a comparative study of spermiogenesis in wild-type and t:t ...a comparative study of spermiogenesis...

/ . Embryo/, exp. Morph. Vol. 44, pp. 243-261, 1978 2 4 3Printed in Great Britain © Company of Biologists Limited 1978

A comparative study of spermiogenesis inwild-type and T:t-bearing mice

By NINA HILLMAN1 AND MARY NADIJCKA1

From the Department of Biology, Temple University, Philadelphia

SUMMARYThe 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 observationsshow that aberrant spermiogenesis occurs in males of all strains and genotypes and that thesame specific types of abnormal spermatids are found in all of the males examined. Nounique morphological defect which could be correlated with the increased transmissionfrequency 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 ofa wild-type allele ( + ), a dominant mutant allele (T), and a series of recessivealleles (tx). With a few exceptions, males which are heterozygous for theserecessive lethal alleles ( + /fz; T/tx) transmit the ^-bearing spermatozoa at afrequency greater than 50 %. Conversely, males heterozygous for the dominantallele (71/ + ) and all heterozygous females (T/tx, + jtx) transmit the alleles in a1:1 ratio (Dunn & Gluecksohn-Schoenheimer, 1939; Dunn, 1960). As a resultof 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 trans-mission frequency was not the result of extra post-meiotic mitoses of ^-bearingspermatids and that specific spermatid defects could not account for the in-creased transmission frequency. Yanagisawa (1965), however, suggested that theincreased transmission frequency could result if spermatozoan abnormalitieswere limited to the + - or T-bearing gametes obtained from heterozygous males(Tjtx; + \tx). Because of the hypothesis proposed in this latter study, we haveundertaken a comparative ultrastructural analysis of mouse spermiogenesis in/^-bearing males (tw32, t6, t12) and in different inbred and outbred strains in orderto establish: first, if aberrant spermatid development occurs in these diverse

1 Authors' address: Department of Biology, Temple University, Philadelphia, Pennsylvania19122, U.S.A.

244 N. HILLMAN AND M. NADIJCKA

males; second, if these defects are the same or different in the groups of malesstudied; and third, if any specific spermatid defect can be correlated with theincreased 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 stockobtained from Dr D. Bennett), +/t% + t12, +/^3 2 , T6/ + , T12j + , Tir32/ + ,C57BL/6J, BALB/c, and randomly breeding Swiss Albino mice. The inbredBALB/c mating pairs were obtained from Dr G. Wolfe in 1964. These latteranimals have been maintained through brother-sister matings. The +/tx andTj + 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 fertilityaccording to the protocol of Dunn & Bennett (1969). Using their criteria, all ofthe males were classified as normal fertile. The averaged transmission frequenciesof the tx alleles from the heterozygous males used in this study was 0-78 for thet* allele, and 0-75 for both the t12 and twSi alleles.

Six males of each strain and genotype were sacrificed by cervical dislocationat 6 months of age. This age was chosen in order to eliminate the documentedcorrelations between the animal's age and the numbers of abnormal spermato-genic cells (Bryson, 1944; Hancock, 1972; Krzanowska, 1972). Testes wereremoved and placed into 3 % glutaraldehyde in 0-1 M-PO4 buffer (pH 7-4). Thetunica albuginea was removed from each testis and the seminiferous tubules cutinto approximately 1 mm segments. These segments were fixed for 2 h inglutaraldehyde, placed into 0-1 M-PO4 buffer for 2 h, postfixed in 1 % osmiumtetroxide (Millonig's, pH 7-3), dehydrated, and embedded in Epon. Ultrathinsections were stained with either lead citrate (Venable & Coggeshall, 1965), orwith both lead citrate and 2 % uranyl acetate (Watson, 1958). The sections wereexamined with a Philips 300 electron microscope.

A detailed description of normal mouse spermatid development is not includedin this report. Several ultrastructural studies of spermiogenesis in variousmammalian species are available (Fawcett & Phillips, 1969; Fawcett, Eddy &Phillips, 1970; Fawcett, Anderson & Phillips, 1971). In addition, there are anumber of reports describing specific stages of, and the development of specificorganelles during, mouse spermiogenesis (Sandoz, 1970; Bennett, Gall,Southard & Sidman, 1971; Bryan & Wolosewick, 1973). An overall descriptionof mouse spermatid development, with particular emphasis placed on spermatidhead development, has been reported by Dooher & Bennett (1973). The presentstudy includes brief descriptions of the development of only those componentstructures which exhibit aberrant morphology. The spermatid staging followsthat 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. Duringthe course of the study it was noted that aberrant spermatids were frequentlyclustered in delimited areas of the seminiferous tubules. Consequently, one thinsection of a tubule would show few abnormalities whereas sections from adifferent area of the same tubule or from other tubules would contain numerousaberrant spermatids. This clustering was also reported by Bryson (1944) andRajasekarasetty (1954) in their light-microscopic studies of mouse spermiogenesisin F-bearing males.

Because aberrant spermatids were not found in each section of the tubule, andbecause sections were randomly selected and examined, it was impossible toestablish either the total number of abnormal cells or the incidence of a specifictype of spermatid abnormality for any individual male. Nevertheless, it waspossible to rank the strains and genotypes in order, beginning with those con-taining the greatest numbers of abnormal cells to those containing the leastnumbers, utilizing the relative ease by which we could find abnormal spermatidsin randomly selected sections. Using this criterion, C57BL/6J and BALB/c malescontained the largest number, and the ^-bearing (T/tx or + \tx) and Swiss Albinomales, the fewest number of aberrant spermatids. Our subjective determinationthat C57BL/6J contained high numbers of abnormal spermatids agrees withJohnson (1974) who reported that C57BL/6J and A/Gr mouse strains containedmore abnormal spermatids (multinucleated spermatids) than did the otherinbred (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 spermatidStage 2, only one proacrosomal vesicle assumes a juxtanuclear position andbecomes closely apposed to the nuclear membrane. During spermatid Stage 3,the convex juxtanuclear membrane of this proacrosomal vesicle becomessituated in a nuclear indentation (Figs. 1 A, B). This delimited area of the nuclearenvelope is characterized by both a layer of condensed chromatin subjacent tothe inner nuclear membrane and an absence of nuclear pores (Sandoz, 1970).

Spermatids in which two proacrosomal vesicles become associated with thepresumptive rostral area of the spermatid nucleus are commonly found (Fig. 2).In these nuclei, the region of the nuclear envelope between the two vesicles doesnot contain condensed chromatin on its inner membrane but usually doescontain nuclear pores.


FIGURES 1-4

A, Acrosome; F, flagellum; IF, implantation fossa; M, mantle; Me, manchette;N, nucleus; PC, proximal centriole; PG, proacrosomal granule; PR, perinuclearring; PV, proacrosomal vesicle.Fig. 1. (A) An electron micrograph of a portion of a normal Stage-3 mouse spermatid.A single proacrosomal vesicle containing a fibrillar proacrosomal granule is situatedin a nuclear identation. x 18900. (B) This insert shows aportion of the proacrosomalvesicle membrane and the subjacent inner and outer membranes of the nuclearenvelope. Note the presence of condensed material associated with the inner nuclearmembrane and the presence of amorphous material between the outer nuclearmembrane and the membrane of the vesicle, x 72000.


The consequence(s) of this duplication has not been determined, but theapparent defect has been found in all of the strains and genotypes examined andhas 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 andregulate to form a normal mature acrosome; or if, conversely, there is noregulation and the duplication results in abnormal acrosomal and nucleardevelopment. If regulation does not occur, and if the initial vesicle-nucleusapposition establishes the future rostral portion of the spermatid head, a dupli-cated acrosomal vesicle may produce two anterior apices. This duplication ofapices could produce those defective spermatids which have either bifid orbifurcated heads.

2. Bifid and bifurcated sperm heads. All of the males contained spermatidswith bifid or bifurcated heads. This abnormality is also found in hop/hop sterilemice (Johnson & Hunt, 1971). A typical example of this type of spermatidabnormality is shown in Fig. 3. In these spermatids, the head has two rostralparts. Each apex may be enclosed by a separate acrosome or the two may beenclosed by a single acrosome. Both apices, however, are always covered by asingle continuous mantle. The extent of bifidity in these spermatids varies andthe cleft frequently extends below the level of the perinuclear ring (Fig. 3).

It is conceivable that most of these aberrant spermatids are derived fromthose Stage-3 spermatids which contain two proacrosomal vesicles and granules.Alternatively, these bizarre spermatids could result from the partial fusion ofthe presumptive caudal regions of two nuclei of a binucleated cell. The fact,however, that these aberrant nuclei have only one implantation fossa and oneflagellum does not support the latter hypothesis. Binucleated spermatids whichdevelop from a binucleated cell usually have two distinct flagella (cf. Figs. 3and 20).

Bifurcation is not limited to the rostral area. Spermatids frequently containnuclei with lateral extensions. These nuclear extensions are always anterior tothe perinuclear ring. Although the spermatid membranes are continuous aroundthe outpocketings, the mantle is often discontinuous at these sites (Fig. 4). Thecausal 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 associationof two proacrosomal vesicles with the nucleus. This defect was found in all of themales examined, x 21000.Fig. 3. An example of a ubiquitous defect, nuclear bifidity, is shown in this micro-graph of a Stage-12 spermatid. In this spermatid the bifidity extends below the levelof the perinuclear ring. Note the presence of a single mantle, implantation fossaand proximal contriole. x 17800.Fig. 4. A longitudinal section of a mature spermatid with a bifurcated nucleus. Al-though the nuclear and acrosomal membranes are always continuous around theseprojections, the mantle is often discontinuous (arrow), x 13000.


late-staged spermatids fills the nucleus and is contiguous with the inner mem-brane of the nuclear envelope. In all of the males, spermatids were found inwhich the condensed chromatin was completely separated from the inner nuclearmembrane or was contiguous with it in some areas and not in others. Thisretraction of the chromatin occurs most frequently in the postacrosomal regionof the nucleus (Fig. 5). This abnormal dehiscence of the nuclear membrane andthe condensed chromatin is also found in spermatids from C57BL\'6J-qkI'qkmice (Bennett et al. 1971).

4. Duplicated implantation fossae and flagella. By spermatid Stage 6 theproximal and distal centrioles, together with the forward basal portion of thesingle axoneme, have migrated to the caudal portion of the spermatid nucleus.The proximal centriole has become orientated perpendicular to the nucleus, andthe circumscribed area of the nuclear membrane above this centriole hasindented to form the implantation fossa. The single flagellum projects into theflagellar canal (Fig. 6).

Spermatid tails which contain two axonemes and their associated flagellarcomponents within a common cytoplasm, bound by a single plasma membranecan be found projecting into the tubule lumen. When such tails are seen incross-section (Fig. 7) we cannot determine if the two axial filament complexesare from a binucleated spermatid which normally has two flagella (Fig. 20), orif the two complexes are both associated with the nucleus of a uninucleatedspermatid. 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, perinuclearring.Fig. 5. A transverse section of a Stage-14 spermatid in which the condensed chro-matin is retracted from the postacrosomal nuclear membrane, x 16000.Fig. 6. A micrograph of a normal Stage-6 spermatid. Note the presence of a singleimplantation fossa and flagellum. x 8800.Fig. 7. A transverse section through a spermatid tail. Two sections of midtails arecontained in a common cytoplasm bounded by a plasma membrane. Withouttracing the tail it is not possible to determine if these duplicated structures are from auninucleated 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 lacksdoublets 4, 5, 6 and 7. The outer dense fibers appear normal, x 32000. (B) Atransverse section through a membrane-limited cytoplasm containing two flagellarendpieces. It is not known if these two tails are from a uninucleated or a binucleatedspermatid. Note the presence of an extra doublet in the one endpiece (arrow) andthe presence of nine pairs of doublets in the surrounding cytoplasm. These doubletsmay have come from the second endpiece which appears to have a disrupted plasmamembrane 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 missingaxial filament components, x 21600.


12


implantation, it is impossible to distinguish between these two alternatives. Wehave observed longitudinal sections which clearly show that some uninucleatedspermatids have two implantation fossae and two axial filament complexes(Fig. 8). These double-tailed spermatids were found in all of the males suggestingthat duplicated fossae and flagella are common abnormalities found in allnormal fertile mice. This same defect is a phenotypic characteristic of spermio-genesis in sterile hop/hop males (Johnson & Hunt, 1971). However, in theselatter males, sperm tail development is either abortive or arrested during theearly spermatid stages.

5. Missing doublets and outer dense fibers. Additional aberrations of thespermatid flagella are missing (Fig. 9 A) or extra (Fig. 9B) doublets and/orouter dense fibers and disorganized flagellar components (Fig. 9C). Thesedefects are the least numerous of the specific aberrations found but are presentin spermatids of all of the males examined. Flagellar disorders (abnormaldistribution and excessive numbers of outer dense fibers and doublets) have alsobeen described in the spermatids of C57BL/'6J-qkjqk mice. In this mutant theflagellar components 'decompose' after Stage 9 or 10 (Bennett et al. 1971).Excessive numbers of doublets and disorganized axonemal components are alsofound in the spermatids of hop I hop sterile males (Johnson & Hunt, 1971). Con-versely, Olds (1973) found no flagellar abnormalities in her study of spermatidsfrom fertile T/tw18, T/f""32 and sterile twl8ltw32 males. However, the presence offlagellar defects in all of the mutant (including 71/?"'32) and wild-type males whichwe studied and the fact that similar defects have been found in the spermatids ofother 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, nuclearenvelope; PG, proacrosomal granule; PR, perinuclear ring; S, Sertoli cell cytoplasm.Fig. 10. (A) A section through a Stage-14 spermatid. Note the presence of Sertolicell cytoplasm (arrow) which is projected into the nucleus. This projection hasoccurred in the rostral area and the cytoplasm is circumscribed by the nuclear,acrosomal and plasma membranes, x 17500. (B) This insert contains a highermagnification 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 normalexcept for the projection of a portion of the manchette into the nucleus. Theindented microtubles are circumscribed by only the nuclear envelope, x 17500.Fig. 12. A longitudinal section through a normal, elongated Stage-8 spermatidnucleus. Note the presence of condensed chromatin associated with the innernuclear membrane and scattered through the nucleoplasm. The perinuclear ring andmanchette are present at this stage, x 8000.Fig. 13. A longitudinal section through an aberrant spermatid. The nucleus is shapedlike aStage-4 nucleus but the chromatin condensation pattern is like thatof a Stage-8nucleus (compare with Fig. 12). The acrosome is aberrant and still contains a fibrousproacrosomal granule. Note, however, that the perinuclear ring and the manchettehave formed and appear normal, x 8000.


ties and argue that some of the axonemal and outer dense fiber defects of theepididymal spermatozoa described by Olds originated during spermiogenesisrather than being effected solely by the epididymal environment.

6. Projections of Sertoli cells and manchette into spermatid nuclei. A verycommon spermatid abnormality in these mice is the projection of Sertoli-cellcytoplasmic extensions which indent the spermatid nucleus. Most frequently theindentations are anterior to the perinuclear ring. In both transverse and longi-tudinal sections of these nuclei the indented Sertoli cell cytoplasm is circum-scribed 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 beingcharacteristic of sterile C57BL/6J-#A:/gfc mice.

We have also found spermatid abnormalities in which the microtubules of themanchette are projected into the nucleus. Because of the level of these indenta-tions, the microtubule projections are circumscribed by only the nuclearenvelope (Fig. 11). We have not determined if Sertoli cell extensions indent themanchette, which in turn projects into the nucleus, or if the manchette projectsindependently into the nucleus. This defect has been found in all of the males andhas also been described in the spermatids of hopjhop sterile mice (Johnson &Hunt, 1971).

7. Non-sequential development of spermatid component parts. Non-sequentialspermatid development occurs when one or more component parts of thespermatid fail to reach the stage of development they normally attain prior tothe formation of additional component parts. The most frequently observednon-sequential development is the delayed or aberrant development of theacrosome 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, pro-acrosomal vesicle.Fig. 14. A longitudinal section through a microheaded Stage-11 spermatid. Theperinuclear ring and the associated microtubules of the mantle appear to be extensiveon 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 theabsence of most of the rostral portion of the head, the disruption and the aberrantreflexion of the nuclear envelope (arrow) and the retraction of the condensedchromatin from the intact portion of the envelope. This type of aberrant spermatidappears to have excessive numbers of microtubules. x 17800.Fig. 16. A longitudinal section of a binucleated Stage-4 spermatid. Note that the twonuclei share a common Golgi apparatus and a common proacrosomal vesicle whichis spaced equidistant from both nuclei, x 7500.Fig. 17. A transverse section of a Stage-8 spermatid which developed from a bi-nucleated cell like that shown in Fig. 16. x 8000.17 EMB 44


perinuclear ring and the manchette. Themanchette is normally present in stage-8spermatids and persists through Stage 15 when it disappears (Dooher & Bennett,1973). In normal Stage-8 spermatids, the nucleus is elongated, patches of denselystaining material are found both adjacent to the inner nuclear membrane andscattered inside the nucleus, the nucleoli have disappeared, the acrosomal cap iscompletely formed and filled with dense material, and the mantle is closelyapposed to the acrosome (Fig. 12). Frequently, one finds cells in which the nucleihave the conformation of younger staged spermatids (e.g. Stage 4) but have losttheir nucleoli and have a chromatin condensation pattern similar to that ofStage-8 spermatids (Fig. 13). The nuclei of these aberrant spermatids always havedefective acrosomal caps (e.g. persistent proacrosomal granules), but they arealways encircled by a normal appearing perinuclear ring and manchette. Thiscommon abnormality suggests, first, that defective acrosomal formation isassociated with, and may be a principle cause for, the abnormal shaping of thespermatid nucleus; second, that the temporal loss of nucleoli and the sub-sequent pattern of chromatin condensation is not dependent upon the con-formation of either the acrosome or the nucleus; and third, that the formationof a normal appearing manchette is not dependent upon the normal conforma-tion of either the acrosome or the nucleus. These findings, in addition todemonstrating that spermatid component parts can develop normally even if thepreceding development of other component parts is aberrant, support the hypo-thesis advanced by Fawcett et al. (1971) that the manchette does not play anactive role in the shaping of the postacrosomal region of the mammalianspermatid head.

8. Manchette abnormalities. We have found cells which appear to containexcessive 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 asingle acrosome, are normally shaped. A single mantle, perinuclear ring andmanchette are present. In serial sections of this spermatid it was found that a singleflagellum was associated with each nucleus, x 10000.Fig. 19. A bizarre Stage-12 spermatid which developed from two nuclei which shareda single proacrosomal vesicle and granule. These aberrant spermatids result whenthe proacrosomal vesicle is not shared equally by the two nuclei. The pattern ofchromatin condensation in each nucleus is normal for Stage-12 spermatids. Notethe large vacuoles in the shared acrosome. x 12600.Fig. 20. An aberrant Stage-12 binucleated spermatid. In addition to the bizarreshape, one nucleus is lacking a flagellum, while the other nucleus contains twoimplantation fossae and flagella. x 11900.Fig. 21. This micrograph of a binucleated Stage-14 spermatid shows two additionaldefects; the retraction of the condensed chromatin from the nuclear membrane, andthe projection of the manchette into the nucleus (arrow), x 18200.

17-2


classes of these cells have been observed. In the first, the spermatid heads areabnormally shaped and are smaller than normal. In longitudinal sections ofthese microheaded spermatids the perinuclear ring appears to be greatlyextended on either one (Fig. 14) or both sides of the nucleus. As a consequenceof this extension, the spermatids appear to contain excessive numbers ofmicrotubules. It is probable, however, that the perinuclear rings and manchettesof spermatids with both normal and abnormal nuclei are of equal size and thatthe manchettes associated with these nuclei contain the same numbers ofmicrotubules. In cells in which the nuclei are smaller, the normal sized peri-nuclear rings and manchettes would be disproportionately large; and conse-quently, the spermatids would appear to contain excessive numbers of micro-tubules.

In the second class the nuclei are completely deformed and their nuclearmembranes are disrupted (Fig. 15). Normally, by Stage 14 the chromatin is con-densed and is contiguous with the entire inner nuclear membrane. In thedefective spermatids at this stage the condensed chromatin is located only in thecentral area of the nucleus. The microtubules of the manchette protrude into thenucleus at those points which are devoid of nuclear envelope, and they appear tobe present in significantly greater numbers than in correspondingly staged,normal spermatids.

A type of spermatid abnormality similar to the latter class has been describedby Dooher & Bennett (1974) in sterile txv2\tu'2 males. These authors suggest thatthe sterility of tw2/tw2 males is related to the formation of spermatids with 'anunusually large number of disorganized microtubules' which appear to depoly-merize prematurely. The tw2 spermatids seldom survive to maturity, and mostare phagocytized by Sertoli cells.

Rattner & Brinkley (1972) have reported that the numbers of microtubules inspermatids are species specific. In order to determine if there is an actualincrease in the number of microtubules in those spermatids which appear tohave excessive numbers, it will be necessary, therefore, to count the microtubulesin both the normal and aberrant cells. Only then will it be possible to state thatthe aberrant spermatids described in either the present report or in those foundin 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 nucleiare developing synchronously (all are Stage 7) and that the nuclei are randomlyorientated. x7700.Fig. 23. A micrograph of a typical tetranucleated cell in which two nuclei are sharinga common proacrosomal vesicle (arrow), while the other two nuclei are developingindependently. Their independent development was established by examining serialsections. x7600.


Binucleated and multinucleated spermatid defects

Testes from males of all genotypes and strains contain both binucleated andmultinucleated spermatids. In binucleated cells the two nuclei frequently sharea single Golgi apparatus, and subsequently, a single proacrosomal vesicle andgranule which forms a single acrosome. Two developmental stages of suchbinucleated spermatids (Stages 4 and 8) are shown in Figs. 16 and 17. Differingspatial relationships between the forming acrosome and the two nuclei result ina wide distribution of nuclear, and subsequent head, morphologies. These rangefrom spermatids having two heads which are conformationally normal exceptfor sharing a common acrosome to spermatids having two conjoined headswhich are highly bizarre (Figs. 18, 19). The aberrant relationship between theacrosome and the two nuclei is often accompanied by other ubiquitous defects.For examples, the two flagella are frequently associated with only one of thetwo nuclei (Fig. 20); the condensed chromatin is not contiguous with thenuclear membranes (Fig. 21); and the manchette (Fig. 21) or the Sertoli cellcytoplasm projects into either or both of the nuclei and/or into the commonacrosome. Similar types of binucleated spermatids have been reported in pink-eyed 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. Accord-ing to Bryan & Wolosewick (1973) this type of binucleated cell gives rise to twonormal spermatozoa.

Multinucleated spermatids have also been observed in the present study, butthey never contain more than four nuclei. Three types of tetranucleated cells arefound. In the first, and at the highest frequency, the four nuclei develop syn-chronously but independently of each other (Fig. 22). In these cells the nucleiare randomly orientated. No structures (e.g. Golgi apparatus, proacrosomalvesicle and granule, centrioles) are shared between or among any of the nuclei.According to Bryan & Wolosewick (1973), this type of multinucleated cell willproduce four normal spermatozoa. In the second type, two of the nuclei share acommon acrosome while the other two nuclei remain independent (Fig. 23). Inthese cells the two nuclei joined by a common acrosome subsequently developaberrantly, showing the same types of abnormalities found in conjoined nucleiof binucleated cells. Finally, two pairs of conjoined nuclei have been observed,each pair sharing a single acrosome. Each joined nuclear pair develops ab-normally, showing the same range of spermatid abnormalities found inbinucleated spermatids.

In his light-microscopic studies of developing spermatogenic cells of mice,Bryan (1971) noted that squashed preparations of viable seminiferous tubulescontained large numbers of multinucleated cells and that there were as manycells with odd numbers of nuclei (Fig. 11 of his report shows a ' spermatid


containing 13 nuclei') as with even numbers of nuclei ('2 to more than 30nuclei'). The strain(s) of animals used in his study was not reported. Later,using electron microscopic preparations, Bryan & Wolosewick (1973) describedthe ultrastructure of binucleated and tetranucleated spermatogenic cells inABP(JAX) mice. In this latter study cells with more then four nuclei were notdescribed. On the basis of their combined observations Bryan & Wolosewicksuggested that multinucleated spermatid development was a common phenom-enon during spermiogenesis. Hunt & Johnson (1971) described the appearanceof multinucleated spermatids in pm and p2bH males using both light andelectron microscopy. In the light-microscopic studies they found that the multi-nucleated spermatids contained as many as 16 nuclei. Later, Johnson (1974),using both light and electron microscopy, examined cells from five inbred andfrom three outbred mouse strains. He found that all of the males containedmultinucleated cells and that the incidence of multinucleated spermatids wasstrain related. Johnson's report, therefore, confirmed the hypothesis thatbinucleated and multinucleated spermatid development was a common phenom-enon in mouse spermiogenesis. His observations, however, differed fromBryan's observations (1971) concerning the incidence of the occurrence of thisabnormality. Johnson found that the frequency of this abnormality wassignificantly lower than that reported by Bryan. Our present observations sup-port Johnson's findings. While all of the males contained multinucleatedspermatids, the frequency of these multinucleated cells was low. Furthermore,we found multinucleated cells more frequently in randomly selected sections ofC57BL/6J and BALB/c testes. This also supports Johnson's observation that theincidence 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 containinglarger numbers of nuclei is not easily resolved. However, since cells with morethan four nuclei were found almost exclusively in their light microscopic andnot in their ultrastructural studies, it is possible that their preparative proceduresfor the former studies caused cellular fusion, thus producing cells with artifactualnumbers of nuclei. However, in spite of these differences in the maximumnumbers of nuclei found in multinucleated spermatozoa, the present observa-tions do support the hypothesis proposed by both Bryan and Johnson: thatmultinucleate spermatid development is a phenomenon common to all geno-types and strains during mouse spermiogenesis.

CONCLUSIONS

1. All of the males examined in the present study produced abnormalspermatids. The same types of aberrations were found in all of the strains andgenotypes studied.

2. The highest incidences of abnormal spermatids were found in C57BL/6J


and BALB/c mice and the lowest, in the randomly breeding Swiss Albino andTjtx, + \tx and T\ + mice.

3. The defects found in the spermatids of these normal fertile males have alsobeen found by other investigators in other mutant mice and in other inbred andoutbred strains of mice.

4. The ubiquitousness of aberrant spermatid development and the strain-related incidence of aberrant spermiogenesis are obvious from this and otherstudies. The effects, therefore, of any specific mutant gene on spermiogenesis canbe determined only after the distribution and range of aberrant spermiogenesisis established for the inbred or outbred strain(s) in the presence and absence ofthe mutant allele(s).

5. The present study shows that the spermatids of + jtx and T\tx have nounique ultrastructural defects which could either result in, or contribute to, theincreased transmission frequency of the ^-bearing allele.

This research was supported by United States Public Health Service Grants Nos. HD 00827and HD 09753. The authors would like to thank Dr Ralph Hillman for his help in the prep-aration of this manuscript and Marie Morris and Geraldine Wileman for their technicalassistance.

REFERENCES

BENNETT, W. I., GALL, A. M., SOUTHARD, J. L. & SIDMAN, R. L. (1971). Abnormal spermio-genesis in Quaking, a myelin-deficient mutant mouse. Biol. Reprod. 5, 30-58.

BRYAN, J. H. D. (197.1). Spermatogenesis revisited. I. On the presence of multinucleatespermatogenic cells in the seminiferous epithelium of the mouse. Z. Zellforsch. mikrosk.Anat. 112, 333-349.

BRYAN, J. H. D. & WOLOSEWICK, J. J. (1973). Spermatogenesis revisited. II. Ultrastructuralstudies of spermiogenesis in multinucleate spermatids of the mouse. Z. Zellforsch. mikrosk.Anat. 138, 155-169.

BRYSON, V. (1944). Spermatogenesis and fertility in Mus musculus as affected by factors at theT locus. / . Morph. 74, 131-186.

DOOHER, G. B. & BENNETT, D. (1973). Fine structural observations on the development ofthe sperm head in the mouse. Am. J. Anat. 136, 339-362.

DOOHER, G. B. & BENNETT, D. (1974). Abnormal microtubular systems in mouse spermatidsassociated with a mutant gene at the T-locus. / . Embryol. exp. Morph. 32, 749-761.

DUNN, L. C. (1960). Variations in the transmission ratios of alleles through egg and sperm inMus musculus. Am. Nat. 94, 385-393.

DUNN, L. C. & BENNETT, D. (1969). Studies of effects of /-alleles in the house mouse onspermatozoa. II. Quasi-sterility caused by different combinations of alleles. / . Reprod. Fert.20, 239-246.

DUNN, L. C. & GLUECKSOHN-SCHOENHEIMER, S. (1939). The influence of taillessness (anury)in the house mouse. II. Taillessness in a second balanced lethal line. Genetics 24, 587-609.

FAWCETT, D. W., ANDERSON, W. A. & PHILLIPS, D. M. (1971). Morphogenetic factors in-fluencing the shape of the sperm head. Devi Biol. 26, 220-251.

FAWCETT, D. W., EDDY, E. N. & PHILLIPS, D. M. (1970). Observations on the fine structureand relationships of the chromatoid body in mammalian spermatogenesis. Biol. Reprod. 2,129-153.

FAWCETT, D. W. & PHILLIPS, D. M. (1969). The fine structure and development of the neckregion of the mammalian spermatozoon. Anat. Rec. 165, 153-184.

HANCOCK, J. L. (1972). Spermatogenesis and sperm defects. In Edinburgh Symposium on theGenetics of the Spermatozoon (ed. R. A. Beatty & S. Gluecksohn-Waelsch), pp. 121-130.Copenhagen: Bogtrykkeriet Forum.

Mouse spermiogenesis 261HUNT, D. M. & JOHNSON, D. R. (1971). Abnormal spermiogenesis in two pink-eyed sterile

mutants in the mouse. / . Embryol. exp. Morph. 26, 111-121.JOHNSON, D. R. (1974). Multinucleate spermatids in the mouse. A quantitative electron

microscope study. Cell Tiss. Res. 150, 323-329.JOHNSON, D. R. & HUNT, D. M. (1971). Hop-sterile, a mutant gene affecting sperm tail

development in the mouse. / . Embryol. exp. Morph. 25, 223-236.KRZANOWSKA, H. (1972). Influence of Y chromosome on fertility in mice. In Edinburgh

Symposium on the Genetics of the Spermatozoon (ed. R. A. Beatty & S. Gluecksohn-Waelsch), pp. 370-386. Copenhagen: Bogtrykkeriet Forum.

OAKBERG, E. F. (1956). A description of spermiogenesis in the mouse and its use in analysisof the cycle of the seminiferous epithelium and germ cell renewal. Am. J. Anat. 99, 391-413.

OLDS, P. (1973). Sperm ultrastructure as an indication of sterility. Schering Workshop onContraception: The Masculine Gender (ed. G. Raspe). Advances in Biosciences 10, 261-270.

RAJASEKARASETTY, M. R. (1954). Studies on a new type of genetically determined quasisterilityin the house mouse. Fert. Steril. 5, 68-97.

RATTNER, J. B. & BRINKLEY, B. R. (1972). Ultrastructure of mammalian spermiogenesis. III.The organization and morphogenesis of the manchette during rodent spermiogenesis./ . Ultrastruct. Res. 41, 209-218.

SANDOZ, D. (1970). Evolution des ultrastructures au cours de la formation de l'acrosome duspermatozoide chez la souris. / . Microscopie 9, 535-558.

VENABLE, J. H. & COGGESHALL, R. (1965). A simplified lead citrate stain for use in electronmicroscopy. / . Cell Biol. 25, 407-408.

YANAGISAWA, K. (1965). Studies on the mechanism of abnormal transmission ratios at theT-locus in the house mouse. IV. Some morphological studies on the mature sperm in malesheterozygous for f-alleles. Jap. J. Genet. 40, 97-104.

WATSON, M. L. (1958). Staining of tissue sections for electron microscopy with heavy metals./ . biophys. biochem. Cytol. 4, 475-478.

(Received 9 September 1977)

a comparative study of spermiogenesis in wild-type and t:t ...a comparative study of spermiogenesis...

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