Implantation Fossa Research Articles

Novel mutations of PMFBP1 in a man with acephalic spermatozoa defects.

BackgroundAcephalic spermatozoa (AS) is a serious but rare reproductive genetic disorder that causes infertility in men. To date, only a few genes associated with AS defects have been identified, including the polyamine modulated factor 1 binding protein 1 (PMFBP1) gene. Consistent with this, PMFBP1 localizes to the head–neck connection, which bridges the implantation fossa and basal body.MethodsA male patient was diagnosed as having an AS defect. Blood samples from all family members and a sample of the patient's semen were collected to determine the genetic causes of his infertility.ResultsCompound heterozygote mutation in the PMFBP1 gene, which is associated with AS defects in the present case: two loss‐of‐function mutations, with one a nonsense mutation c.361C > T p.Gln121Ter, and another a splice donor mutation c.414 + 1G > T. The current study, together with previous studies, suggests that the nonsense mutation is responsible for a truncated PMFBP1 protein during its formation; a splice donor mutation c.414 + 1G > T might lead to new open reading frames, from which the dysfunction of an abnormal PMFBP1 protein might be predicted. Additionally, the expression of outer dense fiber 1 (ODF1) and ODF2 proteins has been experimentally shown to be regulated by the truncated PMFBP1 protein.ConclusionWe herein present a case with AS defects associated with heterozygote mutations of PMFBP1, which have been shown to be rare and pathogenic; the association with an AS defect is a monogenic disorder with a recessive inherited pattern in the patient's family.

SUN5 Interacting With Nesprin3 Plays an Essential Role in Sperm Head-to-Tail Linkage: Research on Sun5 Gene Knockout Mice.

Acephalic spermatozoa syndrome is a rare genetic and reproductive disease. Recent studies have shown that approximately 33–47% of patients with acephalic spermatozoa syndrome have SUN5 mutations, but the molecular mechanism underlying this phenomenon has not been elucidated. In this study, we generated Sun5 knockout mice and found that the head-to-tail linkage was broken in Sun5–/– mice, which was similar to human acephalic spermatozoa syndrome. Furthermore, ultrastructural imaging revealed that the head-tail coupling apparatus (HTCA) and the centrosome were distant from the nucleus at steps 9–10 during spermatid elongation. With the manchette disappearing at steps 13–14, the head and the tail segregated. To explore the molecular mechanism underlying this process, bioinformatic analysis was performed and showed that Sun5 may interact with Nesprin3. Further coimmunoprecipitation (Co-IP) and immunofluorescence assays confirmed that Sun5 and Nesprin3 were indeed bona fide interaction partners that formed the linker of the nucleoskeleton and cytoskeleton (LINC) complex participating in the connection of the head and tail of spermatozoa. Nesprin3 was located posterior and anterior to the nucleus during spermiogenesis in wild-type mice, whereas it lost its localization at the implantation fossa of the posterior region in Sun5–/– mice. Without correct localization of Nesprin3 at the nuclear membrane, the centrosome, which is the originator of the flagellum, was distant from the nucleus, which led to the separation of the head and tail. In addition, isobaric tag for relative and absolute quantitation results showed that 47 proteins were upregulated, and 56 proteins were downregulated, in the testis in Sun5–/– mice, and the downregulation of spermatogenesis-related proteins (Odf1 and Odf2) may also contribute to the damage to the spermatozoa head-to-tail linkage. Our findings suggested that Sun5 is essential for the localization of Nesprin3 at the posterior nuclear membrane, which plays an essential role in the sperm head-tail connection.

Spermiogenesis in the cattle egret (Bubulcus ibis)

Avian species comprise more than half of all vertebrates yet there is a dearth of information regarding spermatid development in this class of animals. This report of spermiogenesis in the cattle egret, Bubulcus ibis, is the first in the order Pelecaniformes. Five sexually mature and reproductively active male cattle egrets were captured in the wild, humanely euthanized, the reproductive organs dissected out, and tissues from the testes routinely prepared for transmission electron microscopy. Twelve steps of spermatid development, using the step-wise system, were determined. Acrosomogenesis in the egret results in a relatively short, solid, bullet-shaped acrosome that ends bluntly anteriorly and flat posteriorly or basally. The nucleus displays remarkable morphological changes, with the anterior end of the mature spermatid becoming flat, lacking a rostrum and an endonuclear canal. A perforatorium does not develop. It is noteworthy that a longitudinal, but not a circular, manchette develops during spermiogenesis in this bird. The proximal centriole is attached to the nucleus, at the implantation fossa, by means of well-formed, electron dense struts of material. An amorphous fibrous sheath develops in the principal piece. The interesting development and peculiar features of the acrosome and nucleus, as well as the absence of the perforatorium and circular manchette in the spermatozoon of the cattle egret, may be of phylogenetic significance.

The coupling apparatus of the sperm head and tail†.

A strong sperm head-tail coupling apparatus (HTCA) is needed to ensure the integrity of spermatozoa during their fierce competition to fertilize the egg. A lot of HTCA-specific components have evolved to strengthen the attachment of the tail to the implantation fossa at the sperm head. Defects in HTCA formation lead to acephalic spermatozoa syndrome and pathologies of some male infertility. Recent studies have provided insights into the pathogenic molecular mechanisms of acephalic spermatozoa syndrome. Here, we summarize the proteins involved in sperm neck development and focus on their roles in the formation of HTCA. In addition, we discuss the fine structures of the sperm neck in different species from an evolutionary view, highlighting the potential conservative mechanism of HTCA formation.

Ultrastructural investigations on spermatogenesis and spermatozoan morphology in the endangered Adriatic sturgeon, Acipenser naccarii (Chondrostei, Acipenseriformes).

Spermatogenesis was investigated in the Adriatic sturgeon, Acipenser naccarii, by light and electron microscopy. The testis of the unrestricted type had a germinal compartment composed of lobules containing germ cells and Sertoli cells, and separated by a basal lamina from the interstitial compartment, in which Leydig and myoid cells were detected for the first time in Acipenseridae. Spermatogenesis occurred in spermatocysts produced when Sertoli cells became associated with type A spermatogonia of subsequent generations, which produced a clone of synchronized aligned spermatogonia. In primary spermatocytes at zygo-pachytene stage, the large spherical nucleus contained synaptonemal complexes. The smaller secondary spermatocytes were ovoid with a central round nucleus and scarce cytoplasm. Spermatids were interconnected by cytoplasmic bridges until early spermiogenesis. Chromatin initially condensed as long, twisted, and nonhomogeneous fibers and finally as a compact structure made of thick filaments. Early spermatids showed the flagellum, the primordia of centriole complex and of "implantation fossa," followed by the acrosomal vesicle formed by Golgi complexes and a fibrous body associated to centriole complex. The spermatozoan head had 10 postero-lateral projections and a trapezoidal nucleus, a cylindrical midpiece with six to eight mitochondria, the centriole complex, and a "9 + 2" flagellum with a pair of lateral fins. Three helical endonuclear canals crossed the nucleus from the acrosome base to the implantation fossa; their spiralization and that of chromatin fibers suggest a spiral twisting of the nucleus during spermiogenesis. The Sertoli cells performed phagocytosis of degenerating spermatids and spermatozoa. Significant interindividual differences were detected in most morphological parameters of spermatozoa. Data on spermatogenesis in A. naccarii and morphometric measurements on mature spermatozoa provide information about the reproductive biology of the species useful not only for phylogenetic studies but also for evaluation of sperm quality for artificial reproduction projects and restocking of this and other critically endangered sturgeon species.

Mouse quiescin sulfhydryl oxidases exhibit distinct epididymal luminal distribution with segment-specific sperm surface associations.

Sulfhydryl oxidation is part of the sperm maturation process essential for the acquisition of sperm fertilization competency and its structural stabilization; however, the specific sulfhydryl oxidases that fulfill these roles have yet to be identified. In this study, we investigate the potential involvement of one atypical thiol oxidase family called quiescin Q6/sulfhydryl oxidase (QSOX) using the mouse epididymis as our model system. With multidisciplinary approaches, we show that QSOX isoform 1 and 2 exhibit complementary distribution throughout the epididymal duct, but that each variant possesses distinct subcellular localization within the epididymal principal cells. While QSOX2 was exclusively present in the Golgi apparatus of the caput and corpus epididymis, QSOX1c, the most profusely express QSOX1 variant, was abundantly present in the cauda luminal fluids. Moreover, immunohistochemistry studies together with proteomic identification in isolated epididymosomes provided evidence substantiating the release of QSOX2, but not QSOX1c, via an apocrine secretory pathway. Furthermore, we demonstrate for the first time, distinct association of QSOX1c and QSOX2 with the sperm acrosome and implantation fossa, during different stages of their epididymal maturation. In conclusion, our study provides the first comprehensive comparisons between QSOX1 and QSOX2 in the mouse epididymis, revealing their distinct epididymal distribution, cellular localization, mechanisms of secretion and sperm membrane association. Together, these data suggest that QSOX1 and QSOX2 have discrete biological functions in male germ cell development.

Ultra-structure of the sperm head-to-tail linkage complex in the absence of the spermatid-specific LINC component SPAG4.

Tight connection between sperm head and tail is crucial for the transport of the male genome and fertilization. The linkage complex, the sperm head-to-tail coupling apparatus (HTCA), originates from the centrosome and anchors to the nuclear membrane. In contrast to its ultra-structural organization, which is already well known for decades, its protein composition largely still awaits future deciphering. SUN-domain proteins are essential components of a complex that links the cytoskeleton to the peripheral nucleoskeleton, which is the nuclear lamina. Here, we studied the impact of the SUN protein SPAG4/SUN4 on the formation of the HTCA. SPAG4/SUN4 is specifically expressed in haploid male germ cells showing a polarized distribution towards the posterior pole in late spermatids that corresponds to the tail attachment site. SPAG4-deficient male mice are infertile with compromised manchette formation and malformed sperm heads. Nonetheless, sperm tails are present demonstrating dispensability of a proper manchette for their formation. Ultra-structural analyses revealed that the development of the sperm head-to-tail linkage complex in the absence of SPAG4 resembles that in the wild type. However, in SPAG4-deficient sperm, the attachment site is diminished with obvious lateral detachment of the HTCA from the nucleus. Our results thus indicate that SPAG4, albeit not essential for the formation of the HTCA per se, is, nevertheless, required for tightening the sperm head-to-tail anchorage by provoking the correct attachment of the lateral parts of the basal plate to the implantation fossa.

Detection of heterozygous mutation in hook microtubule-tethering protein 1 in three patients with decapitated and decaudated spermatozoa syndrome

BackgroundThe mechanism of intramanchette transport is crucial to the transformation of sperm tail and the nuclear condensation during spermiogenesis. Although few dysfunctional proteins could result in abnormal junction between the...

Kdm3a lysine demethylase is an Hsp90 client required for cytoskeletal rearrangements during spermatogenesis.

The lysine demethylase Kdm3a (Jhdm2a, Jmjd1a) is required for male fertility, sex determination, and metabolic homeostasis through its nuclear role in chromatin remodeling. Many histone-modifying enzymes have additional nonhistone substrates, as well as nonenzymatic functions, contributing to the full spectrum of events underlying their biological roles. We present two Kdm3a mouse models that exhibit cytoplasmic defects that may account in part for the globozoospermia phenotype reported previously. Electron microscopy revealed abnormal acrosome and manchette and the absence of implantation fossa at the caudal end of the nucleus in mice without Kdm3a demethylase activity, which affected cytoplasmic structures required to elongate the sperm head. We describe an enzymatically active new Kdm3a isoform and show that subcellular distribution, protein levels, and lysine demethylation activity of Kdm3a depended on Hsp90. We show that Kdm3a localizes to cytoplasmic structures of maturing spermatids affected in Kdm3a mutant mice, which in turn display altered fractionation of β-actin and γ-tubulin. Kdm3a is therefore a multifunctional Hsp90 client protein that participates directly in the regulation of cytoskeletal components.

Morphology and ultrastructure of beluga (Huso huso) spermatozoa and a comparison with related sturgeons

The ultrastructure and morphology of beluga spermatozoa (Huso huso) (Acipenseridae, Chondrostei) were studied by scanning and transmission electron microscopy. Spermatozoa of 51.27±4.71μm total length (mean±SD) were typical of sturgeon and paddlefish spermatozoa. Each consisted of an elongated nucleus (length: 5.84±0.46μm) with distinct acrosome, a cylindrical midpiece (length: 2.10±0.42μm), and a single flagellum (length: 42.21±3.82μm). The acrosome was umbrella shaped (length: 1.12±0.14μm, width: 0.87±0.10μm) with seven to nine posterolateral projections (length: 0.49±0.09μm). Three endonuclear canals, spirally arranged, traversed the nucleus length from the acrosome to the implantation fossa. The flagellum comprised an axoneme with the typical 9+2 organization of microtubules. Two flat fins were present along the flagellum parallel to the plane of the central doublet of microtubules. These fins arose at different positions, 0.57±0.30 and 4.06±1.32μm from the midpiece and terminated 4.18±1.09 and 4.92±1.34μm from the distal end of the flagellum. Principal component analysis revealed spermatozoan ultrastructure and morphology were similar among sturgeon species, and, although assigned to genus Huso, beluga may be closely related to the genus Acipenser.

Morphology and fine structure of the Russian sturgeon, Acipenser gueldenstaedtii (Acipenseridae, Chondrostei) spermatozoa

Summary Spermatozoa morphology and fine structure were studied in the Russian sturgeon, Acipenser gueldenstaedtii (Acipenseridae, Chondrostei) spermatozoon using scanning and transmission electron microscopy. The spermatozoon is composed of an elongated head (length: 6.84 ± 0.04 μm) with an acrosome (1.18 ± 0.01 μm length and 1.05 ± 0.01 μm width), a cylindrical midpiece (length: 1.64 ± 0.03 μm) and a flagellum (length: 49.42 ± 0.37 μm). Eleven to 12 postero-lateral projections (length: 0.65 ± 0.02 μm) were observed at the posterior edge of the acrosome. Three endonuclear canals was observed from the acrosome to the implantation fossa. In the midpiece, 4–6 mitochondria were observed as well as (i) a proximal centriole close to the implantation fossa and (ii) a distal centriole at the base of the axoneme. The flagellum consisted of nine peripheral microtubules and a central pair surrounded by the plasma membrane, which was extended while developing a pair of lateral fins along it. Inter-individual differences in morphological dimensions of spermatozoa were detected. Principal component analysis performed on morphological parameters revealed 74.08% of the variance for the first three components. The acrosome width, midpiece length and posterior width of midpiece were correlated with component 1, flagellar length and total length with component 2, and head length, anterior width of head, posterior width of head and anterior width of midpiece with component 3. Inter-species comparisons in Acipenseridae showed differences in morphological dimensions of spermatozoa. In this context, acrosome, head and midpiece dimensions in the Russian sturgeon were similar to those of Shortnose sturgeon, white sturgeon and Persian sturgeon. Length of the flagellum and total length of spermatozoon in the Russian sturgeon showed similarity to those of Lake sturgeon and Persian sturgeon.

Ultrastructure of the Spermatozoa of the Domestic Duck (Anas platyrhynchos sp.)

The ultrastructure of the spermatozoa of the domestic duck (Anas platyrhynchos sp.) was analysed by transmission and scanning electron microscopies and compared with the results obtained in preliminary studies involving other non-passerine birds. The spermatozoa were characterised by the presence of a short head, short midpiece and long principal piece. The head consisted of a reduced acrosome that contained moderately electron-dense homogenous material. The implantation fossa was observed between the base of the nucleus and the proximal centriole. The midpiece contained electron-dense material associated with the proximal centriole and nuclear membrane, and a long distal centriole surrounded throughout its length by 11-12 elliptical mitochondria. A dense annulus separating the midpiece from the principal piece was visible. Posterior to the annulus, the axoneme was formed surrounded by a dense fibrous sheath, representing the principal piece or flagellum, which was a long segment with a smooth surface and a smaller diameter than the midpiece. The spermatozoon of the domestic duck resembles that of other non-passerine birds, corresponding to a basic type of spermatozoon similar to that of reptiles, called sauropsid type.

Components and development of the centriolar complex during and beyond spermiogenesis in a passeridan bird, the Masked weaver ( Ploceus velatus)

The fate of the proximal centriole in passeridan birds is an area of controversy and relative lack of knowledge in avian spermatogenesis and spermatology. This study examines, for the first time, spatiotemporal changes in the centriolar complex in various phases of spermiogenesis in a passerine bird, the Masked weaver ( Ploceus velatus). It also describes the configuration of the centriolar complex and the relationship between it and the granular body in both intra- and extra-testicular spermatozoa. It is shown that the proximal centriole is retained and attaches, at its free end, to the granular body of spermatids in every step of spermiogenesis, as well as in mature intra-testicular and post-testicular spermatozoa, including those in the lumen of the seminal glomus. As the centriolar complex, along with its attached granular body, approaches the nucleus in the early spermatid, the proximal centriole articulates with the distal centriole at an acute angle of about 45°, and thereafter, both centrioles, still maintaining this conformation, implant, by means of their articulating proximal ends, at the implantation fossa of the nucleus. In the mature spermatid and spermatozoon, the granular body winds itself helically around the centriolar complex in the neck/midpiece region of the cell, and, thus, becomes the granular helix. The significance of this observation must await future studies, including possible phylogenetic re-evaluation and classification of birds.

Morphology and fine structure of Acipenser persicus (Acipenseridae, Chondrostei) spermatozoon: Inter-species comparison in Acipenseriformes

This study describes morphology and fine structure of the Persian sturgeon ( Acipenser persicus) (Acipenseridae, Chondrostei) spermatozoon. The results show that the spermatozoon of A. persicus is differentiated into an elongated head (length: mean ± SD: 7.1 ± 0.5 μm) with an acrosome (length: 1.2 ± 0.2 μm), a cylindrical midpiece (length: 1.8 ± 0.5 μm), a flagellum (length: 50.3 ± 5.9 μm) and a total length of 59.2 ± 6.2 μm. Ten posterolateral projections (PLPs) arise from the posterior edge of the acrosome and there were 3 endonuclear canals that traversed the nucleus from the acrosomal end to the basal nuclear fossa region. Three to six mitochondria were in peripheral midpiece and the proximal and distal centrioles were located near to “implantation fossa” and basement of the flagellum. The axoneme has a typical eukaryotic structure composed of 9 peripheral microtubules and a central pair of single microtubule surrounded by the plasma membrane. Lateral fins were observed along the flagellum. The fins started and ended at 0.5–1 μm from midpiece and at 4–6 μm from the end of flagellum. There were significant differences in the size of almost all measured morphological parameters between males and flagellar, midpiece and nucleus characters were more isolated parameters that can be considered for detecting inter-individual variations. This study showed that sperm morphology and fine structure are similar among sturgeon species, but the dimensions of the parameters may differ.

Spermatogenesis in Boccardiella hamata (Polychaeta: Spionidae) from the Sea of Japan: sperm formation mechanisms as characteristics for future taxonomic revision

AbstractReunov, A.A., Yurchenko, O.V., Alexandrova, Y.N. and Radashevsky, V.I. 2009. Spermatogenesis in Boccardiella hamata (Polychaeta: Spionidae) from the Sea of Japan: sperm formation mechanisms as characteristics for future taxonomic revision. —Acta Zoologica (Stockholm) 91: 477–456.To characterize novel features that will be useful in the discussion and validation of the spionid polychaete Boccardiella hamata from the Sea of Japan, the successive stages of spermatogenesis were described and illustrated. Spermatogonia, spermatocytes and early spermatids are aflagellar cells that develop synchronously in clusters united by a cytophore. At the middle spermatid stage, the clusters undergo disintegration and spermatids produce flagella and float separately in coelomic fluid as they transform into sperm. Spermatozoa are filiform. The ring‐shaped storage platelets are located along the anterior nuclear area. The nucleus is cupped by a conical acrosome. A nuclear plate is present between the acrosome and nucleus. The nucleus is a cylinder with the implantation fossa throughout its length and with the anterior part of the flagellum inside the fossa. There is only one centriole, serving as a basal body of the flagellum, situated in close vicinity of the acrosomal area. A collar of four mitochondria is located under the nuclear base. The ultrastructure of B. hamata spermatozoa from the Sea of Japan appears to be close to that of B. hamata from Florida described by Rice (Microscopic Anatomy of Invertebrates, Wiley‐Liss, Inc., New York, 1992), suggesting species identity of the samples from the two regions. However, more detailed study of Florida’s B. hamata sperm is required for a reliable conclusion concerning the similarity of these two polychaetes. In addition to sperm structure, features such as the cytophore‐assigned pattern of spermatogenic cell development, the synchronous pattern of cell divisions, the non‐flagellate early spermatogenic stages, and the vesicle amalgamation that drives meiotic cell cytokinesis and spermatid diorthosis will likely be useful in future testing of the validity of B. hamata and sibling species throughout the world.

Viability Tests, Active Caspase‐3 and −7, and Chromatin Structure in Ram Sperm Selected Using the Swim‐Up Procedure

Swim-up (SU) is a technique that permits the obtaining of motile sperm. Based on the sperm motility of neat ejaculates, we analyzed functional parameters, cytoplasmic esterases, and mitochondrial dehydrogenases of ram sperm using calcein acetomethylester and resazurin, respectively. Active caspase-3 and -7 and chromatin structure were evaluated in ram sperm before and after the SU process. There were no changes in any of the viability parameters after SU in neat semen samples with less or more than 25% motility. The percentage of active caspase-3 and -7 decreased after SU (68.8 +/- 4.6 vs 54.2 +/- 6.0), whereas a small but significant increase of chromatin structural abnormalities was observed (DNA fragmentation index [DFI], 287.3 +/- 3.1 vs 297.2 +/- 2.4). For the first time, the location of active caspase-3 and -7 was described for ram sperm. Notably, we found active caspases in the implantation fossa region. The presence of active caspases in neat ejaculates and the diminished presence of active caspases in SU-processed ejaculates suggest a role for caspases in motility and possibly in male fertility. The results of this study indicate that the evaluation of more than one cell-function marker is necessary to appropriately evaluate sperm quality. Furthermore, in semen samples with low motility, a lower percentage of sperm with active caspases is obtained after SU, although these sperm present increased values of DFI.

Studies of Male Reproduction in the Greater BilbyMacrotis lagotis

Despite its vulnerable conservation status there is scant information on male Greater Bilby Macrotis lagotis reproduction. Observations of gross anatomy revealed a testis to body weight ratio of 0.08 - 0.17% (n = 4), the presence of a carrot shaped prostate with an oblique coronal segmentation of ventral and dorsal orientated prostatic tissue, an elongated membranous urethra, two bulbourethral glands and a bifurcated urethra in the glans penis (n = I).The testis contained a high proportion of Leydig tissue (37.5 ± 2.7%) and a seminiferous epithelial cell cycle (8 stages identified) with a predominance of pre-meiotic stages (61.4%) and Sertoli cells with unusually large nuclei. A GnRH stimulation test conducted on four different intact sexually mature Bilbies using 2μg Buserelin resulted in maximal plasma androgen secretion 30 to 60 mins after injection. While steps of M. lagotis spermiogenesis were similar to those described in peramelid marsupials, the morphology of the Bilby spermatozoan at spermiation,was radically different to that of the bandicoot sperm cell. Similar to the bandicoot, M. lagotis spermatozoa in the corpus epididymidis dislocated their neck insertion from the primary implantation fossa, so that by the time the sperm cell had reached the cauda epididymidis, the acetabulum had migrated cranially Into a secondary implantation fossa and the nucleus had become streamlined with respect to the flagellum.This study reports the first description of large crystalloid inclusions in the principal cells of the caput epididymidal epithelium, the significance of which remains unknown. Male Bilby reproduction reported in this study supports the present taxonomic position of the Thylacomyidae.

Tail stump sperm defect in Ayrshire bulls: morphogenesis of the defect.

Three cases of tail stump sperm defect have been presented in Finnish Ayrshire bulls. The semen samples of the bulls showed markedly reduced number of sperm with almost total absence of tails. Transmission and scanning electron microscopy of the testicular samples revealed the testicular origin of the defect. The anlagen of the implantation fossa, proximal and distal centrioles, connecting piece, nuclear ring, manchette and annulus were demonstrated, but the axoneme formation was completely blocked. A separate population of spermatids showed nuclear bending and was possibly destroyed by the phagocytic activity of Sertoli cells. The pedigree of the bulls supports the concept that the defect is inherited by a single recessive autosomal gene.

Multiple malformation of bovine spermatozoa with special reference to their lightmicroscopic fluorescing pattern and electron-microscopic structure.

The light microscopic examinations of sperm samples from a sterile Fleckvieh-bull stained by the AT specific fluorochrome Hoechst 33258 showed beneath the constantly occurring paraxial tail numerous other malformations, which appeared under the electron microscope as multiple defects of essential cell organelles: defects of the acrosome as irregular thickening or forming of overturned folds "knobbed acrosome", sometimes with vesicles and inclusions inside, partial separation of the acrosome lamina from the nucleus. The defects of the nucleus were present as double-headed sperms, macro- and microcephalic forms besides bizarre shaped heads with gigantic cavities, often filled with membranes, cytoplasm and canaliculi even as heads with ridges which extended from the base to the apex. The main tail defect was a paraxial inserted tail, sometimes coiled, folded or bent down and combined with duplication or even multiplication of the tail with one or two implantation fossae. This multiplication was followed by extensive disturbances of the tail elements as lack or excessive numbers up to the isolation of tubules, fibers and mitochondria. Often there was no contact between tail and implantation fossa. Another defect was the existence of voluminous cytoplasmic droplets covering not only great parts of the tail but also the total head which included "arch like double membranes" and up to 15 tail segments. The persisting cytoplasmic droplet even as the generally occurring paraxial inserted tail are discussed in connexion with the existing hormonal imbalance as probably genetic damage leading to extensive defects during spermiogenesis.

Ultrastructural study on the fertilisation process in sturgeon ( Acipenser), function of acrosome and prevention of polyspermy

Sturgeon gametes differ from other fish in that their spermatozoa possess acrosome with finger-like posterolateral projections, which undergo exocytosis and filament formation, whereas eggs possess numerous micropyles. The fertilisation process in Acipenser baerii was investigated by fluorescence and electron microscopy. A suitable activation solution containing 2.5 mM CaCl 2, 15 mM Tris, pH 10 was found for detailed description of acrosomal reaction. The acrosome reaction includes the formation of a spear-like fertilisation filament coming from three endonuclear canals and implantation fossa through the acrosomes. It can accelerate the process of polyspermy prevention. Another unique feature of the acrosome was an anchor-like opening of the posterolateral projections. Mature eggs of A. baerii possessed 2–10 micropyles in the animal pole region. The eggs consisted of three principal layers and an outermost jelly coat blocking micropyle, and a layer of cortical granules in unfertilised eggs. With the exposure to freshwater, the jelly like layer separated from the egg surface, whereas the cortical granules swelled. No change between the layers of fertilised and unfertilised eggs, apart from the generation of an increasing perivitelline space by dissolution of the cortical granules, had been observed after the fusion of spermatozoon with an egg. A fertilisation cone blocked a fusion of other spermatozoa with cytoplasmatic projection in the fertilised micropyle.