Association of the developing acrosome with multiple small Golgi units, the Golgi satellites, in spermatids of the musk shrew, Suncus murinus

The spermatozoa of the musk shrew, Suncus murinus, have a fan-like giant acrosome with a diameter of approximately 20 mm. The aim of this study was to investigate how this giant acrosome is constructed in the musk shrew spermatid and, in particular, how the Golgi apparatus involved in acrosome formation behaves. The behaviour of the Golgi apparatus was monitored by confocal laser scanning microscopy with antibody against a Golgi-associated Rab6 small GTPase. In the early Golgi phase, small Golgi units, the Golgi satellites, localized as a large aggregate in the juxtanuclear cytoplasm. As acrosome formation progressed, the Golgi satellites gradually dispersed, associated with proacrosomal vesicles and an acrosomal vesicle, and finally became distributed as multiple small units over the whole surface of an acrosomal cap in the round spermatid. The mode of acrosome formation in musk shrews was distinctly different from that in rats and mice, in which the Golgi apparatus remains as a single unit throughout acrosome formation. In musk shrews, the proacrosomal vesicles formed successively by the Golgi satellites coalesced, one after another, into a potential acrosomal vesicle. This process may result in further enlargement of the acrosome. The results of the present study indicate that Golgi satellites are necessary for the biogenesis and development of the giant acrosome in musk shrew spermatozoa.     

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).     

Equatorial segment protein defines a discrete acrosomal subcompartment persisting throughout acrosomal biogenesis

The equatorial segment of the acrosome underlies the domain of the sperm that fuses with the egg membrane during fertilization. Equatorial segment protein (ESP), a novel 349-amino acid concanavalin-A-binding protein encoded by a two-exon gene (SP-ESP) located on chromosome 15 at q22, has been localized to the equatorial segment of ejaculated human sperm. Light microscopic immunofluorescent observations revealed that during acrosome biogenesis ESP first appears in the nascent acrosomal vesicle in early round spermatids and subsequently segregates to the periphery of the expanding acrosomal vesicle, thereby defining a peripheral equatorial segment compartment within flattened acrosomal vesicles and in the acrosomes of early and late cap phase, elongating, and mature spermatids. Electron microscopic examination revealed that ESP segregates to an electron-lucent subdomain of the condensing acrosomal matrix in Golgi phase round spermatids and persists in a similar electron-lucent subdomain within cap phase spermatids. Subsequently, ESP was localized to electron-dense regions of the equatorial segment and the expanded equatorial bulb in elongating spermatids and mature sperm. ESP is the earliest known protein to be recognized as a marker for the specification of the equatorial segment, and it allows this region to be traced through all phases of acrosomal biogenesis. Based on these observations, we propose a new model of acrosome biogenesis in which the equatorial segment is defined as a discrete domain within the acrosomal vesicle as early as the Golgi phase of acrosome biogenesis.     

Localization of boar sperm proacrosin during spermatogenesis and during sperm maturation in the epididymis.

The localization of proacrosin was determined by using colloidal gold labeling and electron microscopy of boar germ cells during spermiogenesis to post-ejaculation. Proacrosin was first localized in round spermatids during the Golgi phase of spermiogenesis; it was associated with the electron-dense granule, or acrosomal granule that was conspicuous within the acrosome. It remained within the acrosomal granule during the cap and acrosome phases of spermiogenesis. At these stages, there was no apparent association of the proacrosin molecule with the acrosomal membranes. During the maturation phase of spermiogenesis, proacrosin was seen to become dispersed into all regions of the acrosome except the equatorial segment. When sperm from different segments of the epididymis and ejaculated sperm were examined, localization was observed throughout the acrosome except for the equatorial segment. Here proacrosin appeared to be localized on both the inner and outer acrosomal membranes as well as with the acrosomal matrix, although further studies are required to verify the membrane localization. No labeling was seen on the plasma membrane. These data suggest that the synthesis and movement of proacrosin to sites in the acrosome are controlled by an as yet unknown process. The absence of proacrosin on the plasma membrane of mature ejaculated sperm makes it unlikely that this enzyme plays a role in sperm-zona adhesion prior to capacitation.     

Cytochemical and Western Blot Analysis of the Subcompartmentalization of the Acrosome in Rodents using Soybean Lectin

In the present study, the formation and development of the acrosome during spermiogenesis in four different rodent species (rat, mouse, hamster and guinea pig) was compared by means of cytochemical and blotting techniques using a lectin from soybean (SBA). This lectin recognizes specifically the acrosome of the four species at all steps of formation. At the ultrastructural level, SBA-binding pattern was similar in the acrosome of the rat, mouse and hamster. SBA preferentially labelled the electron-lucent area of the acrosome in early spermatids (Golgi and cap phases) and the outer region of the acrosome in mature spermatids (acrosome and maturation phase). The lectin binding pattern was more complex in the guinea pig acrosome. Three different subdomains can be established in the early acrosome of the guinea pig. The lectin bound the three subdomains but mainly a thin fold which spreads over the nucleus during the cap phase. In the acrosome phase, SBA strongly reacted with the principal segment. In contrast, no reactivity was observed in most of this segment in maturation phase spermatids. In this phase, SBA bound preferentially a thin area covering the dorsal region of the apical segment. Lectin blots of detergent-extracted testes indicated that SBA only recognizes proteins of high molecular weight (>100kD) in the four species studied. The results obtained in the present study suggest that the development of acrosomal subdomains is very similar in the mouse, rat and hamster but shows a more complex pattern in the guinea pig.     

Role of microtubule-dependent membrane trafficking in acrosomal biogenesis

The role of microtubule-based trafficking in acrosomal biogenesis was examined by studying the effects of colchicine on spermiogenesis. In electron micrographs of untreated cap-phase mouse spermatids, coated vesicles were always seen on the apex and caudal margins of the developing acrosomal cap. The increase in volume and the accumulation of materials in the acrosome during the Golgi and cap phases were observed to occur via fusion of vesicles at various sites on the growing acrosome. By studying the acid phosphatase localization pattern and colchicine-treated spermatids, the role of clathrin-coated vesicles became clear. Coated vesicle formation at the caudal margin of the acrosome appeared to be responsible for the spreading and shaping of the acrosome over the surface of the nucleus and also established distinct regional differences in the acrosome. In colchicine-treated spermatids, the Golgi apparatus lost its typical membranous stack conformation and disintegrated into many small vesicles. Acrosome formation was retarded, and there was discordance of the spread of the acrosomal cap with that of the modified nuclear envelope. Many symplasts were also found because of the breakdown of intercellular bridges. Colchicine treatment thus indicated that microtubule-dependent trafficking of transport vesicles between the Golgi apparatus and the acrosome plays a vital role in acrosomal biogenesis. In addition, both anterograde and retrograde vesicle trafficking are extensively involved and seem to be equally important in acrosome formation. This work was supported by grants 83-0211-B-002-184 and 93-2320-B-320-012 from the National Science Council, Taiwan, Republic of China.     

Studies on the fine structure of the mammalian testis. I. Differentiation of the spermatids in the cat (Felis domestica)

The differentiation of cat spermatids was studied in thin sections examined with the electron microscope. The Golgi complex of the spermatid consists of a central aggregation of minute vacuoles, partially surrounded by a lamellar arrangement of flattened vesicles. In the formation of the acrosome, one or more moderately dense homogeneous granules arise within vacuoles of the Golgi complex. The coalescence of these vacuoles and their contained granules gives rise to a single acrosomal granule within a sizable membrane-limited vacuole, termed the acrosomal vesicle. This adheres to the nuclear membrane and later becomes closely applied to the anterior two-thirds of the elongating nucleus to form a closed bilaminar head cap. The substance of the acrosomal granule occupies the narrow cleft between the membranous layers of the cap. The caudal sheath is comprised of many straight filaments extending backward from a ring which encircles the nucleus at the posterior margin of the head cap. Attention is directed to the frequent occurrence of pairs of spermatids joined by a protoplasmic bridge and the origin and possible significance of this relationship are discussed.     

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.     

Changes in distribution and molecular weight of the acrosomal protein acrin2 (MC41) during guinea pig spermiogenesis and epididymal maturation

In this study, we examined the localization and characteristics of an intra-acrosomal protein, acrin2 (MC41), during guinea pig spermiogenesis and post-testicular sperm maturation in the epididymis, using the monoclonal antibody MC41. Immunoelectron microscopy demonstrated not only a specific domain localization of acrin2 in the apical segment of the guinea pig sperm acrosome, but also its dynamic behavior according to the spermatid differentiation and passage through the epididymis, as follows: acrin2 was exclusively localized in the membrane of the endoplasmic reticulum of early-stage spermatids but was not detectable in the developing acrosome until spermatids reached the maturation phase. In the final stage of spermiogenesis, acrin2 became localized in the outer acrosomal membrane (OAM)/matrix-associated materials both in the small region posterior to the dorsal matrix and along the ventral margin of the acrosomal apical segment. The acrosomal location of acrin2 in caput epididymidal sperm was almost identical to that observed in the final step spermatids, but during maturation it became progressively more restricted in area until on distal cauda epididymidal sperm it remained only in the dorsal region. In Western blot analysis, the MC41 antibody recognized a 165-kDa protein in the mature sperm extract. Furthermore, it was demonstrated that molecular weight reduction of the protein occurred during sperm passage through the epididymis. These findings indicate that acrin2 changes progressively in both distribution and size during development and maturation of the acrosome.     

STUDIES ON THE FINE STRUCTURE OF THE MAMMALIAN TESTIS : I. DIFFERENTIATION OF THE SPERMATIDS IN THE CAT (FELIS DOMESTICA)

The differentiation of cat spermatids was studied in thin sections examined with the electron microscope. The Golgi complex of the spermatid consists of a central aggregation of minute vacuoles, partially surrounded by a lamellar arrangement of flattened vesicles. In the formation of the acrosome, one or more moderately dense homogeneous granules arise within vacuoles of the Golgi complex. The coalescence of these vacuoles and their contained granules gives rise to a single acrosomal granule within a sizable membrane-limited vacuole, termed the acrosomal vesicle. This adheres to the nuclear membrane and later becomes closely applied to the anterior two-thirds of the elongating nucleus to form a closed bilaminar head cap. The substance of the acrosomal granule occupies the narrow cleft between the membranous layers of the cap. The caudal sheath is comprised of many straight filaments extending backward from a ring which encircles the nucleus at the posterior margin of the head cap. Attention is directed to the frequent occurrence of pairs of spermatids joined by a protoplasmic bridge and the origin and possible significance of this relationship are discussed.     

Changes in distribution of basic nuclear proteins and chromatin organization during spermiogenesis in the greater bandicoot rat, <Emphasis Type="Italic">Bandicota indica</Emphasis>

Male germ cells of the greater bandicoot rat, Bandicota indica, have recently been categorized into 12 spermiogenic steps based upon the morphological appearance of the acrosome and nucleus and the cell shape. In the present study, we have found that, in the Golgi and cap phases, round spermatid nuclei contain 10-nm to 30-nm chromatin fibers, and that the acrosomal granule forms a huge cap over the anterior pole of nucleus. In the acrosomal phase, many chromatin fibers are approximately 50 nm thick; these then thickened to 70-nm fibers and eventually became 90-nm chromatin cords that are tightly packed together into highly condensed chromatin, except where nuclear vacuoles occur. Immunocytochemistry and immunogold localization with anti-histones, anti-transition protein2, and anti-protamine antibodies suggest that histones remain throughout spermiogenesis, that transition proteins are present from step 7 spermatids and remain until the end of spermiogenesis, and that protamines appear at step 8. Spermatozoa from the cauda epididymidis have been analyzed by acid urea Triton X-100 polyacrylamide gel electrophoresis for basic nuclear proteins. The histones, H2A, H3, H2B, and H4, transitional protein2, and protamine are all present in sperm extracts. These findings suggest that, in these sperm of unusual morphology, both transition proteins and some histones are retained, a finding possibly related to the unusual nuclear form of sperm in this species.     

Microtubule configurations and post-translational alpha-tubulin modifications during mammalian spermatogenesis

The mechanisms underlying cell cycle progression and differentiation are tightly entwined with changes associated in the structure and composition of the cytoskeleton. Mammalian spermatogenesis is a highly intricate process that involves differentiation and polarization of the round spermatid. We found that pachytene spermatocytes and round spermatids have most of the microtubules randomly distributed in a cortical network without any apparent centrosome. The Golgi apparatus faces the acrosomal vesicle and some microtubules contact its surface. In round spermatids, at step 7, there is an increase in short microtubules around and over the nucleus. These microtubules are located between the rims of the acrosome and may be the very first sign in the formation of the manchette. This new microtubular configuration is correlated with the beginning of the migration of the Golgi apparatus from the acrosomal region towards the opposite pole of the cell. Next, the cortical microtubules form a bundle running around the nucleus perpendicular to the main axis of the cell. At later stages, the nuclear microtubules increase in size and a fully formed manchette appears at stage 9. On the other hand, acetylated tubulin is present in a few microtubules in pachytene spermatocytes and in the axial filament (precursor of the sperm tail) in round spermatids. Our results suggest that at step 7, the spermatid undergoes a major microtubular reordering that induces or allows organelle movement and prepares the cell for the formation of the manchette and further nuclear shaping. This new microtubular configuration is associated with an increase in short microtubules over the nucleus that may correspond to the initial step of the manchette formation. The new structure of the cytoskeleton may be associated with major migratory events occurring at this step of differentiation.     

Carbendazim-induced abnormal development of the acrosome during early phases of spermiogenesis in the rat testis

Effects of a single, high dose of orally administered carbendazim (100 mg/kg) on acrosome formation in the early phases of spermiogenesis were examined by electron microscopy and immunocytochemistry up to day 7.5 post-treatment. No obvious abnormality of acrosome development was noted in the Golgi phase spermatids on day 1.5 post-treatment. On day 3, step 1 spermatids were seen in stage III seminiferous tubules. In stage V tubules at this post-treatment interval, direct connections between the trans-side saccules of the Golgi stacks and the outer acrosomic membranes were observed in step 5 spermatids. Similar direct connections between these two organelles were also observed in the advanced round spermatids in later stages at days 4.5 and 7.5. On day 4.5, step 1 and 3 spermatids were seen in stage V tubules. On day 7.5, round spermatids with various abnormalities of acrosome development were observed in stage VII tubules, in addition to the discontinuous and granular acrosomes reported previously. These features were not observed in testes of control animals. In the immunocytochemical analysis using an antibody mMN7 that recognizes a protein delivered from the Golgi apparatus to the acrosome, spermatids exposed to carbendazim showed various abnormal immunostaining patterns in the acrosomes. On the other hand, strong immunoreactivity was observed in the Golgi saccules connecting to the acrosomes. These results suggest that in testis treated with carbendazim acrosome development is impaired during the early phases of spermiogenesis, and material supply from the Golgi apparatus to the acrosome is perturbed, which is a possible cause of the abnormal development. Received: 31 March 1998 / Accepted: 28 May 1998     

The Golgi apparatus segregates from the lysosomal/acrosomal vesicle during rhesus spermiogenesis: structural alterations

The acrosome is an acidic secretory vesicle containing hydrolytic enzymes that are involved in the sperm's passage across the zona pellucida. Imaging of the acrosomal vesicle and the Golgi apparatus in live rhesus monkey spermatids was accomplished by using the vital fluorescent probe LysoTracker DND-26. Concurrently, the dynamics of living spermatid mitochondria was visualized using the specific probe MitoTracker CMTRos and LysoTracker DND-26 detected the acrosomal vesicle from its formation through spermatid differentiation. LysoTracker DND-26 also labeled the Golgi apparatus in spermatogenic cells. In spermatocytes the Golgi is spherical and, in round spermatids, it is localized over the acrosomal vesicle, as confirmed by using polyclonal antibodies against Golgin-95/GM130, Golgin-97, and Golgin-160. Using both live LysoTracker DND-26 imaging and Golgi antibodies, we found that the Golgi apparatus is cast off from the acrosomal vesicle and migrates toward the sperm tail in elongated spermatids. The Golgi is discarded in the cytoplasmic droplet and is undetectable in mature ejaculated spermatozoa. The combined utilization of three vital fluorescent probes (Hoechst 33342, LysoTracker DND-26, and MitoTracker CMTRos) permits the dynamic imaging of four organelles during primate spermiogenesis: the nucleus, the mitochondria, the acrosomal vesicle, and the Golgi apparatus.     

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.     

Fine structure of seminiferous tubules in antarctic seals

Summary The fine structure of seminiferous tubules from 5 crabeater, 2 leopard and 2 Ross seals showed that during the nonbreeding season the tubules were essentially similar in possessing spermatogenic and Sertoli cells. However, the tubules of leopard and Ross seals had more primary and secondary spermatocytes and spermatids than the crabeater seals. In general, the tubules were devoid of spermatozoa. The spermatids showed stages of maturation such as Golgi phase of acrosome formation, acrosomal cap formation and condensation of nuclei. Some spermatids degenerated in tubules. Both maturing and degenerating spermatids were closely associated with Sertoli cells. Junctional complexes with plaques of filaments were observed between Sertoli cells and the spermatogenic cells. Sertoli cells, irregular and polygonal, contained highly convoluted nuclei, strands of rough endoplasmic reticulum, smooth endoplasmic reticulum, Golgi complexes, small mitochondria, variable amounts of lipid droplets, lysosomes, lipofuscin granules and highly plicated plasma membranes. In brief, the spermatogenic activity had practically ceased in the testes and the animals probably secreted low levels of testosterone during the nonbreeding season.This research was supported in part by National Science Foundation Grants G.U. 30270 and G.U. 29829X from the Office of Polar Program and by NIH Grant 5 R01 AM11-376     

An isoform of protein disulfide isomerase is expressed in the developing acrosome of spermatids during rat spermiogenesis and is transported into the nucleus of mature spermatids and epididymal spermatozoa

We have purified an isoform of protein disulfide isomerase (EC 5.3.4.1) from rat liver, and raised a specific antibody against the purified protein in rabbit. Immunohistochemical studies using this antibody on rat testis sections, at both light and electron microscopic levels, showed a specific localization of the isoform of protein disulfide isomerase in the developing acrosome of the spermatids. The protein was transferred to the acrosomic vesicle from the Golgi apparatus at late Golgi phase, and remained present in the acrosome of spermatids during cap phase, acrosome phase, and maturation phase. In addition to the acrosome, the protein appeared in the nucleus of spermatids during maturation phase, and was localized in the nucleus of epididymal spermatozoa. By immunoblot analysis, almost all of the isoform of protein disulfide isomerase in the testis was found to be extractable by an isotonic buffer. On the contrary, detergent extraction was required for complete solubilization of the protein in the epididymis. These results suggest that the isoform of protein disulfide isomerase is a new intra-acrosomal soluble protein, and that the protein begins to enter the nucleus of mature spermatids in the testis and tightly binds to the nuclear components in epididymal spermatozoa.     

Ultrastructural and functional variations in the spermatid nucleolus during spermiogenesis in the mouse

The localization, structure, and activity of the nucleolus-organizers (NORs) were studied during spermiogenesis in the mouse by light and electron microscopy procedures including NOR-silver-staining and actinomycin D treatment. After the two meiotic divisions the NORs resume their activity during the Golgi phase of spermatid differentiation (steps 1-3), and the nucleolus displays a specific 'padlock' structure containing the fibrillar components of an active nucleolus. This activity drops during the cap phase (steps 4-7) during which the nucleolus undergoes a segregation process of its components. No nucleolar structure is visible during the acrosomal and maturation phases of spermatid differentiation.