Gamma-tubulin during the differentiation of spermatozoa in various mammals and man

The distribution of gamma-tubulin as a marker of microtubule organizing centres (MTOC) was studied during spermiogenesis in rodents and in rabbit, monkey and man. A polyclonal antibody directed against human gamma-tubulin was used both for indirect immunofluorescence (IIF) and post-embedding immunogold procedures. In all species, gamma-tubulin was detected in the proximal and distal centrioles of round spermatids. In elongating spermatids, gamma-tubulin was predominantly found in the pericentriolar material (PCM) of both centrioles and particularly around the adjunct of the proximal centriole. At this level, some labelling was also associated with manchette microtubules, but other parts of the manchette and the nuclear ring were never labelled. We propose a role for distal centriole gamma-tubulin in axoneme nucleation and centriolar adjunct gamma-tubulin in manchette nucleation. The disappearance of gamma-tubulin in mature spermatozoa indicates that sperm aster nucleation should be dependent on oocyte gamma-tubulin. Remnants of gamma-tubulin in some human spermatozoa suggest that paternal gamma-tubulin also could contribute to sperm aster formation.      

Specific localization of lectins in boar and bull spermatozoa

The roles that complex carbohydrates play in gamete recognition and fertilization are well-documented. This study was designed to identify and map sites of N-linked oligosaccharides in boar and bull sperm. Plant lectins, conjugated with FITC, RITC and gold particles, were used as probes for specific sugar moieties in sperm membranes. Results showed strong labeling of sperm heads by most lectins. However, lectins with specificities for mannosyl, glucosyl, and sialic acid were predominantly distributed over the sperm acrosomal region. Lectins specific for fucosyl residues predominantly stained the postacrosomal regions of both boar and bull sperm. Lectin gold studies revealed that mannosyl and galactosyl-linked residues occupied the acrosomal membrane and plasma membrane in the postacrosomal region, but not the nucleus or nuclear membrane. The cytoplasmic droplet, middle piece and flagellum of boar and bull sperm were moderately stained by lectins. The microquantitative analysis confirmed the fluorescence data. The presence of fucosyl residues predominantly in the postacrosomal regions lends credence to the suggestion that glycoproteins rich in fucosyl-like residues may be a universal carbohydrate moiety involved in recognition processes in mammalian fertilization.     

Extractions reveal specific argentophilic proteins in rat and bull sperm heads

Background: Silver-stainability (argentophilia) of cytoplastmic structures occurring in spermatids have been localized into the organizing perinuclear theca, but the biochemical nature and structural associations of these proteins with the cytoskeletal and membranous elements are unresolved and, therefore, were the aim of the present study. Methods: Light and electron microscopic analysis of the silver-stainability in the rat spermatids and spermatozoa was carried out in the intact testis tissue and epididymal spermatozoa and after their chemical and mechanical extraction. Correlation of argentophilia with specific proteins of rat and bovine spermatids and spermatozoa was investigated using a recently developed technique for silver nitrate staining of proteins on nitro-cellulose. Results: Sequential formation of the silver-stainable domains seemed to proceed from the argentophilic acrosomal ring. Various extractions indicated that argentophilia in the spermatids and spermatozoa was mainly associated with the perinuclear theca and to some extent to the plasma membrane. Hyamine-soluble extract from spermatozoa of rat and bull revealed only a single argentophilic protein of 130 kDa. Hyamine and SDS-soluble extracts of rat testis tissue contained an additional group of argentophilic polypeptides of lower molecular weight (115, 94, 36, 23, and 21 kDa). Conclusions: Reduction in the number of argentophilic proteins appears to be involved in a series of changes in the cyto-architecture of developing spermatids. Tentative cytoskeletal nature of argentophilic proteins remains to be identified. Nevertheless, they may have important physical relations with the higher-order organization of the sperm head cytoskeleton and overlying membranes. © 1994 Wiley-Liss, Inc.     

ILA 147 immunoreactivity of the bull spermatozoa membrane during epididymal maturation

A cytochemical quantitative study was carried out to detect immunostaining of bull spermatozoa during epididymal maturation using an ILA 147 monoclonal antibody and a standard immunoperoxidase method. This antibody recognizes a bovine panleukocyte determinant. Microdensitometric measurements were made on spermatozoa collected from different sites of the male genital tract (caput, corpus and cauda epididymidis and ductus deferens). On the basis of ILA 147 staining, at least 5 subpopulations of sperm cells from each site of the genital tract were found. These subpopulations showed: 1) immunostaining in the acrosome domain and in the cytoplasmic droplet in a proximal position; 2) immunostaining in the acrosome domain and in the distal cytoplasmic droplet; 3) immunostaining in the acrosomal region and lack of a cytoplasmic droplet; 4) immunostaining only in the cytoplasmic droplet; 5) lack of a cytoplasmic droplet and absence of staining of the head. The possible relationship between the presence of ILA 147 on the sperm head and the maturation process was evaluated, and we suggest that the most significant changes in ILA 147 expression occur in the corpus epididymidis. The absence of immunocytochemical staining in some spermatozoa may be related to plasma membrane damage due to spontaneous peroxidative damage of lipids.     

Localization of actin in human, bull, rabbit, and hamster sperm by immunoelectron microscopy

Mammalian spermatozoa have previously been shown to contain actin, but its subcellular localization and function have not been elucidated. In this study, actin has been localized at the ultrastructural level in human, bull, rabbit, and golden hamster spermatozoa by a monoclonal antiactin antibody and a preembedding immunogold labeling technique. Specific labeling was localized 1) around the connecting piece in the neck region of sperm from all four species, although a species-specific pattern was evident; 2) on the external surface of the fibrous sheath of human sperm; 3) in the perinuclear space underlying the postacrosomal sheath of bull and rabbit sperm; and 4) between the plasma membrane and outer acrosomal membrane along the concave margin of the hamster sperm head. SDS-PAGE and Western blots immunostained with the monoclonal antibody confirmed the presence of actin in SDS extracts of Percoll-purified sperm from each species.     

Ultrastructural nuclear defects and increased chromosome aneuploidies in spermatozoa with elongated heads

BACKGROUND: Cellular and molecular mechanisms leading to elongated sperm heads are not known. We have analysed the nuclear status of spermatozoa with elongated heads. METHODS: Fourteen men with at least 30% of spermatozoa with an elongated nucleus were studied and compared with five fertile men as controls. Sperm morphology was analysed by a quantitative ultrastructural analysis. Sperm chromosomal content was assessed by three-colour fluorescence in-situ hybridization (chromosomes X, Y, 18). Y chromosome microdeletion and karyotype were analysed. RESULTS: Elongated sperm head rates of the patients were 46.9% (30-75 versus 0-2% in the control group) by light microscopy and 34.4% by electron microscopy. In all patients, the chromatin was poorly condensed in elongated sperm heads (50% of elongated nuclei). No anomalies of sperm biochemical markers were found. All the men showed normal karyotype (46,XY) and absence of Y chromosome microdeletion. Aneuploidy rates of gonosomes and chromosome 18 were significantly increased in patients (1.64- and 3.6-fold, P = 0.006 and 0.026, respectively). CONCLUSIONS: This study demonstrates that impaired chromatin compaction and slightly increased chromosome aneuploidies are found in spermatozoa with an elongated head, suggesting possible mechanisms such as meiotic non-disjunctions or spermiogenesis anomalies.     

Phagocytosis of spermatozoa by the rabbit vagina

Twenty-four hours after mating in the rabbit, numerous spermatozoa are observed attached to the surgace of the vaginal epithelium. Spermatozoa appear to be attached by their heads to microvilli of the simple columnar cells which compose the vaginal epithelium. Spermatozoa are taken up by the epithelial cells, and they are found within the cells in various stages of degeneration up to seven days after mating. Epithelial cells frequently appear to be filled with numerous vacuoles containing sperm components in various stages of degeneration. It is suggested that some property of the surface of sperm heads may render them particularly susceptible to phagocytosis. Phagocytosis of spermatozoa may not have long-term harmful effects for the epithelium since these epithelial cells presumably have a high rate of turnover.     

Fas ligand in bull ejaculated spermatozoa:A quantitative immunocytochemical study

Apoptosis, or programmed cell death, provides a way to remove redundant cells at the end of their lifespan and thus acts as a homeostatic mechanism, maintaining the correct number of cells in the body by balancing their production and death. In the testis, this process seemed to play a pivotal role in spermatogenesis. It is generally accepted that Sertoli cells control the germ cell population through one of the best-known apoptotic pathways, the Fas/Fas L paracrine signal transduction system, in which a Fas ligand (Fas L) expressed by Sertoli cells induces apoptosis when it binds with its receptor, Fas, expressed by the germ cells. Recently, we demonstrated the presence of Fas antigen in normal ejaculated spermatozoa from fertile bulls and suggested that this molecule might have a non-apoptotic, defensive role against injuries, especially oxidative stress. We have now investigated whether bull mature, fertile spermatozoa express not only the Fas receptor but also its natural ligand Fas L. Our results indicate that the whole sperm population expresses Fas L. We suggest that Fas L in bull spermatozoa, like in murine spermatozoa, might be able to kill activated lymphocytes and protect the male gamete from damage by the self-immune system or the cytotoxic activity of leukocytes in the female genital tract.     

Regional differences in the responsiveness of isolated arteries from cattle,dog and man

Changes in tension of spiral strips from basilar, external carotid, temporal and muscle arteries from cattle, dog and man were monitored isometrically. Dose-response curves for 5-HT, noradrenaline and ergotamine indicated a variation in responsiveness between different arteries of the same species as well as between the same arteries of different species. When tested against 5-HT ergotamine consistently acted as non-competitive dualist but it antagonized responses to noradrenaline only in peripheral arteries from dog and man. In all arteries from the carotid vasculature and in bovine muscle arteries the vasoconstrictor effects of ergotamine and noradrenaline were independent additive. It is suggested that the therapeutic value of ergotamine in the treatment of migraine headache is due to its selective vasoconstrictor activity in the external carotid vasculature mediated through independent additive vasoconstrictor effects of ergotamine and noradrenaline.Dedicated to K. Bucher on the occasion of his 65th birthday.     

Sex determination and gonadal differentiation in man

Sex determination in man and other mammals can be accounted for in terms of three postulates. First, that unless specifically inhibited, the differentiation of the primary gonads goes in the male direction. Second, that the specific inhibiting factor is produced by a structural gene probably located on the X chromosome; this gene need be expressed only in cells of the urogenital ridge. Third, this gene is "switched off" (rendered inactive) by a repressor substance produced by a structural gene located on the Y chromosome.     

Cellular distribution and molecular heterogeneity of MAC393 antigen (clusterin, beta-chain) on the surface membrane of bull spermatozoa

The distribution and size of a surface membrane antigen identified by a monoclonal antibody (MAC9393) have been examined in testicular and epididymal bovine sperm preparations. Western blots indicated a substantial decrease in molecular mass of the antigen during epididymal maturation from approximately 87 kDa in the testis to approximately 35 kDa in the cauda epididymidis. This was accompanied by a change in its cellular localization from the neck and whole head to the acrosomal region. N-terminal microsequencing identified MAC393 antigen as the beta-chain of clusterin. A polyclonal antiserum to the alpha-chain of clusterin recognized both testicular and epididymal forms and revealed that the heterodimer was present on the sperm tail as well as the acrosome. These findings are explained by the co-existence of dimeric and monomeric pools of clusterin on spermatozoa. The polyclonal antiserum recognizes both testicular and epididymal forms of the heterodimer and although the monoclonal antibody binds to the testicular heterodimer, it only recognizes the beta-chain monomer of epididymal clusterin. These findings support previous observations made on human spermatozoa that two forms of clusterin, the beta-chain monomer and the heterodimer, are present on the surface membrane and in seminal plasma.