/ . Embryo/, exp. Morph. Vol. 44, pp. 243-261, 1978
Printed in Great Britain © Company of Biologists Limited 1978
243
A comparative study of spermiogenesis in
wild-type and T:t-bearing mice
By NINA HILLMAN 1 AND MARY NADIJCKA 1
From the Department of Biology, Temple University, Philadelphia
SUMMARY
The results of a comparative ultrastructural study of spermiogenesis in Tftx, +/tx, +/T,
C57BL/6J, BALB/c and randomly breeding Swiss Albino mice are reported. The observations
show that aberrant spermiogenesis occurs in males of all strains and genotypes and that the
same specific types of abnormal spermatids are found in all of the males examined. No
unique morphological defect which could be correlated with the increased transmission
frequency of /x-bearing gametes can be found in males heterozygous for the tx allele.
INTRODUCTION
The T-locus in the house mouse is located on chromosome 17 and consists of
a wild-type allele ( + ), a dominant mutant allele (T), and a series of recessive
alleles (tx). With a few exceptions, males which are heterozygous for these
recessive lethal alleles ( + /fz; T/tx) transmit the ^-bearing spermatozoa at a
frequency greater than 50 %. Conversely, males heterozygous for the dominant
allele (71/ + ) and all heterozygous females (T/tx, + jtx) transmit the alleles in a
1:1 ratio (Dunn & Gluecksohn-Schoenheimer, 1939; Dunn, 1960). As a result
of the increased transmission frequency of P-bearing spermatozoa, litters from
+ \tx inter se matings are composed of more than the expected 25 % homozygous
/ embryos.
In a light-microscopic study Bryson (1944) found that this increased transmission frequency was not the result of extra post-meiotic mitoses of ^-bearing
spermatids and that specific spermatid defects could not account for the increased transmission frequency. Yanagisawa (1965), however, suggested that the
increased transmission frequency could result if spermatozoan abnormalities
were limited to the + - or T-bearing gametes obtained from heterozygous males
(Tjtx; + \tx). Because of the hypothesis proposed in this latter study, we have
undertaken a comparative ultrastructural analysis of mouse spermiogenesis in
/^-bearing males (tw32, t6, t12) and in different inbred and outbred strains in order
to establish: first, if aberrant spermatid development occurs in these diverse
1
Authors' address: Department of Biology, Temple University, Philadelphia, Pennsylvania
19122, U.S.A.
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N. HILLMAN AND M. NADIJCKA
males; second, if these defects are the same or different in the groups of males
studied; and third, if any specific spermatid defect can be correlated with the
increased transmission frequency of ?x-bearing gametes.
MATERIALS AND METHODS
The studies were done on T/te (breeding stock obtained from Dr M. Lyon),
Tit12 (breeding stock obtained from Dr S. Waelsch), T/tlvS2 (breeding stock
obtained from Dr D. Bennett), +/t% + t12, +/^ 3 2 , T6/ + , T12j + , Tir32/ + ,
C57BL/6J, BALB/c, and randomly breeding Swiss Albino mice. The inbred
BALB/c mating pairs were obtained from Dr G. Wolfe in 1964. These latter
animals have been maintained through brother-sister matings. The +/tx and
Tj + males were obtained by crossing the specific T/tx males to BALB/c females.
The males used in the present studies were tested for their level of fertility
according to the protocol of Dunn & Bennett (1969). Using their criteria, all of
the males were classified as normal fertile. The averaged transmission frequencies
of the tx alleles from the heterozygous males used in this study was 0-78 for the
t* allele, and 0-75 for both the t12 and twSi alleles.
Six males of each strain and genotype were sacrificed by cervical dislocation
at 6 months of age. This age was chosen in order to eliminate the documented
correlations between the animal's age and the numbers of abnormal spermatogenic cells (Bryson, 1944; Hancock, 1972; Krzanowska, 1972). Testes were
removed and placed into 3 % glutaraldehyde in 0-1 M-PO4 buffer (pH 7-4). The
tunica albuginea was removed from each testis and the seminiferous tubules cut
into approximately 1 mm segments. These segments were fixed for 2 h in
glutaraldehyde, placed into 0-1 M-PO 4 buffer for 2 h, postfixed in 1 % osmium
tetroxide (Millonig's, pH 7-3), dehydrated, and embedded in Epon. 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.
A detailed description of normal mouse spermatid development is not included
in this report. Several ultrastructural studies of spermiogenesis in various
mammalian species are available (Fawcett & Phillips, 1969; Fawcett, Eddy &
Phillips, 1970; Fawcett, Anderson & Phillips, 1971). In addition, there are a
number of reports describing specific stages of, and the development of specific
organelles during, mouse spermiogenesis (Sandoz, 1970; Bennett, Gall,
Southard & Sidman, 1971; Bryan & Wolosewick, 1973). An overall description
of mouse spermatid development, with particular emphasis placed on spermatid
head development, has been reported by Dooher & Bennett (1973). The present
study includes brief descriptions of the development of only those component
structures which exhibit aberrant morphology. The spermatid staging follows
that established by Oakberg (1956) as modified by Dooher & Bennett (1973).
Mouse spermiogenesis
245
RESULTS AND DISCUSSION
General observations
Testes isolated from all strains and genotypes contained abnormal spermatids.
The identical types of abnormalities were found in all testes examined. During
the course of the study it was noted that aberrant spermatids were frequently
clustered in delimited areas of the seminiferous tubules. Consequently, one thin
section of a tubule would show few abnormalities whereas sections from a
different area of the same tubule or from other tubules would contain numerous
aberrant spermatids. This clustering was also reported by Bryson (1944) and
Rajasekarasetty (1954) in their light-microscopic studies of mouse spermiogenesis
in F-bearing males.
Because aberrant spermatids were not found in each section of the tubule, and
because sections were randomly selected and examined, it was impossible to
establish either the total number of abnormal cells or the incidence of a specific
type of spermatid abnormality for any individual male. Nevertheless, it was
possible to rank the strains and genotypes in order, beginning with those containing the greatest numbers of abnormal cells to those containing the least
numbers, utilizing the relative ease by which we could find abnormal spermatids
in randomly selected sections. Using this criterion, C57BL/6J and BALB/c males
contained the largest number, and the ^-bearing (T/tx or + \tx) and Swiss Albino
males, the fewest number of aberrant spermatids. Our subjective determination
that C57BL/6J contained high numbers of abnormal spermatids agrees with
Johnson (1974) who reported that C57BL/6J and A/Gr mouse strains contained
more abnormal spermatids (multinucleated spermatids) than did the other
inbred (C57BL/Gr; CBA/Gr; AKRfNMRI/Lac) and outbred (p/p mixed;
+ /p25 mixed; +/hop mixed) strains which he examined.
Specific abnormalities
Uninuclear spermatid defects
1. Duplicatedproacrosomal vesicles and granules. Normally, during spermatid
Stage 2, only one proacrosomal vesicle assumes a juxtanuclear position and
becomes closely apposed to the nuclear membrane. During spermatid Stage 3,
the convex juxtanuclear membrane of this proacrosomal vesicle becomes
situated in a nuclear indentation (Figs. 1 A, B). This delimited area of the nuclear
envelope is characterized by both a layer of condensed chromatin subjacent to
the inner nuclear membrane and an absence of nuclear pores (Sandoz, 1970).
Spermatids in which two proacrosomal vesicles become associated with the
presumptive rostral area of the spermatid nucleus are commonly found (Fig. 2).
In these nuclei, the region of the nuclear envelope between the two vesicles does
not contain condensed chromatin on its inner membrane but usually does
contain nuclear pores.
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N. HILLMAN AND M. NADIJCKA
FIGURES
1-4
A, Acrosome; F, flagellum; IF, implantation fossa; M, mantle; Me, manchette;
N, nucleus; PC, proximal centriole; PG, proacrosomal granule; PR, perinuclear
ring; PV, proacrosomal vesicle.
Fig. 1. (A) An electron micrograph of a portion of a normal Stage-3 mouse spermatid.
A single proacrosomal vesicle containing afibrillarproacrosomal granule is situated
in a nuclear identation. x 18900. (B) This insert shows aportion of the proacrosomal
vesicle membrane and the subjacent inner and outer membranes of the nuclear
envelope. Note the presence of condensed material associated with the inner nuclear
membrane and the presence of amorphous material between the outer nuclear
membrane and the membrane of the vesicle, x 72000.
Mouse spermiogenesis
247
The consequence(s) of this duplication has not been determined, but the
apparent defect has been found in all of the strains and genotypes examined and
has also been described in p25Hlp25H and pm/pm
sterile mice (Hunt & Johnson,
1971). It is not known if the two vesicles and their granules ultimately fuse and
regulate to form a normal mature acrosome; or if, conversely, there is no
regulation and the duplication results in abnormal acrosomal and nuclear
development. If regulation does not occur, and if the initial vesicle-nucleus
apposition establishes the future rostral portion of the spermatid head, a duplicated acrosomal vesicle may produce two anterior apices. This duplication of
apices could produce those defective spermatids which have either bifid or
bifurcated heads.
2. Bifid and bifurcated sperm heads. All of the males contained spermatids
with bifid or bifurcated heads. This abnormality is also found in hop/hop sterile
mice (Johnson & Hunt, 1971). A typical example of this type of spermatid
abnormality is shown in Fig. 3. In these spermatids, the head has two rostral
parts. Each apex may be enclosed by a separate acrosome or the two may be
enclosed by a single acrosome. Both apices, however, are always covered by a
single continuous mantle. The extent of bifidity in these spermatids varies and
the cleft frequently extends below the level of the perinuclear ring (Fig. 3).
It is conceivable that most of these aberrant spermatids are derived from
those Stage-3 spermatids which contain two proacrosomal vesicles and granules.
Alternatively, these bizarre spermatids could result from the partial fusion of
the presumptive caudal regions of two nuclei of a binucleated cell. The fact,
however, that these aberrant nuclei have only one implantation fossa and one
flagellum does not support the latter hypothesis. Binucleated spermatids which
develop from a binucleated cell usually have two distinct flagella (cf. Figs. 3
and 20).
Bifurcation is not limited to the rostral area. Spermatids frequently contain
nuclei with lateral extensions. These nuclear extensions are always anterior to
the perinuclear ring. Although the spermatid membranes are continuous around
the outpocketings, the mantle is often discontinuous at these sites (Fig. 4). The
causal factor responsible for these lateral nuclear extensions is not known.
3. Abnormal chromatin condensation. Normally, the condensed chromatin of
Fig. 2. A micrograph of a Stage-3 mouse spermatid showing the aberrant association
of two proacrosomal vesicles with the nucleus. This defect was found in all of the
males examined, x 21000.
Fig. 3. An example of a ubiquitous defect, nuclear bifidity, is shown in this micrograph of a Stage-12 spermatid. In this spermatid the bifidity extends below the level
of the perinuclear ring. Note the presence of a single mantle, implantation fossa
and proximal contriole. x 17800.
Fig. 4. A longitudinal section of a mature spermatid with a bifurcated nucleus. Although the nuclear and acrosomal membranes are always continuous around these
projections, the mantle is often discontinuous (arrow), x 13000.
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N. HILLMAN AND M. NADIJCKA
Mouse spermiogenesis
249
late-staged spermatids fills the nucleus and is contiguous with the inner membrane of the nuclear envelope. In all of the males, spermatids were found in
which the condensed chromatin was completely separated from the inner nuclear
membrane or was contiguous with it in some areas and not in others. This
retraction of the chromatin occurs most frequently in the postacrosomal region
of the nucleus (Fig. 5). This abnormal dehiscence of the nuclear membrane and
the condensed chromatin is also found in spermatids from C57BL\'6J-qkI'qk
mice (Bennett et al. 1971).
4. Duplicated implantation fossae and flagella. By spermatid Stage 6 the
proximal and distal centrioles, together with the forward basal portion of the
single axoneme, have migrated to the caudal portion of the spermatid nucleus.
The proximal centriole has become orientated perpendicular to the nucleus, and
the circumscribed area of the nuclear membrane above this centriole has
indented to form the implantation fossa. The single flagellum projects into the
flagellar canal (Fig. 6).
Spermatid tails which contain two axonemes and their associated flagellar
components within a common cytoplasm, bound by a single plasma membrane
can be found projecting into the tubule lumen. When such tails are seen in
cross-section (Fig. 7) we cannot determine if the two axial filament complexes
are from a binucleated spermatid which normally has two flagella (Fig. 20), or
if the two complexes are both associated with the nucleus of a uninucleated
spermatid. If the axial filament complexes are not traced to their sites of
FIGURES
5-9
A, Acrosome; F, flagellum; IF, implantation fossa; M, mantle; PR, perinuclear
ring.
Fig. 5. A transverse section of a Stage-14 spermatid in which the condensed chromatin is retracted from the postacrosomal nuclear membrane, x 16000.
Fig. 6. A micrograph of a normal Stage-6 spermatid. Note the presence of a single
implantation fossa and flagellum. x 8800.
Fig. 7. A transverse section through a spermatid tail. Two sections of midtails are
contained in a common cytoplasm bounded by a plasma membrane. Without
tracing the tail it is not possible to determine if these duplicated structures are from a
uninucleated or from a binucleated spermatid. x 17400.
Fig. 8. A longitudinal section of a double-tailed Stage-12 uninucleated spermatid.
Note the presence of two implantation fossae and two flagellae. x 12000.
Fig. 9. (A) A transverse section through the midpiece of a spermatid tail which lacks
doublets 4, 5, 6 and 7. The outer dense fibers appear normal, x 32000. (B) A
transverse section through a membrane-limited cytoplasm containing two flagellar
endpieces. It is not known if these two tails are from a uninucleated or a binucleated
spermatid. Note the presence of an extra doublet in the one endpiece (arrow) and
the presence of nine pairs of doublets in the surrounding cytoplasm. These doublets
may have come from the second endpiece which appears to have a disrupted plasma
membrane and to be devoid of all doublets except for the middle pair, x 36000.
(C) This micrograph shows four sections of tails with disorganized and missing
axial filament components, x 21600.
250
12
N. HILLMAN AND M. NADIJCKA
Mouse spermiogenesis
251
implantation, it is impossible to distinguish between these two alternatives. We
have observed longitudinal sections which clearly show that some uninucleated
spermatids have two implantation fossae and two axial filament complexes
(Fig. 8). These double-tailed spermatids were found in all of the males suggesting
that duplicated fossae and flagella are common abnormalities found in all
normal fertile mice. This same defect is a phenotypic characteristic of spermiogenesis in sterile hop/hop males (Johnson & Hunt, 1971). However, in these
latter males, sperm tail development is either abortive or arrested during the
early spermatid stages.
5. Missing doublets and outer dense fibers. Additional aberrations of the
spermatid flagella are missing (Fig. 9 A) or extra (Fig. 9B) doublets and/or
outer dense fibers and disorganized flagellar components (Fig. 9C). These
defects are the least numerous of the specific aberrations found but are present
in spermatids of all of the males examined. Flagellar disorders (abnormal
distribution and excessive numbers of outer dense fibers and doublets) have also
been described in the spermatids of C57BL/'6J-qkjqk mice. In this mutant the
flagellar components 'decompose' after Stage 9 or 10 (Bennett et al. 1971).
Excessive numbers of doublets and disorganized axonemal components are also
found in the spermatids of hop I hop sterile males (Johnson & Hunt, 1971). Conversely, Olds (1973) found no flagellar abnormalities in her study of spermatids
from fertile T/tw18, T/f""32 and sterile twl8ltw32 males. However, the presence of
flagellar defects in all of the mutant (including 71/?"'32) and wild-type males which
we studied and the fact that similar defects have been found in the spermatids of
other mutant mice suggest that these defects are common spermatid abnormal i-
FIGURES
10-13
A, Acrosome; M, mantle; m, microtubules; Me, manchette; N, nucleus; NE, nuclear
envelope; PG, proacrosomal granule; PR, perinuclear ring; S, Sertoli cell cytoplasm.
Fig. 10. (A) A section through a Stage-14 spermatid. Note the presence of Sertoli
cell cytoplasm (arrow) which is projected into the nucleus. This projection has
occurred in the rostral area and the cytoplasm is circumscribed by the nuclear,
acrosomal and plasma membranes, x 17500. (B) This insert contains a higher
magnification of the delimited area of the spermatid head shown in (A), x 36000.
Fig. 11. A longitudinal section of the head of a Stage-9 spermatid which is normal
except for the projection of a portion of the manchette into the nucleus. The
indented microtubles are circumscribed by only the nuclear envelope, x 17500.
Fig. 12. A longitudinal section through a normal, elongated Stage-8 spermatid
nucleus. Note the presence of condensed chromatin associated with the inner
nuclear membrane and scattered through the nucleoplasm. The perinuclear ring and
manchette are present at this stage, x 8000.
Fig. 13. A longitudinal section through an aberrant spermatid. The nucleus is shaped
like aStage-4 nucleus but the chromatin condensation pattern is like thatof a Stage-8
nucleus (compare with Fig. 12). The acrosome is aberrant and still contains a fibrous
proacrosomal granule. Note, however, that the perinuclear ring and the manchette
have formed and appear normal, x 8000.
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N. HILLMAN AND M. NADIJCKA
Mouse spermiogenesis
253
ties and argue that some of the axonemal and outer dense fiber defects of the
epididymal spermatozoa described by Olds originated during spermiogenesis
rather than being effected solely by the epididymal environment.
6. Projections of Sertoli cells and manchette into spermatid nuclei. A very
common spermatid abnormality in these mice is the projection of Sertoli-cell
cytoplasmic extensions which indent the spermatid nucleus. Most frequently the
indentations are anterior to the perinuclear ring. In both transverse and longitudinal sections of these nuclei the indented Sertoli cell cytoplasm is circumscribed by the spermatid plasma, acrosomal and nuclear membranes (Figs. 10 A,
B). In some cases, portions of the mantle are also included in these projections.
These spermatids are similar to those described by Bennett et al. (1971) as being
characteristic of sterile C57BL/6J-#A:/gfc mice.
We have also found spermatid abnormalities in which the microtubules of the
manchette are projected into the nucleus. Because of the level of these indentations, the microtubule projections are circumscribed by only the nuclear
envelope (Fig. 11). We have not determined if Sertoli cell extensions indent the
manchette, which in turn projects into the nucleus, or if the manchette projects
independently into the nucleus. This defect has been found in all of the males and
has also been described in the spermatids of hopjhop sterile mice (Johnson &
Hunt, 1971).
7. Non-sequential development of spermatid component parts. Non-sequential
spermatid development occurs when one or more component parts of the
spermatid fail to reach the stage of development they normally attain prior to
the formation of additional component parts. The most frequently observed
non-sequential development is the delayed or aberrant development of the
acrosome and of the nucleus relative to the temporal appearance of the
FIGURES
14-17
C, Condensed chromatin; G, Golgi apparatus; m, microtubules; Me, manchette;
NE, nuclear envelope; PG, proacrosomal granule; PR, perinuclear ring; PV, proacrosomal vesicle.
Fig. 14. A longitudinal section through a microheaded Stage-11 spermatid. The
perinuclear ring and the associated microtubules of the mantle appear to be extensive
on one side of the nucleus (bracket). This type of defect was found in all males,
x 16100.
Fig. 15. A longitudinal section through an extremely abnormal spermatid. Note the
absence of most of the rostral portion of the head, the disruption and the aberrant
reflexion of the nuclear envelope (arrow) and the retraction of the condensed
chromatin from the intact portion of the envelope. This type of aberrant spermatid
appears to have excessive numbers of microtubules. x 17800.
Fig. 16. A longitudinal section of a binucleated Stage-4 spermatid. Note that the two
nuclei share a common Golgi apparatus and a common proacrosomal vesicle which
is spaced equidistant from both nuclei, x 7500.
Fig. 17. A transverse section of a Stage-8 spermatid which developed from a binucleated cell like that shown in Fig. 16. x 8000.
17
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N. HILLMAN AND M. NADIJCKA
Mouse spermiogenesis
255
perinuclear ring and the manchette. Themanchette is normally present in stage-8
spermatids and persists through Stage 15 when it disappears (Dooher & Bennett,
1973). In normal Stage-8 spermatids, the nucleus is elongated, patches of densely
staining material are found both adjacent to the inner nuclear membrane and
scattered inside the nucleus, the nucleoli have disappeared, the acrosomal cap is
completely formed and filled with dense material, and the mantle is closely
apposed to the acrosome (Fig. 12). Frequently, one finds cells in which the nuclei
have the conformation of younger staged spermatids (e.g. Stage 4) but have lost
their nucleoli and have a chromatin condensation pattern similar to that of
Stage-8 spermatids (Fig. 13). The nuclei of these aberrant spermatids always have
defective acrosomal caps (e.g. persistent proacrosomal granules), but they are
always encircled by a normal appearing perinuclear ring and manchette. This
common abnormality suggests, first, that defective acrosomal formation is
associated with, and may be a principle cause for, the abnormal shaping of the
spermatid nucleus; second, that the temporal loss of nucleoli and the subsequent pattern of chromatin condensation is not dependent upon the conformation of either the acrosome or the nucleus; and third, that the formation
of a normal appearing manchette is not dependent upon the normal conformation of either the acrosome or the nucleus. These findings, in addition to
demonstrating that spermatid component parts can develop normally even if the
preceding development of other component parts is aberrant, support the hypothesis advanced by Fawcett et al. (1971) that the manchette does not play an
active role in the shaping of the postacrosomal region of the mammalian
spermatid head.
8. Manchette abnormalities. We have found cells which appear to contain
excessive numbers of microtubules in all of the testes which we examined. Two
FIGURES
18-21
A, Acrosome; C, condensed chromatin; IF, implantation fossa; m, microtubules;
M, mantle; NE, nuclear envelope.
Fig. 18. A binucleated Stage-9 spermatid. The two nuclei, although joined by a
single acrosome, are normally shaped. A single mantle, perinuclear ring and
manchette are present. In serial sections of this spermatid it was found that a single
flagellum was associated with each nucleus, x 10000.
Fig. 19. A bizarre Stage-12 spermatid which developed from two nuclei which shared
a single proacrosomal vesicle and granule. These aberrant spermatids result when
the proacrosomal vesicle is not shared equally by the two nuclei. The pattern of
chromatin condensation in each nucleus is normal for Stage-12 spermatids. Note
the large vacuoles in the shared acrosome. x 12600.
Fig. 20. An aberrant Stage-12 binucleated spermatid. In addition to the bizarre
shape, one nucleus is lacking a flagellum, while the other nucleus contains two
implantation fossae and flagella. x 11900.
Fig. 21. This micrograph of a binucleated Stage-14 spermatid shows two additional
defects; the retraction of the condensed chromatin from the nuclear membrane, and
the projection of the manchette into the nucleus (arrow), x 18200.
17-2
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N. HILLMAN AND M. NADIJCKA
Mouse spermiogenesis
257
classes of these cells have been observed. In the first, the spermatid heads are
abnormally shaped and are smaller than normal. In longitudinal sections of
these microheaded spermatids the perinuclear ring appears to be greatly
extended on either one (Fig. 14) or both sides of the nucleus. As a consequence
of this extension, the spermatids appear to contain excessive numbers of
microtubules. It is probable, however, that the perinuclear rings and manchettes
of spermatids with both normal and abnormal nuclei are of equal size and that
the manchettes associated with these nuclei contain the same numbers of
microtubules. In cells in which the nuclei are smaller, the normal sized perinuclear rings and manchettes would be disproportionately large; and consequently, the spermatids would appear to contain excessive numbers of microtubules.
In the second class the nuclei are completely deformed and their nuclear
membranes are disrupted (Fig. 15). Normally, by Stage 14 the chromatin is condensed and is contiguous with the entire inner nuclear membrane. In the
defective spermatids at this stage the condensed chromatin is located only in the
central area of the nucleus. The microtubules of the manchette protrude into the
nucleus at those points which are devoid of nuclear envelope, and they appear to
be present in significantly greater numbers than in correspondingly staged,
normal spermatids.
A type of spermatid abnormality similar to the latter class has been described
by Dooher & Bennett (1974) in sterile txv2\tu'2 males. These authors suggest that
the sterility of tw2/tw2 males is related to the formation of spermatids with 'an
unusually large number of disorganized microtubules' which appear to depolymerize prematurely. The tw2 spermatids seldom survive to maturity, and most
are phagocytized by Sertoli cells.
Rattner & Brinkley (1972) have reported that the numbers of microtubules in
spermatids are species specific. In order to determine if there is an actual
increase in the number of microtubules in those spermatids which appear to
have excessive numbers, it will be necessary, therefore, to count the microtubules
in both the normal and aberrant cells. Only then will it be possible to state that
the aberrant spermatids described in either the present report or in those found
in t"'2 homozygous males do in fact contain excessive numbers of microtubules.
FIGURES 22 AND 23
Fig. 22. A section showing three nuclei of a tetranucleated cell. Note that the nuclei
are developing synchronously (all are Stage 7) and that the nuclei are randomly
orientated. x7700.
Fig. 23. A micrograph of a typical tetranucleated cell in which two nuclei are sharing
a common proacrosomal vesicle (arrow), while the other two nuclei are developing
independently. Their independent development was established by examining serial
sections. x7600.
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N. HILLMAN AND M. NADIJCKA
Binucleated and multinucleated spermatid defects
Testes from males of all genotypes and strains contain both binucleated and
multinucleated spermatids. In binucleated cells the two nuclei frequently share
a single Golgi apparatus, and subsequently, a single proacrosomal vesicle and
granule which forms a single acrosome. Two developmental stages of such
binucleated spermatids (Stages 4 and 8) are shown in Figs. 16 and 17. Differing
spatial relationships between the forming acrosome and the two nuclei result in
a wide distribution of nuclear, and subsequent head, morphologies. These range
from spermatids having two heads which are conformationally normal except
for sharing a common acrosome to spermatids having two conjoined heads
which are highly bizarre (Figs. 18, 19). The aberrant relationship between the
acrosome and the two nuclei is often accompanied by other ubiquitous defects.
For examples, the two flagella are frequently associated with only one of the
two nuclei (Fig. 20); the condensed chromatin is not contiguous with the
nuclear membranes (Fig. 21); and the manchette (Fig. 21) or the Sertoli cell
cytoplasm projects into either or both of the nuclei and/or into the common
acrosome. Similar types of binucleated spermatids have been reported in pinkeyed sterile mice (Hunt & Johnson, 1971), in hop /hop sterile mice (Johnson &
Hunt, 1971), and in ABP(JAX) mice (Bryan & Wolosewick, 1973).
Not all binucleated cells, however, give rise to aberrant spermatids. In many,
the two nuclei develop synchronously but independently of each other. According to Bryan & Wolosewick (1973) this type of binucleated cell gives rise to two
normal spermatozoa.
Multinucleated spermatids have also been observed in the present study, but
they never contain more than four nuclei. Three types of tetranucleated cells are
found. In the first, and at the highest frequency, the four nuclei develop synchronously but independently of each other (Fig. 22). In these cells the nuclei
are randomly orientated. No structures (e.g. Golgi apparatus, proacrosomal
vesicle and granule, centrioles) are shared between or among any of the nuclei.
According to Bryan & Wolosewick (1973), this type of multinucleated cell will
produce four normal spermatozoa. In the second type, two of the nuclei share a
common acrosome while the other two nuclei remain independent (Fig. 23). In
these cells the two nuclei joined by a common acrosome subsequently develop
aberrantly, showing the same types of abnormalities found in conjoined nuclei
of binucleated cells. Finally, two pairs of conjoined nuclei have been observed,
each pair sharing a single acrosome. Each joined nuclear pair develops abnormally, showing the same range of spermatid abnormalities found in
binucleated spermatids.
In his light-microscopic studies of developing spermatogenic cells of mice,
Bryan (1971) noted that squashed preparations of viable seminiferous tubules
contained large numbers of multinucleated cells and that there were as many
cells with odd numbers of nuclei (Fig. 11 of his report shows a ' spermatid
Mouse spermiogenesis
259
containing 13 nuclei') as with even numbers of nuclei ('2 to more than 30
nuclei'). The strain(s) of animals used in his study was not reported. Later,
using electron microscopic preparations, Bryan & Wolosewick (1973) described
the ultrastructure of binucleated and tetranucleated spermatogenic cells in
ABP(JAX) mice. In this latter study cells with more then four nuclei were not
described. On the basis of their combined observations Bryan & Wolosewick
suggested that multinucleated spermatid development was a common phenomenon during spermiogenesis. Hunt & Johnson (1971) described the appearance
of multinucleated spermatids in pm and p2bH males using both light and
electron microscopy. In the light-microscopic studies they found that the multinucleated spermatids contained as many as 16 nuclei. Later, Johnson (1974),
using both light and electron microscopy, examined cells from five inbred and
from three outbred mouse strains. He found that all of the males contained
multinucleated cells and that the incidence of multinucleated spermatids was
strain related. Johnson's report, therefore, confirmed the hypothesis that
binucleated and multinucleated spermatid development was a common phenomenon in mouse spermiogenesis. His observations, however, differed from
Bryan's observations (1971) concerning the incidence of the occurrence of this
abnormality. Johnson found that the frequency of this abnormality was
significantly lower than that reported by Bryan. Our present observations support Johnson's findings. While all of the males contained multinucleated
spermatids, the frequency of these multinucleated cells was low. Furthermore,
we found multinucleated cells more frequently in randomly selected sections of
C57BL/6J and BALB/c testes. This also supports Johnson's observation that the
incidence of this abnormality is strain related.
The fact that we found no cells with more than four nuclei while both Bryan
(1971) and Hunt & Johnson (1971) reported the presence of cells containing
larger numbers of nuclei is not easily resolved. However, since cells with more
than four nuclei were found almost exclusively in their light microscopic and
not in their ultrastructural studies, it is possible that their preparative procedures
for the former studies caused cellular fusion, thus producing cells with artifactual
numbers of nuclei. However, in spite of these differences in the maximum
numbers of nuclei found in multinucleated spermatozoa, the present observations do support the hypothesis proposed by both Bryan and Johnson: that
multinucleate spermatid development is a phenomenon common to all genotypes and strains during mouse spermiogenesis.
CONCLUSIONS
1. All of the males examined in the present study produced abnormal
spermatids. The same types of aberrations were found in all of the strains and
genotypes studied.
2. The highest incidences of abnormal spermatids were found in C57BL/6J
260
N. HILLMAN AND M. NADIJCKA
and BALB/c mice and the lowest, in the randomly breeding Swiss Albino and
Tjtx, + \tx and T\ + mice.
3. The defects found in the spermatids of these normal fertile males have also
been found by other investigators in other mutant mice and in other inbred and
outbred strains of mice.
4. The ubiquitousness of aberrant spermatid development and the strainrelated incidence of aberrant spermiogenesis are obvious from this and other
studies. The effects, therefore, of any specific mutant gene on spermiogenesis can
be determined only after the distribution and range of aberrant spermiogenesis
is established for the inbred or outbred strain(s) in the presence and absence of
the mutant allele(s).
5. The present study shows that the spermatids of + jtx and T\tx have no
unique ultrastructural defects which could either result in, or contribute to, the
increased transmission frequency of the ^-bearing allele.
This research was supported by United States Public Health Service Grants Nos. HD 00827
and HD 09753. The authors would like to thank Dr Ralph Hillman for his help in the preparation of this manuscript and Marie Morris and Geraldine Wileman for their technical
assistance.
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