/ . Embryo!, exp. Morph. Vol. 59, pp. 27-37, 1980
Printed in Great Britain © Company of Biologists Limited 1980
27
Sterility in mutant (tLxjtLy) male mice
I. A morphological study of spermiogenesis
By NINA HILLMAN 1 AND MARY NADIJCKA 1
From the Department of Biology, Temple University, Philadelphia
SUMMARY
The results from a comparative ultrastructural study of spermiogenesis in 6-, 10-, 14- and
17-month-old sterile t6/tw32, and fertile T/t«, T/twZ2 and BALB/c, mice are reported. The
studies show that all of the males contained the same types of defective spermatids and that
defects were not limited to specific spermatid stages. Younger males had fewer abnormal
spermatids than older males of the same genotype and at each age the BALB/c and t6/tw32
males appeared to contain more abnormal spermatids than the other males. No unique
spermatid defect or increased frequency of a specific defect was found which can be correlated
with infertility of the t^/tw32 males.
INTRODUCTION
The T/t complex in the house mouse is located on chromosome 17 and
includes a series of recessive tn mutations, referred to as f-haplotypes (Artzt &
Bennett, 1975). Detailed mapping and genetic studies of the recessive mutations
have recently been reported (Lyon, Evans, Jarvis & Sayers, 1979). The categories
of the recessive mutations - lethal (tL), semi-lethal (tSL), and viable (f*7) - are
based on their effect on embryo viability in a homozygous condition. The lethal
recessive mutations have been further subdivided into six classes according to
their abilities to partially complement each other genetically (Bennett, 1975).
A number of the tn mutations have also been shown to cause a reduction in male
fertility when present as a homozygous (V'x/t1}X) or heterozygous (tnx/t"v)
genotype. Various combinations of the recessive mutations always result in
male sterility (Bryson, 1944; Braden & Gluecksohn-Waelsch, 1958; Dunn,
Bennett & Beasley, 1962; Dunn, 1964). These combinations are (1) homozygosity for a semi-lethal mutation (tSL/tSL); (2) heterozygosity for semi-lethal
mutations (tSLx/tSLy); (3) heterozygosity for a lethal and a semi-lethal mutation
(tL/tSL) and (4) heterozygosity for lethal mutations from different complementation groups (iLx/tLy; hereafter referred to as intercomplement males).
Other combinations of the recessive mutations (e.g. tv/tv; tVx/tVv; tL/tv;
tv/tSL) can result in male sterility, quasi-sterility or fertility (Rajasekarasetty,
1954; Braden & Gluecksohn-Waelsch, 1958; Dunn & Bennett, 1969).
Spermiogenesis in t°/tw32 intercomplement males has been described by
1
Authors' address: Department of Biology, Temple University, Philadelphia, Pennsylvania
19122, U.S.A.
28
N. HILLMAN AND M. NADIJCKA
Dooher & Bennett (1977). They noted that spermatid development is completely
normal prior to Stage 11 of development. At Stage 11 and in later developmental stages, however, spermatids show nuclear and head abnormalities.
Dooher & Bennett suggested that these defects are caused by the t mutations,
stating that 'genetic factors other than T/t haplotypes are insignificant in
determining the morphological features of the spermatids'. However, these
investigators did not report the results of comparative ultrastructural studies of
spermiogenesis in control fertile males. Since most of the spermatid defects
described by Dooher & Bennett have been found among the spermatids of other
non-r"-bearing genetic and inbred strains of mice (Johnson & Hunt, 1971;
Bennett, Gall, Southard & Sidman, 1971; Hillman & Nadijcka, 1978), we
undertook the present comparative study of spermiogenesis in correspondinglyaged T/t6, T/tw32, t6/tw32 and BALB/c mice to determine first, if intercomplement males have unique spermatid defects and second, if these defects become
apparent only in the late stages of spermatid development.
MATERIALS AND METHODS
The comparative electron microscopic studies were done on 6-, 10-, 14- and
17-month-old T/t6, T/tw3\ t6/iw32 and BALB/c ( + / + ) mice. The T/t6, T/tw32
and BALB/c males were obtained from inbred stocks maintained by brothersister matings. The intercomplement sterile males were obtained from matings
between T/t6 males and T/tw32 females. This cross produces T/T embryos which
die in utero (Chesley, 1935) and viable T/tL and t6/tw32 offspring. The T/tL
offspring are tailless while the t6/tw32 animals have normal tails. Those animals
which were classified by their phenotype to be intercomplement males were
tested for ten mating units (Bennett & Dunn, 1967) with fertile females to assure
that they were genotypically t6/tw32 males and sterile. The control males were
tested for their levels of fertility and all were classified as being normal fertile
prior to being used for this study. Six males of each genotype were examined at
each age.
The males were sacrified by cervical dislocation and their testes removed and
processed for electron microscopic studies. The protocol used for preparing the
tissues for study followed that described by Hillman & Nadijcka (1978).
Ultrathin sections were stained with either lead citrate (Venable & Coggeshall,
1965) or with both lead citrate and 2 % uranyl acetate (Watson, 1958). The
sections were examined with a Philips 300 electron microscope.
We do not include, in this report, a detailed description of normal mouse
spermatid development. Our criteria for normal spermatid morphology were
obtained from the following studies: Fawcett & Phillips, 1969; Fawcett, Eddy &
Phillips, 1970; Sandoz, 1970; Bennett et al, 1971; Fawcett, Anderson & Phillips,
1971; Bryan & Wolosewick, 1973. The spermatids were classified according to
the staging by Oakberg (1956) as modified by Dooher & Bennett (1973).
Spermatid defects in mutant (tLx/tLy) male mice
29
RESULTS
Spermatid abnormalities
General observations
The same types of abnormal spermatids were found in males representing all
of the genotypes examined. Spermatids at all stages of development showed
aberrant morphologies, with no specific defect limited to the intercomplement
males. Both control and intercomplement males contained aberrant spermatids
which were clustered in the seminiferous tubules. This clustering has been
reported by Bryson (1944), Rajasekarasetty (1954) and Hillman & Nadijcka
(1978). Because of this clustering and because the sections of seminiferous
tubules were randomly selected for microscopic examination, it was not possible
to quantitate the numbers of abnormal spermatids in the intercomplement
males. However, the relative ease of observing aberrant spermatids in the 6month-old intercomplement males suggested that the sterile males contained a
high frequency of abnormal spermatids. This frequency was apparently comparable to that in BALB/c animals, but greater than that in the correspondinglyaged T/tQ and T/tw32 males.
The types of spermatid abnormalities present in the youngest animals (six
months) were found also in 10-, 14- and 17-month-old tytw32, T/tw32, T/t* and
BALB/c mice. However, based on the relative frequencies of finding abnormal
spermatids, there appeared to be increased numbers of defective spermatids in
males from each of the four genotypes when they were examined and compared
at older ages. The BALB/c and intercomplement males appeared to have
higher frequencies of aberrant spermatids than either the T/tw32 or the T/t6
mice at all of the ages examined.
Specific observations
All males contained defective young spermatids. For example, all of the
animals contained Stage-3 spermatids which had duplicated proacrosomal
vesicles and granules. We suggested earlier that this duplication may lead to the
rostral and lateral bifidity and bifurcation of spermatid nuclei (Hillman &
Nadijcka, 1978). Bifid and bifurcated spermatids were present in all males. The
extent of the bifidity and bifurcation varied from only a slight bifidity to a cleft
or bifurcation which extended posteriorly beyond the level of the perinuclear
ring to the caudal-most portion of the nucleus. Males of all genotypes and ages
contained spermatids which showed the complete range of this defect.
An additional early spermatid defect was the presence of binucleated and
multinucleated cells. Of these cells, only those in which two or more nuclei
share a single acrosome give rise to aberrantly-shaped spermatids. These
abnormal cells have been found in numerous inbred and random-bred strains of
mice. The effect of this condition on the morphological development of the
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N. HILLMAN AND M. NADIJCKA
Spermatid defects in mutant (tLx/tLy) male mice
31
Fig. 5. A longitudinal section through two normal and one abnormal Stage-11
spermatids. Note, in the abnormal spermatid, that the apex of the acrosome (arrow)
is adjacent to the perinuclear ring (PR), x 7000.
Fig. 6. This photomicrograph shows sections of two late Stage-9 blunted spermatids. Note that the acrosomal apices (arrows) are abnormally positioned in both
spermatids. x 5600.
FIGURES
1-4
Fig. 1. A section through a normal Stage-6 spermatid. Note the spatial relationship
of the acrosomal cap (A) with the implantation fossa (arrow), x 7000.
Fig. 2. A longitudinal section through a normal Stage-9 spermatid. Note that the
rostral apex (arrow) of the acrosome is midway between the extreme edges of the
acrosomal cap. In serial sections of this spermatid, the implantation fossa of the
flagellum was found to be positioned directly opposite the apex of the acrosome.
x 9400.
Fig. 3. This micrograph shows an abnormal Stage-5 spermatid. Note the misplacement of the acrosomal cap and the future rostrum of the acrosome (arrow)
relative to the implantation fossa (IF) (cf. Fig. 1). x 10600.
Fig. 4. A longitudinal section through an abnormal Stage-9 spermatid. Note that
the apex of the rostrum (arrow) has formed opposite to the implantation fossa but
does not bisect the acrosomal cap. This abnormal positioning of the apex causes the
acrosomal portion of the nucleus to be misshaped and the entire nucleus to become
shortened and wedge-shaped (cf. Fig. 2). x 9400.
3-2
32
N. HILLMAN AND M. NADIJCKA
Spermatid defects in mutant (tLx/tLy) male mice
33
Fig. 11. A longitudinal section through a multiply defective early Stage-14 spermatid.
These defects include nuclear bifidity, retraction of the condensed chromatin from
the nuclear membrane and, possibly, an extensive perinuclear ring with excessive
microtubules. x 13 000.
resultant spermatids has been discussed in detail (Hunt & Johnson, 1971;
Johnson & Hunt, 1971; Bryan & Wolosewick, 1973; Johnson, 1974).
A third spermatid defect, found in all males, was an aberrant spatial relationship of the developing component parts of the spermatid. The most frequently
encountered defect in this category was the incorrect relative positioning of the
Golgi apparatus and its structural derivatives, the proacrosomal vesicle and its
granule, with the positioning of the implantation fossa of the developing
flagellum. Normally the vesicle and the fossa form at opposite poles of the
spermatid nucleus, establishing, respectively, the future rostral and caudal
regions of the elongated nucleus. Subsequent to proacrosomal vesicle formation,
the vesicle spreads in all directions to cover the anterior one-third of the nucleus,
FIGURES
7-10
Fig. 7. A cross section through an aberrantly-shaped Stage-5 spermatid nucleus at
the level of the acrosomal cap. x 10400.
Fig. 8. A micrograph of an abnormal Stage-5 spermatid. The portion of the nucleus
subjacent to the acrosomal cap has a normal configuration. The remainder of the
nucleus is aberrantly shaped. Note the disrupted and reflected nuclear membrane
(arrow), x 7200.
Fig. 9. A section through an aberrant Stage-11 spermatid. Note the presence of
Sertoli cell cytoplasm projecting into both the acrosomal and postacrosomal regions
of the nucleus (arrows). The projection in the rostral region of the nucleus is
circumscribed by the nuclear, acrosomal and plasma membranes, x 14000.
Fig. 10. A section of an extremely bizarre nucleus of a Stage-12 spermatid. Sertoli
cell cytoplasm (arrows) is projected into the nucleus at points both anterior and
posterior to the perinuclear ring (PR), x 14000.
34
N. HILLMAN AND M. NADIJCKA
forming the acrosomal cap. During normal development, the centre of this cap
is positioned directly opposite the implantation fossa (Fig. 1). This central
area of the cap becomes the apex of the acrosome (Fig. 2). In the aberrant
spermatids the acrosomal cap is shifted so that it covers the lateral portion of
the nucleus (Fig. 3). This shift also misaligns the positioning of the presumptive
apex of the developing rostrum relative to the implantation fossa, resulting in
spermatid heads in which the acrosomal portion of the nucleus is blunted and
widened (Fig. 4). Spermatids with blunted, wedge- or club-shaped nuclei were,
in fact, found in all males (Figs. 5 and 6), and it is possible that the initial
defective positioning of the acrosomal cap produced this defect.
Early-staged spermatids with irregular nuclear shapes were present in all
males. In a number of cells, the shape of the entire nucleus was extremely
distorted (Fig. 7) while in other cells only a portion of the nucleus had an
aberrant configuration. For example, Figure 8 shows a Stage-5 spermatid in
which the nuclear area subjacent to the acrosomal cap is normally shaped
whereas the remainder of the nucleus is distorted. The reason for this type of
abnormality is not known; however, it could result from Sertoli cell cytoplasm
projecting into early-staged spermatids, consequently distorting the configuration
of the nuclei. If these projections persist, portions of the nuclei would continue
to develop abnormally, resulting in older spermatids which have indented and
irregularly-shaped heads. Figures 9 and 10 are typical examples of spermatids in
which both the rostral and caudal portions of the nuclei are abnormally shaped.
In both spermatids, indentations of the Sertoli cell cytoplasm are seen projecting
into the nucleus at points both anterior and posterior to the perinuclear ring.
Posterior to the rings, the cytoplasm projects into the otherwise normal
manchettes.
Additional types of head defects were found among the spermatids in animals
of every genotype at all ages. These defects, all of which have been previously
described (Hillman & Nadijcka, 1978), include abnormal chromatin condensation, disrupted nuclear membranes, dehiscence of the nuclear membrane and
the condensed chromatin, non-sequential development of spermatid component
parts and possibly, manchette abnormalities. Besides containing spermatids
which have abnormal protrusions of the microtubules of the manchette into the
nucleus (which may be a passive response to external pressure exerted by Sertoli
cell projections), all males had spermatids whose manchettes appeared to contain
excessive numbers of microtubules. Since the nuclei of the latter spermatids
were always aberrant, the seemingly excess numbers of microtubules may be
illusory.
Frequently a single spermatid demonstrated more than one type of nuclear
and head abnormality (Fig. 11). Moreover, defective development was not
necessarily limited to the head region. All of the males contained spermatids
with duplicated implantation fossae and flagella. Also, all contained mature
spermatids in which the doublets and/or dense fibres of the tail were either
Spermatid defects in mutant (tLx/tLy) male mice
35
missing, duplicated or disorganized. These defects were the same as those
described in wild-type males and males carrying a single recessive tL mutation
(Hillman & Nadijcka, 1978). In all males, tail defects were encountered less
frequently than head defects.
DISCUSSION
Our study shows that the sterility of intercomplement males is not caused by
a unique spermatid defect. The intercomplement males contain a broad spectrum
of spermatid abnormalities (both head and tail defects), all of which can be
found in fertile control animals. The same types of aberrant spermatids are
present in all of the males regardless of their age; however, in older males, these
defects are seen with increasing frequency. The latter observation lends support
to the hypothesis that aberrant spermiogenesis is age-related and cautions
investigators to use correspondingly-aged animals for comparative and definitive
spermiogenesis studies (Bryson, 1944; Hancock, 1972; Krzanowska, 1972).
Our survey also shows that defects in spermatid morphology can be found at
all stages of spermiogenesis. We suggest that the types of defects present in the
younger spermatids could lead to many of the head and nuclear defects which
are apparent in the older spermatids. This suggestion supports the hypothesis
that most abnormalities in spermatid head shapes are caused by aberrantly
formed acrosomes (Hollander, Bryan & Gowen, 1960; Hunt & Johnson, 1971)
or by the impingement of Sertoli-cell cytoplasm, either alone or together with
the manchette, into the developing spermatid nucleus (Bennett et ah, 1971;
Johnson & Hunt, 1971).
Locating defects of the younger spermatid stages often requires the study of
serial sections or the fortuitous selection of sections which are cut in the correct
plane to include several developing component parts of the spermatid (e.g. the
acrosome and the implantation fossa). Because of the inherent limitations
imposed by electron microscopy, defective morphologies of the younger-staged
spermatids are not as easily discerned as are the more obvious developmental
and morphological defects of older spermatids. This difference in the ease of
identifying defects could explain the fact that Dooher & Bennett (1977) observed no abnormalities in the spermatid morphology of tLx/tLy males until
Stage 11 of spermiogenesis. Alternatively, intercomplement males of the
genotype t°/tw3Z may differ from t*/tw32 males in the time of expression of
abnormal spermatid development. However, since f6 and t° belong to the same
complementation group (Bennett, 1975) it is probable that males of these two
genotypes contain the same multiple types of spermatid aberrations.
This research was supported by the United States Public Health Service Grants nos.
HD 00827 and HD 09753. The authors would like to thank Marie Morris and Geraldine
Wileman for their technical assistance.
36
N. HILLMAN AND M. N A D I J C K A
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