Failure of acrosome assembly in a male sterile mouse mutant

Blind-sterile (bs) is a new autosomal recessive mutation of the mouse that causes sterility in males and bilenticular cataracts in both sexes. Sterile bs/bs males exhibited normal copulatory behavior, reduced testis weights, and few or no epididymal sperm. The effects of the bs mutation on spermatogenesis were examined by light and electron microscopy. All sperm present were morphologically abnormal with aberrant head shape. Adult bs/bs testes were characterized by germ cell depletion that resulted in profound alterations of the typical germ cell associations. Only 30% of the tubules contained relatively normal germ cell associations while 39% were extensively depleted, showing only Sertoli cells or Sertoli cells and spermatogonia. The most striking effect of the bs mutation on spermiogenesis was the failure of acrosome formation. Disorganized proacrosomic granules were detected up to step 3 of spermiogenesis by both periodic acid-Schiff staining and ultrastructural analysis. In over 3500 spermatids scored past steps 3-4 of spermiogenesis not a single acrosomal cap or fully developed acrosome was detected. Electron microscopy revealed a thickening of the nuclear envelope of elongating spermatids in the region where the acrosome should have been located; however, no acrosome was present. Chromatin condensation and nuclear elongation did occur in these acrosomeless spermatids, suggesting that caudal growth of the acrosome is not a mechanistic factor in these events.     

Appearance of an intra-acrosomal antigen during the terminal step of spermiogenesis in the rat

Summary In a survey of sperm antigens in the rat, a new intra-acrosomal antigen was found using a monoclonal antibody MC41 raised against rat epididymal spermatozoa. The MC41 was immunoglobulin G₁ and recognized spermatozoa from rat, mouse and hamster. Indirect immunofluorescence with MC41 specifically stained the crescent region of the anterior acrosome of the sperm head. Immuno-gold electron microscopy demonstrated that the antigen was localized within the acrosomal matrix. Immunoblot study showed that MC41 recognized a band of approximately 165000 dalton in the extract of rat sperm from the cauda epididymidis. Immunohistochemistry with MC41 demonstrated that the antigen was first detected in approximately step-2 spermatids, and distributed over the entire cytoplasmic region of spermatids from step 2 to early step 19. The head region became strongly stained in late step-19 spermatids and then in mature spermatozoa. Distinct immunostaining was not found in the developing acrosome of spermatids throughout spermiogenesis. These results suggest that the MC41 antigen is a unique intra-acrosomal antigen which is accumulated into the acrosome during the terminal step of spermiogenesis.     

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.     

Cytochemical characterization of glycoproteins in the developing acrosome of rats. An ultrastructural study using lectin histochemistry, enzymes and chemical deglycosylation.

The composition and distribution of rat acrosomal glycoproteins during spermiogenesis have been investigated at light and electron microscopic level by means of a variety of morphological techniques including the application of lectins conjugated to peroxidase, digoxigenin and colloidal gold, enzyme and chemical deglycosylation procedures and conventional histochemistry. Results obtained with lectin histochemistry in combination with beta-elimination reaction and endoglucosaminidase F/peptide N-glycosidase F digestion suggest that glycoproteins of mature acrosomes contain both N- and O-linked oligosaccharides. N-linked chains of acrosomal glycoproteins contain mannose and external residues of N-acetylglucosamine and galactose. They also have fucose residues linked to the core region of the oligosaccharide side chains. O-linked oligosaccharide chains contain external residues of both galactose and N-acetylgalactosamine. Mannose, fucose, galactose and N-acetylglucosamine residues were detected in acrosomes at all steps of spermiogenesis. N-acetylgalactosamine residues were only observed in the late steps of the spermiogenesis. N-acetylneuraminic acid residues were not detected throughout the acrosomal development. At initial stages of acrosome formation, glycoproteins were preferentially distributed over the acrosomic granules. In cap phase spermatids, lectin binding sites were homogeneously distributed throughout the acrosomes; however, in mature spermatozoa, glycoproteins were predominantly located over the outer acrosomal membrane.     

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.     

ELECTRON MICROSCOPE STUDIES ON SPERM DIFFERENTIATION IN MARINE ANNELID WORMS. II. SPERM FORMATION IN ARENICOLA BRASILIENSIS

The course of spermiogenesis in arenicola brasiliensis was observed with the electron microscope. The spermatogonia floating in the body cavity seem to proliferate and differentiate to mature spermatozoa in the coelomic fluid. More than a hundred spermatids are connected to one large central mass of cytoplasm and spermiogenesis proceeds synchronously in one cluster, which changes into a sperm-disc during maturation. The pre-acrosomal vesicle originates from the Golgi-body and gradually changes into the acrosomal vesicle of peculiar structure like a cup upside down. In the process of differentiation of the acrosome, a part of the material in the acrosomal vesicle is transferred into the space between the vesicle and the nucleus. The posterior one-third of the cylindrical nucleus is surrounded by four middle-piece mitochondria. The flagellar axoneme originates from one of the centrioles, which is located near a posterior pit in the nucleus.     

Intra-acrosomal organization of a 90-kilodalton antigen during spermiogenesis in the rat

The localization of an acrosomal protein was studied using a monoclonal antibody MN7 raised against mouse spermatozoa. MN7 specifically recognized the anterior acrosome of several mammalian (mouse, rat, hamster) spermatozoa fixed with paraformaldehyde. An immunoblot study with periodate treatment showed that MN7 recognized a carbohydrate region of a 90 kDa protein in an extract of mouse and rat cauda epididymal spermatozoa. The change in distribution of the MN7 antigen during acrosome development was investigated in the rat testis using the pre-embedding immunoperoxidase technique. The antigen first appeared in the proacrosomic granules of spermatids in steps 1–2. Small vesicles adjacent to the outer acrosomal membrane and the developing acrosomic system were immunoreactive during steps 4–7. The majority of the antigen was then redistributed to the head-cap portion during steps 8–18, and finally restricted to the anterior acrosome in the step 19-spermatid. These results suggest that the antigen is transported to the acrosome by way of the vesicles that originate from the Golgi apparatus during early spermiogenesis, and are then delivered to the final destination within the acrosome by the intra-acrosomal migration during late spermiogenesis.     

Differential expression of lysosomal associated membrane protein (LAMP-1) during mammalian spermiogenesis

The mammalian acrosome is a secretory vesicle of mature sperms that plays an important role in fertilization. Recent evidence had pointed out that some components found at endosomes in somatic cells are associated with the developing acrosome during the early steps of spermiogenesis. Moreover, the mammalian acrosome contains many enzymes found within lysosomes in somatic cells. In this work, we studied the dynamics of some components of the endosome/lysosome system, as a way to understand the complex membrane trafficking circuit established during spermatogenesis. We show that the cation independent-mannose-6-phosphate receptor (CI-MPR) is transiently expressed in the cytoplasm of mid-stage spermatids (steps 5-11). On the other hand, gamma-adaptin, an adaptor molecule of a complex involved in trafficking from the Golgi to lysosomes, was expressed in cytoplasmic vesicles only in pachytene and Cap-phase spermatids (steps 1-5). Our major finding is that the lysosomal protein LAMP-1 is differentially expressed during spermiogenesis. LAMP-1 appears late in spermatogenesis (Acrosome-phase) contrasting with LAMP-2, which is present throughout the complete process. Both proteins appear to be associated with cytoplasmic vesicles and not with the developing acrosome. None of the studied proteins is present in epididymal spermatozoa. Our results suggest that the CI-MPR could be involved in membrane trafficking and/or acrosomal shaping during spermiogenesis.     

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.     

Assembly of the fluorescent acrosomal matrix and its fate in fertilization in the water strider,Aquarius remigis

Animal sperm show remarkable diversity in both morphology and molecular composition. Here we provide the first report of intense intrinsic fluorescence in an animal sperm. The sperm from a semi‐aquatic insect, the water strider, Aquarius remigis, contains an intrinsically fluorescent molecule with properties consistent with those of flavin adenine dinucleotide (FAD), which appears first in the acrosomal vesicle of round spermatids and persists in the acrosome throughout spermiogenesis. Fluorescence recovery after photobleaching reveals that the fluorescent molecule exhibits unrestricted mobility in the acrosomal vesicle of round spermatids but is completely immobile in the acrosome of mature sperm. Fluorescence polarization microscopy shows a net alignment of the fluorescent molecules in the acrosome of the mature sperm but not in the acrosomal vesicle of round spermatids. These results suggest that acrosomal molecules are rearranged in the elongating acrosome and FAD is incorporated into the acrosomal matrix during its formation. Further, we followed the fate of the acrosomal matrix in fertilization utilizing the intrinsic fluorescence. The fluorescent acrosomal matrix was observed inside the fertilized egg and remained structurally intact even after gastrulation started. This observation suggests that FAD is not released from the acrosomal matrix during the fertilization process or early development and supports an idea that FAD is involved in the formation of the acrosomal matrix. The intrinsic fluorescence of the A. remigis acrosome will be a useful marker for following spermatogenesis and fertilization. J. Cell. Physiol. 226: 999–1006, 2011. © 2010 Wiley‐Liss, Inc.     

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.     

Ubiquitin signals in the developing acrosome during spermatogenesis of rat testis: an immunoelectron microscopic study.

The localization of ubiquitin (UB) signals in the acrosomes of rat spermiogenic cells was investigated by immunoelectron microscopy using two anti-UB antibodies: UB1, reacting with ubiquitinated proteins and free UB; and FK1, recognizing polyubiquitinated proteins but not monoubiquitinated proteins or free UB. Labeling of UB by UB1 (UB1 signal) was detected in the acrosomes at any stage of differentiation. In step 1 spermatids, UB1 signals were detected on the cytoplasmic surface and in the matrix of transport vesicles located between the trans-Golgi network and the acrosome. Weak signals were detected in acrosomal granules within acrosome vesicles that had not yet attached to the nucleus. In step 4-5 spermatids, the acrosome vesicles had enlarged and attached to the nucleus. Strong gold labeling was noted in a narrow space between the outer acrosomal membrane and the developing acrosomal granule, where a dense fibrous material was observed on routine electron microscopy, whereas the acrosomal granule was weakly stained by UB1 antibody. In step 6-8 spermatids, UB1 signals were detected in the fibrous material that expanded laterally to form a narrow electronless dense zone between the acrosomal granule and the outer acrosomal membrane. Labeling in the acrosomal granule increased. In step 9-11 spermatids, UB1 signals were confined to the narrow zone from the tip of the head to the periphery of the ventral fin. The matrix of the acrosome was weakly stained. In epididymal sperm, UB1 labeling in the acrosome decreased without any pretreatment, whereas staining was noted in a spot in the neck region and in the dorsal fin after trypsin digestion. On the other hand, the staining pattern with FK1 was quite different from that with UB1. The trans-Golgi network was weakly stained but the cis-Golgi network was strongly stained. The dense fibrous material just beneath the outer membrane was never stained with FK1. The results suggest that UB on the surface of transport vesicles is involved in anterograde transport from the Golgi apparatus to the acrosome. The physiological role of UB in acrosomes is not clear. Two candidates for monoubiquitinated proteins in the acrosome, which have a UB-interacting motif, were found by cyber screening.     

Cytochemical characterization of glycoproteins in the developing acrosome of rats

Summary The composition and distribution of rat acrosomal glycoproteins during spermiogenesis have been investigated at light and electron microscopic level by means of a variety of morphological techniques including the application of lectins conjugated to peroxidase, digoxigenin and colloidal gold, enzyme and chemical deglycosylation procedures and conventional histochemistry. Results obtained with lectin histochemistry in combination with -elimination reaction and endoglucosaminidase F/peptide N-glycosidase F digestion suggest that glycoproteins of mature acrosomes contain both N- and O-linked oligosaccharides. N-linked chains of acrosomal glycoproteins contain mannose and external residues of N-acetylglucosamine and galactose. They also have fucose residues linked to the core region of the oligosaccharide side chains. O-linked oligosaccharide chains contain external residues of both galactose and N-acetylgalactosamine. Mannose, fucose, galactose and N-acetylglucosamine residues were detected in acrosomes at all steps of spermiogenesis. N-acetylgalactosamine residues were only observed in the late steps of the spermiogenesis. N-acetylneuraminic acid residues were not detected throughout the acrosomal development. At initial stages of acrosome formation, glycoproteins were preferentially distributed over the acrosomic granules. In cap phase spermatids, lectin binding sites were homogeneously distributed throughout the acrosomes; however, in mature spermatozoa, glycoproteins were predominantly located over the outer acrosomal membrane.     

Targeting and fusion proteins during mammalian spermiogenesis.

Regulated exocytosis is controlled by internal and external signals. The molecular machinery controlling the sorting from the newly synthesized vesicles from the Golgi apparatus to the plasma membrane play a key role in the regulation of both the number and spatial location of the vesicles. In this context the mammalian acrosome is a unique vesicle since it is the only secretory vesicle attached to the nucleus. In this work we have studied the membrane trafficking between the Golgi apparatus and the acrosome during mammalian spermiogenesis. During bovine spermiogenesis, Golgi antigens (mannosidase II) were detected in the acrosome until the late cap-phase spermatids, but are not found in testicular spermatozoa (maturation-phase spermatids). This suggests that Golgiacrosome flow may be relatively unselective, with Golgi residents retrieved before spermination is complete. Surprisingly, rab7, a protein involved in lysosome/endosome trafficking was also found associated with the acrosomal vesicle during mouse spermiogenesis. Our results suggest that the acrosome biogenesis is associated with membrane flow from both the Golgi apparatus and the endosome/lysosome system in mammalian spermatids.     

Biochemical and morphological characterization of the intra-acrosomal antigen SP-10 from human sperm

The human sperm protein SP-10 was previously defined as a "primary vaccine candidate" by a World Health Organization Taskforce on Contraceptive Vaccines. By one- and two-dimensional immunoblots, we show that SP-10, extracted from ejaculated human sperm, demonstrated a polymorphism of immunogenic peptides from 18 to 34 kDa, a pattern that was conserved from individual to individual and was not altered by reducing agents. The majority of the antigenic peptides possessed isoelectric points of approximately 4.9. Immunocytochemistry on testis sections indicated that SP-10 was localized to round spermatids and spermatozoa within the adluminal compartment of the seminiferous epithelium. Immunofluorescence showed that SP-10 was not associated with the surface of acrosome-intact, ejaculated sperm. Light and electron microscopic immunocytochemistry localized SP-10 throughout the acrosome, and electron microscopic evidence demonstrated a bilaminar array in association with the inner aspect of the outer acrosomal membrane and the outer aspect of the inner acrosomal membrane. After induction of the acrosome reaction with the ionophore A23187, SP-10 remained displayed on the sperm head in association with the inner acrosomal membrane and equatorial segment. The results indicate that the MHS-10 monoclonal antibody may be used as a marker of acrosome development in the human and as a probe to evaluate acrosome status. The results also support the hypothesis that inhibition of sperm-egg interaction by anti-SP-10 monoclonal antibody may occur as a result of antigen exposure following the acrosome reaction.     

Membrane trafficking machinery components associated with the mammalian acrosome during spermiogenesis.

Active trafficking from the Golgi apparatus is involved in acrosome formation, both by delivering acrosomal contents to the nascent secretory vesicle and by controlling organelle growth and shaping. During murine spermiogenesis, Golgi antigens (giantin, beta-COP, golgin 97, mannosidase II) are detected in the acrosome until the late cap-phase spermatids, but are not found in testicular spermatozoa (maturation-phase spermatids). This suggests that Golgi-acrosome flow may be relatively unselective, with Golgi residents retrieved before spermiation is complete. Treatment of spermatogenic cells with brefeldin A, a drug that causes the Golgi apparatus to collapse into the endoplasmic reticulum, disrupted the Golgi in both pachytene spermatocytes and round spermatids. However, this treatment did not affect the acrosomal granule, and some beta-COP labeling on the acrosome of elongating spermatids was maintained. Additionally, N-ethylmaleimide sensitive factor, soluble NSF attachment proteins, and homologues of the t-SNARE syntaxin and of the v-SNARE VAMP/synaptobrevin, as well as members of the rab family of small GTPases, are associated with the acrosome (but not the acrosomal granule) in round and elongated spermatids. This suggests that rab proteins and the SNARE machinery for membrane recognition/docking/fusion may be involved in trafficking during mammalian acrosome biogenesis.     

Integration of the mouse sperm fertilization-related protein equatorin into the acrosome during spermatogenesis as revealed by super-resolution and immunoelectron microscopy

Spermatids must precisely integrate specific molecules into structurally supported domains that develop during spermatogenesis. Once established, the architecture of the acrosome contributes to the acrosome reaction, which occurs prior to gamete interaction in mammals. The present study aims to clarify the morphology associated with the integration of the mouse fertilization-related acrosomal protein equatorin (mEQT) into the developing acrosome. EQT mRNA was first detected by in situ hybridization in round spermatids but disappeared in early elongating spermatids. The molecular size of mEQT was approximately 65 kDa in the testis. Developmentally, EQT protein was first detected on the nascent acrosomal membrane in round spermatids at approximately step 3, was actively integrated into the acrosomal membranes of round spermatids in the following step and then participated in acrosome remodeling in elongating spermatids. This process was clearly visualized by high-resolution fluorescence microscopy and super-resolution stimulated emission depletion nanoscopy by using newly generated C-terminally green-fluorescent-protein-tagged mEQT transgenic mice. Immunogold electron microscopy revealed that mEQT was anchored to the acrosomal membrane, with the epitope region observed as lying 5–70 nm away from the membrane and was associated with the electron-dense acrosomal matrix. This new information about the process of mEQT integration into the acrosome during spermatogenesis should provide a better understanding of the mechanisms underlying not only acrosome biogenesis but also fertilization and male infertility.     

Differentiation of the acrosomal complex in ostrich (Struthio camelus) spermatids

The acrosomal complex of ostrich sperm consists of a small, cone-shaped acrosome and a slender, cylindrical perforatorium housed within a deep endonuclear canal. The perforatorium is almost exclusively endonuclear in location and is only covered by the acrosome at its point of origin in the apical subacrosomal space. The development of the acrosome is generally similar to that described in other non-passerine birds. Small proacrosomal granules (vesicles) emanating from the Golgi apparatus coalesce to form a large, membrane-bound acrosomal vesicle filled with homogeneous, electron-dense material. The acrosomal vesicle attaches to the nucleus via a shallow depression and subsequently collapses to form the typical cap-like acrosome of non-passerine birds. In ostrich spermatids the endonuclear canal becomes obvious when the collapsed acrosomal vesicle has assumed a dumbbell-shaped appearance. The perforatorium, which originates from moderately electron-dense material contained within the apical subacrosomal space, expands within the deepening endonuclear canal. The material of the perforatorium does not originate in the form of an obvious granule as in chicken and budgerigar spermatids. Indications are that in ostrich spermatids the developing acrosome plays a role in the shaping of the tip of the nucleus. The perforatorium, however, appears to represent a residual structure that has no specifically identified function. © 1996 Wiley-Liss, Inc.     

Proacrosome and acrosome of the spermatozoon in Acanthobdella peledina (Annelida: Clitellata)

Summary

Proacrosome and acrosome of the primitive leech Acanthobdella peledina are described by means of transmission electron microscopy. The proacrosome develops in early spermatids and has the shape of a pot-bellied urn with an opening towards the nucleus. Its wall is formed by a thin vesicle. In its interior, many sections of tubular structures are visible. This urn is seated atop a short, electron-dense tube. The resultant acrosome is unusually elongated, with a helically coiled acrosomal tube forming its base. Above the tube the thin acrosomal vesicle encloses a central space, within which is the acrosomal rod. The acrosomal structures clearly indicate a sister-group relationship to the Euhirudinea, but do not corroborate the notion of close kinship with the Branchiobdellidae.     

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.