Novel aspect of perinuclear theca assembly revealed by immunolocalization of non-nuclear somatic histones during bovine spermiogenesis

The perinuclear theca (PT) is an important accessory structure of the sperm head, yet its biogenesis is not well defined. To understand the developmental origins of PT-derived somatic histones during spermiogenesis, we used affinity-purified antibodies against somatic-type histones H3, H2B, H2A, and H4 to probe bovine testicular tissue using three different immunolocalization techniques. While undetectable in elongating spermatid nuclei, immunoperoxidase light microscopy showed all four somatic histones remained associated to the caudal head region of spermatids from steps 11 to 14 of the 14 steps in bovine spermiogenesis. Immunogold electron microscopy confirmed the localization of somatic histones on two nonnuclear structures, namely transient manchette microtubules of step-9 to step-11 spermatids and the developing postacrosomal sheath of step-13 and -14 spermatids. Immunofluorescence demonstrated somatic histone immunoreactivity in the developing postacrosomal sheath, and on anti-beta-tubulin decorated manchette microtubules of step-12 spermatids. Focal antinuclear pore complex labeling on the base of round spermatid nuclei was detected by electron microscopy and immunofluorescence, occurring before the nucleoprotein transition period during spermatid elongation. This indicated that, if nuclear histone export precedes their degradation, this process could only occur in this region, thereby questioning the proposed role of the manchette in nucleocytoplasmic trafficking. Somatic histone immunodetection on the manchette during postacrosomal sheath formation supports a role for the manchette in PT assembly, signifying that some PT components have origins in the distal spermatid cytoplasm. Furthermore, these findings suggest that somatic histones are de novo synthesized in late spermiogenesis for PT assembly.     

Transition of basic protein during spermatogenesis of <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> (Osbeck, 1765)

According to the ultrastructural characteristic observation of the developing male germ cells, spermatogenesis of the crustacean shrimp, Fenneropenaeus chinensis, is classified into spermatogonia, primary spermatocytes, secondary spermatocyte, four stages of spermatids, and mature sperm. The basic protein transition during its spermatogenesis is studied by transmission electron microscopy of ammoniacal silver reaction and immunoelectron microscopical distribution of acetylated histone H4. The results show that basic protein synthesized in cytoplasm of spermatogonia is transferred into the nucleus with deposition on new duplicated DNA. In the spermatocyte stage, some nuclear basic protein combined with RNP is transferred into the cytoplasm and is involved in forming the cytoplasmic vesicle clumps. In the early spermatid, most of the basic protein synthesized in the new spermatid cytoplasm is transferred into the nucleus, and the chromatin condensed gradually, and the rest is shifted into the pre-acrosomal vacuole. In the middle spermatid, the nuclear basic protein linked with DNA is acetylated and transferred into the proacrosomal vacuole and assembled into the acrosomal blastema. At the late spermatid, almost all of the basic protein in the nucleus has been removed into the acrosome. During the stage from late spermatid to mature sperm, some de novo basic proteins synthesized in the cytoplasm belt transfer into the nucleus without a membrane and almost all deposit in the periphery to form a supercoating. The remnant histone H4 accompanied by chromatin fibers is acetylated in the center of the nucleus, leading to relaxed DNA and activated genes making the nucleus non-condensed.     

Changes in intranuclear chromatin architecture induce bipolar nuclear localization of histone variant H1T2 in male haploid spermatids

Spermiogenesis entails a major biochemical and morphological restructuring of the germ cell packing the DNA into the condensed spermatid nucleus. H1T2 is a histone H1 variant selectively and transiently expressed in male haploid germ cells during spermiogenesis that specifically localizes to a chromatin domain at the apical pole under the acrosome. We explored the mechanisms determining polar localization of H1T2 in spermatids. In acrosome-deficient round spermatids of hrb -/- and gopc -/- mice, H1T2 localization is not altered, indicating that proper acrosome development is not required for specifying nuclear polarity. In contrast, in late round spermatids from trf2 -/- or hmgb2 -/- mice, a bipolar H1T2 localization was observed revealing that polarity is modified by loss of proteins specifying chromatin architecture. Our results show that intranuclear chromatin organization is critical for correct polar localization of H1T2 and that H1T2 can be a useful molecular marker revealing chromatin disorganization in spermatids.     

Acroplaxome, an F-actin-keratin-containing plate, anchors the acrosome to the nucleus during shaping of the spermatid head

Nuclear shaping is a critical event during sperm development as demonstrated by the incidence of male infertility associated with abnormal sperm ad shaping. Herein, we demonstrate that mouse and rat spermatids assemble in the subacrosomal space a cytoskeletal scaffold containing F-actin and Sak57, a keratin ortholog. The cytoskeletal plate, designated acroplaxome, anchors the developing acrosome to the nuclear envelope. The acroplaxome consists of a marginal ring containing keratin 5 10-nm-thick filaments and F-actin. The ring is closely associated with the leading edge of the acrosome and to the nuclear envelope during the elongation of the spermatid head. Anchorage of the acroplaxome to the gradually shaping nucleus is not disrupted by hypotonic treatment and brief Triton X-100 extraction. By examining spermiogenesis in the azh mutant mouse, characterized by abnormal spermatid/sperm head shaping, we have determined that a deformity of the spermatid nucleus is restricted to the acroplaxome region. These findings lead to the suggestion that the acroplaxome nucleates an F-actin-keratin-containing assembly with the purpose of stabilizing and anchoring the developing acrosome during spermatid nuclear elongation. The acroplaxome may also provide a mechanical planar scaffold modulating external clutching forces generated by a stack of Sertoli cell F-actin-containing hoops encircling the elongating spermatid nucleus.     

Changes in nuclear proteins of rat testis cells separated by velocity sedimentation.

The technique of velocity sedimentation at unit gravity has been used to separate rat testis cell suspensions into fractions enriched in particular cell types. Changes in the nuclear proteins from the various fractions have been characterized by polyacrylamide gel electrophoresis, and correlated with the changing morphology of the nucleus during spermatogenesis. The most striking alterations in both protein composition and nuclear morphology occur during spermatid maturation as both histone and non-histone proteins are replaced by highly basic, low molecular weight, spermatidal proteins. This replacement process is accompanied by a quantitative reduction in both histone and non-histone proteins. The synthesis of at least three basic proteins has been identified with late stage spermatids. One of these proteins is a highly basic sperm-specific protein containing high levels of cyst(e)ine and arginine. A second protein synthesized in late stage spermatids is lysine rich, while the third protein contains cyst(e)ine and co-migrates with histone F2a1 on acid-urea polyacrylamide gels. The changes in protein composition of rat testis nuclei after irradiation or hypophysectomy reflect the resulting changes in the cellular composition of the testis. After selective elimination of the germinal cells by irradiation, the electrophoretic pattern of acid-soluble proteins from the testis is very similar to that of somatic tissue. Thus, the cellular specificity of nuclear proteins demonstrated here using cell separation techniques is also apparent following treatments which selectively alter the cellular composition of the testis.     

Microelectrophoretic analysis of basic protein changes during spermiogenesis in the dogfish Scylliorhinus caniculus (L.)

Spermatogenesis in the dogfish is characterized by the synchronous development of germinal cells inside follicles. This particularity has permitted studies on precise stages of cell differentiation, especially on the evolution of chromatin structure. A microelectrophoretic method has been devised for the determination of the basic nuclear protein content of accurately identified homogeneous stages of spermatid differentiation. No significant difference was observed during the first stages of spermiogenesis, i.e., in round spermatids, where a typical histone complement was present. At the beginning of nuclear elongation, two new basic protein fractions appeared and coexisted for some time with typical histones; they replaced somatic histones progressively. Later, during elongation, four proteins of high electrophoretic mobility appeared and gradually replaced the intermediary basic proteins. In elongated spermatids, DNA was found tightly packed by these four proteins: three are arginine- and cysteine-rich (Z1, Z2 and S4), the fourth is arginine-rich (Z3). At first, these fractions are all soluble in 0.25 M HCl but during sperm maturation only one (Z3) remains acid-soluble, the others being extractable only after reducing and alkylating treatments. This modification of solubility of Z1, Z2 and S4 corresponded to the oxidation of cysteine residues to form ---S---S--- crosslinks in chromatin of mature sperm cells. Thus spermiogenesis of the dogfish shows two basic nuclear protein transitions which both occur during nuclear elongation.     

The acroplaxome is the docking site of Golgi-derived myosin Va/Rab27a/b- containing proacrosomal vesicles in wild-type and Hrb mutant mouse spermatids

Acrosome biogenesis involves the transport and fusion of Golgi-derived proacrosomal vesicles along the acroplaxome, an F-actin/keratin 5-containing cytoskeletal plate anchored to the spermatid nucleus. A significant issue is whether the acroplaxome develops in acrosomeless mutant mice. Male mice with a Hrb null mutation are infertile and both spermatids and sperm are round-headed and lack an acrosome. Hrb, a protein that contains several NPF motifs (Asn-Pro-Phe) and interacts with proteins with Eps15 homology domains, is regarded as critical for the docking and/or fusion of Golgi-derived proacrosomal vesicles. Here we report that the lack of an acrosome in Hrb mutant spermatids does not prevent the development of the acroplaxome. Yet the acroplaxome in the mutant contains F-actin but is deficient in keratin 5. We also show that the actin-based motor protein myosin Va and its receptor, Rab27a/b, known to be involved in vesicle transport, are present in the Golgi and Golgi-derived proacrosomal vesicles in wild-type and Hrb mutant mouse spermatids. In the Hrb mutant, myosin-Va-bound proacrosome vesicles tether to the acroplaxome, where they flatten and form a flat sac, designated pseudoacrosome. As spermiogenesis advances, round-shaped spermatid nuclei of the mutant display several nuclear protrusions, designated nucleopodes. Nucleopodes are consistently found at the acroplaxome- pseudoacrosome site. Our findings support the interpretation that the acroplaxome provides a focal point for myosin-Va/ Rab27a/b-driven proacrosomal vesicles to accumulate, coalesce, and form an acrosome in wild-type spermatids and a pseudoacrosome in Hrb mutant spermatids. We suggest that nucleopodes develop at a site where a keratin 5-deficient acroplaxome may not withstand tension forces operating during spermatid nuclear shaping.     

Histone synthesis and replacement during spermatogenesis in the mouse.

Separation of labelled nuclei by sedimentation velocity at unit gravity (Staput method) was used to study the timing of histone synthesis and replacement by testis-specific basic nuclear protein (TSP) during spermatogenesis in the mouse. Animals were injected (intratesticularly) with 1.25 micronCi per testis 3H-arginine or 2.5 micronCi per testis 3H-lysine, testis nuclei were separated, and the acid extract of each nuclear fraction was analyzed by acrylamide gel electrophoresis. The distribution of labelled histones and TSP in separated nuclei was assessed 2 h after incorporation. Changes in the labelled histone and TSP content of nuclei during subsequent differentiation (1--34 days post-label) was followed in fractions of separated testis cell nuclei and in nuclei of cauda epididymal spermatozoa. Analysis of total histone and (TSP) content indicated quantitative changes during development. Nuclei from primary spermatocytes had relatively larger amounts of histones H1 and H4. Spermatid nuclei showed a relative reduction in histones H1 and H4, coincident with the appearance of TSP in these nuclei. These results suggested that synthesis and/or removal of certain histones must occur in late primary spermatocyte and early spermatid stages of spermatogenesis. Results of labelling experiments indicated several periods of histone synthesis during spermatogenesis: (1) closely associated with the last DNA synthesis(i.e., in early primary spermatocytes), (2) late in meiotic prophase (i.e., in pachytene primary spermatocytes) and (3) simultaneous with TSP synthesis (i.e., in late spermatids). Histone H1 was more heavily labelled toward the end of the primary spermatocyte period. Histone H4 was more heavily labelled in the early primary spermatocyte period, and again at the time of TSP synthesis in spermatids. Histones synthesized before the pachytene primary spermatocyte stage appeared to be replace, but histones synthesized later in spermatogenesis appeared to be at least partially retained in epididymal spermatozoa. These results suggested that repeated specific alterations in the protein complement of the nucleus are an integral part of spermatogenic differentiation in the mouse.     

Development of the acrosome and alignment,elongation and entrenchment of spermatids in procarbazine-treated rats

Intraperitoneally administered procarbazine caused, among other features previously reported (Russell et al., 1983), specific defects in the acrosome of cap phase spermatids of the rat seminiferous epithelium. The effect of procarbazine was to fragment and eventually cause resorption of the acrosomes of a small number of steps 5–9 spermatids. Although the acrosome was lost, dose union of the leaflets of the nuclear envelope underlying the acrosomal sac was maintained as was the marginal fossa and acrosomal zonule. Spermatids at steps 8 and 9 of development, which had lost their acrosomes, showed nuclei which were eccentric within the cell—a feature which normally occurs at these steps of spermiogenesis in acrosome intact cells. Even without an acrosomal sac, the plasma membrane of these cells (in stage VIII) became orientated to the region of the nuclear membrane which would have underlaid the acrosome. Although abundant, Sertoli ectoplasmic specialization did not become aligned with the spermatid head. The spermatid failed to become orientated within the seminiferous epithelium and failed to enter the crypts within the Sertoli cell as usually occurs during the elongation process. Thus, the presence of an acrosome is not likely related to the formation of an eccentric nucleus or the alignment of the surface of the nucleus which would normally underlay the acrosome with the cell's plasma membrane (internal alignment). The presence of an acrosome may be related to the alignment of the spermatid head with the ectoplasmic specialization, which in turn may influence the orientation and positioning of the late spermatids within the seminiferous epithelium (external alignment) and their position within recesses of the Sertoli cell. This study also suggests a role for the manchette in the process of elongation of the spermatid.     

Spermiogenesis and chromatin condensation in the common tree shrew, <Emphasis Type="Italic">Tupaia glis</Emphasis>

We have investigated the cellular characteristics, especially chromatin condensation and the basic nuclear protein profile, during spermiogenesis in the common tree shrew, Tupaia glis. Spermatids could be classified into Golgi phase, cap phase, acrosome phase, and maturation phase. During the Golgi phase, chromatin was composed of 10-nm and 30-nm fibers with few 50-nm to 60-nm knobby fibers. The latter were then transformed into 70-nm knobby fibers during the cap phase. In the acrosome phase, all fibers were packed into the highest-order knobby fibers, each about 80–100 nm in width. These chromatin fibers became tightly packed in the maturation phase. In a mature spermatozoon, the discoid-shaped head was occupied by the acrosome and completely condensed chromatin. H3, the core histone, was detected by immunostaining in all nuclei of germ cell stages, except in spermatid steps 15–16 and spermatozoa. Protamine, the basic nuclear protein causing the tight packing of sperm chromatin, was detected by immunofluorescence in the nuclei of spermatids at steps 12–16 and spermatozoa. Cross-immunoreactivity of T. glis H3 and protamine to those of primates suggests the evolutionary resemblance of these nuclear basic proteins in primate germ cells. This work was supported by the Thailand Research Fund (Senior Research Fellowship to Prof. Prasert Sobhon).     

Transformation of sperm histone during formation and maturation of rat spermatozoa.

Changes of chromosomal basic proteins of rats have been followed during transformation of spermatids into spermatozoa in the testis and during maturation of spermatozoa in the epididymis. Rat testis chromatin has been fractionated on the basis of differing sensitivity to shearing, yielding a soluble fraction and a condensed fraction. The sperm histone is found in the condense fraction. Somatic-type histones are found in both fractions. The somatic-type histones in the condensed fraction contains much more lysine-rich histone I, than does the somatic-type histones in the soluble fraction. This may suggest that the lysine-rich histone I is the last histone to be displaced during the replacement of somatic-type histones by sperm histone. After extensive shearing followed by sucrose centrifugation, the condensed portion of testis chromatin can be further fractionated into two morphologically distinctive fractions. One is a heavy fraction possessing an elongated shape typical of the head of late spermatids. The other is a light fraction which is presumably derived from spermatids at earlier stages of chromatin condensation and which is seen as a beaded structure in the light microscope. Sperm histone of testis chromatin can be extractable completely by guanidinium chloride without a thiol, wheras 2-mercaptoethanol is required for extraction of sperm histone from caput and cauda epididymal spermatozoa. The light fraction of the condensed testis chromatin contains unmodified and monophospho-sperm histone. The sperm histones of the heavy fraction is mainly of monophospho and diphospho species, whereas unmodified and monophosphosperm histones are found in caput and cauda epididymal spermatozoa. Labeling of cysteine sulfhydryl groups of sperm histone releases by 2-mercaptoethanol treatment shows that essentially all of the cysteine residues of sperm histone in testis chromatin are present as sulfhydryl groups, while those of sperm histone isolated from mature (cauda epididymal) spermatozoa are present as disulfide forms and approximately 50% of the cysteine residues of sperm histone obtained from caput epididymal spermatozoa are in disulfide forms. These results suggest that phosphorylation of sperm histone is involved in the process of chromatin condensation during transformation of spermatozoa in the epididymis.     

Biosynthesis of specific histones during meiotic prophase of mouse spermatogenesis

A kinetics study has demonstrated histone synthesis occurring at two distinct phases during meiotic prophase of mouse spermatogenesis. These two periods have been delineated by quantifying the synthesis of DNA and basic nuclear proteins in spermatogenic cells at discrete intervals following the intratesticular injection of [3H] thymidine and [14C] arginine, respectively. One phase of histone synthesis occurs coincident with DNA synthesis in preleptotene spermatocytes. By contrast, a second phase of histone synthesis occurs during midprophase of meiosis, independent of semiconservative DNA synthesis. The [14C] arginine incorporated into the basic nuclear proteins of pachytene spermatocytes is conserved during spermiogenesis and then subsequently discarded within the residual bodies, which are formed during late spermiogenesis. Fluorographic analyses of isotopically labeled basic nuclear proteins in pachytene spermatocytes has shown that only the somatic complement of histones are synthesized during the preleptotene period, whereas the second phase involves the synthesis of proteins H1t, H2S, and "A". In addition, several nonhistone basic nuclear proteins are synthesized concomitant with the germ cell-specific histones. Thus, the data clearly demonstrate that pachytene spermatocytes actively synthesize a number of novel chromatin-associated polypeptides.     

Mouse round spermatids developed in vitro from preexisting spermatocytes can produce normal offspring by nuclear injection into in vivo-developed mature oocytes

It has been shown that mature oocytes injected with nuclei from round spermatids collected from mouse testis can generate normal offspring and that round spermatids can develop in vitro. An undetermined issue is whether spermatids developed in vitro are capable of generating fertile offspring by nuclear injection into oocytes. Herein, we report the production of normal and fertile offspring by nuclear injection using haploid spermatid donors derived from mouse primary spermatocyte precursors cocultured with Sertoli cells. Cocultured spermatogonia and spermatocytes were characterized by their nuclear immunoreactive patterns determined by an antibody to phosphorylated histone H2AX (gamma-H2AX), a marker for DNA double-strand breaks. Cocultured round spermatid progenies display more than one motile flagellum, whose axonemes were recognized by antitubulin immunostaining. Flagellar wavelike movement and flagellar-driven propulsion of round spermatids developed in vitro were documented by videomicroscopy (http://www.sci.ccny.cuny.edu/ approximately kier). We also show that breeding of male and female mouse offspring generated by spermatid nuclear injection produced fertile offspring. In addition to their capacity to produce fertile offspring, cocultured, flagellated round spermatids can facilitate the analysis of the mechanisms of centriolar polarity, duplication, assembly, and flagellar growth, including the intraflagellar transport of cargo proteins.     

C-terminal kinesin motor KIFC1 participates in acrosome biogenesis and vesicle transport

We have identified a possible role for the KIFC1 motor protein in formation of the acrosome, an organelle unique to spermatogenesis. KIFC1, a C-terminal kinesin motor, first appears on membrane-bounded organelles (MBOs) in the medulla of early spermatids followed by localization to the acrosomal vesicle. KIFC1 continues to be present on the acrosome of elongating spermatids as it flattens on the spermatid nucleus; however, increasing amounts of KIFC1 are found at the caudal aspect of the spermatid head and in distal cytoplasm. The KIFC1 motor is also found in the nucleus of very immature round spermatids just prior to its appearance on the acrosome. In some cases, KIFC1 appears localized just below the nuclear membrane adjacent to the subacrosomal membrane. We demonstrate that KIFC1 is associated with importin beta and colocalizes with this nuclear transport factor on curvilinear structures associated with the spermatid nuclei. These data support a model in which KIFC1, perhaps in association with nuclear factors, assists in the formation and/or elongation of the spermatid acrosome. This article represents the first demonstration of a direct association of a molecular motor with the spermatid acrosome, the formation of which is essential for fertilization.     

Haploid genome reactivation and recovery by cell hybridization

DNA replication in haploid spermatid nuclei has been induced by hybridization of mouse early spermatids to proliferating HeLa cells. Use of polyethylene glycol rather than inactivated Sendai virus as the cell fusion agent was found to be essential to the production of large numbers of heterokaryons containing spermatid nuclei. DNA replication was detected in the heterokaryons by autoradiography. Density of silver grains over spermatid nuclei closely approximated the grain density over labelled HeLa nuclei in the same heterokaryons. Mouse centromeric heterochromatin appeared to be labelled last during the spermatid DNA synthetic period. On the average, HeLa nuclei in heterokaryons began DNA synthesis before spermatid nuclei. Results indicated, however, that DNA synthesis by HeLa nuclei might not be a prerequisite for spermatid DNA synthesis. These experiments demonstrate induction of DNA synthesis in spermatid nuclei, the first major step toward reactivation and recovery of their haploid genome by cell hybridization.     

High mobility group proteins in ram spermatids

The four major high mobility group proteins HMG 1, 2, 14 and 17, HMG 19B and histone H1(0) were identified in the ram testis by their extraction and solubility characteristics and by their electrophoretic mobilities. HMG 14 and 17 were isolated by chromatography and amino acid analysis revealed that they were similar to their calf thymus analogues. A protein, named 2R and co-extracted with HMG 14, was also purified and analysed. Electrophoretic analyses of the proteins extracted by 0.75 M perchloric acid (PCA) or by 0.35 M NaCl from round and non-round spermatids, separated by centrifugal elutriation, showed that the four major HMG proteins disappear from nuclei in the oldest round spermatids, at the time the nuclear content of protein 2R and histone H1(0) increases in spermatids. Ubiquitin and HMG 19B were present in the round and elongating spermatids, but not in elongated spermatids which contained only protamine. The relation was considered between several protein changes and genetic inactivation and structural reorganization of the spermatid chromatin.     

Assembly of spermatid acrosome depends on microtubule organization during mammalian spermiogenesis

The acrosome is a secretory vesicle attached to the nucleus of the sperm. Our hypothesis is that microtubules participate in the membrane traffic between the Golgi apparatus and acrosome during the first steps of spermatid differentiation. In this work, we show that nocodazole-induced microtubule depolarization triggers the formation of vesicles of the acrosomal membrane, without detaching the acrosome from the nuclear envelope. Nocodazole also induced fragmentation of the Golgi apparatus as determined by antibodies against giantin, golgin-97 and GM130, and electron microscopy. Conversely, neither the acrosome nor the Golgi apparatus underwent fragmentation in elongating spermatids (acrosome- and maturation-phase). The microtubule network of round spermatids of azh/azh mice also became disorganized. Disorganization correlated with fragmentation of the acrosome and the Golgi apparatus, as evaluated by domain-specific markers. Elongating spermatids (acrosome and maturation-phase) of azh/azh mice also had alterations in microtubule organization, acrosome, and Golgi apparatus. Finally, the spermatozoa of azh/azh mice displayed aberrant localization of the acrosomal protein sp56 in both the post-acrosomal and flagellum domains. Our results suggest that microtubules participate in the formation and/or maintenance of the structure of the acrosome and the Golgi apparatus and that the organization of the microtubules in round spermatids is key to sorting acrosomal proteins to the proper organelle.     

Studies on sex-organ development. Changes in nuclear and chromatin composition and genomic activity during spermatogenesis in the maturing rooster testis.

We developed a technique to separate nuclei of rooster testis by centrifugation through a discontinuous sucrose density gradient and by sedimentation at unit gravity. Four different major fractions obtained from testicular nuclei and one from the vas deferens were characterized according to their velocity of sedimentation, morphology and DNA content. The ratios (w/w) of basic proteins, non-histone proteins and RNA to DNA decreased during spermiogenesis both in nuclei and chromatin. Changes in the electrophoretic patterns of histones and non-histone proteins were detected especially in the elongated spermatids. The lack of uptake of [3H]uridine in elongating and elongated spermatids and in spermatozoa was demonstrated by radioautography and by the detection of labelled RNA extracted from different fractions of nuclei. Template activity for RNA synthesis and the binding of actinomycin D by testicular nuclei reached a peak in the elongated spermatid stage, when the histones are replaced by the protamine.     

Spermatogenesis in the Watase's shrew, Crocidura watasei--a light electron microscopic study.

Spermatogenesis in the Watase's shrew, Crocidura watasei, was investigated by light and transmission electron microscopy. The cycle of the seminiferous epithelium was divided into 12 stages using the development of spermatids as a main criterion. The steps of spermatids were characterized by morphological changes of the nucleus and acrosomal structure. The relative frequencies of the stages 1 to x 11 were 11.0, 10.3, 6.8, 10.6, 24.0, 6.4, 4.4, 7.9, 6.4, 4.9, 3.7 and 3.6%, respectively. Four types of spermatogonia (A1, A2, In and B) could be discerned by the observation of whole mount samples. The development of spermatids was divided into four phases (Golgi, cap, acrosome and maturation phases), as in other mammals. In Golgi phase of the spermatid, several acrosomal granules were encountered. In cap phase, the acrosome gradually spread over the nuclear surface. In early acrosome phase, the acrosome began to elongate and reached the maximal length in step 8 spermatids. The acrosome of step 8 spermatids was twice as long as that of spermatozoa. In late acrosome phase, the acrosome was on the way of shrinkage. Finally, the fan-shaped acrosome was formed in maturation phase. These findings suggested that the process of acrosomal formation was quite characteristic in the Watase's shrew in that the spermatid acrosome elongated most prominently in the mammals hitherto examined.