PART 3: FEMALE REPRODUCTIVE SYSTEM
CONTENTS
1.
2.
3.
4.
5.
6.
Normal female reproductive tract histology
Endocrine control of the oestrous cycle
Morphological changes during the oestrous cycle
Normal background variation of structure and common spontaneous lesions
Common morphological responses to endocrine disruption
References and bibliography
INTRODUCTION
i.
The female laboratory rat, like most placental mammals, demonstrates intrinsic reproductive cyclicity,
characterised by the regular occurrence of an oestrous cycle. During this cycle, numerous well defined and
sequential alterations in reproductive tract histology, physiology and cytology occur, initiated and regulated by
the hypothalamic-pituitary-ovarian (HPO) axis. The changes in vaginal cytology are discussed separately in
more detail in Part 5 of the Guidance Document.
ii.
The oestrous cycle consists of four stages, termed prooestrus, oestrus, metoestrus (or dioestrus 1) and
dioestrus (or dioestrus 2). Because rats are continuously polyoestrous (i.e., cycle constantly throughout the year),
dioestrus is immediately followed by the prooestrus phase of the next cycle. Anoestrus, a period of
reproductive quiescence between oestrous cycles, is thus not usually observed in healthy, cycling female rats.
Oestrous cyclicity only ceases during pseudopregnancy, pregnancy, and lactation, although a fertile
postpartum oestrus does occur within 24 hours after birth.
iii.
Sexual maturity in female rats usually occurs between 30 and 50 days of age. Kennedy and Mitra
(1963) reported the mean age of puberty in female rats, based on the occurrence of vaginal opening (VO) and
first oestrus, as 38 days. Recent studies evaluating the rodent pubertal female assay have recorded similar
mean VO ages in control rats of 32 and 35 days (Hyung et al, 2002; Goldman et al, 2000). Following puberty, the
oestrous cycle occurs regularly every 4-6 days for a variable proportion (25-70%) of the animal’s lifespan,
depending on the strain of rat. Sprague-Dawley rats, for example, demonstrate oestrous cycle abnormalities as
early as 6 months of age. In contrast, Fischer 344 rats show far greater reproductive robustness, typically
cycling normally until 15 to 18 months of age.
ix.
The length of each stage of the oestrous cycle, as reported by Mandl (1951), and Long and Evans (1922),
is shown in Figure 1.1. These workers found that within a given population of normally cycling rats, dioestrus
showed the greatest variation in duration and thus exerted the most influence over oestrous cycle length.
-1-
Mandl (1951)
Long and Evan
(1922)
Figure 1.1 – Duration (in hours) of the four stages of the oestrous cycle. Note the short duration of metoestrus.
1.
NORMAL FEMALE REPRODUCTIVE TRACT HISTOLOGY
Ovary
1.1
The paired ovaries of the rat are grape-like structures that vary in gross appearance and size,
depending on the stage of the oestrous cycle. The subgross anatomy of the rodent ovary is shown in Figure 1.2.
Covering its surface is a single layer of modified peritoneal mesothelium, the ovarian surface epithelium (OSE),
which is continuous with the broad ligament (mesovarium) that supports the ovary. The OSE of a single ovary
can range from squamous to cuboidal, columnar or pseudostratified columnar in type; this regional variation in
OSE morphology accompanies the cyclical changes that occur within the underlying ovarian parenchyma
during the oestrous cycle (Figures 1.3a and 1.3b).
Figure 1.2 – Subgross anatomy of the
normal rodent ovary (mouse, H&E
x4). The cortex (C) contains numerous
follicles at various stages of maturation.
The medulla (M), which is not always
present in histological sections, contains
lymphatics, nerves and numerous blood
vessels.
C
M
CL
F
-2-
cortex
medulla
corpus luteum
developing follicles
Figure 1.3a – Ovarian surface epithelium (OSE) – columnar type (rat, H&E x40).
OSE
TA
C
ovarian surface epithelium
tunica albuginea
cortex
Figure 1.3b – Ovarian surface epithelium (OSE) – simple squamous type (rat, H&E
x40).
-3-
1.2
The mesovarium completely encloses each ovary and oviduct within a single compartment, the ovarian
bursa (Figure 1.3c). In rodents, the ovarian bursa communicates with the peritoneal cavity via a small slit-like
opening.
Figure 1.3c – Rete ovarii (RO) and ovarian bursa (OB) (rat, H&E x10).
1.3
The ovarian stroma forms the body of the ovary and is composed of spindle-shaped, fibroblast-like cells
and delicate collagen fibres admixed with ground substance. The stroma directly beneath the OSE is dense and
fibrous, and forms a narrow and variably distinct zone termed the tunica albuginea. The ovarian stroma beneath
the tunica albuginea is divided into a peripheral cortex and central medulla (Figure 1.2), although the latter is
not always visible in histological sections of ovary.
1.4
The rete ovarii may be observed histologically within the rodent ovary. This structure arises from cells
of mesonephric origin which migrate into the developing gonad during embryogenesis. In the adult rat, the
rete ovarii is composed of several groups of anastomosing tubules embedded within the ovarian stroma and
lined by a cuboidal or columnar epithelium (Figure 1.3c).
1.5
In sexually mature rats, the cortex contains numerous follicles at various stages of development. Five
stages of follicular maturation (folliculogenesis) are described:
i.
Primordial follicle – This represents the earliest stage of follicular development. Primordial follicles form
during early foetal development and are typically located within the peripheral cortex, just beneath the
tunica albuginea. Each primordial follicle consists of a primary oocyte surrounded by a simple squamous
follicular epithelium. Envelopment of the primary oocyte by follicular cells arrests development of the
germ cell at the first meiotic division. During each oestrous cycle a cohort of “resting” primordial follicles
starts to develop into primary follicles; this process occurs occurs independently of hormonal stimulation
up until the formation of early tertiary follicles.
ii.
Primary follicle – The squamous follicular cells surrounding the primordial follicle differentiate into a
single layer of columnar cells, forming a primary follicle (Figure 1.4).
-4-
Figure 1.4 – Primary (A) and early secondary follicle (B) (rat, H&E x40).
O
ZG
TA
OSE
primary oocyte
developing zona granulosa
tunica albuginea
ovarian surface epithelium (cuboidal)
iii.
Secondary follicle – Proliferation of the columnar cell monolayer results in the formation of a multilayered
zone of granulosa cells, the zona granulosa, around the oocyte. This is accompanied by the development of a
thick glycoprotein and acid proteoglycan coat, the zona pellucida, between the oocyte and the zona
granulosa (Figures 1.4 and 1.5). As the secondary follicle continues to grow, multiple fluid-filled spaces
form within the zona granulosa; this stage is termed a vesicular follicle. Ovarian stromal cells surrounding
the developing follicle become arranged into concentric layers and form the theca folliculi, or theca (Figures
1.5 and 1.6). This layer is separated from the zona granulosa by a basement membrane.
iv.
Tertiary follicle – The cystic spaces within the zona granulosa coalesce and form a large central cavity, the
follicular antrum. This cavity is filled with fluid, the liquor folliculi, and surrounded by the zona granulosa.
The primary oocyte is eccentrically positioned within the tertiary follicle and resides within a mound of
granulosa cells, called the cumulus oophorus, that protrudes into the antrum. The granulosa cells
immediately surrounding the oocyte are termed the corona radiata (Figure 1.7).
The theca of the tertiary follicle is divisible into two zones: a theca interna and theca externa. The theca
interna consists of polygonal cells with vacuolated cytoplasm and open faced, vesicular nuclei. These cells
demonstrate the typical ultrastructural characteristics of steroid producing cells (e.g. numerous cytoplasmic
lipid droplets, large numbers of mitochondria, and an extensive smooth endoplasmic reticulum), and are
the main site of synthesis of androstenedione (a sex steroid intermediate). In contrast, the cells of the theca
externa are spindle-shaped and merge with the surrounding ovarian stroma; they serve no endocrine
function.
-5-
Figure 1.5 – Secondary follicle (mouse, H&E x20).
O
ZP
VS
primary oocyte
zona pellucida
vesicular spaces
ZG
TF
zona granulosa
theca folliculi
Figure 1.6 – Vesicular follicle (mouse, H&E x20). Vesicular spaces (VS) are
clearly visible within the zona granulosa (ZG).
-6-
Figure 1.7 – Tertiary follicle (rat, H&E x20). The large follicular antrum (FA) and
eccentrically positioned primary oocyte (O) characterise this stage.
O
CR
ZG
v.
primary oocyte
corona radiata
zona granulosa
CO
FA
TF
cumulus oophorus
follicular antrum
theca folliculi
Preovulatory (Graafian) follicle – A small number of tertiary follicles enter a preovulatory stage and
undergo further morphological changes. The follicular antrum continues to enlarge, causing attenuation of
the surrounding zona granulosa. Degeneration of the granulosa cells of the cumulus oophorus occurs; this
causes the primary oocyte to detach from the zona granulosa and float freely within the follicular antrum
(Figure 1.8). The primary oocyte completes the first meiotic division just prior to ovulation and forms the
secondary oocyte.
Figure 1.8 – Preovulatory (Graafian) follicle
(rat, H&E x40). The primary oocyte (O) floats
freely within the follicular antrum (FA).
O
ZP
CR
FA
ZG
-7-
primary oocyte
zona pellucida
corona radiata
follicular antrum
zona granulosa
1.6
Following extrusion of the secondary oocyte from the Graafian follicle, the granulosa and thecal cells of
the follicle remnant undergo hypertrophy and, to a lesser extent, hyperplasia (Figure 1.9). This process, termed
lutenisation, occurs under the influence of lutenising hormone (LH) and prolactin, the two major luteotrophic
hormones in rodents. Lutenisation is accompanied by degeneration of the basement membrane separating the
theca interna and zona granulosa, and infiltration of the postovulatory follicle by blood vessels from the theca
interna. The resulting mature corpus luteum (“yellow body”) is a large eosinophilic structure that may bulge out
from the ovarian surface or obscure the ovarian corticomedullary junction, depending on its location (Figure
1.10).
Figure 1.9 – Corpus luteum (rat, H&E
x40). The luteal cells (LC) comprising
the corpus luteum are plump and
polygonal; they contain large nuclei and
moderate amounts of eosinophilic
cytoplasm. Cytoplasmic vacuoles form
within luteal cells as the corpus luteum
matures and subsequently degenerates.
Numerous blood vessels (BV) are
present, consistent with its function as a
temporary endocrine gland.
Figure 1.10 – Corpora lutea (CL)
(rat, H&E x10). Note the marked
protrusion of these large postovulatory
follicles beyond the surface of the ovary.
Another pair of corpora lutea are present
within the body of the ovary.
-8-
1.7
Each corpus luteum matures during the oestrous cycle in which it is formed before regressing over the
course of several subsequent cycles. Consequently, at least three sets of corpora lutea are present within the
ovaries of normally cycling rats. Degenerating corpora lutea progressively shrink in size and are characterised
by increased amounts of fibrous tissue and yellow-brown lipofuscin pigment (Figure 1.11). The fibrous tissue
mass that constitutes the corpus luteum during the final stages of regression is termed the corpus albicans
(“white body”); this undergoes complete regression in the rat, leaving no fibrous tissue remnant within the
ovary.
Figure 1.11 – Degenerating corpus luteum (rat,
H&E x20). Note the invading fibroblasts (F) and
lipofuscin pigment (L).
1.8
Only a small number of primordial follicles within the cohort progress through folliculogenesis to form
Graafian follicles and ovulate. The remainder undergo follicular degeneration, or atresia, at various stages
during follicular maturation. In rodents, degenerating tertiary follicles (Figures 1.12 and 1.13) eventually give
rise to interstitial glands within the ovarian stroma, although these are more obvious in the mouse than the rat.
These glands are composed of aggregates of theca interna cells, often arranged around remnants of degenerate
zonae pellucida (Figure 1.14). They are transitory structures, eventually breaking up into small clusters of
interstitial cells that become scattered throughout the medulla.
Figure 1.12 – Degenerate (atretic) tertiary follicle
(TF) (rat, H&E x10). A viable vesicular follicle (VF)
is also present.
-9-
Figure 1.13 – Degenerate (atretic) tertiary follicle (rat, H&E x40). Pyknotic nuclei
and karyorrhectic nuclear debris are scattered throughout the degenerate zonular
granulosa (ZG).
TF
FA
ZG
theca folliculi
follicular antrum
degenerating zona granulosa
Figure 1.14 – Interstitial glands (mouse, H&E x20). Theca interna cells surround
remnants of degenerate zonae pellucida (ZP).
- 10 -
Uterus
1.9
The rat uterus is duplex, i.e. it comprises two uterine horns that join together and open into the vagina
via two separate cervices. The basic histological organisation of the uterus is shown below in Figures 1.15:
Figure 1.15 – Uterine horn (rat, H&E x10,
longitudinal section).
En
Ep
LP
G
endometrium
surface epithelium
lamina propria
endometrial gland
My
C
L
myometrium
circular layer (inner)
longitudinal layer (outer)
Pe
perimetrium
Figure 1.16 – Endometrium (rat, H&E x20). The variation in
surface epithelium height is an artefact of sectioning.
Ep
LP
G
surface epithelium
lamina propria
endometrial gland
BV
P
blood vessel
pigment
The inner mucosa, or endometrium, consists of a surface
columnar epithelium overlying a thick lamina propria
containing numerous blood vessels and endometrial
glands. The middle muscular layer, or myometrium, is
composed of an inner circular and outer longitudinal
smooth muscle layer. The myometrium is covered by
the perimetrium, a thin connective tissue layer overlain
by a simple serosa. Variable numbers of lymphocytes
and other leucocytes are present within the superficial
lamina propria of the endometrium.
Pigmented
stromal cells, containing fine to coarse green-brown
granules composed of ferritin, haemosiderin and
lipofuscin, may also be observed within the lamina
propria of postpartum and mature rodents (Figure
1.16).
- 11 -
Vagina
1.10
The basic trilaminar arrangement of tissues observed in the uterus is conserved in the vagina, which
consists of an inner mucosa, middle muscularis and outer adventitia (Figures 1.17 and 1.18). The vaginal
mucosa comprises a lamina propria covered by a stratified squamous epithelium (Figure 1.19). The mucosa
undergoes profound cyclic changes over the course of the oestrous cycle; these alterations are discussed in
more detail below. The smooth muscle bundles that form the muscularis are also disposed in inner circular and
outer longitudinal layers, although these are ill-defined in comparison with those forming the myometrium.
The outer longitudinal layer of the muscularis merges with the adventitia, a thin outer connective tissue layer.
Figure 1.17 – Transverse section through
vagina and urethra (rat, H&E x4).
V
U
Lu
Ep
vagina
urethra
vaginal lumen
surface epithelium
Lu
Ep
LP
M
Ad
vaginal lumen
surface epithelium
lamina propria
muscularis
adventitia
Figure 1.18 – Transverse section through vagina (rat, H&E x10).
- 12 -
Figure 1.19 – Transverse section through vagina (rat, H&E
x20). During prooestrus, the vaginal epithelium consists of four
layers (Table 3.1). The stratum granulosum (SG) comprises the
superficial 2-3 cell layers immediately beneath the stratum
corneum (SC). The stratum germinativum (SGerm) is the only
layer that is present throughout the oestrous cycle.
SM
SC
SG
SGerm
- 13 -
stratum mucification
stratum corneum
stratum granulosum
stratum germinativum
2.
ENDOCRINE CONTROL OF THE OESTROUS CYCLE
Introduction
2.1
The cyclic changes that occur in the female reproductive tract are initiated and regulated by the
hypothalamic-pituitary-ovarian (HPO) axis. Although folliculogenesis occurs independently of hormonal
stimulation up until the formation of early tertiary follicles, the gonadotrophins lutenising hormone (LH) and
follicle stimulating hormone (FSH) are essential for the completion of follicular maturation and development of
mature preovulatory (Graafian) follicles.
The typical reproductive hormonal patterns observed over the course of the rat oestrous cycle are shown
schematically in Figure 2.1 below. The sites of production and key functions of these hormones are
summarised in Table 2.1.
O
PL
LH
FSH
P
oestrogen
prolactin
lutenising hormone
follicle stimulating hormone
progesterone
Figure 2.1 – Reproductive hormone fluctuations during the rat oestrous cycle. Oestrogen and
lutenising hormone levels peak during prooestrus. Ovulation follows approximately 10-12 hours later
and coincides with the progesterone peak. Follicle stimulating hormone peaks twice in the rat, with the
secondary FSH peak occurring at the time of ovulation or shortly after (see text for further details).
D
Oe
dioestrus
oestrus
PO
M
- 14 -
prooestrus
metoestrus
Pituitary gonadotrophin secretion drives follicular maturation and oestrogen secretion
2.2
Levels of LH and FSH begin to increase just after dioestrus. Both hormones are secreted by the same
secretory cells (gonadotrophs) in the pars distalis of the anterior pituitary (adenohypophysis). FSH stimulates
development of the zona granulosa and triggers expression of LH receptors by granulosa cells. LH initiates the
synthesis and secretion of androstenedione and, to a lesser extent, testosterone by the theca interna; these
androgens are utilised by granulosa cells as substrates in the synthesis of oestrogen. Pituitary release of
gonadotrophins thus drives follicular maturation and secretion of oestrogen during prooestrus.
2.3
Gonadotrophin secretion by the anterior pituitary is regulated by lutenising hormone-releasing hormone
(LHRH), produced by the hypothalamus. LHRH is transported along the axons of hypothalamic neurones to
the median eminence, where it is secreted into the hypothalamic-hypophyseal portal system and transported to
the anterior pituitary. The hypothalamus secretes LHRH in rhythmic pulses; this pulsatility is essential for the
normal activation of gonadotrophs and subsequent release of LH and FSH.
Ovulation is triggered by an oestrogen-mediated preovulatory LH surge
2.4
The increase in oestrogen observed during prooestrus initiates several characteristic morphological
changes in the uterus and vagina (Table 2.1). This rise in oestrogen also suppresses release of LHRH by the
hypothalamus, as well as directly inhibiting pituitary secretion of both LH and FSH. Negative feedback control
of pituitary FSH secretion is also achieved by the peptide inhibin, produced by the granulosa cells of the
maturing follicle. The various hormonal interactions of the HPO axis are summarised in Figure 2.2 below.
2.5
Oestrogen levels rise during the morning, peak around midday and then fall during the afternoon of
prooestrus. Once peak levels are reached, the inhibition of LHRH and gonadotrophin secretion by this ovarian
steroid ceases; oestrogen instead starts promoting hypothalamic LHRH release, as well as augmenting anterior
pituitary responsiveness to LHRH. This positive oestrogenic modulation of hypothalamic-pituitary function
results in a preovulatory LHRH surge and corresponding surge in LH (Figure 2.2).
2.6
The LH surge, which closely follows the oestrogen peak, occurs during the afternoon of prooestrus and
triggers ovulation approximately 10-12 hours later. FSH levels peak twice in the rat; the first (preovulatory)
peak is LHRH-dependent and occurs in concert with the LH peak. This is followed by a second (postovulatory)
rise in FSH that occurs at the time of ovulation or shortly after. This secondary FSH elevation is thought to be
LHRH-independent, reflecting reduced inhibin synthesis by the postovulatory follicle.
The rat corpus luteum is short-lived and regresses in the absence of prolactin stimulation
2.7
Progesterone levels start to increase during prooestrus and peak during ovulation. Like oestrogen,
progesterone feedback control of hypothalamic-pituitary function may be negative or positive, depending on
the stage in the oestrous cycle (Figure 2.2). Following ovulation, progesterone synergises with oestrogen to
inhibit gonadotrophin secretion.
Conversely, during prooestrus, rising progesterone levels trigger
hypothalamic LHRH secretion, stimulating gonadotrophs in the anterior pituitary and reinforcing the
preovulatory LH surge.
2.8
After ovulation, lutenisation of the follicular granulosa and thecal cells occurs resulting in the
formation of the corpus luteum. In the rat, the corpus luteum secretes progesterone autonomously for
approximately 48 hours, before becoming non-functional and degenerating over the course of several
subsequent oestrous cycles.
- 15 -
2.9
Prolongation of corpus luteum function requires continued pituitary secretion of prolactin, the major
luteotrophic hormone in the rat. Prolactin levels peak and fall simultaneously with the preovulatory LH surge
in normally cycling rats. Copulation during oestrus causes vaginocervical stimulation which triggers, via a
neuroendocrine reflex, twice daily prolactin release by lactotrophs in the adenohypophysis. This matinginduced prolactin secretion disrupts normal oestrous cyclicity by maintaining the newly formed corpus luteum
in a functional state, thus prolonging dioestrus.
2.10
The continued secretion of progesterone during this lengthened dioestrus phase initiates development
of the endometrium in preparation for implantation of the fertilised ovum, as well as initiating and maintaining
vaginal mucification (Table 2.1). If the mating is sterile or implantation fails to occur, the corpus luteum
regresses, terminating the prolonged dioestrus phase (referred to as pseudopregnancy) after 12-14 days, thus
allowing resumption of normal reproductive cyclicity.
Table 2.1 – Major reproductive hormones in the rat: sources and key functions.
HORMONE
SOURCE
LUTENISING
HORMONERELEASING
HORMONE
(LHRH)
Hypothalamus
LUTENISING
HORMONE
(LH)
FOLLICLE
STIMULATING
HORMONE
(FSH)
OESTROGEN
KEY FUNCTIONS


Pulsatile secretion of LHRH stimulates gonadotrophs in anterior pituitary
Triggers release of lutenising hormone (LH) and follicle stimulating
hormone (FSH)
Anterior pituitary
(adenohypophysis)



Stimulates production of androstenedione and testosterone by theca interna
Follicular granulosa cells utilise androgens in synthesis of oestrogen
LH surge triggers ovulation
Anterior pituitary
(adenohypophysis)


Stimulates development of follicular granulosa cells
Upregulates granulosa cell LH receptor expression


Negative and positive feedback control of hypothalamic-pituitary function
Stimulates:
- proliferation and cornification of vaginal epithelium
- increased vascular permeability
- neutrophil infiltration of vaginal epithelium and uterine endometrium
- proliferation of endometrial stromal fibroblasts and epithelial cells
- hypertrophy of uterine myometrium


Negative and positive feedback control of hypothalamic-pituitary function
Stimulates:
- mucification of vaginal epithelium†
- activation and differentiation of endometrial stromal fibroblasts†
- endometrial gland secretion
- hypertrophy of uterine myometrium


Vaginocervical stimulation triggers prolactin secretion by lactotrophs
Major luteotrophic factor in the rat – required for maintenance of corpus
luteum

Negative feedback control of pituitary FSH secretion
Preovulatory
(Graafian) follicle
PROGESTERONE
Postovulatory
follicle
(corpus luteum)
PROLACTIN
Anterior pituitary
(adenohypophysis)
INHIBIN
Preovulatory
(Graafian) follicle
†requires
- 16 -
priming with oestrogen
Figure 2.2 – Hormonal interactions of the HPO axis. Pulsatile release of lutenising hormone-releasing hormone
(LHRH) by the hypothalamus drives pituitary secretion of gonadotrophins. The ovarian steroids oestrogen and
progesterone exert feedback control at the level of the hypothalamus and pituitary.
- 17 -