Hazards in the Work Place to consider over 100
chemicals with the intent of estimating their imminent hazards
(AMA Council on Scientific Affairs, 1985).
of Missouri, and workers in Sweden who handle organic solvents
(toluene, benzene, and xylene) suffer from low sperm counts, abnormal sperm, and varying degrees of infertility. Diethylstilbestrol
(DES), lead, chlordecone, methyl mercury, and many cancer
chemotherapeutic agents have been shown to be toxic to the male
and female reproductive systems and possibly capable of inflicting genetic damage to germ cells (cf. Barlow and Sullivan, 1982;
Office of Technology Assessment Report, 1985).
In the past few years, there has been an ongoing debate about
a decline in human sperm counts (Carlsen et al., 1992). Carlsen
and coworkers (1992) reported that there has been a decline in semen quality over the past 50 years. Additionally, they reported an
increased incidence in genitourinary abnormalities, including testicular cancer, cryptorchidism, and hypospadias. The outcome of
these studies has been challenged by several groups of investigators (Bromwich et al., 1994; Lipshultz, 1996; Thomas, 1998). The
original evidence using meta-analysis failed to support the hypothesis that sperm counts declined significantly between 1940
and 1990 (Bromwich et al., 1994). Sharpe and Skakkeback (1993)
have hypothesized that the increasing incidence of reproductive
abnormalities in the human male may be related to increased
estrogen exposure in utero. Their hypothesis includes an increased exposure to DES and the possible presence of environmental
estrogen mimics (e.g., DDT). A number of natural and anthropogenic substances exhibit weak estrogen properties (Muller et al.,
1995; Cooper and Kavlock, 1997). There are many naturally occurring phytoestrogens (e.g., coumestans and isoflavonoids) that
possess weak estrogen-binding properties (Thomas, 1997). Many
botanically derived substances possess estrogen-like activity, including soy proteins. Not only are there compounds in the environment that possess estrogenic properties, but there are also environmental antiandrogens (e.g., vinclozolin) (Kelce and Wilson,
1997).
The etiology of many adverse reproductive outcomes among
humans is poorly understood. Generally, studies of epidemiologic
and reproductive outcomes have focused upon maternal factors
(Olshan and Faustman, 1993). Relatively speaking, only recently
have studies begun to examine the role of chemical perturbation
of paternal exposures. Male-mediated developmental toxicity has
received some recent attention. It has become increasingly clear
that reproductive toxicity involves both the male and female
(Mattison et al., 1990). Also noteworthy is the fact that nonreproductive endocrine organs can be adversely affected by drugs and
chemicals (Thomas, 1994).
Endocrine Disruptors
Large numbers and large quantities of endocrine-disrupting chemicals (e.g., o, p-DDT) have been released into the environment since
World War II (cf. Colborn, et al., 1993). Exposure to endocrinedisrupting chemicals has been linked with abnormal thyroid function in birds and fish; diminished fertility in birds, fish, shellfish,
and mammals; and demasculatinization and feminization in fish,
gastropods, and birds (Vos et al., 2000). The significance of endocrine-disrupting chemicals, also known as environmental estrogens or xenoestrogens, is unknown at this time. In general, the
mechanism(s) of endocrine disruption caused by non-heavy metal
agents is due to competition for receptors or inhibition of steroidogenesis.
Concern for reproductive hazards is not new; it dates back to
the Roman Empire. Lead, found in high concentration in pottery
and water vessels, probably played a role in the increased incidence of stillbirths. Lead is now known to be an abortifacient as
well as capable of producing teratospermias. In the United States,
male factory workers occupationally exposed to 1,2-dibromo-3chloropropane (DBCP) became sterile, as evidenced by oligospermia, azoospermia, and germinal cell aplasia. Factory workers
in battery plants in Bulgaria, lead mine workers in the U.S. state
Reproductive Hazards
The potential hazard of chemicals to reproduction and the risks to
humans from chemical exposure are difficult to assess because of
the complexity of the reproductive process, the unreliability of laboratory tests, and the quality of human data. In the human, it is estimated that one in five couples are involuntarily sterile; over onethird of early embryos die, and about 15 percent of recognized
pregnancies abort spontaneously. Among the surviving fetuses at
birth, approximately 3 percent have developmental defects (not always anatomic); with increasing age, over twice that many become
detectable. It should be obvious that even under normal physiologic conditions, the reproductive system does not function in a
very optimal state. Not surprisingly, the imposition of chemicals
(or drugs) on this system can further interfere with a number of biological processes or events.
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GENERAL REPRODUCTIVE
BIOLOGY
rate during gametogenesis is called nondisjunction and can result
in gonadal agenesis. Klinefelter’s syndrome is characterized by
testicular dysgenesis with male morphology and an XXY karyotype; Turner’s syndrome includes ovarian agenesis with female
morphology (XO karyotype).
Hermaphroditism (true and pseudo) may occur secondary to
nondisjunction of sex chromosomes during the initial cleavage mitosis of the egg. Such a condition is usually due to an XY karyotype and sometimes to sex mosaics of XY/XX or XY/XO. Pseudohermaphrodites are characterized by secondary sex characteristics
that differ from those predicted by genotype.
Chemically induced nondisjunction is a common genetic abnormality. Nondisjunction of Y chromosomes may be detected by
the presence or absence of fluorescent bodies on the chromatin of
sperm (YFF spermatozoa). YFF sperm are increased in patients
treated with certain antineoplastic agents and irradiation.
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The developing gonad is very sensitive to chemical insult, with
some cellular populations being more vulnerable than others to an
agent’s toxic actions. Further, the developing embryo is uniquely
sensitive to changes in its environment, whether such changes are
caused by exposure to foreign chemicals or certain viruses. The
toxicologist must be mindful of the teratogenic potential of a chemical as well as be aware of its potential deleterious actions on maternal biochemical processes. The development of normal reproductive capacity may offer particularly susceptible targets for
toxins. Environmental factors might alter the genetic determinants
of gonadal sex, the hormonal determinants of phenotypic sex, fetal gametogenesis, and reproductive tract differentiation as well as
postnatal integration of endocrine functions and other processes essential for the propagation of the species. The effects of environmental agents on sexual differentiation and development of reproductive capacity are largely unknown. Of the chemicals that have
been studied, it is noteworthy that they possess a wide diversity in
molecular structure and that they may affect specific cell populations within the reproductive system.
Sexual Differentiation
An understanding of reproductive physiology requires consideration of the process of sexual differentiation or the pattern of development of the gonads, genital ducts, and external genitalia (cf.
Simpson, 1980; De La Chapelle, 1987; Goldberg, 1988).
The transformation of androgens into estrogens through the
aromatization occurs in the hypothalamus during fetal life; this is
one of the key events leading to sexual differentiation. During the
last decade, the increased evidence of disorders of male sexual differentiation (e.g., hypospadias, cryptorchidism, micropenis) has
raised the possibility that certain environmental chemicals might
be detrimental to normal male genital development in utero. Male
sexual differentiation is critically dependent on the physiologic action of androgens. Thus, an imbalance of the androgen/estrogen
ratio can affect sexual differentiation. Environmental xenoestrogens that mimic estrogens (e.g., certain herbicides, pesticides,
plasticizers, nonylphenols, etc.) or environmental antiandrogens
[e.g., p,p-DDE (the major metabolite of DDT) vinclozolin, linuron, etc.] that perturb endocrine balance might cause demasculinizing and feminizing effects in the male fetus. Disturbed male
sexual differentiation has, in some instances, been purported to be
caused by increased exposure to environmental xenoestrogens
and/or antiandrogens.
Gonadal Sex A testes-determining gene [sex-determining region
of the Y chromosome (SRY)] on the Y chromosome is responsible
for determining gonadal sex (cf. De La Chapelle, 1987, Swain and
Lovell-Badge, 1999). It converts undifferentiated gonads into a
testes. The organization of the gonadal anlage into the seminiferous or spermatogenic tubules of the male may be mediated by the
SRY gene. The testes produces two separate hormones: the müllerian inhibiting factor (MIF) and testosterone. Testosteroneinduced masculine differentiation is modulated by androgen receptors regulated by genes on the X chromosome. Alterations of
the sex chromosomes may be transmitted by either one of the
parents (gonadal dysgenesis) or may occur in the embryo itself.
Failure of the sex chromosomes of either of the parents to sepa-
675
Genotypic Sex The normal female chromosome complement is
44 autosomes and 2 sex chromosomes, XX. The two X chromosomes contained in the germ cells are necessary for the development of a normal ovary. Autosomes are also involved in ovarian
development, differentiation of the genital ducts, and external
genitalia characteristic of a normal female. This requires the involvement of a single X chromosome with genetic cellular events.
Generally, the second X chromosome of a normal XX female is
genetically inactive in nongonadal cells, although it has been shown
that the tip of the short arm of the chromosome is genetically active.
The Y chromosome is consistent with the male determinant.
The normal male has a chromosome complement of 44 autosomes
and 2 sex chromosomes, X and Y. An additional X chromosome
does not change the male phenotype conferred by the Y chromosome, but the gonads are often dysfunctional (Klinefelter’s syndrome, XXY genotype). Genetic coding on the X chromosome may
be involved in transforming the gonad into a testis.
The presence of chromatin material on the short (p) arm of
the (Yp) chromosome directs the development of the testes.
Chromatin material (Yq) on the long (q) arm directs the development of spermatogenesis.
Phenotypic (Genital) Sex During the early stages of fetal development, sexual differentiation does not require any known hormonal products. The differentiation of the genital ducts and the
external genitalia, however, requires hormones. The onset of testosterone synthesis by the male gonad is necessary for the initiation
of male differentiation. Although the testes are required in male
differentiation, the embryonic ovaries are not needed to attain the
female phenotype. Female characteristics develop in the absence
of androgen secretion.
Two principal types of hormones are secreted by the fetal
testes—an androgenic steroid responsible for male reproductive
tract development and a nonsteroid factor that causes regression of
the mullerian ducts. Sertoli cells are the likely source of MIF or
antimüllerian hormone (AMH). Leydig cell differentiation and regression correspond well with the onset and subsequent decline in
testosterone synthesis by the fetal testis. Thus, the embryonic testis
suppresses the development of the mullerian ducts, allows the development of the wolffian duct and its derivatives, and thereby imposes the male phenotype on the embryo.
Three periods for testosterone production are important to sexual differentiation. The first period occurs on days 14 to 17 of gestation in the rat and weeks 4 to 6 in the human. The second period
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and dihydrotestosterone. The testes also secrete small amounts of
estrogens. The ovaries, depending on the phase of the menstrual
cycle, secrete various amounts of estrogens and progesterone.
Estradiol is the principal steroid estrogen secreted by the ovary in
most mammalian species. The ovary is also the chief source of
progesterone. The corpus luteum and the placenta are also primary
sites of secretion of progesterone.
Gametogenic and secretory functions of either the ovary or
testes are dependent on the secretion of adenohypophyseal gonadotropins, follicle stimulating hormone (FSH), and luteinizing
hormone (LH). In the male, LH is also referred to as ICSH (interstitial cell–stimulating hormone). FSH in the female stimulates follicular development and maturation in the ovary. FSH in the male
stimulates the process of spermatogenesis. Sertoli cells are the target cells for the action of FSH in the testes of mammals. FSH receptors are present on the Sertoli cells, and the gene for the FSH
receptor is predominantly expressed in these cells (cf. Heckert and
Griswold, 1993). The secretion of pituitary FSH and LH is modulated by gonadal hormones. Sex steroids secreted by the testes or
ovaries regulate the secretion of pituitary gonadotropins. The Sertoli cell of the testes secretes small amounts of estrogen and a proteinaceous hormone called inhibin. Inhibin aids in the modulation
of spermatogenesis. ICSH (LH) provokes the process of steroidogenesis in the testes (cf. Herbert et al., 1995).
The onset of puberty results in the cyclic secretion of pituitary gonadotropins in the female. This establishes the normal menstrual cycle. In males, puberty is advanced by the continuous and
noncyclic secretion of gonadotropins.
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occurs about day 17 of gestation to about 2 weeks postnatal age
in the rat and from month 4 of pregnancy to 1 to 3 months of postnatal age in the human. The third period follows a long period of
testicular inactivity in both species until testosterone production is
reinitiated between 40 and 60 days of age in the rat and between
12 to 14 years of age in the human.
The dynamics of testosterone and dihydrotestosterone production and cellular interactions are an important prerequisite to
knowing which chemicals might affect sexual differentiation. Factors that reduce the ability of testosterone to be synthesized and
activated, to enter the cell, and/or to affect the cell nucleus’s ability to regulate the synthesis of androgen-dependent proteins would
have a potential to alter sexual differentiation. Some chemicals are
capable of exerting a testosterone-depriving effect on the developing systems. These include effects on the feedback regulation of
gonadotropin secretion, gonadotropin effectiveness, testosterone
and dihydrotestosterone synthesis, and plasma binding as well as
cytoplasmic receptor and nuclear chromatin binding.
Insufficient amounts of androgens can feminize the male fetus with otherwise normal testes and an XY karyotype. Slight deficiencies affect only the later stages of differentiation of the external genital organs and result in microphallus, hypospadias (the
urethra opens on undersurface of penis), and a valviform appearance of the scrotum with masculine general morphology. However,
a severe androgen deficiency (or resistance) allows the mullerian
system to persist and results in external genital organs of a female
type (vagina and uterus) that coexist with ectopic testes and normal male efferent ducts. A lack of androgen receptors can also lead
to a testicular feminization type of syndrome even when normal
levels of testosterone are present. Finally, sexual behavior also appears to be “imprinted” in the central nervous system by androgens from the testis and could be affected by endogenous and exogenous chemicals.
Estrogens exert an important developmental effect. Nearly 30
years have elapsed since the association between maternal DES administration and vaginal adenocarcinoma in female offspring was
reported. DES, a synthetic estrogen, was used extensively in the
treatment of both humans and livestock. Other nonsteroidal estrogens, namely, the insecticides chlordecone and DDT (and its
metabolites) exhibit uterotropic actions in experimental animals
(cf. Thomas, 1975). The estrogenicity of chlordecone was first described in workers at a pesticide-producing plant (Cohn et al.,
1978). Several so-called xenoestrogens — including herbicides,
fungicides, insecticides, and nematocides—have been identified
(cf. Colborn, et al., 1993). Similarly, polychlorinated biphenyls
(PCBs) are uterotropic. Zearalenone, a plant mycotoxin, also exhibits female sex hormone properties. The effects of environmental hormone-disrupters (namely xenoestrogens) are not restricted
to females. Indeed, vinclozolin, a dicarboximide fungicide, has
metabolites that can act as androgen antagonists (Kelce et al.,
1994).
GONADAL FUNCTION
Central Modulation
Regardless of gender, the gonads possess a dual function: an endocrine function involving the secretion of sex hormones and a
nonendocrine function or the production of germ cells (gametogenesis). The testes secrete male sex steroids, including testosterone
Testicular Function
There are several subpopulations of cells in the mammalian testes,
all of which are subject to some degree of local regulation (cf.
Maddocks, et al., 1990; Spiteri-Grech and Nieschlag, 1993). These
local regulatory factors include peptide growth factors, proopiomelanocortin derivatives, neuropeptides, and steroids (Table
20-1). There are many complex cell-to-cell communications, any
one of which could serve as a site for chemical or heavy metal perturbation. Many agents that can affect either spermatogenesis or
steroidogenesis can also affect leukocytes and other testesregulating factors produced by cells of the immune system (cf.
Murdoch, 1994). The paracrine or local regulation of testicular
function is an interesting concept, yet the nature of the testicular
architecture and the multiple interactions that can occur at various
cellular levels renders this biological system not only complex, but
very difficult to study from both a physiologic or a toxicologic
standpoint. Nevertheless, these local testicular factors are very important in modulating the paracrine control of the male gonad
(Table 20-2). Paracrine and autocrine factors from Sertoli and germ
cells are important in the functioning of both cell types (Griswold,
1995).
Apoptosis Apoptosis and necrosis constitute two distinct forms
of cell death that differ in morphology, mechanism, and incidence
(Fig. 20-1) (see also Chap. 10, “Developmental Toxicology”).
Necrosis includes membrane disruption, respiratory hypovia, membrane collapse, cell swelling, and rupture in pathologic tissue.
Apoptosis, on the other hand, describes the scattered, apparently
random cell deaths in normal cells (cf. Nakano, 1997). The ca-
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Table 20-1
Growth Factors Isolated from the Testis
PROPOSED MITOGENIC
GROWTH FACTOR
IGF-I
IGF-II
TGF-
TARGET IN THE TESTIS
Sertoli cells
Germ cells
Sertoli cells
Peritubular cells
Sertoli cells
Sertoli cells
NI
Sertoli cells
Peritubular cells
NI
NI
NI
Sertoli cells
Germ cells?
Rete testis fluid
Sertoli cells
Germ cells
NI
Germ cells
NI
NI
Sertoli cells?
NI
NI
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Inhibin
SGF
bFGF
TGF-
TESTICULAR ORIGIN
SCSGF
(TGF-?)
EGF
RTFGF
tlL-I
NGF-
KEY:
IGF I and II, insulin-like growth factors I and II; TGF, transforming growth factor; SCSGF, Sertoli cell secreted growth
factor; EGF, epidermal growth factor; RTFGF, rete testis fluid–derived growth factor; tlL-1, testicular interleukin-1–like
factor; NGF, nerve growth factor. NI, no information available.
SOURCES: Derived from Maddocks et al., 1990; Spiteri-Grech and Nieschlag, 1993; and Shioda et al., 1994.
pacity for cell “suicide” appears to be present in most, if not all
tissues to maintain a homeostatic state.
During the fertile phase of the life span of mammals, the function of the gonads is hormonally controlled. In addition to the proliferation of somatic and germ cells in the ovary and in the testes
during normal gonadal development, degeneration of gonadal cells
plays an important physiologic role and leads to a depletion of a
majority of germ cells in both sexes (cf. Billig, et al., 1996; Robertson and Orrenius, 2000). In the testes, morphologic signs of germ
cell degeneration during spermatogenesis were recognized nearly
100 years ago (Regaud, 1900).
The biochemical pathway(s) that cause apoptosis are not
known, but DNA lysis is involved (Thompson, 1994). Several specific genes/gene products have been associated with the apoptosis
that follows androgen depletion. For example, one prominent
marker is TRPM-2 (a sulfated glycoprotein-2, or “clusterin”),
which increases shortly after androgen removal. Likewise, transforming growth factor beta (TGF-) increases after androgen
removal. In the female, the role of TGF- in uterine apoptosis is
unclear.
There is increasing evidence suggesting that apoptosis rather
than necrosis predominates in many cytolethal toxic injuries
(Raffray and Cohen, 1997). Tissue selectivity of toxicants can stem
from the apoptotic or necrotic thresholds at which different cells
die.
Physiologically, apoptosis serves to limit the number of germ
cells in the seminiferous epithelium (cf. Billig et al., 1995). During
the process of spermatogenesis, clonal proliferation of germ cells
occurs during the many mitotic divisions, leading to a significantly
expanding germ cell population. Left unchecked, the number of
germ cells would quickly outgrow the supportive capacity of the
Sertoli cell. Hence, a delicate balance exists in the testes between
proliferation and apoptosis. About three-fourths of the potential
population of mature germ cells in the testes may be lost by active
elimination (DeRooÿ and Lok, 1987).
The Sertoli cell of the testes has been suggested to be a controlling factor in germ cell apoptosis (cf. Roberts, et al., 1997). Further, germ cell viability depends on Sertoli cell factors as well as
the intimate contact between these two testicular cell types. Sertoli
cells appear to regulate germ cell apoptosis directly through a
paracrine mechanism. The Sertoli cell may mediate the death of
fas-bearing germ cells via the expression of fasL on its cell
membrane.
A number of chemicals reportedly increase the incidence of
apoptosis in the rodent testes (Ku et al., 1995; Billig et al., 1995;
Richburg and Boekelheide, 1996; Nakagawa et al., 1997). In rats,
the Sertoli cell toxicant mono-2-ethylhexyl phthalate (MEHP)
causes an early and progressive detachment of germ cells from the
seminiferous epithelium (Richburg and Boekelheide, 1996). This
MEHP-induced distruption of the Sertoli cell–germ cell physical
interaction has been suggested to alter normal Sertoli cell–directed
regulation of germ cell apoptosis. Mitomycin C, an antibiotic that
inhibits DNA synthesis, induces apoptosis with fragmentation of
nuclear DNA in mouse spermatogenic cells, especially spermatogonia (Nakagawa et al., 1997). Why spermatogenic germ cells have
a relatively high level of apoptosis is unknown, but it could be a
mechanism for eliminating cells with abnormal chromosomes.
Spermatogenesis Spermatogenesis is a unique process in which
the timing and stages of differentiation are known with a considerable degree of certainty. The dynamics of the process of spermatogenesis as well as the kinetics have been studied extensively
and have recently been reviewed by Foote and Berndtson (1992).
In producing spermatozoa by the process of spermatogenesis, the
germinal epithelium plays a dual role: It must produce millions of
spermatozoa each day and also continuously replace the popula-
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Table 20-2
Local Factors Modulating the Function of Testicular Cells
Germ Cell (GC)
MODULATING
CELL OF
FACTORS
ORIGIN
(SC)†
(SC)
(SC)
(SC)
(LC)
CELL OF
FACTORS
ORIGIN
—
—
Inhibits
—
Stimulates
NGF
(SC)
ACTION*
—
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IGF-1
IL-1
Inhibin
SCF
Activin
SECRETORY
ACTION*
Leydig Cell (LC)‡
IGF-1
IL-1
Inhibin
SCF
TGF-
IGF-1
TGF-
TGF-
(SC)
(SC)
(SC)
(SC)
(SC)
(PT)
(PT)
(PT)
-Endorphin
ACTH
CRF
Testosterone
Activin
Testosterone
Oxytocin
Activin
Stimulates
Stimulates/inhibits?
Stimulates
Stimulates
Inhibits
Stimulates
Inhibits
Inhibits
(SC)
(SC)
(SC)
(SC)
(SC)
(PT)
(PT)
(PT)
Inhibits
Stimulates
—
Stimulates
?
Stimulates
Stimulates
Stimulates
(SC)
Stimulates
(PT)
(PT)
(PT)
(GC)
(GC)
(GC)
(LC)
(LC)
(LC)
(LC)
(LC)
(LG)
(LC)
(LC)
Stimulates
Inhibits
Inhibits
—
—
Stimulates
Stimulates/inhibits?
Stimulates
Inhibits
Stimulates/inhibits?
Stimulates
Stimulates
Stimulates
Inhibits
Peritubular Cell (PT)
TGF-
Testosterone
Oxytocin
IGF-1
(SC)
(LC)
(LC)
(Serum)
Stimulates
Stimulates
Stimulates
—
P-MOD-S
Sertoli Cell (SC)§
NGF
P-MOD-S
ACTH
MSN
CRH
Testosterone
-endorphin
(GC)
(PT)
(LC)
(LC)
(LC)
(LC)
(LC)
TGF-
TGF-
NGAG
IGF-1
IL-1
SGF
LHRH
SCF
TGF-
IL-1-
IGF-1
Inhibin
-FGF
Estrogen
Stimulates
Stimulates
Stimulates
Stimulates
—
Stimulates
Inhibits
*Stimulates denotes known stimulatory action; inhibits denotes known inhibitory action on respective cell(s).
†Letters in parentheses denote cell of origin of the various factor (e.g., SC, Sertoli cell; GC, germ cell, etc.).
‡Leydig cells (LC) are also influenced by serum-derived factors (e.g., glucocorticoids, ANF, etc.), ICSH, and autoregulatory factors (e.g., estrogen, angiotensis II, -endorphin,
CRF, etc.).
§Sertoli cells (SC) are also influenced by serum-derived factors (e.g., retinol, EGF, insulin, etc.), FSH, and autoregulatory factor (e.g., IGF-1, -FGF, etc.).
SOURCE: Thomas, 1995a, with permission.
tion of cells that give rise to the process, the spermatogonia (cf.
Amann, 1989).
Sperm are among the smallest cells in humans, where its
length is about 50 m or only about one-half the diameter of the
ovum, the largest cell of the female organism. The relative volume
of a sperm is about 1/100,000 that of the egg. The sperm has a
head, a middle piece, and a tail, which correspond, respectively, to
the following functions: activation and genetics, metabolism, and
motility.
Whereas only a few hundred human ova are released as cells
ready for fertilization in a life-time, millions of motile sperm are
formed in the spermatogenic tubules each day. Several physiologic
factors affect the regulation of sperm motility (e.g., spermine, spermidine, “quiescence” factor, cAMP, motility stimulating factor)
(Lindemann and Kanous, 1989).
Spermatogenesis starts at puberty and continues almost
throughout life. The primitive male germ cells are spermatogonia,
which are situated next to the basement membrane of the seminif-
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679
Figure 20-1. Comparison of cellular events during apoptosis and necrosis. (Modified from Raffray and Cohen, 1997, with permission.)
erous tubules. Following birth, spermatogonia are dormant until
puberty, when proliferative activity begins again. The onset of
spermatogenesis accompanies functional maturation of the testes.
Two major types of spermatogonia are present—type A, which
generates other spermatogonia, and type B, which becomes a mature sperm. The latter type develops into primary spermatocytes,
which undergo meiotic divisions to become secondary spermatocytes. The process of meiosis results in the reduction of the normal complement of chromosomes (diploid) to half this number
(haploid) (Fig. 20-2). Meiosis ensures the biologic necessity of evo-
lution through the introduction of controlled variability. Each gamete must receive one of each pair of chromosomes. Whether it
receives the maternal or paternal chromosome is a matter of chance.
In the male, meiosis is completed within several days. In the female, meiotic division begins during fetal life but is then suspended
until puberty. Meiosis may be the most susceptible stage for chemical insult (cf. Herbert, et al., 1995).
Secondary spermatocytes give rise to spermatids. Spermatids
complete their development into sperm by undergoing a period of
transformation (spermiogenesis) involving extensive nuclear and
Figure 20-2. Cellular replication (mitosis) and cellular reductive divisions (meiosis) involved in spermatogenesis, oogenesis, and fertilization.
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ries of cellular associations to appear at one point within a tubule
is termed the duration of the cycle of the seminiferous epithelium.
The duration of one such cycle depends on the cell turnover rate
of spermatogonia and is thus equal to it. Thus, the duration of the
cycle of seminiferous epithelium varies among mammals, being a
low of about 9 days in the mouse to a high of about 16 days in humans (Table 20-3) (Galbraith, et al., 1982). Spermatids, emanating
from spermatogonia committed to differentiate approximately 4.5
cycles earlier, are continuously released from the germinal
epithelium.
Maturation changes occur in the sperm as they traverse along
the tubules of the testes and the epididymides. During this passage,
sperm acquire the capacity for fertilization and become more
motile. There is a progressive dehydration of the cytoplasm, decreased resistance to cold shock, changes in metabolism, and variations in membrane permeability. Each ejaculate contains a spectrum of normal sperm as well as those that are either abnormal or
immature.
Normalcy of spermatogenesis can be evaluated from two
standpoints: the number of spermatozoa produced per day and the
quality of spermatozoa produced. The number of spermatozoa produced per day is defined as daily sperm production (Amann, 1981).
The efficiency of sperm production is the number of sperm produced per day per gram of testicular parenchyma. The efficiency
of sperm production in humans is only about 20 to 40 percent of
that in other mammals (Amann, 1986). Sperm production in a
young man is about 7 million sperm per day per gram; by the fifth
to ninth decade of life, it drops to approximately one-half or about
3.5 million per day per gram (cf. Johnson, 1986).
Blazak et al. (1985) — using several parameters including
sperm production, sperm number, sperm transit time, and sperm
motility—have provided an assessment of the effects of chemicals
on the male reproductive system. These authors concluded that
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cytoplasmic reorganization. The nucleus condenses and becomes
the sperm head; the two centrioles give rise to the flagellum or axial filament. Part of the Golgi apparatus becomes the acrosome,
and the mitochondria concentrate into a sheath located between
two centrioles.
Seminiferous tubules contain germ cells at different stages of
differentiation and also Sertoli cells. In a cyclic fashion, spermatogonia A of certain areas of a tubule become committed to divide synchronously, and the cohorts of the resulting cells differentiate in unison. Thus, a synchronous population of developing
germ cells occupies a defined area within a seminiferous tubule.
Cells within each cohort are connected by intercellular bridges.
The anatomic relationships of the mammalian testes reveal
that the process of spermatogenesis occurs within the seminiferous tubules (Fig. 20-3). The germ cells, along with the Sertoli cells,
are contained within the membranous boundaries of the seminiferous tubules. Conversely, the Leydig cells are situated in the interstitium or outside the seminiferous tubules. Several different
species display a single cellular association of the seminiferous
tubules; in humans, however, such cellular associations differ and
are intermingled in a mosaic-like pattern. Several cellular associations, varying among species, may be detected. Each cellular association contains four or five types of germ cells organized in a
specific, layered pattern. Each layer represents one cellular generation. Fourteen cellular associations are observed in the seminiferous epithelium in the rat (LeBlond and Clermont, 1952; Heller
and Clermont, 1964).
Presuming a fixed point within the seminiferous tubule could
be viewed in the developing germ cell, there would be a sequential appearance of each of these cellular associations that would be
characteristic of the particular species. This progression through
the series of cellular associations would continue to repeat itself in
a predictable fashion. The interval required for one complete se-
Figure 20-3. Schematic cross section of seminiferous tubules of testes.
Morphology of the Sertoli cell along with the cellular events involved in spermatogenesis (spermatogonium
through spermatid).
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Table 20-3
Criteria for Spermatogenesis in Laboratory Animals and Humans
RABBIT
(NEW
Duration of cycle of seminiferous
epithelium (days)
MOUSE
RAT
8.6
12.9
ZEALAND
DOG
MONKEY
WHITE)
(BEAGLE)
(RHESUS)
10.7
13.6
9.5
HUMAN
16.0
1.5
4.7
8.3
1.7
3.5
Fraction of a lifespan as:
B-type spermatogonia
Primary spermatocyte
Round spermatid
0.11
1.00
0.41
0.10
1.00
0.40
0.08
1.00
0.43
Testes weight (g)
0.2
3.7
6.4
Daily sperm production:
Per gram testis (106/g)
Per male (106)
28
5
24
86
25
160
20
300
23
1100
4.4
125
49
440
1600
?*
5700
420
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Life span of:
B-type spermatogonia (days)
L Z spermatocytes (days)
P D spermatocytes (days)
Golgi spermatids (days)
Cap spermatids (days)
Sperm reserves in caudia
(at sexual rest: only 106)
Transit time (days) through
(at sexual rest):
Caput corpus epididymides
Cauda epididymides
KEY:
3.1
5.6
2.0
7.8
12.2
2.9
5.0
1.3
7.3
10.7
2.1
5.2
3.0
5.1
3.0
9.7
4.0
5.2
13.5
6.9
3.0
2.9
6.0
9.5
1.8
3.7
0.19
1.00
0.48
12.0
?
?
6.3
9.2
15.6
7.9
1.6
0.19
1.00
0.35
0.25
1.00
0.38
49
34
4.9
5.6
1.8
3.7
L, leptotene; Z, zygotene; P, pachytene; D, diplotene.
*A question mark indicates unclear or inadequate data.
SOURCE: Galbraith WM, Voytek P, Ryon MG: Assessment of Risks to Human Reproduction and to Development of the Human Conceptus from Exposure to Environmental
Substances. Oak Ridge, TN: Oak Ridge National Laboratory, U.S. Environmental Protection Agency, 1982. Available as order number DE82007897 from the National
Technical Information Service, Springfield, VA.
testes weights and epididymal sperm numbers were unreliable indicators of sperm production.
Sertoli Cells The Sertoli cell is now recognized as playing an
important role in the process of spermatogenesis (cf. Foster, 1992;
Griswold, 1995). In early fetal life, the Sertoli cells secrete antimüllerian hormone (AMH). Their exact physiologic role is not understood, but after puberty, these cells begin to secrete the hormone
inhibin, which may aid in modulating pituitary FSH.
Germ cell development occurs in close association with the
Sertoli cells, which provide them with structural support, nutrients,
and regulatory/paracrine factors. Brinster and Zimmerman (1994)
described the interaction between germ cells and Sertoli cells
within the seminiferous tubules. Recent experiments indicate that
spermatogenesis can be restored in infertile testes through germ
cell tranplantation (Ogawa et al., 1999).
The Sertoli cell junctions form the blood-testis barrier that
partitions the seminiferous epithelium into a basal compartment
containing spermatogonia and early spermatocytes and an adluminal compartment containing more fully developed spermatogenic
cells. An ionic gradient is maintained between the two tubular compartments. Nutrients, hormones, and other chemicals must pass either between or through Sertoli cells in order to diffuse from one
compartment to another. Germinal cells are found either between
adjacent pairs of Sertoli cells or inside their luminal margin (see
Fig. 20-4).
Sertoli cells secrete a number of hormones and/or proteins.
These secretory products can be used to measure Sertoli function
in the presence of chemical insult. The Sertoli cells secrete tissue
plasminogen activator, androgen-binding protein (ABP), inhibin,
AMH, transferrin, and other proteases. ABP is a protein similar to
plasma sex steroid–binding globulin (SSBG). In rodents, ABP acts
as a carrier for testosterone and dihydrotestosterone. Sertoli cells
probably synthesize estradiol and estrone in response to FSH
stimulation.
Normal spermatogenesis requires Sertoli cells. Many chemicals affecting spermatogenesis act indirectly through their effect
on the Sertoli cell [e.g., dibromochloropropane (DBCP), monoethylhexyl phthalate (MEHP)] rather than directly on the germ
cells. Tetrahydrocannabinol (THC) acts at several sites in the reproductive system, including the Sertoli cell, where it acts by inhibiting FSH-stimulated cAMP accumulation (Heindel and Keith,
1989).
Interstitium (Leydig Cells) The Leydig or interstitial cells are
the primary sites of testosterone synthesis (Fig. 20-5) (cf. Ewing,
1992). These cells are closely associated with the testicular blood
vessels and the lymphatic space. The spermatic arteries to the testes
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4/18/01
Figure 20-4. Cellular sites of action of chemical or drugs in the testes.
Secretion may occur to both the adluminal and basal compartments in the seminiferous tubules. The type, amount,
and detection of Sertoli cell secretion may be influenced by the stage of testicular development endocrine status and the stage of cycle of the seminiferous epithelium (Griswold, 1995). Note the anatomic proximity of the
Sertoli cell and the germ cell. See also Tables 20-1 and 20-2 for various hormonal and growth factor interactions between the various subpopulations of testicular cells.
are tortuous; their blood flows parallel to blood in the pampiniform plexus of the spermatic veins but in the opposite direction
(Fig. 20-5). This anatomic arrangement seems to facilitate a countercurrent exchange of heat, androgens, and other chemicals.
LH stimulates testicular steroidogenesis (Zirkin and Chen,
2000). Androgens are essential to spermatogenesis, epididymal
sperm maturation, the growth and secretory activity of accessory
sex organs, somatic masculinization, male behavior, and various
metabolic processes. Surprisingly, there are a large number of di-
verse chemicals/drugs that can cause Leydig cell hyperplasia/neoplasia. This chemically induced proliferation of Leydig cells is particularly evident in the rodent (Table 20-4) (Thomas, 1995b; Cook
et al., 1999).
A number of nongenotoxic agents can produce Leydig cell
hyperplasia in rats, mice, and dogs (Cook et al., 1999). Androgen
receptor antagonists (e.g., flutamide), 5-reductase inhibitors
(e.g., finasteride), testosterone biosynthesis inhibitors (e.g., cimetidine, metronidazole, vinclozolin, etc.), aromatase inhibitors (e.g.,
formestane), dopamine agonists (e.g., mesulergine), estrogen
agonists/antagonists (e.g., DES, tamoxifen, etc.), and GnRH agonists (e.g., leuprolide, etc.) are all capable of producing Leydig cell
hyperplasia. It is obvious that they exhibit vastly different modes
of action. Agents that produce Leydig cell hyperplasia in experimental animals can also be grouped according to their chemical
activity (e.g., antihypertensives, calcium channel blockers, fungicides, goitrogens, etc.) as well as by their chemical class (e.g.,
flurochemicals, nitroaromatics, organochlorines, etc.). Nongenotoxic compounds that induce Leydig cell tumors in rats most likely
have little relevance to humans under most exposure conditions
because humans are quantitatively less sensitive than rats (Cook et
al., 1999).
Posttesticular Processes
Figure 20-5. Schematic representation of secretory elements from the
Sertoli cell.
Extratesticular sites include the epididymis and the endothelial cells of the
pampiniform plexus.
The end product of testicular gametogenesis is immature sperm.
Posttesticular processes involve ducts that move maturing sperm
from the testis to storage sites where they await ejaculation. A number of secretory processes exist that control fluid production and
ion composition; secretory organs contribute to the chemical composition (including specific proteins) of the semen.
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Table 20-4
Chemicals/Drugs Causing Leydig Cell Hyperplasia/Neoplasia in Rodents
AGENT CLASS OR BIOLOGIC ACTIVITY
Cadmium
Estrogen
Linuron
S0Z-200-110, isradine
Flutamide
Gemfibrozil
Finasteride
Cimetidine
Hydralazine
Carbamazepine
Vidarabine
Mesulegine
Clomiphene
Perfluoroctanoate
Dimethylformide
Diethylstilbestrol
Nitrosamine
Methoxychlor
Oxolinic acid
Reserpine
Metronidazole
Cyclophosphamide
Methylcholanthrene
Heavy metal
Hormone
Herbicide
Calcium channel blocker
Antiandrogen
Hypolidemic agent
5-reductase inhibitor
Histamine (H2) receptor blocker
Antihypertensive agent
Anticonvulsant/analgesic
Antiviral agent
Dopamine (D2) agonist-antagonist
Treatment of infertility
Industrial ingredient (plasticizers, lubricant/wetting agent(s)
Industrial use (tannery & leathergoods, metal dyes)
Synthetic hormone
Industrial uses
Pesticide with estrogenic properties
Antimicrobial agent
Antihypertensive
Antiprotozoal
Antineoplastic
Experimental carcinogen
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AGENT/CHEMICAL/DRUG
SOURCES:
Ewing, 1992; Bosland, 1994; Prentice et al., 1992; Thomas, 1995b; Cook et al., 1999.
Efferent Ducts The fluid produced in the seminiferous tubules
moves into a system of spaces called the rete testis. The chemical
composition of the rete testis fluid is unique and has a total protein concentration much lower than that of the blood plasma. The
efferent ducts open into the caput epididymis.
Although the rete testis fluid normally contains inhibin, ABP,
transferrin, myoinositol, steroid hormones, amino acids, and various enzymes, only ABP and inhibin appear to be specific products
and useful indicators of the functional integrity of the seminiferous epithelium or Sertoli cells (Mann and Lutwak-Mann, 1981).
However, relative concentrations of other constituents may indicate alterations in membrane barriers or active transport processes.
The concentration of chemicals in the rete testis fluid relative to
unbound plasma concentration has been used to estimate the
permeability of the blood-testis barrier for selected chemicals
(Okumura et al., 1975).
Epididymides The epididymis is a single, highly coiled duct
measuring approximately 5 m in humans. It is arranged anatomically into three parts called the caput, the corpus, and the cauda
epididymides (cf. Cooper, 1986).
From the rete testis, testicular fluid first enters efferent ducts
and then the epididymides. Here the sperm are subjected to a changing chemical environment as they move through the organ.
The first two sections together (the caput and the corpus) are
regarded as making up that part of the epididymis involved with
sperm maturation, whereas the terminal segment (the cauda) is regarded as the site of sperm storage. There are, however, differences
in the position and extent of the segments in various species of
mammals.
From 1.8 to 4.9 days are required for sperm to move through
the caput to the corpus epididymis, where maturation takes place.
In contrast, the transit time for sperm through the cauda epididymis
in sexually rested males differs greatly among species and ranges
from 3.7 to 9.7 days. Average sperm transit time for a 21- to
30-year-old man is 6 days. The number of sperm in the caput and
corpus epididymis is similar in sexually rested males and in males
ejaculating daily. The number of sperm in the cauda epididymis is
more variable, being lower in sexually active males.
Active transport processes affect the amount of fluid flowing
through the epididymis. Because much of the fluid produced by
the testis is apparently absorbed in the epididymis, the relative concentration of sperm is increased.
Hence, important functions of the epididymis are reabsorption of rete testis fluid, metabolism, epithelial cell secretions, sperm
maturation, and sperm storage. The chemical composition of the
epididymal plasma plays an important role in both sperm maturation and sperm storage. Environmental chemicals perturb these
processes and can produce adverse effects.
Accessory Sex Organs The anatomic relationship of accessory
sex organs in the male rodent is depicted in Fig. 20-6. Most mammals possess seminal vesicles (exceptions: cats and dogs) and most
have prostate glands. However, the physiologic and anatomic characteristics of the prostate gland may vary considerably among
mammals (Wilson, 1995).
The seminal plasma functions as a vehicle for conveying the
ejaculated sperm from the male to the female reproductive tract.
This plasma is produced by the secretory organs of the male reproductive system, which, along with the epididymides, include the
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and boars is ejaculated in much larger quantities. Sperm move from
the distal portion of the epididymis through the vas deferens (ductus deferens) to the urethra. Vasectomy is the surgical removal of
the vas deferens or a portion of it. The semen of some animals, including rodents and humans, tends to coagulate on ejaculation. The
clotting mechanism (e.g., “copulatory plug”) involves enzymes and
substrates from different accessory organs.
Although all male mammals have prostates, the organ differs
anatomically, physiologically, and chemically among species, and
lobe differences in the same species may be pronounced. The rat
prostate is noted for its complex structure and its prompt response
to castration and androgen stimulation. The human prostate is a
tubuloalveolar gland made up of two prominent lateral lobes that
contribute about one-third of the ejaculate.
Prostatic secretion in humans and many other mammalian
species contains acid phosphatase, zinc, and citric acid. The prostatic secretion is the main source of acid phosphatase in human
semen; its concentration provides a convenient method for assessing the functional state of the prostate. The human prostate also
produces spermine. Prostate-specific antigen (PSA) is a 33-kDa
protein synthesized primarily by the prostatic epithelium (Polascik
et al., 1999). It is a tumor marker for prostate cancer. Certain proteins and enzymes (acid phosphatase, -glutamyl transpeptidase,
glutamicoxaloacetic transaminase), cholesterol, inositol, zinc, and
magnesium have also been proposed as indicators of prostatic secretory function. Radioactive zinc (65Zn) uptake by rodent prostate
glands has been used as an index for androgenic potency (Gunn
and Gould, 1956). An ionic antagonism exists between zinc and
cadmium. Cadmium can induce metallothionein in the prostate
glands of experimental animals (Waalkes et al., 1982; cf. Waalkes,
et al., 1992).
The anatomic structure of the seminal vesicle varies among
animals. The seminal vesicle is a compact glandular tissue arranged
in the form of multiple lobes that surround secretory ducts. Like
the prostate, the seminal vesicle is responsive to androgens and is
a useful indicator of Leydig cell function. The vesicular glands can
be used as a gravimetric indicator for androgens.
In humans, the seminal vesicle contributes about 60 percent
of the seminal fluid. The seminal vesicles also produce more than
half of the seminal plasma in laboratory and domestic animals such
as the rat, guinea pig, and bull. In the human, bull, ram, and boar
(but not the cat), most of the seminal fructose is secreted by the
seminal vesicles; consequently, in these species the chemical assay of fructose in semen is a useful indicator of the relative contribution of the seminal vesicles to whole semen. Seminal vesicle
secretion is also characterized by the presence of proteins and enzymes, phosphorylcholine, and prostaglandins. PSA occurs in high
concentration in seminal fluid.
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Figure 20-6. Anatomic relation of components of rodent sex accessory
glands.
D.D., ductus deferens; B.L., bladder; V.P., ventral prostate; L.P., lateral
prostate; C.G., coagulating gland (also called the anterior prostate); S.V.,
seminal vesicle; D.P., dorsal prostate. (From Hayes, 1982, with permission.)
prostate, seminal vesicles, bulbourethral (Cowper’s) glands, and
urethral (Littre’s) glands. Any abnormal function of these organs
can be reflected in altered seminal plasma characteristics. Seminal
plasma is normally an isotonic, neutral medium, which, in many
species, contains sources of energy such as fructose and sorbitol,
that are directly available to sperm. Functions of the other constituents, such as citric acid and inositol, are not known. In general, the secretions from the prostate and seminal vesicles contribute little to fertility (Mann and Lutwak-Mann, 1981).
The accessory sex organs are androgen-dependent. They serve
as indicators of the Leydig cell function and/or androgen action.
The weights of the accessory sex glands are an indirect measure
of circulating testosterone levels. The ventral prostate of rats has
been used to study the actions of testosterone and to investigate
the molecular basis of androgen-regulated gene function.
Human semen emission initially involves the urethral and
Cowper’s glands, with the prostatic secretion and sperm coming
next and the seminal vesicle secretion delivered last. There is a
considerable overlap between the presperm, sperm-rich, and
postsperm fractions. Therefore even if an ejaculate is collected in
as many as six (split ejaculate) fractions, it is rarely possible to obtain a sperm-free fraction consisting exclusively of prostatic or
vesicular secretions.
Acid phosphatase and citric acid are markers for prostatic
secretion; fructose is an indicator for seminal vesicle secretion. It
is estimated that about one-third of the entire human ejaculate is
contributed by the prostate and about two-thirds by the seminal
vesicles. Both the vas deferens and the seminal vesicles apparently
synthesize prostaglandins. Semen varies both in volume and composition between species. Human, bovine, and canine species have
a relatively small semen volume (1 to 10 mL); semen of stallions
Erection and Ejaculation These physiologic processes are controlled by the central nervous system (CNS) but are modulated by
the autonomic nervous system. Parasympathetic nerve stimulation
results in dilatation of the arterioles of the penis, which initiates
an erection. Erectile tissue of the penis engorges with blood, veins
are compressed to block outflow, and the turgor of the organ
increases. In the human, afferent impulses from the genitalia and
descending tracts, which mediate erections in response to erotic
psychic stimuli, reach the integrating centers in the lumbar segments of the spinal cord. The efferent fibers are located in the pelvic
splanchnic nerves (Andersson and Wagner, 1995).
Ejaculation is a two-stage spinal reflex involving emission and
ejaculation. Emission is the movement of the semen into the ure-
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thra; ejaculation is the propulsion of the semen out of the urethra
at the time of orgasm. Afferent pathways involve fibers from receptors in the glans penis that reach the spinal cord through the internal pudendal nerves. Emission is a sympathetic response effected
by contraction of the smooth muscle of the vas deferens and seminal vesicles. Semen is ejaculated out of the urethra by contraction
of the bulbocavernosus muscle. The spinal reflex centers for this
portion of the reflex are in the upper sacral and lowest lumbar segments of the spinal cord; the motor pathways traverse the first to
third sacral roots of the internal pudendal nerves.
Little is known concerning the effects of chemicals on erection or ejaculation (Woods, 1984). Pesticides, particularly the
organophosphates, are known to affect neuroendocrine processes
involved in erection and ejaculation. Many drugs act on the autonomic nervous system and affect potency (Table 20-5) (see also
Papadopoulas, 1980; Buchanan and Davis, 1984; Stevenson and
Umstead, 1984; Keene and Davies, 1999). Impotence, the failure
to obtain or sustain an erection, is rarely of endocrine origin; more
often, the cause is psychological. The occurrence of nocturnal or
early-morning erections implies that the neurologic and circulatory
pathways involved in attaining an erection are intact and suggests
the possibility of a psychological cause.
Normal penile erection depends upon the relaxation of smooth
muscles in the corpora cavernosa. In response to sexual stimuli,
cavernous nerves and endothelial cells release nitric oxide, which
stimulates the formation of cyclic guanosine monophosphate
(GMP) by guanylate cyclase. The drug sildenafil (Viagra) is used
to treat erectile dysfunction; its mechanism of action resides in its
ability to selectively inhibit cGMP-specific phosphodiesterase
type 5. By selectively inhibiting cGMP catabolism in cavernosal
smooth muscle cells, sildenafil restores the natural erectile response
(cf. Goldstein, et al., 1998; Lu, 2000).
685
Ovarian Function
Oogenesis Ovarian germ cells with their follicles have a dual origin; the theca or stromal cells arise from fetal connective tissues
of the ovarian medulla, the granulosa cells from the cortical mesenchyme (Fig, 20-7).
Figure 20-7. Schematic representation of ovarian morphology.
About 400,000 follicles are present at birth in each human
ovary. After birth, many undergo atresia, and those that survive are
continuously reduced in number. Any agent that damages the
oocytes will accelerate the depletion of the pool and can lead to
reduced fertility in females. About one-half of the number of
oocytes present at birth remain at puberty; the number is reduced
to about 25,000 by 30 years of age. About 400 primary follicles
will yield mature ova during a woman’s reproductive life span.
During the approximately three decades of fecundity, follicles in
various stages of growth can always be found. After menopause,
follicles are no longer present in the ovary.
Follicles remain in a primary follicle stage following birth until puberty, when a number of follicles start to grow during each
ovarian cycle. However, most fail to achieve maturity. For the
follicles that continue to grow, the first event is an increase in size
of the primary oocytes. During this stage, fluid-filled spaces appear among the cells of the follicle, which unite to form a cavity
or antrum, otherwise known as the graafian follicle.
Primary oocytes undergo two specialized nuclear divisions,
which result in the formation of four cells containing one-half the
Table 20-5
Drug-Induced Impotence
AGENT
Narcotics
Morphine
Ethanol
Psychotropics
Chlorpromazine
Diazepam
Tricyclic antidepressants
MAO inhibitors
Hypotensives
Methyldopa
Clonidine
Reserpine
Guanethidine
Hormones/antagonists
Estrogens
Cyproterone
KEY:
CNS
ANS
ENDO
?
?
CNS, central nervous system; ANS, autonomic nervous system; ENDO, endocrine.
Millar, 1979, Buchanan and Davis, 1984.
SOURCES:
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number of chromosomes (Fig. 20-2). The first meiotic division occurs within the ovary just before ovulation, and the second occurs
just after the sperm fuses with the egg. In the first stage of meiosis, the primary oocyte is actively synthesizing DNA and protein
in preparation for entering prophase. The DNA content doubles as
each of the prophase chromosomes produces its mirror image. Each
doubled chromosome is attracted to its homologous mate to form
tetrads. The members of the tetrads synapse or come to lie side by
side. Before separation, the homologous pairs of chromosomes exchange genetic material by a process known as crossing over. Thus,
qualitative differences occur between the resulting gametes. Subsequent meiotic stages distribute the members of the tetrads to the
daughter cells in such a way that each cell receives the haploid
number of chromosomes. At telophase, one secondary oocyte and
a polar body have been formed, which are no longer genetically
identical.
The secondary oocyte enters the next cycle of division very
rapidly; each chromosome splits longitudinally; the ovum and the
three polar bodies now contain the haploid number of chromosomes and half the amount of genetic material. Although the nuclei of all four eggs are equivalent, the cytoplasm is divided unequally. The end products are one large ovum and three rudimentary
ova (polar bodies), which subsequently degenerate. The ovum is
released from the ovary at the secondary oocyte stage; the second
stage of meiotic division is triggered in the oviduct by the entry of
the sperm.
Ovarian Cycle The cyclic release of pituitary gonadotropins involving the secretion of ovarian progesterone and estrogen is depicted in Fig. 20-8. These female sex steroids determine ovulation
and prepare the female accessory sex organs to receive the male
sperm. Sperm, ejaculated into the vagina, must make their way
through the cervix into the uterus, where they are capacitated.
Sperm then migrate into the oviducts, where fertilization takes
place. The conceptus then returns from the oviducts to the uterus
and implants into the endometrium.
Postovarian Processes
Female accessory sex organs function to bring together the ovulated ovum and the ejaculated sperm. The chemical composition
and viscosity of reproductive tract fluids, as well as the epithelial
morphology of these organs, are controlled by ovarian (and trophoblastic) hormones.
Oviducts The oviducts provide the taxis of the fimbria, which is
under muscular control. The involvement of the autonomic nervous system in this process, as well as in oviductal transport of both
the male and female gametes, raises the possibility that pharmacologic agents known to alter the autonomic nervous system may
alter function and therefore fertility.
Uterus Uterine endometrium reflects the cyclicity of the ovary
as it is prepared to receive the conceptus. The myometrium’s major
role is contractile. In primates, at the end of menstruation, all but
the deep layers of the endometrium are sloughed. Under the influence of estrogens from the developing follicle, the endometrium
increases rapidly in thickness. The uterine glands increase in length
but do not secrete to any degree. These endometrial changes are
called proliferative. After ovulation, the endometrium becomes
slightly edematous, and the actively secreting glands become
Figure 20-8. Hormonal regulation of menstrual function.
FSH, follicle stimulating hormone; GnRH, gonadotropin releasing hormone: LH, luteinizing hormone.
tightly coiled and folded under the influence of estrogen and progesterone from the corpus luteum. These are secretory (progestational) changes (Fig. 20-8).
When fertilization fails to occur, the endometrium is shed and
a new cycle begins. Only primates menstruate. Other mammals
have a sexual or estrus cycle. Female animals come into “heat” (estrus) at the time of ovulation. This is generally the only time during which the female is receptive to the male. In spontaneously
ovulating species (e.g., rodents), the endocrine events are comparable with those in the menstrual cycle. In the rabbit, ovulation is
a reflex produced by copulation.
Cervix The mucosa of the uterine cervix does not undergo cyclic
desquamation, but there are regular changes in the cervical mucus.
Estrogen, which makes the mucus thinner and more alkaline, promotes the survival and transport of sperm. Progesterone makes the
mucus thick, tenacious, and cellular. The mucus is thinnest at the
time of ovulation and dries in an arborizing, fernlike pattern on a
slide. After ovulation and during pregnancy, it becomes thick and
fails to form the fern pattern. Disruptions of the cervix may be expressed as disorders of differentiation (including neoplasia), disturbed secretion, and incompetence. Exfoliative cytologic (Papanicolaou’s stain) and histologic techniques are currently used to
assess disorders of differentiation. Various synthetic steroids (e.g.,
oral contraceptives) can affect the extent and pattern of cervical
mucus.
Vagina Estrogen produces a growth and proliferation of vaginal
epithelium. The layers of cells become cornified and can be readily identified in vaginal smears. Vaginal cornification has been used
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togen, estrogen, and progesterone, which are needed to achieve independence from the ovary in maintaining the pregnancy. Rapid
proliferation of the cytotrophoblast serves to anchor the growing
placenta to the maternal tissue.
The developing placenta consists of proliferating trophoblasts,
which expand rapidly and infiltrate the maternal vascular channels.
Shortly after implantation, the syncytiotrophoblast is bathed by maternal venous blood, which supplies nutrients and permits an exchange of gases. Histotrophic nutrition involves yolk sac circulation; hemotrophic nutrition involves the placenta. Placental
circulation is established quite early in women and primates and
relatively much later in rodents and rabbits. Interestingly, placental dysfunction due to vascular compromise caused by cocaine
leads to increased fetal risk, causing growth retardation and
prematurity. Fetal loss due to abruptio placentae may occur (cf.
Doering, et al., 1989).
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as an index for estrogens. Progesterone stimulation produces a thick
mucus and the epithelium proliferates, becoming infiltrated with
leukocytes. The cyclic changes in the vaginal smear in rats are easily recognized. The changes in humans and other species are similar but less apparent. Analysis of vaginal fluid or cytologic studies of desquamated vaginal cells (quantitative cytochemistry)
reflects ovarian function. Vaginal sampling of cells and fluid might
offer a reliable and easily available external monitor of internal
function and dysfunction. Alteration in vaginal flora can indicate
a toxicologic condition associated with the use of vaginal tampons
[namely toxic shock syndrome (TSS)].
Fertilization During fertilization, the ovum contributes the maternal complement of genes to the nucleus of the fertilized egg and
provides food reserves for the early embryo. The innermost of the
egg is the vitelline membrane. Outside the ovum proper lies a thick,
tough, and highly refractile capsule termed the zona pellucida,
which increases the total diameter of the human ovum to about
0.15 mm. Beyond the zona pellucida is the corona radiata, derived
from the follicle; it surrounds the ovum during its passage in the
oviduct.
Formation, maturation, and union of a male and female germ
cell are all preliminary events leading to a combined cell or zygote.
Penetration of ovum by sperm and the coming together and pooling
of their respective nuclei constitute the process of fertilization.
Only minutes are required for the sperm to penetrate the zona
pellucida after passing through the cumulus oophorus in vitro, and
probably less in vivo. The sperm traverse along a curved oblique
path. Entering the perivitelline space, the sperm head immediately
lies flat on the vitellus; its plasma membrane fuses with that of the
vitellus and then embeds into the ovum. The cortical granules of
the egg disappear, the vitellus shrinks, and the second maturation
division is reinitiated, which results in extrusion of the second polar body. A specific factor in the ovum appears to trigger the development of the male pronucleus; the chromatin of the ovum
forms a female pronucleus.
As syngamy approaches, the two pronuclei become intimately
opposed but do not fuse. The nuclear envelopes of the pronuclei
break up; nucleoli disappear, and chromosomes condense and
promptly aggregate. The chromosomes mingle to form the
prometaphase of the first spindle, and the egg divides into two blastomeres. From sperm penetration to first cleavage usually requires
about 12 h in laboratory animals.
From a single fertilized cell (the zygote), cells proliferate and
differentiate until more than a trillion cells of about a hundred different types are present in the adult organism.
Implantation The developing embryo migrates through the
oviduct into the uterus. Upon contact with the endometrium, the
blastocyst becomes surrounded by an outer layer or syncytiotrophoblast, a multinucleated mass of cells with no discernible boundaries, and an inner layer of individual cells, the cytotrophoblast.
The syncytiotrophoblast erodes the endometrium, and the blastocyst implants. Placental circulation is then established and trophoblastic function continues. The blastocysts of most mammalian
species implant about day 6 or 7 following fertilization. At this
stage, the differentiation of the embryonic and extraembryonic (trophoblastic) tissues is apparent.
Trophoblastic tissue differentiates into cytotrophoblast and
syncytiotrophoblast cells. The syncytiotrophoblast cells produce
chorionic gonadotropin, chorionic growth hormones, placental lac-
687
Placentation Morphologically, the placenta may be defined as
the fusion or opposition of fetal membranes to the uterine mucous
membrane (cf., Slikker and Miller, 1994). In humans, the placenta
varies considerably throughout gestation. The integral unit of the
placenta is the villous tree. The core of the villous tree contains
the fetal capilaries and associated endothelium.
Placentation varies considerably among various domestic animals, experimental animals, and primates (Slikker and Miller,
1994). Humans and monkey possess a hemochorial placenta. Pigs,
horses, and donkeys have an epitheliochorial type of placenta,
whereas sheep, goats, and cows have a syndesmochorial type of
placenta. In laboratory animals (e.g., rat, rabbit, and guinea pig),
the placenta is termed a hemoendothelial type. Among the various
species, the number of maternal and fetal cell layers ranges from
six (e.g., pig, horse) to a single one (e.g., rat, rabbit). Primates, including humans, have three layers of cells in the placenta that a
substance must pass across. Thus, the placentas of some species
are “thicker” than others.
Generally, the placenta is quite impermeable to chemicals/drugs with molecular weights of 1000 Da or more. Most medications have molecular weights of 500 Da or less. Hence, molecular size is rarely a factor in denying a drug’s entrance across the
placenta and into the embryo/fetus. Placental permeability to a
chemical is affected by placental characteristics including thickness, surface area, carrier systems, and lipid-protein concentration
of the membranes. The inherent characteristics of the chemical itself, such as its degree of ionization, lipid solubility, protein binding, and molecular size also affect its transport across the placenta.
INTEGRATIVE PROCESSES
Hypothalamo-Pituitary-Gonadal Axis
FSH and LH are glycoproteins synthesized and released from a
subpopulation of the basophilic gonadotropic cells of the pituitary
gland. Hypothalamic neuroendocrine neurons secrete specific releasing or release-inhibiting factors into the hypophyseal portal system, which carries them to the adenohypophysis, where they act
to stimulate or inhibit the release of anterior pituitary hormones.
Luteinizing hormone-releasing hormone (LHRH) acts on gonadotropic cells, thereby stimulating the release of FSH and LH.
LHRH and follicle stimulating hormone-releasing hormone
(FSHRH) appear to be the same substance. Native and synthetic
forms of LHRH stimulate the release of both gonadotrophic hor-
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Puberty
rate of secretion of LHRH, resulting in increases in LH. As puberty approaches, a pulsatile pattern of LH and FSH secretion is
observed. The gonad itself is not required for activating FSH or
LH at the onset of puberty. It is a CNS phenomenon. Female puberty is affected by a wide range of influences including climate,
race, heredity, athletic activity, and degree of adiposity.
SEXUAL BEHAVIOR AND LIBIDO
Physiologic processes that account for sexual behavior are poorly
understood. The external environment greatly affects sexual behavior, and libido components of reproductive activity depend on
a close interplay between neural and endocrine events. For example, a correlation of behavior and receptivity for insemination is
attained by complex neuroendocrine mechanisms involving the
brain, the pituitary, and sex steroid hormones. This complexity
varies even among higher vertebrates. Thus, in reproductive studies involving rodents, the investigator must determine whether the
animals actually mate. In the rat, this can be determined by inspecting females each day for vaginal plugs. The number of mountings, thrusts, and ejaculations each can be quantified as indicators
of reproductive behavior. It is also important to determine whether
the male animal mounts females or other males. If the male copulates and is still sterile, indicators of male fertility such as testicular function should be considered. Failure to copulate suggests either a neuromuscular and/or behavioral defect in the experimental
animal.
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mones; thus, it has been proposed to call this compound gonadotropin-releasing hormone (GnRH).
The neuroendocrine neurons have nerve terminals containing
monoamines (norepinephrine, dopamine, serotonin) that impinge
on them. Reserpine, chlorpromazine, and monoamine oxidase
(MAO) inhibitors modify the content or actions of brain
monoamines that affect gonadotropins.
FSH probably acts primarily on the Sertoli cells, but it also
appears to stimulate the mitotic activity of spermatogonia. LH
stimulates steroidogenesis. A defect in the function of the testis
(in the production of spermatozoa or testosterone) will tend to be
reflected in increased levels of FSH and LH in serum because of
the lack of the “negative feedback” effect of testicular hormones
(Fig. 20-9).
The hypothalamo-pituitary-gonadal feedback system is a very
delicately modulated hormonal process. Several sites in the endocrine process can be perturbed by drugs (e.g., oral contraceptives) and by different chemicals (Fig. 20-9). Gonadotoxic agents
may act on neuroendocrine processes in the brain or they may act
directly on the target organ (e.g., gonad). Toxicants that adversely
or otherwise alter the hepatic and/or renal biotransformation of
endogenous sex steroid might be expected to interfere with the
pituitary feedback system (cf. Cooper, et al., 1998).
From the early newborn period to the onset of puberty, the testes
remain hormonally dormant. After birth, the androgen-secreting
Leydig cells in the mammalian fetal testes become quiescent, and
a period follows in which the gonads of both sexes await final maturation of the reproductive system.
The onset of puberty begins with secretion of increasing levels of gonadotropins. The physiologic trigger for puberty is poorly
understood, but somehow a hypothalamic gonadostat changes the
GENERAL TOXICOLOGIC/
PHARMACOLOGIC PRINCIPLES
Many of the principles that govern absorption, distribution, metabolism, and excretion of a chemical or drug also apply to the reproductive system. There are, however, some rather unique barriers that affect a chemical’s action on the mammalian reproductive
system. The maternal-fetal interface occurring at the placenta represents a barrier to chemicals coming in contact with the developing embryo. Unfortunately, the placenta is not so restrictive as to
prevent most chemicals from crossing the placenta. Most chemicals are not denied entrance into a number of compartments or secretions of the reproductive tract. Indeed, xenobiotic and certain
drugs can be readily detected in uterine secretions, in milk of the
lactating mother, and in seminal fluid (Mann and Lutwak-Mann,
1981). No specialized barriers appear to prevent chemicals or drugs
from acting on the ovary. Several drugs are known to interfere with
ovarian function (Table 20-6) (Gorospe and Reinhard, 1995).
Unlike the female gonad, the male gonad has a somewhat specialized barrier. This specialized biological barrier is referred to as the
blood–testis barrier.
Blood–Testis Barrier
Figure 20-9. Hormonal relationship between the adenohypopysealhypothalamic-gonadal axis.
Inhibitory actions () and stimulatory actions () are depicted along with
sites of chemical/drug perturbation (Large black arrows).
There are a number of specialized anatomic barriers in the body.
Tissue permeability barriers include the blood–brain barrier, the
blood–thymus barrier, and the blood–bile barrier. Important barriers within the endocrine system are the placental barrier and the
blood–testis barrier. The blood–testis barrier is situated somewhere
between the lumen of an interstitial capillary and the lumen of a
seminiferous tubule (Neaves, 1977). Several anatomically related
features intervene between the two luminal spaces, including the
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689
Table 20-6
Inhibitors of Steroidogenic Enzymes
INHIBITOR
Cholesterol side chain
cleavage
Aromatase
Aminoglutethimide, 3-methoxybenzidine, cyanoketone,
estrogens, azastene, danazol
4-Acetoxy-androstene-3,17-dione,
4-hydroxy-androstene-3,17-dione,
1,4,6-androstatriene-3,17-dione,
6-bromoandrostene-3,17-dione,
7(4amino)phenylthioandrostenedione,
-testolactone, fenarimol,* MEHP†
Danazol, metyrapone, furosemide and other diuretics‡
Danazol, spironolactone
Danazol, spironolactone
Danazol, spironolactone
Danazol
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ENZYME
11-Hydroxylase
21-Hydroxylase
17-Hydroxylase
17,20-Desmolase
17-Hydroxysteroid
dehydrogenase
3-Hydroxysteroid
dehydrogenase
c-17-L-20-lyase
Danazol
Ketoconozole§
*See Hirsch et al., 1987.
†See Davis et al., 1989.
‡See Bicikova et al., 1996.
§See Effendy and Krause, 1989.
SOURCE: Modified from Haney, 1985, with permission.
capillary endothelium, capillary basal lamina, lymphatic endothelium, myoid cells, basal lamina of the seminiferous tubule, and Sertoli cells. The barrier that impedes or denies the free exchange of
chemicals/drugs between the blood and the fluid inside the seminiferous tubules is located in one or more of these structures. The
apparent positioning of distances relative to transepithelial permeability can affect the passage (or blockage) of a substance through
the blood–testis barrier. These epithelial cell anatomic relationships can affect the tightness of fit between cells and the extent to
which a chemical’s passage can occur. Such junctions or cell unions
are often leaky and may allow for a substance’s passage. These socalled gap junctions may even be less developed in the immature
or young mammalian testes, hence affording greater opportunities
for foreign chemicals to permeate the seminiferous tubule.
Steinberger and Klinefelter (1993) have developed a twocompartment model for culturing testicular cells that similates a
blood–testis barrier. This culture model has been proposed to study
Sertoli cell and Leydig cell dysfunction in vitro.
Setchell and coworkers (1969) first demonstrated that immunoglobulins and iodinated albumin, inulin, and a number of
small molecules were excluded from the seminiferous tubules by
the blood–testis barrier. Dym and Fawcett (1970) suggested that
the primary permeability barrier for the seminiferous tubules was
composed of the surrounding layers of myoid cells while specialized Sertoli cell-to-Sertoli cell junctions within the seminiferous
epithelium constituted a secondary cellular barrier. Certain classes
of adhesive molecules (e.g., E- and N-cadherin, - and -catenin,
plakoglobin, etc.) may act to promote Sertoli-to-Sertoli cell
adhesion and tight junction formation (Byers, et al., 1994) (see also
Fig. 20-4).
Okumura and coworkers (1975) quantified permeability
rates for nonelectrolytes and certain chemicals/drugs. Low-
molecular-weight molecules (e.g., water, urea) can readily cross
the blood–testis barrier; larger-sized substances (e.g., inulin) are
impeded. The degree of lipid solubility and ionization are important determinants as to whether a substance can permeate the
blood – testis barrier. A number of factors are known to affect
the permeability of the blood–testis barrier, including ligation of
the efferent ductules, autoimmune orchiditis, and vasectomy (cf.
Sundaram and Witorsch, 1995).
Biotransformation of Exogenous
Chemicals
Testes The mammalian gonad is capable of metabolizing a host
of foreign chemicals that have traversed the blood–testis barrier.
While mixed-function oxidases and epoxide-degrading enzymes
may not be as active as hepatic systems, they are nevertheless
present in the testes. Cytochrome P450, in general, is quite sensitive to the effects of a number of chemicals. Gonadal cytochrome
P450 is no exception. Arylhydrocarbon hydroxylase (AHH) is present in testicular microsomes (Lee et al., 1981). Consequently, the
pathways for steroidogenesis contain a number of enzymes that are
affected by chemicals or drugs (Table 20-6). Like the process of
steroidogenesis in the gonads, the adrenal cortex is also vulnerable to chemical insult (cf. Colby, 1988). Both the parent compound
and its metabolite(s) can adversely affect the gonad (Table 20-7).
Whether biotransformation occurs gonadally or extragonadally, the
end result can be interference with spermatogenesis and/or
steroidogenesis. Their mechanisms of toxicity vary considerably.
The microsomal ozidation of n-hexane yields 2,5-hexanedione
(2,5-HD). N-hexane, an environmental toxicant, causes peripheral
polyneuropathy and testicular atrophy (Boekelheide, 1987, 1988).
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Table 20-7
Biotransformation of Drugs, Chemicals, and Their Metabolites—Ability to Exert Toxic
Actions on the Male Gonad
PARENT COMPOUND
REFERENCE
Desethylamiodarone
Holt et al., 1984
N-Methyletetrazolethiol*
Comereski et al., 1987
Isomers of 2-ethyl hexanol
(?)†
Mono-ethylhexyl phthalate
and 2-ethyl hexanol (?)†
Dichloropropene(s)
derivatives
(?)†
2-Methoxyacetaldehyde
Ritter et al., 1987
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Amiodarone
(antiarrhythmic drug)
Cephalosporin analogs
(antimicrobial drug)
Valproic acid
(antiepileptic drug)
Diethylhexyl phthalate
(DEHP; plasticizer)
Dibromochloropropane‡
(DBCP; fungicide)
METABOLITE
Ethylene glycol monoethyl
ether
(industrial solvent)
n-Hexane
(environmental toxicant)
Acrylamide
(industrial use)
Vinclozolin
(fungicide)
Thomas et al., 1982
Torkelson et al., 1961
Foster et al., 1986
2,5-Hexanedione
Boekelheide, 1987
N-Methylacrylamide,
N-isopropylacrylamide
Butanoic acid derivative
and an enanilide
metabolite
Sakamoto and
Hashimoto, 1986
Kelce et al., 1994
*Only substituent is a testicular toxin, not cephalosporin.
†Questionable testicular toxin but probably teratogenic.
‡Radiometabolites of (3H)-DBCP are not preferentially labeled in the testes.
SOURCES: Modified from Thomas and Ballantyne, 1990; Shemi et al., 1987.
The testicular toxicity is separate from its neurotoxicity. 2,5-HD
produces gonadal toxicity by altering testicular tubulin. The HD
testicular toxicity results from alterations in Sertoli cell microtubules and the altered microtubules result from pyrole-dependent
cross-linking (cf. Li and Heindel, 1998). HD toxicity is slow in
onset. Initially, HD affects the cross-linking of cytoskeletal elements leading to altered protein secretions and trafficking in the
Sertoli cell. Consequently, there is altered Sertoli cell–germ cell
contacts and a loss of Sertoli cell paracrine support of the germ
cells (Richburg, et al., 1994; Li and Heindel, 1998).
Ethylene glycol monoethyl ether, along with its metabolites,
is a gonadal toxin (Nagano et al., 1979; Wang and Chapin, 2000).
Metabolites such as 2-methoxy-ethanol (2-ME) and 2-ethoxyethanol (2-EE) induce testicular toxicity. They may cause testicular atrophy, decreased sperm motility, and an increased incidence of abnormal sperm. Most likely, the metabolism of
monoalkyl glycol ethers occurs via alcohol and aldehyde dehydrogenases, leading to the formation of methoxyacetic acid (MAA).
MAA is believed to be the ultimate toxic metabolite of 2-ME. However, methoxyacetaldehyde (MALD), an intermediate metabolite
of 2-ME, can also produce testicular lesions (Foster et al., 1986;
Feuston et al., 1989). Although the site of action was thought to
be upon the late spermatocyte, it now appears that the Sertoli cells
are the prime target for 2-ME (cf. Li and Heindel, 1998). It is unclear whether the mechanism of testicular toxicity of 2-ME induces
germ cell death by reducing the available purine bases causing decreased RNA synthesis in spermatocytes or that 2-ME induces
germ cell death by interfering with interregulating signal trans-
duction pathways within either Sertoli cells or germ cells, causing
a disruption of cell-to-cell communication (Li and Heindel, 1998).
Dinitrobenzene (DNB) or 1,3 dinitrobenzene can cause testicular toxicity in rats. The toxicity is species- and age-dependent
and can be partially reversible (cf. Li and Heindel, 1998). DNB
causes vacuolization of the Sertoli cells and the detachment of germ
cells (Foster et al., 1992). The mechanism of toxicity of DNB is
unclear, but both Sertoli cells and germ cells may be affected.
Several heavy metals are known to adversely affect testicular
function (cf. Thomas, 1995a). Cadmium causes testicular toxicity,
which consists of a loss of endothelial tight junctional barriers,
leading to edema, increased fluid pressure, ischemia, and tissue
necrosis. Cadmium’s effect on Sertoli cell tight junctions (namely
the blood–tubule barriers) may be due to its actions on the actin
filaments associated with these junctions (cf. Li and Heindel,
1998). Like other Sertoli toxicants (e.g., HD, DNB, 2-ME, and
phthalates), testicular toxicity is age-related. Some species are more
sensitive than others. Cadmium-induced capillary toxicity (e.g.,
pampiniform plexus) leads to necrosis and ischemia. Cadmium, at
least at low doses, appears to be a stage-specific Sertoli cell
toxicant.
Esters of o-phthalic acid (phthalate esters or PAEs) are used
extensively in medical devices and other consumer products as
plasticizers. Because the PAEs are not convalently bound to the
plastic, they can leach into the environment (cf. Thomas and
Thomas, 1984). There are several different PAEs exhibiting varying degrees of testicular toxicity, particularly in rats and mice.
Diethyl hexyl phthalate (DEHP) and its metabolite monoethyhexyl
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damage to pachytene and dividing spermatocytes and round spermatids in rats (Lloyd et al., 1988). Ethane dimethane sulfonate
(EDS) effectively eradicates Leydig cells and endogenous testosterone (cf. Bremner, et al., 1994).
Metabolites of cephalosporin reportedly cause testicular toxicity in rats (Comereski et al., 1987). Testicular degeneration from
analogs of cephalosporin is most likely to occur with cefbuperazone, cefamandole, and cefoperazone. Cyclosporine can also inhibit testosterone biosynthesis in the rat testes (Rajfer et al., 1987).
Amiodarone and its desethyl metabolite can be detected in high
concentrations in the testes and semen, but their effects on spermatogenesis or sperm motility are not known (Holt et al., 1984).
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phthalate (MEHP) cause early sloughing of spermatids and spermatocytes and severe vacuolization of Sertoli cell cytoplasm. Gray
and Beamand (1984) proposed that the mechanism of DEHPinduced testicular atrophy involves a membrane alteration leading
to separation of germ cells (spermatocytes and spermatids) from
the underlying Sertoli cells. The action of MEHP has been attributed to its ability to reduce FSH binding to Sertoli cell membranes
(Grasso, et al., 1993). The separation of spermatocytes and spermatids interferes with the transfer of nutrients from the Sertoli cells,
leading to death and disintegration of the germ cells. MEHP, and
not DEHP, is most likely the proximate testicular toxicant (Albro,
et al., 1989). MEHP increases germ cell detachment from the Sertoli cell. MEHP is the only phthalate monoester that reduced Sertoli cell ATP levels. It specifically inhibits FSH-stimulated cAMP
accumulation in Sertoli cell cultures (cf. Li and Heindel, 1998).
The collapse of vimentin filaments in Sertoli cells by MEHP appears to lead to a loss of Sertoli–germ cell contacts (Richburg and
Boekelheide, 1996). Finally, DEHP is not only a reproductive toxicant in the male (e.g., rodents) but it can significantly suppress
preovulatory follicle granulosa cell estradiol production (Davis et
al., 1994a).
Many other chemicals can produce testicular toxicity, but less
information is generally available about their mechanism(s) than
some of the more well-studied Sertoli cell toxicants (e.g., HD,
DNB, PAE, cadmium, and glycol ethers). Vinclozolin, a fungicide
that undergoes biotransformation, produces at least two major
metabolites that can effectively act as antagonists to the androgen
receptor (Kelce et al., 1994). Epichlorohydrin, a highly reactive
electrophile used in the manufacture of glycerol and epoxy resins,
produces spermatozoal metabolic lesions (cf. Toth et al., 1989).
Tri-o-cresyl phosphate (TOCP), an industrial chemical used as a
plasticizer in lacquers and varnishes, decreases epididymal sperm
motility and density. TOCP interferes with spermatogenic
processes and sperm motility directly and not via an androgenic
mechanism or decreased vitamin E (Somkuti et al., 1987).
The male reproductive system can be adversely affected by
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (cf. Bjerke and
Peterson, 1994; Zacharewski and Safe, 1998). TCDD can alter
germ cells at all developmental stages in the testes (Chahoud et al.,
1992). Dioxin can reduce Leydig cell volume, but at doses that do
not appear to affect spermatogenesis (Johnson et al., 1992). In rodents, TCDD is both embryotoxic and teratogenic (cf. Dickson and
Buzik, 1993). TCDD has an avidity for the estrogen receptor (cf.
Hruska and Olson, 1989) and other receptors (e.g., Ah receptor).
Recently, the safety assessment of the polychlorinated biphenyls
(PCBs), with particular reference to reproductive toxicity, has been
reviewed (Battershill, 1994; Birnbaum, 1998).
2-Methoxylethanol (2-ME), an industrial solvent, is toxic to
both the male and female reproductive system (Mebus et al., 1989).
2-ME must be metabolized to 2-methoxyacetic acid (2-MAA) by
alcohol and aldehyde dehydrogenases in order to attain its testicular toxicity. All stages of spermatocyte development and some
stages of spermatid development are affected by 2-ME, but it seems
to be more selective in destroying early- and late-stage pachytene
primary spermatocytes. 2-ME is also embryotoxic and teratogenic
in several species (Hanly et al., 1984). 2-ME (also known as methyl
cellosolve) when applied dermally can produce a decline in epididymal sperm and testicular spermatid counts in rats (Feuston et
al., 1989). Ethanol also causes delayed testicular development and
may affect the Sertoli cell and/or the Leydig cell (Anderson et al.,
1989). Trifluoroethanol and trifluoroacetaldehyde produce specific
691
Ovary Like the testes, the ovary has the metabolic capability to
biotransform certain exogenous substrates. Furthermore, the
process of ovarian steroidogenesis, like that of the testes and the
adrenal cortex (cf. Colby, 1988), is susceptible to different agents
that interfere with the biosynthesis of estrogens (see Table 20-6).
Less is known about how chemicals or drugs interfere with ovarian metabolism. The ovary has not been studied as extensively because of its more difficult and complex hormonal relationships.
Nevertheless, several chemotherapeutic agents can inhibit ovarian
function (Table 20-8). Recently, Faustman et al. (1989) have studied the toxicity of direct-acting alkylating agents on rodent embryos. Their findings failed to reveal any specific structure/activity patterns among various alkylating agents. Like the testes,
mixed-function oxidases and various cytochrome systems are
found in the ovary. Primordial oocyte toxicity as well as toxicity
at other sites can be affected by certain chemicals or drugs (Haney,
1985).
DNA Repair
Alkylating Agents Depending on the species, there are varying
degrees of capacity for spermatogenic cells to repair DNA damage
due to environmental toxicants (Lee, 1983). It is well known that
ultraviolet and x-rays can damage DNA molecules; lethal mutation
(i.e., cell deaths) and mutation resulting from transformed cells can
also occur. Spermatogenic cells can be used to study unscheduled
DNA synthesis (Dixon and Lee, 1980). Unscheduled DNA repair
in spermatogenic cells is dose- and time-dependent. Spermiogenic
cells are less able to repair DNA damage resulting from alkylating
agents. This DNA repair system provides a protective mechanism
from certain toxicants; it is also a sensitive index of chromosome
damage.
Drug-induced unscheduled DNA synthesis in mammalian
oocytes reveals that female gametes possess an excision repair capacity (Pedersen and Brandriff, 1980). Unlike mature sperm, the
Table 20-8
Chemotherapeutic Agents and Ovarian Dysfunction
Prednisone
Vincristine
Vinblastine
6-Mercaptopurine
Nitrogen mustard
Cyclophosphamide
Chlorambucil
SOURCES:
Copyright © 2001 by The McGraw-Hill Companies
Busulfan
Methotrexate
Cytosine arabinoside
L-Asparginase
5-Fluorouracil
Adriamycin
Haney, 1985, with permission. See also Gorospe and Reinhard, 1995.
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mature oocyte maintains a DNA repair ability. However, this ability decreases at the time of meiotic maturation.
TARGETS FOR CHEMICAL
TOXICITY
CNS
The gonads are also targets for a host of drugs and chemicals
(Table 20-11) (Chapman, 1983; Thomas and Keenan, 1986). The
majority of these agents are representatives of major chemical
classes of cancer chemotherapeutic agents, particularly the alkylating agents. A number of endocrine agents are of value in the
treatment of certain cancers. Antiestrogens (e.g., tamoxifen), aromatase inhibitors (e.g., aminoglutethimide), GnRH agonists and
antagonists, and antiandrogens (e.g., flutamide) can interfere with
the endocrine system (cf., Lonning and Lien, 1993). Procarbazine,
an antineoplastic drug, causes severe damage to the acrosomal
plasma membrane and the nucleus of the sperm head in hamsters
(Singh et al., 1989). Alkylating agents are effective against rapidly
dividing cells. Not surprisingly, the division of germ cells is also
affected, leading to arrest of spermatogenesis.
Different cell populations of the mammalian testis exhibit
somewhat different thresholds of sensitivity to different toxicants
(Fig. 20-5). Thus, the germ cells are most sensitive to chemical insult (i.e., spermatogenesis). The Sertoli cells possess a somewhat
intermediate sensitivity to chemical inhibition; Leydig cells are
quite resistant to environmental toxicants. Cell-specific testicular
toxicants have been employed to evaluate the distribution of creatine in the rete testis (Moore et al., 1992). Creatine is associated
with cells of the seminiferous epithelium: elevated urinary excretion of creatine may provide a noninvasive marker for testicular
toxicity in vivo.
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Lead Different occupations can result in varying degrees of chromosomal aberration (Table 20-9). In particular, lead toxicity can
induce a variety of chromatid and chromosome breaks. Lead is one
of the earliest substances associated with deleterious effects on the
reproductive system (Thomas and Brogan, 1983). Lead poisoning
has been associated with reduced fertility, miscarriages, and stillbirth since antiquity (Lancranjan et al., 1975). Lead salts are among
the oldest known spermicidal agents; lead has long been known to
be an abortifacient (cf. Hildebrand et al., 1973). Lead exposure
results in a general suppression of the hypothalamic-pituitarytesticular axis in rats (Klein et al., 1994) and possibly in men occupationally exposed to this heavy metal (Rodamilans et al., 1988)
(Table 20-10).
Gonads
There are several sites of interference by chemicals upon the mammalian reproductive system (Fig. 20-9). Drugs and chemicals can
act directly on the CNS, particularly the hypothalamus and the adenohypophysis (cf. Cooper et al., 1998). A number of drugs (e.g.,
tranquilizers, sedatives, etc.) can modify the CNS, leading to alterations in the secretion of hypothalamic-releasing hormones
and/or gonadotropins. Synthetic steroids (namely 19-nortestosterones) are very effective in suppressing gonadotropin secretion
and hence block ovulation.
Sertoli Cells (See also “Biotranformation of Exogenous
Chemicals—Testes,” above.) Dibromochloropropane (DBCP), a
fungicide, causes infertility in a number of species, including hu-
Table 20-9
Occupational Exposure to Lead and Its Relationship to Chromosomal Aberrations
EXPOSED SUBJECTS
Positive findings
Lead oxide factory workers
Chemical factory workers
Zinc plant workers
Blast-furnace workers, metal
grinders, scrap workers
Battery plant workers and lead
foundry workers
Lead oxide factory workers
Battery melters, tin workers
Ceramic, lead, and battery
workers
Smelter workers
Battery plant workers
TYPE OF ABERRATION
Chromatid and chromosome breaks
Chromatid gaps, breaks
Gaps, fragments, rings, exchanges,
dicentrics
Gaps, breaks, hyperploidy, structural
abnormalities
Gaps, breaks, fragments
Chromatid and chromosome aberrations
Dicentrics, rings, fragments
Breaks, fragments
Gaps, chromatid and chromosome
aberrations
Chromatid and chromosome aberrations
Negative findings
Policemen
Lead workers
Shipyard workers
Smelter workers
Volunteers (ingested lead)
Children (near a smelter)
SOURCE:
Thomas and Brogan, 1983, with permission.
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CHAPTER 20 TOXIC RESPONSES OF THE REPRODUCTIVE SYSTEM
Table 20-10
Some Actions of Lead on the Male Reproductive System
SPECIES
EFFECT
Mouse
Mouse
Human
SOURCE:
Infertility
Germinal epithelial damage
Oligospermia and testicular degeneration
Decreased sperm motility and prostate
hyperplasia
Infertility
Abnormal sperm
Teratospermia, hypospermia, and
asthenospermia
Bell and Thomas, 1980, with permission.
mans. DBCP causes sterility, but it may do so by acting through
the Sertoli cell. DBCP may also inhibit sperm carbohydrate metabolism at the NADH dehydrogenase step in the mitochondrial
electron transport chain (Greenwell et al., 1987). Despite DBCP’s
propensity to cause degeneration of the seminiferous tubules, toxicokinetic studies fail to reveal any preferential uptake by the testes
(Shemi et al., 1987). DBCP gonadotoxicity appears to be genderspecific, since only testicular injury has been reported; it does not
cause comparable adverse effects in the female rat (Shaked et al.,
1988). Analogs of DBCP cause testicular necrosis as well as DNA
damage in the rat (Soderlund, et al., 1988).
The production of lactate and pyruvate are indicators of Sertoli cell function (Williams and Foster, 1988). Either dinitrobenzene (DNB) or mono-(2-ethylhexyl)phthalate (MEHP) can affect
lactate (and pyruvate) production by rat Sertoli cell cultures. The
Sertoli cell appears to be a prime target for the toxic actions of
DNB (Blackburn, et al., 1988). Chapin et al. (1988) have also indicated that MEHP adversely affects the mitochondria of the Sertoli cell in vitro. Likewise, dinitrotoluene (DNT) has a locus of
toxic action that is the Sertoli cell (Bloch et al., 1988). Dinitrobenzene initially damages Sertoli cells with a subsequent degeneration and exfoliation of germ cells.
Steroidogenesis Steroid biosynthesis can occur in several endocrine organs including the adrenal cortex, ovary, and the testes.
Other peripheral tissues and the CNS contain enzymatic systems
Table 20-11
Drugs That Are Gonadotoxic in Humans
MALES
FEMALES
Busulfan
Chlorambucil
Cyclophosphamide
Nitrogen mustard
Doxorubicin
Corticosteroids
Cytosine-arabinoside
Methotrexate
Procarbazine
Vincristine
Vinblastine
Busulfan
Chlorambucil
Cyclophosphamide
Nitrogen mustard
SOURCE:
also capable of steroid synthesis. Pregnenolone is the common precursor of all steroid hormones produced by the adrenal cortex (e.g.,
mineratocorticoids and glucocorticoids), the ovary (e.g., estrogens
and progesterone), and the testes (e.g., androgens). Specific subpopulations of cells in the mammalian gonad are capable of synthesizing steroids. In the ovary, the granulosa cells secrete estrogens in response to FSH. The thecal cells of the ovary secrete
progesterone (as does the corpus luteum) (see Fig. 20-7). In the
testes, the Leydig cell (or the interstitial cell) in response to LH
(or ICSH) secretes androgens (e.g., testosterone and dihydrotestosterone).
Several drugs, hormones, and chemicals can affect steroidogenesis (see Table 20-7) by interfering or inhibiting specific enzymes (see also “Biotransformaton of Exogenous Chemicals,”
above). Also, anti-LH peptides can affect Leydig cell steroidogenesis. LHRH analogs (e.g., buserelin) can interfere with both ovarian and testicular function (Donaubauer et al., 1987).
The liver and the kidney contain enzyme systems that affect
the biological half-life of steroids and other hormones. Hence,
xenobiotics that interfere with excretory processes might be expected to alter the endocrine system. For example, a number of hepatic steroid hydroxylases can be induced by either organophosphates or organochlorine pesticides. Such hydroxylation reactions
can be expected to render the endogenous steroid more polar and
hence more readily excreted by the kidney.
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Rat
Rat
Rat
Rat
693
EVALUATING REPRODUCTIVE
CAPACITY
A number of hormone assays are available to assess endocrine function (Thomas and Thomas, 2001). The endocrine system of the
female is more complex and dynamic than that of the male. Hence,
evaluating reproductive function in the female is more difficult.
Immediate distinctions must also be made between the pregnant
and the nonpregnant female. Regardless of gender, both behavioral
and physiologic factors must be considered in evaluating reproductive toxicity. The physiologic events involved in reproduction
involve inherent time factors that are species-specific. Often, evaluating the potential of a chemical or drug to affect the reproductive system is costly and time-consuming. Furthermore, many of
the endpoints used to evaluate the reproductive system are not always reliable and have limitations (Table 20-12).
The fact that such a wide variety of chemicals and drugs can
perturb the reproductive system adds another dimension of difficulty in attempting to evaluate reproductive toxicity. Not only is
there considerable diversity in chemical configuration of the toxicant, but sites and mechanisms of action can be very different. It
is obvious that several classes of therapeutic agents can affect both
the male and the female reproductive systems.
TESTING MALE REPRODUCTIVE
CAPACITY
General Considerations
Vinblastine
Chapman, 1983, with permission.
A host of tests have been used or proposed for evaluating the male
reproductive system (Table 20-13). Several cellular sites or
processes are vulnerable to chemical and/or drug insult. Perturbation of many of the endocrine or biochemical events associated
with the male reproductive system seldom occurs after a single exposure to a toxicant(s). Rather, multiple exposure extended over
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Table 20-12
Advantages and Limitations of Standard Reproductive Procedures
ENDPOINT
LIMITATIONS
VALUE
Insensitive
Testicular histology
Testis weights
Subjective; not quantitative
Less sensitive than sperm
counts; affected by edema
Integrates all reproductive
functions
Information on target cell
Rapid; quantitative
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Fertility
SOURCE:
Meistrich, 1989, with permission.
Table 20-13
Potentially Useful Tests of Male Reproductive Toxicity for Laboratory Animals
and/or Humans*
Testis
Size in situ
Weight
Spermatid reserves
Gross and histologic evaluation
Nonfunctional tubules (%)
Tubules with lumen sperm (%)
Tubule diameter
Counts of leptotene spermatocytes
Epididymis
Weight and histology
Number of sperm in distal half
Motility of sperm, distal end (%)
Gross sperm morphology, distal
end (%)
Detailed sperm morphology, distal
end (%)
Biochemical assays
Accessory sex glands
Histology
Gravimetric
Semen
Total volume
Gel-free volume
Sperm concentration
Total sperm/ejaculate
Total sperm/day of abstinence
Sperm motility, visual (%)
Sperm motility, videotape (% and
velocity)
Gross sperm morphology
Detailed sperm morphology
Fertility
Ratio exposed: pregnant females
Number of embryos or young per
pregnant female
Ratio viable embryos: corpora lutea
Number 2–8 cell eggs
Sperm per ovum
In vitro
Incubation of sperm in agent
Hamster egg penetration test
Other tests considered
Tonometric measurement of
testicular consistency
Qualitative testicular histology
Stage of cycle at which
spermiation occurs
Quantitative testicular histology
Sperm motility
Time-exposure photography
Multiple-exposure photography
Cinemicrography
Videomicrography
Sperm membrane characteristics
Evaluation of sperm metabolism
Fluorescent Y bodies in
spermatozoa
Flow cytometry of spermatozoa
Karyotyping human sperm
pronuclei
Cervical mucus penetration test
Endocrine
Luteinizing hormone
Follicle stimulating hormone
Testosterone
Gonadotropin-releasing hormone
*See Galbraith et al., 1982, for complete table and discussion of the relative usefulness of these tests.
SOURCE: Dixon, 1986, with permission.
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CHAPTER 20 TOXIC RESPONSES OF THE REPRODUCTIVE SYSTEM
mors may be due to its ability to reduce the cytotoxicity of cadmium in interstitial cells. Different heavy metals seem to exert their
toxic effects upon different subpopulations of testicular cells (Table
20-15). Mechanisms of heavy metal toxicity vary and include not
only different cell sensitivities but also direct versus indirect actions. Furthermore, it appears that primary damage to one cell type
may secondarily affect other cell types in the testes.
The sensitivity of the various parameters used to evaluate the
male reproductive system varies considerably. There are advantages as well as limitations to a number of standard reproductive
procedures. Testicular weight is a rapid quantitative index, but this
measurement is less sensitive than sperm counts and is affected by
water imbibition (edema). In normal males, the number of sperm
produced per day per testis is largely determined by testicular size.
In many mammals, testis size is correlated to daily sperm production. Fertility as an index is quite insensitive, although it does incorporate all reproductive functions. Fertility profiles using serial
mating studies to assess the biological status of sperm cells have
been a useful test for both dominant lethal mutations (Epstein et
al., 1972) and male reproductive capacity (Lee and Dixon, 1972).
Testicular histology provides information on target cell morphology, although it too is subjective and not particularly quantitative.
Histologic evaluation of the seminiferous tubules can establish cellular integrity and provide information about the process of spermatogenesis (Fig. 20-10). Good tissue fixation is essential for detecting the more subtle changes in the seminiferous epithelium (cf.
Creasy, 1997). It is more difficult to detect morphologic changes
in Leydig cells and to some extent Sertoli cells. Leydig cell function is better determined by evaluating androgen levels (or gonadotropins) or, in the case of Sertoli cells, by the measurement of
androgen-binding protein (ABP).
In order to undertake a meaningful histologic evaluation of
the testes, it is necessary to understand the spermatogenic cycle
and to identify its various stages. The use of seminiferous tubule
staging is very important to the evaluation of testicular injury
(Creasy, 1997). If damage is detected, it must be characterized. For
example, Sertoli cell damage is frequently recognized by inter- or
intracellular vacuoles or by swelling of the basal Sertoli cell cyto-
Table 20-14
Dietary Deficiency(s) and Spermatogenic Arrest
SPECIES
Manganese
Vitamin A
Vitamin B (pyridoxine)
Vitamine E
Zinc
Rats and rabbits
Mice, rats, and guinea pigs
Rats
Rats, hamsters, and guinea pigs
Mice, rats, dogs, and sheep
SOURCE:
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DEFICIENCY
Mann and Lutwak-Mann, 1981, with permission.
some length of time are most likely required to detect male reproductive toxicity. Most of the tests are invasive and hence limited
to animals and not generally acceptable for use in humans. Indeed,
in humans, the noninvasive approaches involve sperm counts,
blood gonadotrophin levels, and a nonbarren marriage. Testicular
biopsy can be used in selected circumstances to evaluate spermatogenesis (i.e., infertility/sterility), but this procedure is obviously invasive. Azoospermia can be caused by certain chemical
agents, genetic disorders (e.g., Klinefelter’s syndrome), infections
(e.g., mumps), irradiation, and hormonal defects. Dietary deficiencies are well known to cause spermatogenic arrest (Table
20-14). Similarly, lead can produce infertility, sterility, and varying
abnormalities in sperm function and morphology (Table 20-10).
Pogach et al., (1989) have reported that cisplatin causes Sertoli cell
dysfunction in rodents. These changes in Sertoli cell function appear to be responsible for cisplatin-induced impairment in spermatogenesis. Other heavy metals such as cobalt, iron, cadmium,
mercury, molybdenum, and silver can adversely affect spermatogenesis and accessory sex organ function. Dietary zinc deficiency
can produce sterility (Prasad et al., 1967). Likewise, chemically induced zinc depletion (e.g., phthalates) can produce testicular damage, as evidenced by sterile seminiferous tubules (Thomas et al.,
1982). In experimental animals, zinc prevents cadmium carcinogenicity in the rat testes (Koizumi and Waalkes, 1989). The major
preventive effect of zinc against cadmium-induced testicular tu-
Table 20-15
Summary of Cellular Site(s) of Action of Excess Heavy Metals on the Male Reproductive System
Evidence for Testicular
Toxicity (Primary or
Secondary)
EVIDENCE FOR HYPOTHALAMIC/
METAL
Cadmium
Zinc
Lead
Chromium
Cobalt
Platinum
Vanadium
GC
LC
SC
None
✆
✆
✆
None
Possible suppression of FSH and LH
None
None
None
None
•
•
✆
•
•?
✆
•
✆?
•
✆
•
ADENOHYPOPHYSIAL EFFECTS
695
MECHANISM/COMMENT
Hypoxia/ischemia
(Endothelial Cells)
Toxicity due to deficiency
Endocrine and paracrine toxicity
Unknown?
Toxicity due to general hypoxia
Inhibits DNA synthesis
•-Evidence for direct cellular action
✆ -Evidence for some direct action, but possibly secondarily mediated
-Deficiency of metal causes cellular toxicity
SOURCE: Thomas, 1995a, with permission.
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4-17-2001
Figure 20-10. Histology section of rat testes.
Above: Normal H&E section revealing morphologic integrity of seminiferous tubules. Below: Chemically induced testicular damage resulting in vacuolation of seminiferous tubules. Note partially sterile tubules. (From
Thomas and Thomas, 1994, with permission.)
plasm. Morphologic changes in the Leydig cell may be more difficult to detect. Degeneration and necrosis of germ cells can be recognized by the normal criteria of nuclear pyknosis and cytoplasmic eosinophilia. Some quantitative assessment of the histology of
the testes includes measuring tubular diameter and cell counts of
spermatocytes or round spermatids.
Thus, there are essentially two approaches to establishing whether or not a chemical is able to exert an adverse effect on
spermatogenesis: (1) evaluation of testicular morphology (i.e.,
pathology) and (2) functional evaluation of spermatogenesis
(Sharpe, 1998). Included in the assessment are the detection of abnormalities in spermatogenesis/testicular morphology, stage-
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dependent germ cell degeneration, and impairment of normal
sperm release.
Flow Cytometry
Ca, Cd, Co, Cr, Cu, Fe, Mg, Mn, Mo, Ni, Pb, Rb, Se, Vd and
Zn—can be detected in seminal plasma (Abou-Shakra et al., 1989).
Both quantitative and qualitative characteristics of more than
one ejaculate must be evaluated to ensure that conclusions concerning testicular function are valid. Since semen represents contributions from accessory sex glands as well as the testes and epididymides, only the total number of sperm in an ejaculate is a
reliable estimate of sperm production. The number of sperm introduced into the pelvic urethra during emission and the volume
of fluid from the accessory sex glands are independent. The potential sources of error in measuring ejaculate volume, concentration, and the seminal characteristics necessary to calculate total
sperm per ejaculate must be considered (Amann, 1981).
There have been recent advances in the automation of semen
analysis. Semiautomated measures of sperm motility may be categorized as indirect or direct methods. Indirect methods of sperm
analysis estimate mean swimming speed of cells by measuring
properties of the whole sperm suspension. Spectrometry or turbidimetric methods record changes in optical density. Direct methods involve visual assessment of individual sperm cells and stem
from early efforts to quantitate sperm swimming speed. Such direct measurements may include photographic methods like timedexposure photography, multiple-exposure photography, and cinematography. Computer-aided sperm motion analysis (CASMA)
may be applied to morphology, physiology, motility, or flagellar
analysis. CASMA allows visualization of both digitized static and
dynamic sperm images. Semen analysis and fertility assessment
should recognize statistical power and experimental design for toxicologic studies (Williams et al., 1990).
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Flow cytometric analyses of the testes can be used to evaluate specific cell populations (Selden et al., 1989). This technique has the
advantage of being able to assess simultaneously multiple characteristics on a cell-to-cell basis, with the results being rapidly correlated for each cell type or property. Cell size, cell shape, cytoplasmic granularity and pigmentation, along with measurements of
surface antigens, lectin binding, DNA/RNA, and chromatin structure are among some of the intrinsic and extrinsic parameters that
can be evaluated. The toxic effects of thiotepa on mouse spermatogenesis have been determined using dual-parameter flow cytometry. The dual parameters of DNA stainability versus RNA content provide excellent resolution of testicular cell types (Evenson
et al., 1986). Flow cytometry has also been used to study the effects of methyl-benzimidazol-2-yl-carbamate (MBC) on mouse
germ cells. MBC exposure results in an altered ratio of testicular
cell types, abnormal sperm head morphology, and altered sperm
chromatin structure (Evenson et al., 1987).
Oxidative damage to spermatogenic cells has also been associated with reproductive dysfunction in laboratory animals, and this
too can provide an index for assessing risk. Angioli et al., (1987)
have proposed an in vitro spermatogenic cell model for assessing
reproductive toxicity; it involves the ability of bleomycin to reduce
oxidative changes in male germ cell populations.
Penetration of zona-free hamster eggs by human sperm has
also been suggested as a useful chemical test to assess male fertility. Recently, this assay has also been recommended as a prognostic indicator in in vitro fertilization programs (Nahhas and
Blumenfeld, 1989).
697
Sex Accessory Organs
The epididymis and the sex accessory organs can also be used to
evaluate the status of male reproductive processes. While the epididymis has an important physiologic role in the male reproductive tract, it is less useful as a parameter for assessing gonadotoxins. Its histologic integrity may be examined, but the most
meaningful determinations are the number of sperm stored within
the cauda epididymis and a measure of sperm motility and morphology. Epididymal sperm may be extruded onto a glass slide and
viewed under the microscope for motility and abnormalities. Sex
accessory organs, usually the prostate (e.g., ventral lobes in the
rodent) and the seminal vesicles (empty), provide a rapid and
quantitative measure of the male reproductive processes that
are androgen-dependent. Chemical indicators in sex accessory
glands such as fructose and citric acid have also been used to evaluate male sex hormone function (cf. Mann and Lutwak-Mann,
1981).
Semen Analyses
Semen analysis can be used as an index of testicular and posttesticular organ function. Semen can be collected from a number of
experimental and domestic animals using an artificial vagina. Electroejaculatory techniques and chemically induced ejaculations have
also been employed to produce semen samples, particularly in
animal husbandry. In humans, several trace elements—including
Sperm Counts and Motility
Several factors affect the number of sperm in an ejaculate, including age, testicular size, frequency, degree of sexual arousal, and
seasons (particularly in domestic animals) (cf. Thomas, 1996). Although ejaculatory frequency or the interval since the last ejaculation alters the total number of sperm per ejaculate, ejaculation frequency does not influence daily sperm production. However,
because of epididymal storage, frequent ejaculation is necessary if
the number of sperm counted in ejaculated semen is to reflect sperm
production accurately. If only one or two ejaculates are collected
weekly, a 50 percent reduction in sperm production probably would
remain undetected. Ejaculates should be collected daily (or every
other day) over a period of time. The analysis of isolated ejaculate
or even several ejaculates collected at irregular intervals cannot estimate sperm production or output. The first several ejaculates in
each series contain more sperm than subsequent ejaculates because
the number of sperm available for ejaculation is being reduced.
In experimental animals (e.g., rodents), epididymal sperm
may be extruded, diluted with saline in a hemocytometer, and
counted. Sperm motility may also be assessed. Sperm morphology
may be evaluated using either wet preparations or properly prepared stained smears, which require an appropriate classification
scheme (Wyrobek, 1983; Wyrobek et al., 1983). Chromosomal
analyses can be used in the laboratory or the clinic to diagnose certain genetic diseases.
Androgens and Their Receptors
The androgen receptor (AR) is a member of the steroid/nuclear receptor superfamily, all members of which share a basic and func-
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cles) contain steroid-modifying enzymes that can activate, inactivate, and alter the receptor specificity of androgens. For example,
steroid 5-reductase converts testosterone to 5-dihydrotestosterone, which is a more potent androgenic ligand. Many factors
can affect androgenic actions (Table 20-16).
Other Secretory Biomarkers
Efforts have been made to identify so-called testicular marker enzymes as indicators of normal or abnormal cellular differentiation
in the gonad (Hodgen, 1977; Shen and Lee, 1977; Chapin et al.,
1982). At least eight enzymes—hyaluronidase (H), lactate dehydrogenase isoenzyme-X (LDH-X), and the dehydrogenases of sorbitol (SDH), -glycerophosphate (GPDH), glucose-6-phosphate
(G6PDH), malate (MDH), glyceraldehyde-3-phosphate (G3PDH),
and isocitrate (ICDH)—have been studied with regard to their usefulness as predictors of gonadal toxicity. Several genes are expressed exclusively in male germ cells (cf. Heckert and Griswold,
1993).
A number of secretory products of the Sertoli cell hold some
potential for evaluating male reproductive function. Of the several
secretory products of the Sertoli cell (e.g., transferrin, ceruloplasmin, tissue plasminogen activator, sulfated glycoproteins), androgen-binding protein (ABP) has perhaps received the most attention
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tional homology. AR action is highly specific in spite of the homology between AR and other steroid receptors. The AR is composed of three functional domains. The two predominant naturally
occurring ligands of the AR are testosterone and dihydrotestosterone. AR exists as a phosphoprotein in different cell types (cf.
MacLean et al., 1997).
Androgen receptors for testosterone and dihydrotestosterone
(DHT) have also been used to evaluate the effects of various gonadotoxins. A number of divalent metal ions (Zn, Hg, Cu, Cd, etc.)
can inhibit androgen-receptor binding in rodent prostate glands
(Donovan et al., 1980). In addition to heavy metals interfering with
androgen binding, DDT and p,p-DDE are potent androgen receptor antagonists and can affect male reproduction (Kelce et al.,
1995). The major metabolite of DDT, namely, p, p-DDE, inhibits
androgen binding to the androgen receptor as well as androgeninduced transcriptional activity. Hydroxyflutamide and p, p-DDE
were equally effective in inhibiting androgen-induced transcriptional activity.
Hormonally active androgens promote reproductive and anabolic (myotropic) functions. Both reproductive and anabolic effects
of androgens are mediated by their interaction with AR (cf. Roy
et al., 1999). Hormonally active androgens are C-19 steroids with
an oxo-functional group at the C-3 position and a hydroxy group
at 17. Androgen target cells (e.g., prostate gland, seminal vesi-
Table 20-16
Factors Affecting Androgen Effectiveness
TARGET
EFFECT
EXAMPLE
Hypothalamic-pituitary
interaction
Feedback control of LHRHmediated gonadotropin
secretion
Estrogens, progestins
Gonadotropin action
Disrupt reproductive control
processes involving
gonadotropins
LH-FSH antibodies
Androgen synthesis
Inhibit key enzymes, e.g.,
cholesterol desmolase,
17-hydroxylase, 3 -hydroxysteroid oxidoreductase,
5-reductase
Steroid analogues,
diphenylmethylanes
(amphenone B,DDD),
pyridine derivatives (SU
series), disubstituted
glutaric acid imides
(glutethimides), triazines,
hydrazines,
thiosemicarbazones
DHT synthesis
Inhibit 5-reductase in target
tissue
Androstene-17-carboxylic
acid, progesterone
Plasma binding
Alter ratio of bound and free
androgen in systemic
circulation
Estrogens
Cytoplasmic receptors
Alter effect on target tissue by
affecting binding to
cytoplasmic receptors
Cyproterone acetate, 17methyl--testosterone,
flutamide
DHT cellular binding
Block DHT effect on target
tissue
Cyproterone acetate,
spironolactone,
dihydroprogesterone,
RU-22930
SOURCE:
Dixon, 1982, with permission.
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uate primordial germ cell proliferation, migration, ovarian differentiation, and folliculogenesis (Ways et al., 1980; Thompson,
1981).
Serial oocyte counts can monitor oocyte and/or follicle destruction in experimental animals (Pedersen and Peters, 1968). This
approach is a reliable means of quantifying the effects of chemicals on oocytes and follicles.
Follicular growth may be assayed in experimental animals using (3H)-thymidine uptake, ovarian response to gonadotropins, and
follicular kinetics (Hillier et al., 1980). These approaches identify
both direct and indirect effects on follicular growth and identify
drugs and other environmental chemicals that are ovotoxic
(Mattison and Nightingale, 1980).
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as a potential indicator for detecting gonadal injury. Sertoli cell
ABP and testicular transferrin may be affected by similar regulatory agents (e.g., FSH, insulin) (Skinner et al., 1989). Leydig cell
cultures can also be considered as a potential indicator to evaluate
endocrine function of the gonad (Brun et al., 1991). Pig Leydig
cell culture can be used to discriminate between specific and nonspecific inhibitors of steroidogenesis. Leydig cells, like Sertoli
cells, secrete a number of proteins, peptides, and other substances
[e.g., -endorphin, corticotropin-releasing factor (CRF)] (Eskeland
et al., 1989). The testes contain various neuropeptides and growth
factors. These include LHRH, TRH, POMC, oxytocin, vasopressin
and still other peptide precursors (cf. Shioda et al., 1994; SpiteriGrech and Nieschlag, 1993). Many of these factors are involved in
the autocrine or paracrine regulation of the testes (Table 20-2).
Other than the inhibitory actions of DBCP on Sertoli cell ABP secretions, neither this cell and its secretions nor the Leydig cell has
been used in reproductive toxicology evaluation.
TESTING FEMALE REPRODUCTIVE
CAPACITY
General Considerations
The evaluation of mammalian reproductive processes is far more
complex in the female than in the male. Female reproductive
processes involve oogenesis, ovulation, the development of sexual
receptivity, coitus, gamete and zygote transport, fertilization, and
implantation of the concepters. All these processes or events offer
potential opportunities for chemical or drug interference.
Evaluation of the female reproductive tract for toxicologic perturbations not surprisingly may overlap with testing methods for
assessing teratogenicity and mutagenicity. Indeed, reproductive
endpoints that indicate dysfunction in the female (Table 20-17), including perinatal parameters, often overlap with developmental
toxicity endpoints (Table 20-18). The neonate is particularly sensitive to a variety of drugs and chemicals (Thomas, 1989).
Gross pathology (e.g., gravimetric responses—ovary, uterus,
etc.) and histopathology are important to reproductivity and should
be evaluated (Ettlin and Dixon, 1985). Both light microscopy and
electron (transmission and scanning) microscopy may be useful in
assessing ovarian and pituitary ultrastructure. As in the male (Table
20-13), there are a number of useful tests to evaluate the female
reproductive system (Table 20-17). These tests can be performed
on a wide variety of endpoints, at different anatomic sites, and can
include biochemical, hormonal, or morphologic parameters.
Oogenesis/Folliculogenesis
Methods to assess directly the effects of test compounds on oogenesis and/or folliculogenesis include histologic determination of
oocytes and/or follicle number (Dobson et al., 1978). Chemical effects on oogenesis can be measured indirectly by determining the
fertility of the offspring (McLachlan et al., 1981; Kimmel et al.,
1995; Davis and Heindel, 1998). Other indirect measures of ovarian toxicity in animals include assessment of age at vaginal opening, onset of reproductive senescence, and total reproductive capacity (Gellert, 1978; Khan-Dawood and Satyaswaroop, 1995).
Morphologic tests can quantify and assess primordial germ
cell number, stem cell migration, oogonial proliferation, and urogenital ridge development. In vitro techniques can be used to eval-
699
Estrogens and Their Receptors
The rat, mouse, and human estrogen receptor (ER) exists as two
subtypes, ER and ER, which differ in the C-terminal ligandbinding domain and in the N-terminal transactivation domain
(Kuiper et al., 1998). Estrogen influences the growth, differentiation, and functioning of several target organs. Such organs include
the mammary gland, uterus, vagina, ovary, and several male reproductive system organs (e.g., testes, prostate gland, etc.). Estrogens affect osteogenesis and the CNS and seem to play a role in
the cardiovascular system’s homeostasis. Estrogens migrate in and
out of cells but are retained with high affinity and specificity in
certain target tissues by an intranuclear binding protein called the
estrogen receptor (ER). The newly discovered ER is an important sex hormone receptor not only in the female, but also in the
male. It has been suggested that a possible physiologic ligand for
ER in the male is 5-androstane-3,17-diol. This testosterone
metabolite binds more firmly to ER than to ER. ER and ER
are differentially expressed along the length of the male reproductive tract. ER are expressed in the Sertoli cell, the Leydig cell, and
the epididymis and accessory sex organs. ER is expressed in Sertoli cells and in most germ cells. The presence of aromatase activity in these two cells suggests that estrogens may be involved in
the modulation of spermatogenesis. ER is expressed in the rodent
and human testes (van Pelt et al., 1999). ER is localized in the
nuclei of Leydig cells in fetal and adult rodent testes.
The biological activity of estrogens (and progesterone) is manifest through high-affinity receptors located in the nuclei of specific
target cells (Vegeto et al., 1996). The receptors for estrogen (and
progesterone) are members of a large superfamily of nuclear proteins. Nuclear hormone receptors are single polypeptides organized into discrete functional domains (i.e., regions A through F).
It is understood that the activation of steroid hormone receptors (e.g., estrogen) regulates the transcriptional activity of specific
genes, hence mediating classic or genomic actions of steroid hormones. However, not all steroid effects can be explained by such
a classic model of steroid-target cell interaction. Instead, signalgenerating steroid receptors on the cell surface have been referred
to as nonclassic, nongenomic steroid effects (Revelli et al., 1998).
There are several cell types within the reproductive system wherein
estrogens exert early physiologic effects that are too rapid to be
mediated by the sequence of genomic activation. Signal transduction mechanisms involving nongenomic steroid effects are particularly evident in spermatozoa. Most nongenomic actions of steroids
seem to involve Ca2 as a second messenger. It is possible that
nongenomic and genomic actions may synergize, resulting in both
rapid onset and long-lasting or persistent actions.
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Table 20-17
Potentially Useful Tests of Female Reproductive Toxicity
Body Weight
Uterus
Cytology and histology
Luminal fluid analysis
(xenobiotics, proteins)
Decidual response
Dysfunctional bleeding
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Ovary
Organ weight
Histology
Number of oocytes
Rate of follicular atresia
Follicular steroidogenesis
Follicular maturation
Oocyte maturation
Ovulation
Luteal function
Oviduct
Histology
Gamete transport
Fertilization
Transport of early embryo
Hypothalamus
Histology
Altered synthesis and release of
neurotransmitters,
neuromodulators, and
neurohormones
Cervix/vulva/vagina
Cytology
Histology
Mucus production
Mucus quality (sperm
penetration test)
Pituitary
Histology
Altered synthesis and release of
trophic hormones
Fertility
Ratio exposed: pregnant
females
Number of embryos or young
per pregnant female
Ratio viable embryos: corpora
lutea
Ratio implantation: corpora lutea
Number 2–8 cell eggs
Endocrine
Gonadotropin
Chorionic gonadotropin levels
Estrogen and progesterone
In Vitro
In vitro fertilization of
superovulated eggs, either
exposed to chemical in culture or
from treated females
SOURCE:
Modified from Dixon, 1986, with permission.
Table 20-18
Developmental Toxicity Endpoints
Type I changes
(Outcomes permanent, life-threatening, and frequently associated with gross
malformations)
Reduction of number of live births (litter size)
Increased number of stillbirths
Reduced number of live fetuses (litter size)
Increased number of resorptions
Increased number of fetuses with malformations
Type II changes
(Outcomes nonpermanent, non-life-threatening, and not associated with malformations)
Reduced birth weights
Reduced postnatal survival
Decreased postnatal growth, reproductive capacity
Increased number of fetuses with retarded development
SOURCES:
Frankos, 1985; Collins et al., 1998.
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(see also Chap. 10, “Developmental Toxicology”). It may be seen
that there is reasonable harmonization between the various regulatory agencies. This condensed table (Table 20-19) does not reveal the protocol for mating procedures, F1 mating, second mating, and other experimental design conditions, but they too are
similar among the various regulatory agencies. The reproductive
study guidelines of the U.S. Food and Drug Administration (FDA)
harmonize with those of the Environmental Protection Agency
(EPA) and the [OECD (Organization for Economic Cooperation
and Development)]. Some of the guidelines of the [ICH (International Conference on Harmonization)] are blank because these
guidelines are intended to be generic (Collins et al., 1998). While
guidelines are reviewed periodically to keep abreast of changing
science and technology, the FDA’s Redbook II has reduced the
number of generations from three to two. The number of litters/
generations has been decreased from two to one. Monitoring
of estrous cycle, time of vaginal opening, and time of preputial
separation are new requirements. The amount of histopathology,
particularly of the pups, has been increased (Collins et al.,
1998).
Reproduction (multigenerational) study test guidelines have
also undergone some revisions (Collins et al., 1998). The FDA
prefers that either the rat or the rabbit be used, with the choice of
species based on pharmacokinetic differences. The EPA, OECD,
and ICH recommend the most relevant species, but again the rat
or the rabbit is often preferred. At least three dose levels are recommended. All adults must undergo necropsy with examination of
the uterus and placenta.
The minimal reproductive study recommended consists of two
generations, with one litter per generation (Collins et al., 1999).
The guideline contains optional procedures for inclusion of additional litters per generation, additional generations, a test for teratogenic and developmental toxicity effects, optional neurotoxicity screening, and optional immunotoxicity screening.
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Serum levels of estrogen or estrogenic effects on target tissues are indicators of normal follicular function. Tissue and organ
responses include time of vaginal opening in immature rats, uterine weight, endometrial morphology, and/or serum levels of FSH
and LH. Granulosa cell culture techniques provide direct screens
of the ability of chemicals to inhibit cell proliferation and/or
estrogen production (Zeleznik et al., 1979). The biosynthesis of
estradiol and its metabolism to estrone and estriol by the ovary
constitutes another indicator of the reproductive process. The
peripheral catabolism of these steroids is principally a function of
the liver.
Nuclear and cytoplasmic estrogen/progesterone may provide
important toxicologic applications. Estradiol and progesterone receptors are especially important since chemicals (e.g., DDT and
other organochlorine pesticides) compete for these receptors and
perhaps alter their molecular conformation (Thomas, 1975).
701
Ovulation/Fertilization/Implantation
Ovulation differs among various mammalian species. Some animals ovulate spontaneously upon copulation (e.g., the rabbit),
whereas other species (e.g., humans and subhuman primates) have
a hormonally dependent cycle. Several steroidal and nonsteroidal
agents can interfere with this neuroendocrine process of ovulation.
In the estrus cycle of rodents, ovulation occurs at intervals of 4 to
5 days. Ovulation occurs during estrus and can be readily detected
by cornification of vaginal epithelium. The rat’s estrus cycle can
be divided into four stages and can be recognized by changes in
vaginal cytology: proestrus, estrus, metestrus, and diestrus.
The processes of fertilization and implantation can be affected
by both chemicals and drugs. The formation, maturation, and union
of germ cells compose a complex physiologic event that is sensitive to foreign substances. Fertilization can also be achieved in vitro
with sperm and ova extradited from a variety of different mammalian species including humans.
Reproductive performance is best assessed by pregnancy, and
this represents a successful index for evaluating endocrine toxicity (or lack thereof). The mating study using rats is a fundamental
procedure that determines total reproductive capacity.
REPRODUCTIVE TESTS AND
REGULATORY REQUIREMENTS
General Considerations
The history of reproductive guidelines has recently been reviewed
(Collins et al., 1998). Over the years several attempts have been
made to standardize testing methods. Testing procedures to simulate human exposure have taken two different paths. One is based
on the premise that specific injury from a chemical/drug can be
more readily established by administering it only during certain
periods of gestation. The second path was devised for compounds
likely to involve chronic exposure and for which there may be a
concentration factor when administered during several generations.
Over the years, many efforts have been undertaken to harmonize
testing guidelines (Christian, 1992; 2001.)
Endpoints—Females
Endpoints in studies of female reproductive toxicity include the
following:
• Female fertility index
[(number of pregnancies/number of matings) 100]
• Gestation index
[(number of litters—live pups/number of pregnancies) 100]
• Live-born index
[(number of pups born alive/total number of pups born) 100]
• Weaning index
[(number of pups alive at day 21/number of pups alive and kept
on day 4) 100]
• Sex ratio and percentage by sex
• Viability index
[(number of pups alive on day 7/number of pups alive and kept
on day 4) 100]
Endpoints—Males
The endpoints of male reproductive toxicity include the following:
Guidelines
Testing guidelines for evaluating reproductive and developmental
toxicity in females are outlined in Table 20-19 (Collins et al., 1998)
• Evaluation of testicular spermatid numbers
• Sperm evaluation for motility, morphology and numbers
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Two, one litter/generation
Rodent
5–9 weeks
30/sex
Two
Rat (preferred)
5–9 weeks
At least 20 pregnant
Two
Rat (preferred)
6–9 weeks
At least 20 pregnant
Dose levels
Route of administration
Minimum three dose levels
Oral (preferred) diet,
drinking water, gavage
8–11 weeks before mating,
throughout mating and
pregnancy
Minimum three dose levels
Oral (preferred) diet,
drinking water, gavage
10 weeks before mating;
dosing continued during
mating and pregnancy
Minimum three dose levels
Oral (preferred) diet,
drinking water, gavage
10 weeks before mating;
dosing continued during
mating and pregnancy
Dosing schedule
SOURCE:
Condensed and modified from Collins et al., 1998, with permission. See original table for more detailed information.
Copyright © 2001 by The McGraw-Hill Companies
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ICH (U.S. FDA, 1994)
Two
Rat (preferred)
Sufficient to allow
meaningful interpretation
Minimum three dose levels
Determined by intended
human usage
Treat males and females
before mating, during
mating and through
implantation. Other
treatment regimens
required.
Page 702
Number of generations
Animal species
Age of animals
Number of animals
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Table 20-19
Comparison of Reproductive Guidelines
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that are perhaps representative of a molecular approach to determining mechanisms of toxicologic action.
HUMAN RISK FACTORS
AFFECTING FERTILITY
General Considerations
Most humans are exposed to a vast number of chemicals that may
be hazardous to their reproductive capacity (Faber and Hughes,
1995). Many chemicals have been identified as reproductive hazards in laboratory studies (Clegg et al., 1986; 2001; Working,
1988). Although the extrapolation of data from laboratory animals
to humans is inexact, a number of these chemicals have also been
shown to exert detrimental effects on human reproductive performance. The list includes drugs, especially steroid hormones and
chemotherapeutic agents; metals and trace elements; pesticides;
food additives and contaminants; industrial chemicals; and consumer products.
Fertility in humans, like that in experimental animals, is susceptible to toxic effects from environmental and/or industrial chemicals. Infertility is a problem of increasing concern in several industrialized countries. Levine (1983) has suggested methods for
detecting occupational causes of male infertility. The decrease in
sperm quality purportedly having occurred over the past 50 years
(Carlsen et al., 1992) has been refuted as being due to the lower
reference standards (cf. Bromwich et al., 1994). Furthermore, a
comparison of the production of spermatozoa from the testes of
different species reveals that the output of sperm in humans is approximately four times less than that in other mammals in terms
of the number of sperm produced per gram of tissue (Amann and
Howard, 1980).
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Sperm motility can be assessed by microscopic techniques or
with a computer-assisted sperm analysis (CASA) system (Seed et
al., 1996). Sperm (minimum 200 per sample) from the cauda epididymis or proximal vas deferens should be examined as a fixed
wet preparation and classified as either normal or abnormal (Clegg
et al., 2001). Total sperm counts in the cauda epididymis can be
assessed (Robb et al., 1978).
Both the FDA and the EPA have established study protocols
to assess the reproductive risks of chemicals and drugs. The FDA
imposes guidelines for drugs that includes three different protocols
(namely segments) on development, fertility, and general reproductive performance:
Segment I: Fertility and Reproduction Function in Males and
Females
Segment II: Developmental Toxicology and Teratology
Segment III: Perinatal and Postnatal Studies
Segment I studies are initial studies often leading to additional
protocols such as developmental protocols. By using pregnant animals (e.g., segment III) that are treated for the last third of their
period of gestation, including lactation and weaning, assessment
can be made about the effects of chemical and/or drug exposure
on late fetal development, particularly lactation and offspring
survival.
The National Toxicology Program (NTP) adopted the Fertility
Assessment by Continuous Breeding (FACB) protocol in the early
1980s. The FACB protocol was introduced by McLachlan et al.
(1981) and was designed to reduce the time for reproductive toxicity testing yet still provide data comparable with those obtained
from other testing systems. FACB tests take no longer than the improved and shortened EPA test designs. The FACB protocol uses
more animals per group and in general increases the statistical
power of the assay. Morrissey et al. (1988) have evaluated the effectiveness of continuous breeding reproduction studies. This subtle modification of increasing the statistical power of the assay is
important, since fertility is an especially important indicator of reproductive toxicity and is one of the least sensitive indicators in
the assessment of the reproductive system (Schwetz et al., 1980).
Experimental design for toxicologic studies, particularly in studies
involving semen analysis and fertility assessment, must recognize
statistical power (Williams et al., 1990).
Reproductive toxicity studies extending over multiple generations are scientifically and logistically difficult to manage, interpret, and finance (Johnson, 1986). While the FDA segment tests
are collectively very meaningful in assessing reproductive toxicity
(or safety), none of these batteries of tests can replace the other,
and the multigeneration evaluation has considerable scientific merit
for justifying their expense. However, current multigeneration protocols could be revised in order to improve on the toxicologic information collected. FDA reproductive testing guidelines require
preclinical animal testing for each new drug depending on how
women might be exposed to the drug itself. The FDA further categorizes drugs on five different levels, depending on potential risk
(e.g., category A, no evidence of human development toxicity, to
category D or X, demonstrated birth defects) (cf. Frankos, 1985).
It is evident that a number of test systems are available to assess the degree of change in the reproductive system. Some such
tests employ many animals and follow their reproductive histories
for more than one generation, whereas others employ cell systems
703
Male
It has also been suggested that the human male is more vulnerable to environmental and occupational toxins than other mammals
(Overstreet, 1984; Overstreet et al., 1988). Reproductive hazards
and reproductive risks have led to the formulation of protection
policies in certain occupations (Perrolle, 1993; Sattler, 1992;
Thomas and Barsano, 1994). The somewhat fragile nature of the
male reproductive system to occupational exposure to the fungicide DBCP was reaffirmed when Whorton et al. (1977) described
its injurious effects on the testes. Fortunately, recovery from severe oligospermia after DBCP exposure has been reported by Lantz
et al. (1981). Levine et al. (1983), however, have indicated that reproductive histories are superior to sperm counts in assessing male
infertility caused by DBCP.
It has been extremely difficult to directly correlate human exposure to occupational chemicals with alterations in the reproductive system. A particularly complicating factor in this lack of correlation is that the normal reproductive processes seldom operate
at a physiologic optimum. For example, as many as 15 percent of
all married couples in the United States are defined as being clinically infertile (MacLeod, 1971), whereas another 25 percent of the
women exhibit impaired fecundity (Mosher, 1981). At least 30 percent of early human conceptions and up to 15 percent of recognized pregnancies are terminated by spontaneous abortion (cf.
Haney, 1985). Of the 15 percent of spontaneous abortions that terminate recognized pregnancies, about 25 percent involve abnormalities related to genetic etiologies and another 7 percent are
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Female
animal models. Clegg et al. (2001), Buiatti et al. (1984), and Paul
(1988) have reviewed several factors that are important in assessing risk to the male reproductive system. It is considerably easier
to extrapolate controlled drug studies in animals to exact therapeutic regimens in humans than it is to simulate a chemical’s exposure in an animal to a presumed environmental exposure in humans. Occupational exposures are inexact, and environmental
levels are even more difficult to document (cf. Lemasters and
Selevan, 1984). Exposures usually involve mixtures of chemicals,
and individuals may not be aware of all the chemicals with which
they come into contact. Thus, the effect of individual chemicals is
difficult to assess, and cause-and-effect relationships are nearly impossible to establish.
Ulbrich and Palmer (1995) have undertaken a large survey of
medicinal products in an effort to match human (male) and several
experimental animals with respect to reproductive endpoints (e.g.,
spermatogenesis, sperm counts, sperm motility, etc.). The survey
included a wide range of medicinal products, both hormonal and
nonhormonal formulations. Sperm analysis results were comparable to results obtained by histopathology and/or organ weight
changes following drug administration. Validation of sperm analyses is problematic but provides a realistic alternative to histopathology and organ weight when the latter are impractical.
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caused by so-called environmental agents. By far, most of these
abortions are due to unknown factors, and this constitutes about 7
percent of the cases of spontaneous abortions.
It is noteworthy that chronic illness may have a profound affect on gonadal function (Turner and Wass, 1997). Several systemic
illnesses can reduce spermatogenesis, including thyrotoxicosis, hypothyroidism, renal failure, mumps, and Crohn’s disease. A large
number of nonhormonal diseases can likewise decrease serum
testosterone as well as gonadotrophins. Aging, nutritional deficiencies, and obesity can affect fertility. Thus, a host of both endocrine and nonendocrine diseases can affect male fertility.
Many factors can affect the normalcy of the female reproductive
system, as evidenced by variations in the menstrual process. Hence,
physiologic, sociologic, and psychological factors have been linked
with menstrual disorders. Factors that are known to affect menstruation yet are for the most part completely unrelated to occupational settings include age, body weight extremes, liver disease,
thyroid dysfunction, intrauterine contraceptive devices, stress, exercise, and marital status. It is, therefore, obvious that a number of
factors can affect menstruation and that these factors do not even
include such things as therapeutic drugs (Selevan et al., 1985), socalled recreational drugs, or potentially toxic substances present in
occupational environments. Even the choice of control populations
in studies involving the adverse effects on the reproductive system
can affect the risk estimates.
EXTRAPOLATION OF ANIMAL
DATA TO HUMANS
The exclusive use of animal experimental results to predict outcomes in humans still represents an uncertainty. This uncertainty
can be somewhat relieved if findings from multiple species are
known, particularly subhuman primates, and there are epidemiologic studies that help substantiate laboratory experiments. While
there are many general similarities among mammals with respect
to their response to drugs and/or chemicals, there are nevertheless
some notable differences. Many of these species differences can
be attributed to toxicokinetics, especially biotransformation.
Greater predictability can be seen in results from well-validated
EPIDEMIOLOGIC STUDIES
Epidemiology is increasingly important in establishing cause-andeffect relationships (Scialli and Lemasters, 1995). Epidemiology
and risk assessment are inextricably related. Reproductive surveillance programs are important underpinnings for monitoring endocrine processes. By closely monitoring worker exposures to
industrial/environmental toxicants, safer conditions will be established.
If exposure to a chemical has occurred in a human population
or if concern surrounds the use of a certain chemical, epidemiologic studies may be used to identify effects on reproduction.
Sheikh (1987) has pointed out factors that are important in selecting control populations for studying adverse reproductive effects
on occupational environments. The design of epidemiologic studies may involve either retrospective or prospective gathering of
data. Statistical aspects to be considered in epidemiologic studies
include power, sample size, significance level, and magnitude of
effect.
REFERENCES
Abou-Shakra FR, Ward NI, Everard DM: The role of trace elements in male
infertility. Fertil Steril. 52:307–310, 1989.
Albro PW, Chapin RE, Corbett JT, et al: Mono-2-ethylhexyl phthalate, a
metabolite of di-(2-ethylhexyl) phthalate, causally linked to testicular
atrophy in rats. Toxicol Appl Pharmacol 100:193–200, 1989.
AMA Council on Scientific Affairs: Effects of toxic chemicals on the reproductive system. JAMA 253:3431–3437, 1985.
Amann RP: A critical review of methods for evaluation of spermatogenesis from seminal characteristics. J Androl 2:37–58, 1981.
Amann RP: Detection of alterations in testicular and epididymal function
in laboratory animals. Environ Health Perspect 70:149–158, 1986.
Amann RP: Structure and function of the normal testis and epididymis. J
Am Coll Toxicol 8:457–471, 1989.
Amann RP, Howard SS: Daily spermatozoal production and epididymal
spermatozoal reserves of the human male. J Urol, 124:211–219, 1980.
Anderson RA Jr, Berryman SH, Phillips JF, et al: Biochemical and structural evidence for ethanol-induced impairment of testicular development: Apparent lack of Leydig cell involvement. Toxicol Appl Pharmacol, 100:62–85, 1989.
Andersson, KE, Wagner G: Physiology of penile erection. Physiol Rev
75:191–236, 1995.
Angioli MP, Ramos K, Rosenblum IY: Interactions of bleomycin with reduced and oxidized ion in rat spermatogenic cells. In Vitro Toxicol
1:45–54, 1987.
Baker DB, Landrigan PJ: Occupationally related disorders. Med Clin North
Am, 74:441–460, 1990.
Copyright © 2001 by The McGraw-Hill Companies
Retrieved from: www.knovel.com
2996R_ch20_673-709
4-17-2001
9:38 AM
Page 705
CHAPTER 20 TOXIC RESPONSES OF THE REPRODUCTIVE SYSTEM
phthalate on rat Sertoli cell-enriched primary cultures. Toxicol Appl
Pharmacol 92:467–479, 1988.
Chapin RE, Norton RM, Popp JA, et al: The effects of 2,5-hexanedione on
reproductive hormones and testicular enzyme activities in the F-344
rat. Toxicol Appl Pharmacol 62:262–272, 1982.
Chapman RM: Gonadal injury resulting from chemotherapy. Am J Ind Med
4:149–161, 1983.
Christian MS: Harmonization of reproductive guidelines: Perspective from
the International Federation of Teratology Societies. J Am Coll Toxicol 11:299–302, 1992.
Christian, MS: Test methods for assessing female reproductive and developmental toxicity, in Hayes AW (ed): Principles and Methods of Toxicology, 4th ed. Philadelphia: Taylor & Francis, 2001, pp 1301–1381.
Clegg ED, Sakai CS, Voytek PE: Assessment of reproductive risks. Biol
Reprod 34:5–16, 1986.
Clegg ED, Perreault, SD, Klinefelter GR: Assessment of male reproductive toxicity, in Hayes AW (ed): Principles and Methods of Toxicology, 4th ed. Philadelphia: Taylor & Francis, 2001, pp 1263–1300.
Colborn T, vom Saal FS, Soto AM: Developmental effects of endocrinedisrupting chemicals in wildlife and humans. Environ Health Perspect
101:378–384, 1993.
Colby H: Adrenal gland toxicity: Chemically-induced dysfunction. J Am
Coll Toxicol 7:45–69, 1988.
Collins TFX, Sprando RL, Hansen DL, et al: Testing guidelines for evaluation of reproductive and developmental toxicity of food additives in
females. Int J Toxicol 17:299–325, 1998.
Collins TFX, Sprando RL, Shackelford ME, et al: Food and Drug Administration proposed testing guidelines for reproduction studies. Regul
Toxicol Pharmacol 30:29–38, 1999.
Comereski CR, Bergman CL, Buroker RA: Testicular toxicity of N-methyltetrazolethiol cephalosporin analogues in the juvenile rat. Fund Appl
Toxicol 8:280–289, 1987.
Cook JC, Klinefelter GR, Hardisty JF, et al: Rodent Leydig cell tumorigenesis: A review of the physiology, pathology, mechanisms, and relevance to humans. Crit Rev Toxicol 29:2, 169–261, 1999.
Cooper RL, Goldman JM, Tyrey L: The hypothalamus and pituitary as targets for reproductive toxicants, in Korach KS (ed): Reproductive and
Developmental Toxicology. New York: Marcel Dekker, 1998, pp 195–
210.
Cooper RL, Kavlock RJ: Endocrine disruptors and reproductive development: A weight-of-evidence overview. J Endocrinol 152:159–166,
1997.
Cooper TG: The Epididymis, Sperm Maturation and Fertilization. Berlin:
Springer-Verlag, 1986.
Creasy DM: Evaluation of testicular toxicity in safety evaluation studies:
The appropriate use of spermatogenic staging. Toxicol Pathol 25:119–
131, 1997.
Davis BJ, Heindel JJ: Ovarian toxicants: multiple mechanisms of action,
in Korach KS (ed): Reproduction and Developmental Toxicology. New
York: Marcel Dekker, 1998, pp 375–398.
Davis BJ, Maronpot RR, Heindel JJ: Di-(2-ethylhexyl) Phthalate suppresses
estradiol and ovulation in cycling rats. Toxicol Appl Pharmacol
128:216–223, 1994a.
Davis BJ, Weaver R, Gaines LJ, et al: Mono-(2-ethylhexyl) phthalate suppresses estradiol production independent of FSH-cAMP: Stimulation
in rat granulosa cells. Toxicol Appl Pharmacol 128:224–228, 1994b.
De La Chapelle A: The Y-chromosomal and autosomal testis-determining
genes, in Goodfellow PN, Craig IW, Smith JC, Wolfe J (eds): The sexdetermining factor. Development 101 (suppl):33–38, 1987.
De Rooij D, Lok D: Regulation of the density of spermatogonia in the seminiferous epitheliium of the Chinese hamster, II. Anat Rec 217:131–
136, 1987.
Dickson LC, Buzik SC: Health risks of “dioxins”: A review of environmental and toxicological considerations. Vet Hum Toxicol 35:68–77,
1993.
Dixon RL: Potential of environmental factors to affect development of reproductive system. Fund Appl Toxicol 2:5–12, 1982.
Co
py
rig
hte
dM
ate
ria
l
Barlow SM, Sullivan FM: Reproductive Hazards of Industrial Chemicals.
London: Academic Press, 1982.
Barsano CP, Thomas JA: Endocrine disorders of occupational and environmental origin. Occup Med 7:479–502, 1992.
Battershill JM: Review of the safety assessment of polychlorinated biphenlyls (PCBs) with particular reference to reproductive toxicity. Hum
Exp Toxicol 13:581–587, 1994.
Bell JU, Thomas JA: Effects of lead on the reproductive system, in Singhal RL, Thomas JA (eds): Basic and Clinical Toxicity of Lead. Baltimore: Urban & Schwarzenberg, 1980, pp 169–189.
Biciková M, Hill M, Hampl R, et al: Inhibition of rat renal and testicular
11-hydroxysteroid dehydrogenase by some antihypertensive drugs,
diuretics, and epitestosterone. Horm Metab Res 29:465–468, 1997.
Billig H, Chun SY, Eisenhauer K, et al: Gonadal cell apoptosis: Hormoneregulated cell demise. Hum Reprod Update 2:2, 103–117, 1996.
Billig H, Furuta I, Rivier C, et al: Apoptosis in testis germ cells: Developmental changes in gonadotropin dependence and localization to selective tubule stages. Endocrinology 136:5–12, 1995.
Birnbaum LS: Developmental effects of dioxins, in Korach K (ed): Reproductive and Developmental Toxicology. New York: Marcel Dekker,
1998, pp 87–112.
Bjerke DJ, Peterson RE: Reproductive toxicity of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in male rats: Different effects of in utero
versus lactational exposure. Toxicol Appl Pharmacol 127:241–249,
1994.
Blackburn DM, Gray AJ, Lloyd SC, et al: A comparison of the effects of
the three isomers of dinitrobenzene on the testis in the rat. Toxicol
Appl Pharmacol 92:54–64, 1988.
Blazak WF, Ernest TL, Stewart BE: Potential indicators of reproductive
toxicity: Testicular sperm production and epididymal sperm number,
transit time, and motility in Fischer 344 rats. Fund Appl Toxicol
5:1097–1103, 1985.
Bloch E, Gondos B, Gatz M, et al: Reproductive toxicity of 2,4-dinitrobenzene in the rat. Toxicol Appl Pharmacol, 94:466–472, 1988.
Boekelheide, K: 2,5-Hexanedione alters microtubule assembly: I. Testicular atrophy, not nervous system toxicity, correlates with enhanced tubulin polymerization. Toxicol Appl Pharmacol, 88:370–382, 1987.
Boekelheide, K: Rat testis during 2,5-hexanedione intoxication and recovery: II. Dynamic of pyrrole reactivity, tubulin content, and microtubule
assembly. Toxicol Appl Pharmacol 92:28–33, 1988.
Bond MB: Role of corporate policy on reproductive hazards of the workplace. J Occup Med 28:193–199, 1986.
Bremner WJ, Millar MR, Sharpe RM, et al: Immunohistochemical localization of androgen receptors in the rat testis: Evidence for stagedependent expression and regulation by androgens. Endocrinology
135:1227–1353, 1994.
Brinster RL, Zimmerman JW: Spermatogenesis following male germ-cell
transplantation. Proc Natl Acad Sci USA 91:11298–11302, 1994.
Bromwich P, Cohen J, Stewart J, et al: Decline in sperm counts: An artifact of changed reference range of “normal”? Br Med J 309:10–20,
1994.
Brun HP, Leonard JF, Moronvalle V, et al: Pig Leydig cell culture: A useful in vitro test for evaluating the testicular toxicity of compounds.
Toxicol Appl Pharmacol 108:307–320, 1991.
Buchanan JF, Davis LJ: Drug-induced infertility. Drug Intell Clin Pharm
18:122–132, 1984.
Buiatti E, Barchielli A, Geddes M, et al: Risk factors in male infertility: A
case-control study. Arch Environ Health 39:266–270, 1984.
Byers SW, Sujarit S, Jegou B, et al: Cadherins and cadherin-associated molecules in the developing and maturing rat testis. Endocrinology
134:630–639, 1993.
Carlsen E, Giwercman A, Keiding N, et al: Evidence for decreasing quality of semen during past 50 years. Br Med J 305:609–612, 1992.
Chahoud I, Hartmann J, Rune GM, et al: Reproductive toxicity and toxicokinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Arch Toxicol
66:567–572, 1992.
Chapin RE, Gray TJB, Phelps JL, et al: The effects of mono-(2-ethylhexyl)-
705
Copyright © 2001 by The McGraw-Hill Companies
Retrieved from: www.knovel.com
2996R_ch20_673-709
706
4-17-2001
9:38 AM
Page 706
UNIT 4 TARGET ORGAN TOXICITY
Frankos VH: FDA perspectives on the use of teratology data for human
risk. Fund Appl Toxicol 5:615–625, 1985.
Galbraith WM, Voytek P, Ryon MG: Assessment of Risks to Human Reproduction and to Development of the Human Conceptus from Exposure to Environmental Substances. Soringfield, VA: Oak Ridge National Laboratory, U.S. Environmental Protection Agency, National
Technical Information Service, 1982.
Gellert RJ: Uterotrophic activity of polychlorinated biphenyls (PCB) and
induction of precocious reproductive aging in neonatally treated female rats. Environ Res 16:123–130, 1978.
Gilfillan SC: Lead poisoning and the fall of Rome. J Occup Med 7:53–60,
1965.
Goldberg EH: H-Y antigen and sex determination. Philos Trans R Soc Lond
[Biol] 322:73–81, 1988.
Goldstein I, Lue TF, Padma-Nathan H, et al: Oral sildenafil in the treatment of erectile dysfunction. N Engl J Med 338:1397–1404, 1998.
Gorospe WC, Reinhard M: Toxic effects on the ovary of the nonpregnant
female, in Witorsch RJ (ed): Reproductive Toxicology, 2d ed. New
York: Raven Press, 1995, pp 141–157.
Grasso P, Heindel JJ, Powell, CJ, et al: Effects on mono(2-ethylhexyl)
phthalate, a testicular toxicant, on follicle-stimulating hormone binding to membranes from cultured rat sertoli cells. Biol Reprod 48:454–
459, 1993.
Gray TJB, Beamand JA: Effect of some phthalate esters and other testicular toxins on primary cultures of testicular cells. Food Cosmet Toxicol
22:123–131, 1984.
Greenwell A, Tomaszewski KE, Melnick RL: A biochemical basis for
1,2-dibromo-3-chloropropane-induced male infertility: Inhibition of
sperm mitochondrial electron transport activity. Toxicol Appl Pharmacol 91:274–280, 1987.
Griswold MD: Interactions between germ cells and Sertoli cells in the testis.
Biol Reprod 52:211–216, 1995.
Gunn SA, Gould TC: Difference between dorsal and lateral components of
dorsolateral prostate in Zn65 uptake. Proc Soc Exp Biol Med 92:17–
20, 1956.
Haney AF: Effects of toxic agents on ovarian function, in Thomas JA,
Korach KS, McLachlan JA (eds): Endocrine Toxicology. New York:
Raven Press, 1985.
Hanly JR Jr, Yano BL, Nitschke KD, et al: Comparison of the teratogenic
potential of inhaled ethylene glycol monomethyl ether in rats, mice
and rabbits. Toxicol Appl Pharmacol 75:409–422, 1984.
Hayes AW: Principles and Methods of Toxicology. New York: Raven Press,
1982.
Heckert L, Griswold MD: Expression of the FSH receptor in the testis. Rec
Progr Horm Res 48:61–82, 1993.
Heindel JJ, Keith WB: Specific inhibition of FSH-stimulated cAMP accumulation by delta-9 tetrahydro-cannabinol in cultures of rat Sertoli
cells. Toxicol Appl Pharmacol 101:124–134, 1989.
Heller CG, Clermont Y: Kinetics of the germinal epithelium in man. Recent
Progr Horm Res 20:545–575, 1964.
Herbert DC, Supakar PC, Roy AR: Male reproduction, in Witorsch RJ (ed):
Reproductive Toxicology, 2d ed. New York: Raven Press, 1995, pp
3–21.
Hildebrand DC, Der R, Griffin WT, et al: Effect of lead acetate on reproduction. Am J Obstet Gynecol 115:1058–1065, 1973.
Hillier SG, Zeleznik AJ, Knazek RA, et al: Hormonal regulation of preovulatory follicle maturation in the rat. J Reprod Fertil 60:219–229,
1980.
Hirsch KS, Weaver DE, Black LJ, et al: Inhibition of central nervous system aromatase activity: A mechanism for Fenarinol-induced infertility in the male rat. Toxicol Appl Pharmacol 91:235–245, 1987.
Hodgen GD: Enzyme markers of testicular function, in Johnson AD, Gomes
WR (eds): The Testis. Vol 4. Advances in Physiology, Biochemistry,
and Function. New York: Academic Press, 1977.
Holt DW, Adams PC, Campbell RWJ, et al: Amiodarone and its desethyl
metabolite: Tissue distribution and ultrastructural changes in amiodarone-treated patients. Br J Clin Pharmacol 17:195–196, 1984.
Co
py
rig
hte
dM
ate
ria
l
Dixon RL: Toxic responses of the reproductive system, in, Klaassen CD,
Amdur MO, Doull J (eds): Casarett and Doull’s Toxicology: The Basic Science of Poisons, 3d ed. New York: Macmillian, 1986.
Dixon RL, Lee IP: Pharmacokinetic and adaptation factors in testicular toxicity. Fed Proc 39:66–72, 1980.
Dobson RL, Koehler CG, Felton JS, et al: Vulnerability of female germ
cells in developing mice and monkeys to tritium, gamma rays, and
polycyclic aromatic hydrocarbons, in Mahlum DD, Sikor MR, Hackett
PL, Andrew FD (eds): Developmental Toxicology of Energy-Related
Pollutants. Conference 771017. Washington, DC: U.S. Department of
Energy Technical Information Center, 1978.
Doering PL, Davidson CL, LaFauce L, et al: Effects of cocaine on the human fetus: A review of clinical studies. Drug Intellig Clin Pharm
23:639–645, 1989.
Donaubauer AH, Kramer M, Krein K, et al.: Investigations of the carcinogenicity of the LH-RH analog buserelin (HOE 766) in rats using the
subcutaneous route of administration. Fund Appl Toxicol 9:738–752,
1987.
Donovan MP, Schein LG, Thomas JA: Inhibition of androgen-receptor interaction in mouse prostate gland cytosol by divalent metal ions. Mol
Pharmacol 17:156–162, 1980.
Dym M, Fawcett DW: The blood-testis barrier in the rat and the physiological compartmentation of the seminiferous ephithelium. Biol
Reprod 3:300–326, 1970.
Eckols K, Williams J, Uphouse L: Effects of chlordecone on progesterone
receptors in immature and adult rats. Toxicol Appl Pharmacol
100:506–516, 1989.
Effendy I, Krause W: In vivo effects of terbinafine and ketoconazole on
testosterone plasma levels in healthy males. Dermatologica 178:103–
106, 1989.
Epstein SS, Arnold E, Andrea J, et al: Detection of chemical mutagens by
the dominant lethal assay in mice. Toxicol Appl Pharmacol 23:288–
325, 1972.
Eskeland NL, Lugo DI, Pintar JE, et al: Stimulation of beta-endorphin secretion by corticotropin-releasing factor in primary rat Leydig cell cultures. Endocrinology 124:2914–2919, 1989.
Ettlin RA, Dixon RL: Reproductive toxicology, in Mottet NK (ed): Environmental Pathology, Chemicals. New York: Oxford University Press,
1985.
Evenson DP, Baer RK, Jost LK, et al: Toxicity of thiotepa on mouse spermatogenesis as determined by dual-parameter flow cytometry. Toxicol
Appl Pharmacol 82:151–163, 1986.
Evenson DP, Janca FC, Jost LK: Effects of the fungicide methyl-benzimidazol-2-yl carbamate (MBC) on mouse germ cells as determined by
flow cytometry. J Toxicol Environ Health 20:387–399, 1987.
Ewing LL: The Leydig cell, in Scialli AR, Clegg ED (eds): Reversibility
in Testicular Toxicity Assessment. Boca Raton, FL: CRC Press, 1992.
Faber KA, Hughes CL Jr: Clinical aspects of reproductive toxicology, in
Witorsch RJ (ed): Reproductive Toxicology 2d ed. New York: Raven
Press, 1995, pp 217–240.
Fabrio S: On predicting environmentally-induced human reproductive hazards: An overview and historical perspective. Fund Appl Toxicol
5:609–614, 1985.
Faustman EM, Kirby Z, Gage D, et al: In vitro developmental toxicity of
five direct acting alkylating agents in rodent embryos: Structureactivity patterns. Teratology 40:199–210, 1989.
Feuston MH, Bodnar, KR, Kerstetter SL, et al: Reproductive toxicity of
2-methoxyethanol applied dermally to occluded and nonoccluded sites
in male rats. Toxicol Appl Pharmacol 100:145–161, 1989.
Foote RH, Berndtson WE: The germinal cells, in Scialli AR, Clegg ED
(eds): Testicular Toxicity Assessment. Boca Raton, FL: CRC Press,
1992.
Foster PMD: The Sertoli cell, in Scialli AR, Clegg ED (eds): Reversibility
in Testicular Toxicity Assessment. Boca Raton, FL: CRC Press, 1992.
Foster PMD, Blackburn DM, Moore RB, et al: Testicular toxicity of
2-methoxyacetaldehyde, a possible metabolite of ethylene glycol
monomethyl ether in the rat. Toxicol Lett 32:73–80, 1986.
Copyright © 2001 by The McGraw-Hill Companies
Retrieved from: www.knovel.com
2996R_ch20_673-709
4-17-2001
9:38 AM
Page 707
CHAPTER 20 TOXIC RESPONSES OF THE REPRODUCTIVE SYSTEM
and Developmental Toxicology. New York: Marcel Dekker, 1998, pp
655–691.
Lindemann CB, Kanous KS: Regulation of mammalian sperm motility.
Arch Androl 23:1–22, 1989.
Lipshultz L: “The debate continues” —The continuing debate over the possible decline in semen quality. Am Soc Reprod Med 65:909–911, 1996.
Lloyd SC, Blackburn DM, Foster PMD: Trifluoroethanol and its oxidative
metabolites: Comparison of in vivo and in vitro effects in rat testis.
Toxicol Appl Pharmacol 92:390–401, 1988.
Lonning PE, Lien EA: Pharmacokinetics of anti-endocrine agents. Cancer
Surv 17:343–370, 1993.
Lu T: Erectile dysfunction N Engl J Med 348:1802–1813, 2000.
MacLean HE, Warne GL, Zajac JD: Localization of functional domains in
the androgen receptor. J Steroid Biochem Mol Biol 62:4, 233–242,
1997.
MacLeod J: Human male infertility. Obstet Gynecol Surv 26:335–351,
1971.
Maddocks S, Parvinen M, Soder O, et al: Regulation of the testis. J Reprod Immunol 18:33–50, 1990.
Mann T, Lutwak-Mann C: Male Reproductive Function and Semen: Themes
and Trends in Physiology, Biochemistry, and Investigative Andrology.
New York: Springer-Verlag, 1981.
Mattison DR, Nightingale MS: The biochemical and genetic characteristics of murine ovarian aryl hydrocarbon (benzo[a]pyrene)hydroxylase
activity and its relationship to primordial oocyte destruction by polycyclic aromatic hydrocarbons. Toxicol Appl Pharmacol 56:399–408,
1980.
Mattison DR, Plowchalk DR, Meadows MJ, et al: Reproductive toxicity:
Male and female reproductive systems as targets for chemical injury.
Med Clin North Am 74:391–411, 1990.
McElveen JC Jr: Reproduction hazards in the workplace: Some legal considerations. J Occup Med 28:103–110, 1986.
McLachlan JA, Newbold RR, Korach KS, et al: Transplacental toxicology:
Prenatal factors influencing postnatal fertility, in Kimmel CA, BuelkeSam J (eds): Developmental Toxicology. New York: Raven Press, 1981.
Mebus CA, Welsch F, Working PK: Attenuation of 2-methoxyethanolinduced testicular toxicity in the rat by simple physiological compounds. Toxicol Appl Pharmacol 99:110–121, 1989.
Meistrich ML: Evaluation of reproductive toxicity by testicular sperm head
counts. J Am Coll Toxicol 8:551–567, 1989.
Millar JGB: Drug-induced impotence. Practitioner 223:634–639, 1979.
Moore NP, Creasy DM, Gray TJB: Urinary creatine profiles after administration of cell-specific testicular toxicants to the rat. Arch Toxicol
66:435–442, 1992.
Morrissey RE, Lamb IV JC, Schwetz BA, et al: Association of sperm, vaginal cytology, and reproductive organ weight data with results of continuous breeding reproduction studies in Swiss (CD-1) mice. Fundam
Appl Toxicol 11:359–371, 1988.
Mosher WD: Contraceptive utilization: United States. Vital Health Stat
23:1–58, 1981.
Müller AMF, Makropoulos V, Bolt HM: Toxicological aspects of pestrogenmimetic xenobiotics present in the environment. Toxicol Environ News
2:68–73, 1995.
Murdoch WJ: Immunoregulation of mammalian fertility. Life Sci 55:1871–
1886, 1994.
Nagano K, Nakayama E, Koyano M, et al: Testicular atrophy of mice induced by ethylene glycol monoakyl ethers. Jpn J Ind Health 21:29–
35, 1979.
Nahhas F, Blumenfeld F: Zona-free hamster egg penetration assay and prognostic indicator in an IVF program. Arch Androl 23:33–37, 1989.
Nakagawa S, Nakamura N, Fujioka M, et al: Spermatogenic cell apoptosis induced by mitomycin C in the mouse testis. Toxicol Appl Pharmacol 147. 204–213, 1997.
Nakano R: Apoptosis: Gene-directed cell death. Horm Res 48(suppl 3):2–
4, 1997.
Neaves WB: The blood-testis barrier, in Johnson AD, Gomes WR (eds):
The Testis. Vol 6. New York: Academic Press, 1977, pp.125–153.
Co
py
rig
hte
dM
ate
ria
l
Hruska RE, Olson JR: Species differences in estrogen receptors and in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure. Toxicol Lett
48:289–299, 1989.
Johnson EM: The scientific basis for multigeneration safety evaluations. J
Am Coll Toxicol 5:197–201, 1986.
Johnson L, Dickerson R, Safe SH, et al: Reduced Leydig cell volume and
function in adult rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin
without a significant effect on spermatogenesis. Toxicology 76:103–
118, 1992.
Keene LC, Davies PH: Drug-related erectile dysfunction. Adverse Drug
React Toxicol Rev 18:1, 5–24, 1999.
Keke WR, Stone CR, Laws SL, et al: Persistent DDT metabolite p,p-DDE
is a potent androgen receptor antagonist. Nature 375, 581–585, 1995.
Kelce WR, Monosson E, Gamcsik MP, et al: Environmental hormone disruptors: Evidence that vinclozolin developmental toxicity is mediated
by antiandrogenic metabolites. Toxicol Appl Pharmacol 126:276–285,
1994.
Kelce WR, Wilson EM: Clinical, functional and molecular implications of
environmental antiandrogens. J Mol Med 75:198–207, 1997.
Khan-Dawood FS, Satyaswaroop PG: Toxic effects of chemicals and drugs
on the sex accessory organs in the nonpregnant female, in Witorsch
RJ (ed): Reproductive Toxicology, 2d ed. New York: Raven Press, 1995,
pp 159–173.
Kimmel GL, Clegg ED, Crisp TM: Reproductive toxicity testing: A risk
assessment perspective, in Witorsch RJ (ed): Reproductive Toxicology,
2d ed. New York: Raven Press, 1995, pp 75–98.
Klein D, Wan YY, Kamyab S, et al: Effects of toxic levels of lead on gene
regulation in the male axis: Increase in messenger ribonucleic acids
and intracellular stores on gonadotrophs within the central nervous
system. Biol Reprod 50:802–811, 1994.
Koizumi T, Waalkes MP: Effects of zinc on the distribution and toxicity of
cadmium in isolated interstitial cells of the rat testis. Toxicology
56:137–146, 1989.
Ku WW, Wine RN, Chae BY, et al: Spermatocyte toxicity of
2-methoxyethanol (ME) in rats and guinea pigs: Evidence for the induction of apoptosis. Toxicol Appl Pharmacol 134:100–110, 1995.
Kuiper GGJM, Lemmen JG, Carlsson B, et al: Interaction of estrogenic
chemicals and phytoestrogens with estrogen receptor . Endocrinology 139:4252–4263, 1998.
Lancranjan I, Papescu HI, Gavanescu O, et al: Reproductive ability of
workmen occupationally exposed to lead. Arch Environ Health
30:396–401, 1975.
Lantz GD, Cunningham GR, Huckins C, et al: Recovery from severe
oligospermia after exposure to dibromochloropropane. Fertil Steril
35:46–53, 1981.
Lave LB: Toxic substances control in the 1990s: Are we poisoning ourselves with low-level exposures? Annu Rev Public Health 11:69–87,
1990.
LeBlond CP, Clermont Y: Definition of the stages of the cycle of the seminiferous epithelium of the rat. Ann NY Acad Sci 55:548–571, 1952.
Lee IP, Dixon RL: Effects of procarbazine on spermatogenesis studied by
velocity sedimentation cell separation and serial mating. J Pharmacol
Exp Ther 181:219–226, 1972.
Lee IP, Suzuki K, Nagayama J: Metabolism of benzo(a)pyrene in rat
prostate glands following 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure. Carcinogenesis 2:823–831, 1981.
Lee JP: Adaptive biochemical repair response toward germ cell DNA damage. Am J Ind Med 4:135–147, 1983.
Lemasters GK, Selevan SG: Use of exposure data in occupational
reproductive studies. Scand J Work Environ Health 10:1–6,
1984.
Levine RJ: Methods for detecting occupational causes of male infertility.
Scand J Work Environ Health 9:371–376, 1983.
Levine RJ, Blunden PB, DalCorso RD, et al: Superiority of reproductive
histories to sperm counts in detecting infertility at a DBCP manufacturing plant. J Occup Med 25:591–597, 1983.
Li LH, Heindel JJ: Sertoli cell toxicants, in Korach KS (ed): Reproduction
707
Copyright © 2001 by The McGraw-Hill Companies
Retrieved from: www.knovel.com
2996R_ch20_673-709
708
4-17-2001
9:38 AM
Page 708
UNIT 4 TARGET ORGAN TOXICITY
Roy AK, Lavrovsky CS, Song S, et al: Regulation of androgen action. Vitam
Horm 55:309–352, 1999.
Safe SH: Dietary and environmental estrogens and antiestrogens and their
possible role in human disease. Environ Sci Pollut Res 1(1):29–33,
1994.
Sakamoto J, Hashimoto K: Reproductive toxicity of acrylamide and related
compounds in mice—Effects on fertility and sperm morphology. Arch
Toxicol 59:201–205, 1986.
Sattler B: Rights and realities: A critical review of the accessibility of
information on hazardous chemicals. Occup Med 7:189–196,
1992.
Schrag SD, Dixon RL: Occupational exposure associated with male reproductive dysfunction. Annu Rev Pharmacol Toxicol 25:567–592,
1985.
Schwetz BA, Roa KS, Park CN: Insensitivity of tests for reproductive problems. J Environ Pathol Toxicol 3:81–98, 1980.
Scialli AR, Lemasters GK: Epidemiologic aspects of reproductive toxicology, in Witorsch RJ (ed): Reproductive Toxicology, 2d ed. New York:
Raven Press, 1995, pp 241–263.
Seed J, Chapin RE, Clegg ED, et al: Consensus report: Methods for assessing sperm motility, morphology, and counts in the rat, rabbit and
dog. Reprod Toxicol 10:237–244, 1996.
Selden JR, Robertson RT, Miller JE, et al: The rapid and sensitive detection of perturbations in spermatogenesis: Assessment by quantitative
dual parameter (DNA/RNA) flow cytometry. J Am Coll Toxicol 8:507–
523, 1989.
Selevan SG, Lindhohm ML, Hornung RW, et al: A study of occupational
exposure to antineoplastic drugs and fetal loss in nurses. N Engl J Med
313:1173–1178, 1985.
Setchell BP, Vogimayr JK, Waites GMH: A blood-testis barrier restricting
passage from blood lymph into rete testis fluid but not into lymph. J
Physiol 200:73–85, 1969.
Shaked I, Sod-Moriah UA, Kaplanski J, et al: Reproductive performance
of dibromochloropropane-treated female rats. Int J Fertil 33:129–133,
1988.
Sharpe RM: Toxicity of spermatogenesis and its detection, in Korach K
(ed): Reproductive and Developmental Toxicology. New York: Marcel
Dekker, 1998, pp 625–654.
Sharpe RM, Shakkenback NE: Are oestrogens involved in falling sperm
counts and disorders of the male reproductive tract? Lancet 341:1392–
1395, 1993.
Sheikh K: Choice of control population in studies of adverse reproductive
effects of occupational exposures and its effect on risk estimates. Br
J Ind Med 44:244–249, 1987.
Shemi D, Sod-Moriah UA, Kaplanski J, et al: Gonadotoxicity and kinetics
of dibromochloropropane in male rats. Toxicol. Lett 36:209–212,
1987.
Shen RS, Lee IP: Developmental patterns of enzymes in mouse testis. J
Reprod Fertil 48:301–305, 1977.
Shioda S, Legradi G, Leung W, et al.: Localization of pituitary adenylate
cyclase-activating polypeptide and its messenger ribonucleic acid in
the rat testis by light and electron microscopic immunocytochemistry
and in situ hybridization. Endocrinology 135:818–825, 1994.
Simpson JL: Genes, chromosomes, and reproductive failure. Fertil Steril
33:107–116, 1980.
Singh H, Kozel T, Jackson S: Effect of procarbazine on sperm morphology in Syrian hamsters. J Toxicol Environ Health 27:107–121, 1989.
Skinner MK, Schlitz SM, Anthony CA: Regulation of Sertoli cell differentiated function: Testicular transferrin and androgen-binding protein
expression. Endocrinology 124:3015–3024, 1989.
Slikker W Jr, Miller RK: Placental metabolism and transfer role in developmental toxicology, in Kimmel CA, Buelke JS (eds), Developmental Toxicology, 2d ed. New York: Raven Press, 1994.
Soderlund EJ, Brunborg G, Omichinski JG, et al: Testicular necrosis and
DNA damage caused by deuterated and methylated analogues of
1,2-dibromo-3-chloropropane in the rat. Toxicol Appl Pharmacol
94:437–447, 1988.
Co
py
rig
hte
dM
ate
ria
l
Nisbet IC, Karch NJ: Chemical Hazards to Human Reproduction. Park
Ridge, NJ: Noyes Data, 1983.
Office of Technology Assessment Report. Reproductive Health Hazards in the
Workplace. Washington, DC: U.S. Government Printing Office, 1985.
Ogawa T, Dobrinski I, Avarbock MR, Brinster RL: Transplantation of male
germ line stem cells restores fertility in infertile mice. Nature Med
5:29–34, 1999.
Okumura K, Lee IP, Dixon RL: Permeability of selected drugs and chemicals across the blood-testis barrier of the rat. J Pharmacol Exp Ther
194:89–95, 1975.
Olshan AF, Faustman EM: Male-mediated developmental toxicity. Annu
Rev Public Health 14:159–181, 1994.
Overstreet JW: Reproductive risk assessment. Teratogenesis Carcinog Mutagen 4:67–75, 1984.
Overstreet JW, Samuels SJ, Day P, et al: Early indicators of male reproductive toxicity. Risk Anal 8:21–26, 1988.
Papadopoulas C: Cardiovascular drugs and sexuality. Arch Intern Med
140:1341–1345, 1980.
Paul ME: Reproductive fitness and risk. Occup Med 3:323–340, 1988.
Pedersen I, Peters H: Proposal for a classification of oocytes and follicles
in the mouse ovary. J Reprod Fertil 17:555–557, 1968.
Pedersen RA, Brandriff B: Radiation- and drug-induced DNA repair in
mammalian oocytes and embryos, in Generoso WM, Shelby MD,
DeSerres FJ (eds): DNA Repair and Mutagenesis in Eukaryotes. New
York: Plenum Press, 1980.
Perrolle JA: Reproductive hazards: A model protection policy for the chemical industry. Occup Med 8:755–786, 1993.
Pogach LM, Lee Y, Gould S, et al: Characterization of cis-platinum-induced
Sertoli cell dysfunction in rodents. Toxicol Appl Pharmacol 98:350–
361, 1989.
Polascik TJ, Osterling JE, Partin AW: Prostate specific antigen: A decade
of discovery—What we have learned and where we are going. J Urol
162:293–306, 1999.
Prasad AS, Obeleas D, Wolf P, et al: Studies on zinc deficiency: Changes
in trace element and enzyme activities in tissues of zinc-deficient rats.
J Clin Invest 46:549–557, 1967.
Raffray M, Cohen GM: Apoptosis and necrosis in toxicology: A continuum of distinct modes of cell death? Pharmacol Ther 75:3, 153–177,
1997.
Rajfer J, Sikka SC, Lemmi C, et al: Cyclosporine inhibits testosterone
biosynthesis in the rat testis. Endocrinology 121:586–589, 1987.
Regaud CP: Degenerescence des cellules seminales chez mammiferes en
l’abscence de tout etat pathologique. CR Séances Soc Biol 52:268–
270.
Revelli A, Massobrio M, Tesarik J: Nongenomic actions of steroid hormones in reproductive tissues. Endocrinol Rev 19:3–17, 1998.
Richburg JH, Boekelheide K: Mono-(2-ethylhexyl) phthatate rapidly alters
both Sertoli cell vimentin filaments and germ cell apoptosis in young
rat testes. Toxicol Appl Pharmacol 137:42–50, 1996.
Richburg JH, Redenbach DM, Boekelheide K: Seminferous tubule fluid secretion is a Sertoli cell microtubule-dependent process inhibited by
2,5-Hexanedione exposure. Toxicol Appl Pharmacol 128:302–309,
1994.
Ritter EJ, Scott WJ Jr, Randall JL, et al: Teratogenicity of di(ethylhexyl)
phthalate, 2-ethylhexanol, 2-ethylhexanoic acid, and valproic acid, and
potentiation by caffeine. Teratology 35:41–46, 1987.
Robb GW, Amann RP, Killian GJ: Daily sperm production and epididymal
sperm reserve of pubertal and adult rats. J Reprod Fertil 54:103–107,
1978.
Roberts RA, Nebert DW, Hickman JA, et al: Perturbation of the
mitosis/apoptosis balance: A fundamental mechanism in toxicology.
Fundam Appl Toxicol 38:107–115, 1997.
Robertson JD, Orrenius S: Molecular mechanisms of apoptosis induced by
cytotoxic chemicals. Crit Rev Toxicol 30:609–627, 2000.
Rodamilans M, Osaba MJ Mtz, To-Figueras J, et al: Lead toxicity on endocrine testicular function in an occupationally exposed population.
Hum Toxicol 7:125–128, 1988.
Copyright © 2001 by The McGraw-Hill Companies
Retrieved from: www.knovel.com
2996R_ch20_709
5/22/01
9:37 AM
Page 709
CHAPTER 20 TOXIC RESPONSES OF THE REPRODUCTIVE SYSTEM
Toth GP, Zenick H, Smith MK: Effects of epichlorohydrin on male and female reproduction in Long-Evan rats. Fundam Appl Pharmacol 13:16–
25, 1989.
Tuchmann-Duplessis H, David G, Haegel P: Illustrated Human Embryology. Vol I. New York: Springer-Verlag, 1972.
Turner HE, Wass JAH: Gonadal function in men with chronic illness. Clin
Endocrinol 47:379–403, 1997.
Ulbrich B, Palmer AK: Detection of effects on male reproduction—A literature survey. J Am Coll Toxicol 14:293–327, 1996.
Van Pelt AMM, de Rooij DG, van der Burg B, et al: Ontogeny of estrogen
receptor- expression in rat testis. Endocrinology 140:478–483,
1999.
Vegeto E, Wagner BL, Imhof MO, et al: The molecular pharmacology of
ovarian steroid receptors. Vitam Horm 52:99–128, 1996.
Vos, JG, Dybing, E., Greim, HA, et al: Health effects of endocrinedisrupting chemicals on wildlife, with special reference to the
European situation. Crit Rev Toxicol 30:71–133, 2000.
Waalkes MP, Coogan TP, Barter RA: Toxicological principles of metal carcinogenesis with special emphasis on cadmium. Crit Rev Toxicol
22:175–201, 1992.
Waalkes MP, Donovan MP, Thomas JA: Cadmium-induced prostate metallothionein in the rabbit. Prostate 3:23–25, 1982.
Wang W, Chapin RE: Differential gene expression detected by suppression
substractive hybridization in ethylene glycol monomethyl etherinduced testicular lesion. Toxicol Sci 56:165–174, 2000.
Ways SC, Blair PB, Bern HA, et al: Immune responsiveness of adult mice
exposed neonatally to diethylstilbestrol, steroid hormones, or vitamin
A. J Environ Pathol 3:207–227, 1980.
Whorton DM, Kraus RM, Marshall S, et al: Infertility in male pesticide
workers. Lancet 2:1259–1267, 1977.
Williams J, Foster PMD: The production of lactate and pyruvate as sensitive indices of altered rat Sertoli cell function in vitro following the
addition of various testicular toxicants. Toxicol Appl Pharmacol
94:160–170, 1988.
Williams J, Gladen BC, Schrader, SM, et al: Semen analysis and fertility
assessment in rabbits: Statistical power and design considerations for
toxicology studies. Fundam Appl Toxicol 15, 651–665, 1990.
Wilson MJ: Toxicology of the male accessory sex organs and related glands,
in Witorsch RJ (ed): Reproductive Toxicology, 2d ed. New York: Raven
Press, 1995, 123–139.
Woods JS: Drug effects on human sexual behavior, in Woods NF (ed): Human Sexuality in Health and Illness, 3d ed. St. Louis: Mosby, 1984.
Working PK: Male reproductive toxicology: Comparison of the human to
animal models. Environ Health Perspect 77:37–44, 1988.
Wyrobek AJ: Methods for evaluating the effects of environmental chemicals on human sperm production. Environ Health Perspect 48:53–59,
1983.
Wyrobek AJ, Gordon LA, Burkhart JG, et al: An evaluation of the mouse
sperm morphology test and other sperm tests in nonhuman mammals.
A report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutat Res 115:1–72, 1983.
Zacharewski T, Sate SH: Antiestrogenic activity of TCDD and related compounds, in Korach KS (ed): Reproduction and Developmental Toxicology. New York: Marcel Dekker, 1998, pp 431–448.
Zeleznik AJ, Hillier SG, Knazek RA, et al: Production of long-term steroid
producing granulosa cell cultures by cell hybridization. Endocrinology 105:156–162, 1979.
Zenick H, Clegg ED: Tissues in risk assessment in male reproduction toxicology. J Am Coll Toxicol 5:249–261, 1986.
Zirkin BR, Chen H: Regulation of Leydig cell steroidogenic function during aging. Biol Reprod 63:977–981, 2000.
Co
py
rig
hte
dM
ate
ria
l
Somkuti SG, Lapadula DM, Chapin RE, et al: Reproductive tract lesions
resulting from subchronic administration (63 days) of tri-o-cresyl
phosphate in male rats. Toxicol Appl Pharmacol 89:49–63, 1987.
Spiteri-Grech J, Nieschlag E: Paracrine factors relevant to the regulation
of spermatogenesis. J Reprod Fertil 98:1–4, 1993.
Stevenson JG, Umstead, GS: Sexual dysfunction due to antihypertensive
agents. Drug Intell Clin Pharm 18:113–121, 1984.
Sullivan FM: The European Community classification of chemicals for reproductive toxicity. Toxicol Lett 64/65:183–189.
Sundaram K, Witorsch RJ: Toxic effects on the testes, in Witorsch RJ (ed):
Reproductive Toxicology, 2d ed. New York: Raven Press, 1995, pp
99–121.
Swain, A., Lovell-Badge, R. Mammalian sex determination: A molecular
drama. Genes Dev 13:755:767, 1999.
Thomas JA: Actions of chemicals and other factors on Leydig cell growth
and proliferation, in Endocrine Toxicology. New York: Raven Press,
1995b.
Thomas JA: Actions of drugs/chemicals on nonreproductive endocrine organs. Toxic Subst J 13:187–200, 1994.
Thomas JA: Effects of chemicals/drugs on spermatogenesis, in Hamamah
S, Mieusset R (eds): Male Gametes: Production and Quality. Paris:
INSERM, 1996, pp 151–158.
Thomas JA: Effects of pesticides on reproduction, in Thomas JA, Singhal
RL (eds): Molecular Mechanisms of Gonadal Hormone Action. Baltimore: University Park Press, 1975, pp 205–223.
Thomas JA: Phytoestrogens and hormonal modulation: A mini-review. Environ Nutr Int eract 1:5–12, 1997.
Thomas JA: Reproductive hazards and environmental chemicals: A review.
Toxic Subst J 2:318–348, 1981.
Thomas JA: Sperm counts, phytoestrogens, and estrogen-mimic substances,
in Dunaif GE, Olin SS, Scimeca, et al (eds): Human Diet and Endocrine Modulation (Estrogenic and Androgenic Effects). Washington
DC: ILSI Press, 1998, pp 257–263.
Thomas JA: Testes-specific metal toxicology, in Goyer RA, Waalkes MP,
Klassen CD (eds): Organ Specific Metal Toxicology. Orlando, FL: Academic Press, 1995a.
Thomas JA, Barsano CP: Occupational reproductive risks, in Encyclopedia of Environmental Control Technology. 1994, pp 195–215.
Thomas JA, Barsano CP: Pharmacologic and toxicologic responses in the
neonate. J Am Coll Toxicol 5:957–962, 1989.
Thomas JA, Barsano CP: Survey of reproductive hazards. J Am Coll Toxicol 5:203–207, 1986.
Thomas JA, Brogan WC: Some actions of lead on the sperm and on the
male reproductive system. Am J Ind Med 4:127–134, 1983.
Thomas JA, Curto KA, Thomas MJ: MEHP/DEHP gonadal toxicity and
effects on rodent accessory sex organs. Environ Health Perspect
45:85–92, 1982.
Thomas JA, Keenan EJ: Principles of Endocrine Pharmacology. New York:
Plenum Press, 1986.
Thomas JA, Thomas MJ: Biological effects of di-(2-ethylhexyl) phthalate and other phthalic acid esters. Crit Rev Toxicol 13:283–317,
1984.
Thomas MJ, Thomas JA: Hormone assays and endocrine function, in Hayes
AW (ed), Principles and Methods of Toxicology. Philadelphia: Taylor
& Francis, 2001, pp 1383–1414.
Thompson E Jr: The effects of estradiol upon the thymus of the sexually
immature female mouse. J Steroid Biochem 14:167–174, 1981.
Thompson EB: Apoptosis and steroid hormones. Mol Endocrinol 8:665–
673, 1994.
Torkelson RR, Sadek SE, Rowe VK: Toxicologic investigations of 1,2dibromo-3-chlorpropane. Toxicol Appl Pharmacol 3:545–557, 1961.
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