Proacrosomal Granules Research Articles

Ultrastructure developments during spermiogenesis in Polydora ciliata (Annelida: Spionidae), a parasite of mollusca

Spionid worms of Polydora ciliata inhabit the shells of many commercially important bivalves and cause disease in molluscan aquaculture. Their sperm structure is closely related to their fertilization method. To give an insight into the sperm structure and spermatogenesis, ultrastructure details of the subcellular components of germ cells during spermiogenesis of Polydora ciliata are detected by transmission electron microscopy (TEM). In P. ciliata, during spermiogenesis, chromatin is regularly arranged as dense fibrils and becomes more condensed when the nucleus elongates. Microtubules do not surround the nucleus during its elongation. The Golgi phase is characterized by the formation of proacrosomal granules within the Golgi apparatus. The proacrosomal granules fuse to form a single, spherical acrosomal vesicle that migrates to the anterior pole of the cell. At the time of nuclear condensation, mitochondria become reduced in number but increased in size, causing deep indentation at the base of the nucleus. The mid-piece has a few mitochondria. The cap phase includes the spreading of the acrosomal granule over the surface of the nucleus of the differentiating spermatid. The acrosomal phase of spermiogenesis is typically associated with changes in the shape of the nucleus, acrosome and tail. The relationship of sperm ultrastructure to spermiogenesis in spionidae species was discussed.

Spermiogenesis and Taxonomical Values of Sperm Ultrastructures in Male Mercenaria stimpsoni (Heterodonta: Veneridae)

Spermatid differentiations during spermiogenesis and sperm ultrastructures in male Mercenaria stimpsoni were investigated by transmission electron microscopic observations. In the early stage of the spermatid during spermiogenesis, a few granules and a proacrosomal granule, which is formed by the Golgi complex, become a proacrosomal vesicle. Consequently, it becomes an acrosome by way of the process of acrosome formation. The morphologies of the sperm nucleus type and the acrosome of this species have a curved cylindrical type and cap shape, respectively. The spermatozoon is approximately <TEX>$48-51{\mu}m$</TEX> in length including a curved cylinderical sperm nucleus (about <TEX>$4.18{\mu}m$</TEX> long), an acrosome (about <TEX>$0.52{\mu}m$</TEX> in length) and tail flagellum (<TEX>$42-45{\mu}m$</TEX> long). As some ultrastructural characteristics of the acrosomal vesicle, the peripheral parts of two basal rings show electron opaque part (region), while the apex part of the acrosome shows electron lucent part (region). These charateristics of the sperm belong to the family Veneridae in the subclass Heterodonta, unlike a characteristic of the subclass Pteriomorphia showing all part of the acrosome being composed of electron opaque part (region). Therefore, it is easy to distinguish the families or the subclasses by the acrosome structures. Exceptionally, In particular, a cylinder-like nucleus of the sperm is curved (the angle of the nucleus is about <TEX>$80^{\circ}$</TEX>), as seen in some species of Veneridae (range from <TEX>$0^{\circ}$</TEX> to <TEX>$80^{\circ}$</TEX>). The number of mitochondria in the midpiece of the sperm of this species are four, as one of common characteristics appeared in most species except for a few species in Veneridae in the subclass Heterodonta. Cross-sectioned axoneme of the sperm tail flagellum shows a 9+2 structure.

SPACA7 Is a Novel Male Germ Cell-Specific Protein Localized to the Sperm Acrosome That Is Involved in Fertilization in Mice1

Sperm acrosome associated 7 (SPACA7) is a novel protein of unknown function with no homology to any known protein. Spaca7 transcripts are detected only in testis and predict a 158-residue mature polypeptide with one potential N-glycosylation site and no cysteines. Orthologs are present in various species, including mice and humans. We developed a polyclonal antibody to mouse SPACA7 to study its expression and function. Western blotting and immunofluorescence microscopy detected SPACA7 only in testis, and it was detected in testis starting at Postnatal Day 21 and into adulthood. Immunofluorescence staining of testicular germ cells detected weak SPACA7 expression as early as zygotene spermatocytes. Higher expression was observed in round spermatids, where SPACA7 was localized to a perinuclear spot adjacent to the Golgi and to the acrosome of elongating spermatids and spermatozoa. Immunogold electron microscopy demonstrated that SPACA7 is localized within the proacrosomal granule of round spermatids and the acrosome of spermatozoa. Finally, we showed that SPACA7 was retained within the acrosome of epididymal sperm and was released upon the acrosome reaction. To assess if SPACA7 was involved in fertilization, in vitro fertilization assays in the presence of anti-SPACA7 IgG were performed. Anti-SPACA7 inhibited fertilization of cumulus-intact eggs and prominently delayed cumulus dispersal. However, anti-SPACA7 did not inhibit fertilization of cumulus-free eggs. Our findings indicate that release of SPACA7 from the acrosome accelerates cumulus dispersal and facilitates fertilization via unknown mechanisms. This study is the first to document the expression of endogenous SPACA7 and a function for this novel acrosomal protein.

PICK1 and ICA69 Control Insulin Granule Trafficking and Their Deficiencies Lead to Impaired Glucose Tolerance

Diabetes is a metabolic disorder characterized by hyperglycemia. Insulin, which is secreted by pancreatic beta cells, is recognized as the critical regulator of blood glucose, but the molecular machinery responsible for insulin trafficking remains poorly defined. In particular, the roles of cytosolic factors that govern the formation and maturation of insulin granules are unclear. Here we report that PICK1 and ICA69, two cytosolic lipid-binding proteins, formed heteromeric BAR-domain complexes that associated with insulin granules at different stages of their maturation. PICK1-ICA69 heteromeric complexes associated with immature secretory granules near the trans-Golgi network (TGN). A brief treatment of Brefeldin A, which blocks vesicle budding from the Golgi, increased the amount of PICK1 and ICA69 at TGN. On the other hand, mature secretory granules were associated with PICK1 only, not ICA69. PICK1 deficiency in mice caused the complete loss of ICA69 and led to increased food and water intake but lower body weight. Glucose tolerance tests demonstrated that these mutant mice had high blood glucose, a consequence of insufficient insulin. Importantly, while the total insulin level was reduced in PICK1-deficient beta cells, proinsulin was increased. Lastly, ICA69 knockout mice also displayed similar phenotype as the mice deficient in PICK1. Together, our results indicate that PICK1 and ICA69 are key regulators of the formation and maturation of insulin granules.

Molecular cytogenetic and genetic aspects of globozoospermia: a review

Infertility is estimated to affect up to 15% of couples of reproductive age. Among the male factors, globozoospermia (also called round-headed sperm syndrome) is a rare type of teratozoospermia accounting for <0.1% of male infertility. Lack of acrosome, whose production is a postmeiotic event in spermatogenesis, and round sperm head are its main characteristics. The acrosomeless spermatozoon is unable to go through the zona pellucida and fuse with the oolemma of the oocyte, and fertilisation failures have been attributed to a deficiency in oocyte activation capacity, even when intracytoplasmic sperm injection (ICSI) is attempted. The pathogenesis of this anomaly is still unclear but genetic factors are likely to be involved. DNA fragmentation rate has been reported for 16 globozoospermic males, usually using the terminal uridine nick-end labelling (TUNEL) assay. Most of the patients had a DNA fragmentation index (DFI) higher than that in fertile men. The rate of aneuploidy for some specific chromosomes was increased in 12 among the 26 globozoospermic males reported in the literature. The same results (high DFI and aneuploidy rates) were observed in infertile males compared to fertile men, notably in those with oligoasthenozoospermia or teratozoospermia, independently of the origins. Mutations or deletions in three genes, SPATA16, PICK1 and DPY19L2, have been shown to be responsible for globozoospermia. Proteins coded by the first two genes localise to the Golgi apparatus and the proacrosomal granules that are transported in the acrosome. It is likely that other proteins involved in the acrosome formation remain to be identified.

Germ Cell Development During Spermatogenesis and Taxonomic Values in Mature sperm Morphology in Male Argopecten irradians irradians (Pteriomorphia: Pectinidae) in Southern Korea

Ultrastructural studies of germ cell development during spermatogenesis and taxonomic values in mature sperm morphology of Argopecten irradians irradians were investigated by transmission electron microscopic observations. In the early stage of spermatid during spermiogenesis, a few granules and proacrosomal granules are formed by the Golgi complex. In the late stage of spermatid during spermiogenesis, a proacrosomal vesicle becomes an acrosomal vesicle in the acrosome through spermiogenesis. The sperm is approximately 45-48 μm in length including a jar-shaped sperm nucleus (about 1.45 μm long), an acrosome (about 0.34 μm long) and tail flagellum. The axoneme of the sperm tail shows a 9+2 structure. As one of common characteristics of mature sperm morphologies in Pectinidae species in subclass Pteriomorphia, mature spermatozoon consists of the cone-shaped acrosomal vesicle and subacrosomal material on the invaginated jar-shaped nucleus. The acrosomal vesicle of this species is composed of electron high dense opaque part (material) from the base to the tip, as have seen in the species in the subclass Pteriomorphia. Exceptionally, five mitochondria are found in the sperm midpiece of this species, unlike four in most species of Pectinidae in subclass Pteriomorphia. However, the acrosomal vesicle of spermatozoa of A. irradians irradians resemble to those of other investigated Pectinidae species in subclass Pteriomorphia. Therefore, we can use sperm morphology as a tool in the resolution of taxonomic relationships within the Pectinidae species. These morphological charateristics of acrosomal vesicle belong to the family Pectinidae in the subclass Pteriomorphia.

Ultrastructures of Germ Cells During Spermatogenesis and Taxonomic Values in Sperm Morphology in Male Mya arenaria oonogai (Heterodonta: Myidae)

The ultrastructures of germ cells during spermatogenesis and sperm morphology in male Mya arenaria oonogai, which was collected on the coastal waters of Samcheonpo, south coast of Korea, were investigated by transmission electron microscopic observations. In the early stage of the spermatid during spermiogenesis, a few granules and a proacrosomal granule, which is formed by the Golgi complex, appear on the spermatid nucleus, and then it becomes a proacrosomal vesicle. Consequently, it becomes an acrosome by way of the process of acrosome formation. The morphologies of the sperm nucleus type and the acrosome of this species have a curved cylindrical type and cone shape, respectively. The spermatozoon is approximately <TEX>$48-50{\mu}m$</TEX> in length including a curved cylinderical sperm nucleus (about <TEX>$2.65{\mu}m$</TEX> long), an acrosome (about <TEX>$0.64{\mu}m$</TEX> in length) and tail flagellum (<TEX>$40-45{\mu}m$</TEX> long). As some ultrastructural characteristics of the acrosomal vesicle, the peripheral parts of two basal rings show electron opaque part (region), while the apex part of the acrosome shows electron lucent part (region). These charateristics of the sperm belong to the family Myidae or some species of Veneridae in the subclass Heterodonta, unlike a characteristic of the subclass Pteriomorphia showing all part of the acrosome being composed of electron opaque part (region). Therefore, it is easy to distinguish the families or the subclasses by the acrosome structures. Exceptionally, In particular, a cylinder-like nucleus of the sperm is curved (the angle of the nucleus is about <TEX>$20^{\circ}$</TEX>), as seen in some species of Veneridae (range from <TEX>$0^{\circ}-80^{\circ}$</TEX>). The number of mitochondria in the midpiece of the sperm of this species are four, as one of common characteristics appeared in most species except for a few species in Veneridae in the subclass Heterodonta. Cross-sectioned axoneme of the sperm tail flagellum shows a 9+2 structure: the axoneme of the sperm tail flagellum consists of nine pairs of peripheral microtubules at the periphery and a pair of central doublets at the center.

Deficiency in the Omega-3 Fatty Acid Pathway Results in Failure of Acrosome Biogenesis in Mice1

An omega-3 fatty acid, docosahexaenoic acid (DHA), is enriched in testicular membrane phospholipids, but its function is not well understood. The Fads2 gene encodes an enzyme required for the endogenous synthesis of DHA. Using Fads2-null mice (Fads2-/-), we found in our preceding studies that DHA deficiency caused the arrest of spermiogenesis and male infertility, both of which were reversed by dietary DHA. In this study, we investigated a cellular mechanism underlying the DHA essentiality in spermiogenesis. Periodic acid-Schiff staining and acrosin immunohistochemistry revealed the absence of acrosomes in Fads2-/- round spermatids. Acrosin, an acrosomal marker, was scattered throughout the cytoplasm of the Fads2-/- spermatids, and electron microscopy showed that proacrosomal granules were formed on the trans-face of the Golgi. However, excessive endoplasmic reticulum and vesicles were present on the cis-face of the Golgi in Fads2-/- spermatids. The presence of proacrosomal vesicles but lack of a developed acrosome in Fads2-/- spermatids suggested failed vesicle fusion. Syntaxin 2, a protein involved in vesicle fusion, colocalized with acrosin in the acrosome of wild-type mice. In contrast, syntaxin 2 remained scattered in reticular structures and showed no extensive colocalization with acrosin in the Fads2-/- spermatids, suggesting failed fusion with acrosin-containing vesicles or failed transport and release of syntaxin 2 vesicles from Golgi. Dietary supplementation of DHA in Fads2-/- mice restored an intact acrosome. In conclusion, acrosome biogenesis under DHA deficiency is halted after release of proacrosomal granules. Misplaced syntaxin 2 suggests an essential role of DHA in proper delivery of membrane proteins required for proacrosomal vesicle fusion.

Myosin Va Participates in Acrosomal Formation and Nuclear Morphogenesis during Spermatogenesis of Chinese Mitten Crab Eriocheir sinensis

BackgroundThe Chinese mitten crab Eriocheir sinensis belongs to the Class Crustacea, Decapoda, Brachyura. The spermatozoon of this species is of aflagellated type, it has a spherical acrosome surrounded by the cup-shaped nucleus, which are unique to brachyurans. For the past several decades, studies on the spermatogenesis of the mitten crab mainly focus on the morphology. Compared with the extensive study of molecular mechanism of spermatogenesis in mammals, relatively less information is available in crustacean species. Myosin Va, a member of Class V myosin, has been implicated in acrosome biogenesis and vesicle transport during spermatogenesis in mammals. In the present study we demonstrate the expression and cellular localization of myosin Va during spermatogenesis in E. sinensis.Methodology/Principal FindingsWestern blot demonstrated that myosin Va is expressed during spermatogenesis. Immunocytochemical and ultrastructural analyses showed that myosin Va mainly localizes in the cytoplasm in spermatocytes. At the early stage of spermiogenesis, myosin Va binds to the endoplasmic reticulum vesicle (EV) and proacrosomal granule (PG). Subsequently, myosin Va localizes within the proacrosomal vesicle (PV) formed by PG and EV fusion and locates in the membrane complex (MC) at the mid spermatid stage. At the late spermatid stage, myosin Va is associated with the shaping nucleus and mitochondria. In mature spermatozoon, myosin Va predominates in acrosomal tubule (AT) and nucleus.Conclusions/SignificanceOur study demonstrates that myosin Va may be involved in acrosome biogenesis and nuclear morphogenesis during spermatogenesis in E. sinensis. Considering the distribution and molecular characteristics of myosin Va, we also propose a hypothesis of AT formation in this species. It is the first time to uncover the role of myosin Va in crustacean spermatogenesis.

PICK1 deficiency causes male infertility in mice by disrupting acrosome formation

Protein interacting with C kinase 1 (PICK1) is a peripheral membrane protein involved in protein trafficking, a function that has been well characterized in neurons. Here, we report that male mice deficient in PICK1 are infertile and have a phenotype resembling the human disease globozoospermia. The primary defect in the testes of Pick1-knockout mice was fragmentation of acrosomes in the early stages of spermiogenesis. This fragmentation was followed by defects in nuclear elongation and mitochondrial sheath formation, leading to round-headed sperm, reduced sperm count, and severely impaired sperm motility. We found that PICK1 interacted with Golgi-associated PDZ- and coiled-coil motif-containing protein (GOPC) and the primary catalytic subunit of protein kinase 2 (CK2alpha'), proteins whose deficiencies lead to globozoospermia in mice. PICK1 was highly expressed in round spermatids and localized to Golgi-derived proacrosomal granules. GOPC colocalized with PICK1 in the Golgi region and facilitated formation of PICK1-positive clusters. Furthermore, there was an increase in apoptosis in the seminiferous tubules of Pick1-/- mice, a phenotype also seen in CK2alpha'-deficient mice. Our results suggest that PICK1 is involved in vesicle trafficking from the Golgi apparatus to the acrosome and cooperates with other proteins such as GOPC and CK2alpha' in acrosome biogenesis.

The origin and assembly of a zona pellucida binding protein, IAM38, during spermiogenesis

IAM38 is the most prominent protein of the inner acrosomal membrane extracellular coat (IAMC) of spermatozoa. It is retained by the inner acrosomal membrane after the acrosome reaction and is implicated in secondary sperm-zona binding (Yu et al., Dev Biol2006;290:32-43). Its gene knockout, Zpbp1(-/-), prevents acrosome compaction and leads to acrosome fragmentation and sterility in male mice (Lin et al., Mol Cell Biol2007;27:6794-6805). Because of this protein's important role in acrosomal formation and fertilization, the authors were interested in tracing its origin and expression during spermiogenesis. The authors reasoned that a detailed immunocytochemical investigation, utilizing anti-IAM38 antibodies as probes at the ultrastructural level, could reveal the processes involved in assembly of the IAMC and the compaction of the acrosome. High level of the IAM38 transcript was already detectable at postnatal day 20, corresponding to the time in mouse testis development when round spermatids first appear and acrosomal formation is initiated. IAM38 protein was first seen during this time (Golgi phase of spermiogenesis) within the dense core of the proacrosomal granules (PG) of round spermatids. After PG fusion to form the acrosomic vesicle (AV) the immunoreactivity was concentrated in the centrally located acrosomic granule (AG) of the AV, until acrosomal-nuclear docking occurred. Subsequently, in the cap phase, IAM38 migrated from the AG to the extracellular side of the acrosomal membrane, eventually disappearing altogether from the AG. During the elongation phase IAM38 labeling disappeared from the outer acrosomal membrane (OAM) in the apical and principal segments of the acrosome but remained lining the OAM in the equatorial segment (ES) as well as the entire IAM of the acrosome. Compaction of the ES in the maturation phase of spermiogenesis coincided with the IAM38-labeled extracellular coat of the inner and outer acrosomal membranes coming together in close proximity of each other to form a "zipper-like" structure found in the ES of mature spermatozoa. In summary, the results set a precedent, showing for the first time that glycoproteins, which originate in the acrosomic granule can be transferred to the acrosomal membrane during acrosome formation. Furthermore, the localization of IAM38 on the extracellular side of both the inner and outer acrosomal membrane during the cap phase of acrosomal development supports the role of this prominent protein in acrosomal compaction.

Spermatid Differentiation and Sperm Ultrastructure of the Granular Ark, Tegillarca granosa (Bivalvia: Arcidae)

This study describes spermatogenesis and sperm ultrastructure of the granular ark, Tegillarca granosa using light and electron microscopy. In the active spermatogenic season, the testis comprises many spermatogenic follicles that contain germ cells in different developmental stages. Primary spermatocytes in the pachytene stage are characterized by synaptonemal complexes. The early spermatids are characterized by the appearance of several Golgi bodies, increased karyoplasmic electron density, and tubular mitochondria. The mass of proacrosomal granules consists of numerous heterogeneous granules with high electron density that are about 20 nm in diameter. From the midstage of spermiogenesis, the well-developed mitochondria in the cytoplasm aggregate posterior to the nucleus and surround the proximal and distal centrioles. The proacrosomal granules condense and form a single acrosome with a thin envelope. During late spermiogenesis, the acrosome begins to elongate becoming conical. The sperm is approximately <TEX>$35.0{\mu}m$</TEX> long and consists of a head, midpiece, and tail. The head comprises a round nucleus and a conical acrosome. A micro fibrous axial rod is observed between the nucleus and acrosome. The midpiece has a calyx-like structure with five mitochondria, and the tail, which has the typical "9+2" microtubular system, originates from the distal centriole.

Ultrastructural examination of spermiogenesis within the testis of the ground skink, Scincella laterale (Squamata, Sauria, Scincidae)

Although the events of spermiogenesis are commonly studied in amniotes, the amount of research available for lizards (Sauria) is lacking. Many studies have described the morphological characteristics of mature spermatozoa in lizards, but few detail the ultrastructural changes that occur during spermiogenesis. The purpose of this study was to gain a better understanding of the subcellular events of spermiogenesis within the temperate ground skink (Scincella laterale). The morphological data presented here represent the first complete ultrastructural study of spermiogenesis within the Scincidae clade. Samples of testes from 20 specimens were prepared using standard techniques for transmission electron microscopy. Many of the ultrastructural changes occurring during spermiogenesis within the ground skink are similar to that of other saurians. However, there were a few unique characteristics that to date have not been described during spermiogenesis in other lizards. For example, during early round spermatid development within the ground skink testis, proacrosomal granules begin to form within the acrosomal vesicle before making contact with the apex of the nucleus. Also, a prominent microtubular manchette develops during spermiogenesis; however, the circular component of the manchete is absent in this species of skink. This developmental difference in manchette formation may lead to the more robust and straight mature spermatozoa that are common within the Scincidae family. These anatomical character differences may be valuable nontraditional sources that along with more traditional sources (i.e., mitochondrial DNA) may help elucidate phylogenetic relationships, which are historically considered controversial at best, among species within Scincidae and Sauria.

Regulation of acrosome formation inmice expressing green fluorescent protein as a marker

Transgenic mice expressing enhanced green fluorescent protein under acrosin promoterwere used to study the role of the Golgi complex and of the cytoskeleton during early development of the acrosomic system in exactly defined stages of the seminiferous epithelial cycle during in vitro differentiation. First acrosin expression was found uniformly in the cytoplasm of stage IV pachytene spermatocytes. The steady-state level increased up to stage X pachytene spermatocytes, and in diakinetic primary spermatocytes, acrosin started to accumulate into the Golgi complex. During step 2 of spermiogenesis, several small fluorescent proacrosomic granules were seen in various parts of the Golgi complex, and they fused to a solid acrosomic system at step 3. In cultured stage I–III seminiferous tubule segments, nocodazole slowed down acrosin incorporation and increased the distance of the acrosomic system from the nucleus. Follicle stimulating hormone had an opposite effect by increasing density of the acrosomic system together with activation of the surrounding microtubule network. The observations suggest that microtubules have an important function during the early differentiation of the acrosomic system.

Ultrastructure of gametogenesis in a chemosynthetic mytilid bivalve ( Bathymodiolus childressi ) from a bathyal, methane seep environment (northern Gulf of Mexico)

The ultrastructural features of gametogenesis have been described in the methane-seep mytilid bivalve Bathymodiolus childressi Gustafson, Turner, Lutz & Vrijenhoek, 1998 collected from the Gulf of Mexico in August 1995. This is the first ultrastructural description of gametogenesis in any methane-seep bivalve. B. childressi is gonochoric, and both the testis and ovary consist of acini surrounded by inter-acinal tissue composed of adipogranular cells that serve a nutrient storage function. Oocytes develop in close association with squamous follicle cells although the follicle cells do not appear to play a primary role in yolk synthesis. During the vitellogenic phase, biosynthesis of four types of organelles occurs, including lipid droplets, yolk granules, cortical granules, and unknown inclusions, which are exocytosed as part of egg envelope biogenesis. Vitellogenesis appears to be largely autosynthetic involving the Golgi complex and rough endoplasmic reticulum. Sperm differentiation resembles that reported in other mytilid bivalves and includes the differentiation of proacrosomal granules and a flagellum during the early stages of spermatogenesis. Mature sperm have a cap-like acrosome, substantial subacrosomal material, a bullet-shaped nucleus, and four to five mitochondria in the midpiece. The general features of gametogenesis closely resemble those reported in shallow-water, seasonally reproducing mytilids. Despite the relatively stable methane seep environment and presumed continuous availability of nutrients, reproduction parallels that of littoral mytilids, suggesting some phylogenetic constraints on the capacity for variability in gametogenic processes in the family Mytilidae.

A poorly differentiated germ cell tumor (seminoma) in a Long Evans rat.

A large neoplasm that replaced 1 testis of a Long Evans Rat was noted at the final necropsy of a dietary 2-yr study. By light microscopy, the morphological features were consistent with a poorly differentiated seminoma. Ultrastructurally, the cells were polygonal, had a round nucleus, had straight cellular boundaries, and bore no resemblance to Sertoli cells. Although there was little evidence of spermatocytic differentiation, the presence of proacrosomal granules and vesicles, prominent Golgi apparatus, tight intercellular junctions, and a few centriolar pairs without axoneme development, in conjunction with the absence of lipid droplets or abundant smooth endoplasmic reticulum, supported the diagnosis of seminoma rather than Leydig cell tumor. The cells were S-100- and vimentin-positive, although cytokeratin- and alpha-fetoprotein-negative. Seminomas are extremely rare neoplasms in rats; this is the first report in this strain and the first extensive analysis of a rat seminoma without spermatocytic differentiation.

The early haploid male gamete develops a capacity for fertilization after the coalescence of the proacrosomal granules.

We compared the fertilizing capacity of rabbit testicular round spermatids (i) before appearance of the proacrosomal granules (stage -1), (ii) with two to three proacrosomal granules attached to the nucleus (stage -2) and (iii) with one granule attached to the nucleus (stages -3 to -5). Testicular samples were minced and observed via confocal scanning laser microscopy, which allows intracellular observation at high magnification and the subsequent identification of acrosomal granules and round spermatids. Nuclei of round spermatids of stage -1 (group A), -2 (group B), and -3 to -5 (group C) were isolated and injected into mature rabbit oocytes. At 24 and 72 h post-injection the number of cleaved oocytes and morulae/blastocysts, respectively, were significantly lower in groups A and B than group C. These results suggest that the rabbit round spermatid develops fertilizing potential after the stage at which the coalescence of the acrosomal granules occurs.

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.

Sorting of the Domain-Specific Acrosomal Matrix Protein AM50 during Spermiogenesis in the Guinea Pig

We previously identified an insoluble 50-kDa acrosomal matrix protein (AM50) localized to the ventral region of the guinea pig sperm apical segment. AM50 is converted to a 42-kDa polypeptide and released during matrix dispersion in acrosome-reacting sperm. This study examines the sorting pathways and assembly processes which generate the domain-specific distribution of AM50 in the acrosome. AM50 was expressed during early acrosome development and localized to the matrix of proacrosomal granules and the acrosomal vesicle. Initial sorting of AM50 occurred in Golgi phase spermatids, where it became concentrated in the matrix surrounding the acrosomal granule. AM50 remained restricted to the apical segment of acrosome phase spermatids and was finally sorted to the ventral matrix of the apical segment in maturation phase spermatids. By reducing SDS-PAGE testicular AM50 exhibited a slightly higher M r of 52 kDa than the 50-kDa form of cauda epididymal spermatozoa. Nonreducing SDS-PAGE demonstrated that testicular and epididymal AM50 were assembled into homomeric complexes of 480 and 450 kDa respectively. Cauda epididymal sperm apical segments contained a second disulfide-cross-linked homomeric complex of 520 kDa composed of a 68-kDa subunit. These studies indicate that AM50 is first assembled into a disulfide-cross-linked complex and subsequently processed into mature AM50. These data also suggest that disulfide-linked complexes of different structural proteins may assemble into distinct acrosomal matrix compartments.

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.