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Query: UNIPROT:P06889 (Mol)
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The epididymal tubule is a dynamic structure, in which spermatozoa undergo distinct physiological and morphological changes. The epithelial cells lining the ductuli vary dramatically in their histochemical and cytological properties according to the region of the tubule in which they are located. Additionally, regional variation is observed regarding the biosynthetic, secretory, and absorptive properties of the epithelial cells. Using in situ histochemical analysis, we document here the region-specific expression of a variety of genes that are transcriptionally active in the adult rat epididymis. Radiolabeled antisense riboprobes were used to localize, within the efferent duct/caput epididymis, transcripts encoding protein B/C, protein D/E (acidic epididymal glycoprotein), sulphated glycoprotein 1, sulphated glycoprotein 2, cellular retinol-binding protein, and the neuroendocrine peptide precursor proenkephalin. Each species of mRNA exhibits a unique pattern of hybridization, revealing that gene transcription within the efferent duct/caput epididymis is also highly region specific. This observation may partially elucidate the molecular basis underlying the phenomenon of regional alterations in the composition of protein factors within the tubule lumen.
Mol Reprod Dev 1991 Sep
PMID:In situ histochemical analysis of region-specific gene expression in the adult rat epididymis. 178 83

In this study, human oocytes obtained after ovarian hyperstimulation for in vitro fertilization (IVF) and gamete intrafallopian transfer (GIFT) were utilized to evaluate sperm/zona pellucida binding potential. Three groups of oocytes were evaluated: 1) uninseminated; 2) inseminated-unfertilized; and 3) fertilized-uncleaved. All oocytes had undergone germinal vesicle breakdown at the time of retrieval and were salt-stored (pH 7.2) for not more than 30 days. Sperm binding was recorded under hemizona assay (HZA) conditions using spermatozoa from eight fertile men (HZA control) and from 1) four teratozoospermic (HZA test) and 2) four normozoospermic (HZA test) infertile men. First, the mean numbers (+/- SD) of sperm tightly bound for fertile controls and teratospermic men to hemizonae from uninseminated oocytes were 69.7 +/- 16 and 14.5 +/- 7, respectively (P = 0.02). Likewise, hemizonae from uninseminated oocytes bound 102.0 +/- 19 and 114.0 +/- 28, respectively, for fertile controls and normospermic men (P = 0.5). Second, hemizonae obtained from inseminated-unfertilized IVF oocytes bound 44.2 +/- 12 and 19.7 +/- 6 for fertile controls and teratospermic men, respectively (P = 0.02). This category of oocytes bound 100.5 +/- 7 and 108.5 +/- 11 sperm, respectively, for fertile controls and normospermic semen (P = 0.3). Third, HZA results of fertilized but uncleaved oocytes showed a mean number of tightly bound sperm of 6.0 +/- 4 compared with 65.0 +/- 1 in control, uninseminated oocytes using fertile sperm. These results demonstrate that uninseminated and inseminated-unfertilized human oocytes, salt-stored under controlled pH conditions, give reliable information regarding sperm binding potential under HZA conditions.
Mol Reprod Dev 1991 Sep
PMID:Comparison of sperm binding potential of uninseminated, inseminated-unfertilized, and fertilized-noncleaved human oocytes under hemizona assay conditions. 178 88

Clusterin (sulfated glycoprotein-2) is a heterodimeric glycoprotein synthesized and secreted by rat Sertoli cells. An antigenically similar form is synthesized and secreted by the epididymis. The goal of this study was to define the epididymal regions in which clusterin is present and the regions in which clusterin is secreted and interacts with developing spermatozoa. Seminiferous tubule (STF), caput, corpus, and cauda fluids were collected by micropuncture and/or microperfusion and two-dimensional Western blot analysis was performed with a polyclonal antibody directed against Sertoli cell clusterin. Clusterin was found in both STF and epididymal fluid. STF contained predominantly the clusterin heavy chain (45 kd); however, a 70 Kd heterodimer was present under nonreducing conditions. Two subunits of clusterin with lower molecular weights (41 kd, heavy chain; 32 kd, light chain) and higher isoelectric points were present in the luminal fluid of all epididymal regions. The intraluminal levels of the heavy and light chains decreased from caput to cauda. Analysis by two-dimensional gel electrophoresis of proteins secreted directly into the epididymal luminal fluid revealed that clusterin was secreted by caput epithelium and not by the corpus and cauda epithelium. Western blots of membrane extracts from testicular, caput, and cauda spermatozoa revealed that testicular clusterin was associated with testicular sperm and epididymal clusterin with predominantly caput sperm. Our findings suggest that clusterin is secreted into the caput epididymal lumen, where it binds to sperm and then dissociates from sperm to be endocytosed by cells of the distal epididymal epithelium.
Mol Reprod Dev 1991 Sep
PMID:In vivo secretion and association of clusterin (SGP-2) in luminal fluid with spermatozoa in the rat testis and epididymis. 178 89

Flow cytometry is a potential method for the separation of X and Y bearing spermatozoa, on the basis of their relative DNA content evaluated by the fluorescence emission intensity due to specific fluorochrome DNA staining. However, spermatozoa DNA is highly condensed and nuclei exhibit flat non spherical shape, which can produce artefacts impeding accurate analysis. In order to avoid these limitations, decondensation of DNA performed by enzymatic treatment and a modification of the flow cytometer that orients the spermatozoa relative to the laser beam are generally used. In this work, we describe alternative methods and materials for selection of 1) decondensed and thus dead spermatozoa without orientation, sorted on the basis of only the 10% spermatozoa containing the least DNA (expected Y) and the 10% spermatozoa containing the more DNA (expected X), or 2) native spermatozoa homogeneously oriented using a simultaneous measurement of Axial light loss (extinction) and Forward angle light scatter. For testing enrichment of each selected fraction we have worked out a molecular hybridization procedure using X and Y specific DNA probes. We analyse and sort bull spermatozoa on these basis: the purity obtained for these fractions is 80% without orientation after enzymatic treatment, and 70% on live spermatozoa "optically" oriented.
Mol Reprod Dev 1991 Nov
PMID:Improvement of flow cytometry analysis and sorting of bull spermatozoa by optical monitoring of cell orientation as evaluated by DNA specific probing. 179 4

Within the seminiferous tubules, the Sertoli cells create an impermeable blood-testis barrier and an unique intratubular microenvironment that fosters the development of spermatozoa. The functional differentiation of spermatozoa therefore requires vectorial secretion by Sertoli cells of substances that cannot cross the blood-testis barrier. We investigated the role of epidermal (EGF) and insulin-like growth factors I and II (IGF-I and IGF-II) in the regulation of vectorial secretion of transferrin by Sertoli cells. In order to study the regulation of vectorial transferrin secretion, we modified culture conditions in the twin chamber culture system to maximise gradients of transferrin secretion. Sertoli cells were plated at high density (3-4 x 10(6) cells/well) into chambers of near equal volume, cultured at 37 degrees C and maintained in simple, fully defined media omitting standard supplements (insulin, EGF, FSH) which affect vectorial transferrin secretion. Using this optimised culture system, maximum gradients of transferrin secretion occurred between days 2 and 3 of culture with preferential secretion (mean ratio 3.7 +/- 0.2) directed towards the apical compartment. The transferrin ratio (ratio of transferrin secreted into the upper over the lower chamber) was decreased by insulin and FSH but not by retinoic acid or testosterone, yet all four stimuli increased total transferrin secretion. IGF-I and IGF-II were effective at physiological concentrations (ED50 = 1 ng/ml) in lowering transferrin ratio and were 100-fold more potent than insulin suggesting that insulin effects on vectorial transferrin secretion by Sertoli cells is mediated through type 1 IGF receptors. EGF also reduced the transferrin ratio (ED50 = 50 ng/ml) as well as stimulating total transferrin secretion. The hormonally mediated reduction in transferrin ratio was consistently due to enhanced secretion of transferrin into the lower chamber. In the first demonstration of a highly polarised response of Sertoli cells to hormonal stimuli, the effects of insulin, FSH and EGF on vectorial transferrin secretion were effected primarily via the basal membrane of the Sertoli cell and operated independent of mechanisms controlling total transferrin secretion. These results establish a potential role for epidermal and insulin-like growth factors in the paracrine regulation of vectorial secretion by the Sertoli cell, in particular the developmental regulation of vectorial transferrin secretion by Sertoli cells. These findings also indicate that previous studies which included insulin and EGF routinely in culture media have systematically underestimated apically directed transferrin secretion.
Mol Cell Endocrinol 1991 Oct
PMID:Effect of epidermal and insulin-like growth factors on vectorial secretion of transferrin by rat Sertoli cells in vitro. 179 90

The goat spermatozoa membranes isolated after treatment with octa (ethylene glycol) mono n-dodecyl ether (C12E8) followed by discontinuous sucrose density gradient centrifugation have been found to contain an ATPase that is stimulated by externally added Ca2+ only. The membrane fraction has also found to contain Mg(2+)-dependent Ca(2+)-ATPase activity, however the former activity is about 2 fold higher than the latter. The molecular weight of the enzyme is found to be about 97,000 on SDS-polyacrylamide gel. The optimum concentration of Ca2+ required for maximum activity is 3 mM for both Mg(2+)-dependent and Mg(2+)-independent Ca(2+)-ATPase. Histidine and imidazole buffers are found to be the most suitable for dependent and independent enzyme activities respectively. ATP with an optimum concentration of 4 mM is observed to be the best substrate than any other nucleotides. The inhibitors like trifluoperazine and vanadate and group specific probes e.g. DTNB and TNBS inhibit these two enzymes but at different rates. Ca(2+)-uptake study shows that the uptake in the presence of Ca2+ and ATP is higher than in the presence of Mg2+, Ca2+ and ATP. The findings lead us to believe that the Mg(2+)-independent Ca(2+)-ATPase has some role in Ca2+ transport like Mg(2+)-dependent enzyme.
Mol Cell Biochem 1991 May 15
PMID:Biochemical characterization of a calcium ion stimulated-ATPase from goat spermatozoa. 183 Jan 26

The aim of this work was to study the role of different parameters involved in the motility of human spermatozoa. Human spermatozoa were totally demembranated with 0.05% Triton X-100, and the demembranation was checked using electron microscopy. We have shown that, with a concentration of ATP-Mg lower than 2 mM, a pH effect was observed with a dose-dependent motility reactivation at pH 7.1, with 14% +/- 2.0% motile cells at 1 mM ATP-Mg and a straight line velocity (VSL) of 12.0 +/- 1.4 microns/sec. However, at pH 7.8, more than 65% of the spermatozoa were reactivated with as low as 0.02 mM ATP-Mg and 77.8% +/- 2.5% of them were motile at 1 mM ATP-Mg and had a VSL of 23.4 +/- 3.9 microns/sec. The depletion of free calcium by the addition of 0.5 mM EGTA in the reactivation medium (RM) improved the percentage of motile cells and the VSL most markedly at low ATP-Mg and low pH. If no MgSO4 was added in RM, cells were not motile at pH 7.8, but 30-40% reactivated at pH 7.1. If 5 mM Ca2+ was added to the RM, up to 88% of the cells became reactivated at both pHs, but the beat frequencies were very low, suggesting different mechanisms of reactivation when Mg2+ or when Ca2+ is present in the RM.
Mol Reprod Dev 1991 Jun
PMID:Effects of pH on the reactivation of human spermatozoa demembranated with Triton X-100. 187 24

C1q is a component of the classical complement pathway that can react with the Fc-fragment of immunoglobulins and with other proteins, such as fibronectin, laminin, and a specific C1q receptor present on several cell types. Given its role in many adhesion systems, mainly related to phagocytosis, we tested the effects of C1q on the interaction between human spermatozoa and zona-free hamster eggs. The presence of C1q in the medium used for gamete coincubation resulted in promotion of sperm-oolemma adhesion and an inhibition of penetration. The number of adherent sperm per egg at 5 micrograms/ml concentration was 90 +/- 35 vs. 29 +/- 7 for the control (P less than 0.001). At 1 microgram/ml, the lower concentration at which C1q had an effect, the number of penetrating sperm/egg was 0.6 vs. 1.7 for the control without C1q (P less than 0.01), and the percent of penetrated eggs was 28% vs. 85%. At 50 micrograms/ml, the percent of penetrated eggs was 7%, with a penetration index of 0.07. The addition of C1q to the medium resulted in sperm agglutination, which varied between sperm donors. The presence of C1q receptors, as detected by anti-C1qR monoclonal antibodies (Mabs), was demonstrated both on zona-free hamster eggs by immunobead rosetting and on human spermatozoa by immunobead binding and indirect immunofluorescence. Mabs directed against different epitopes of C1qR had different effects on gamete interaction, with a partial inhibition of penetration mediated by some of them. The binding of C1q to antibody-free human spermatozoa was also demonstrated both by means of indirect immunofluorescence and utilizing 125I-C1q.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Reprod Dev 1991 Jun
PMID:Complement component C1q and its receptor are involved in the interaction of human sperm with zona-free hamster eggs. 187 25

The surface membrane of mammalian spermatozoa is known to undergo considerable conformational and organizational changes during epididymal maturation. However, much less is known about remodelling of intracellular membranes. In this communication we have used specific immunological markers to study the behavior of several antigens both on and within rat spermatozoa as they mature in the epididymis. Four monoclonal antibodies (McAbs) designated 5B1, 1B5, 2D6, and 1B6 were used to probe testicular and caput and cauda epididymal spermatozoa by indirect immunofluorescence and immunogold labeling techniques. None of the McAbs bound to testicular spermatozoa; in all cases, they became reactive only on spermatozoa which had reached the caput epididymis. McAb 5B1 was restricted to the outer acrosomal membrane (OAM) of the acrosomal cap domain. The epitope first appeared on antigen(s) with molecular mass (Mr) of approximately 200 kDa in immature spermatozoa, but later in mature spermatozoa the antigen(s) had Mr of approximately 160 kDa. The antigen(s) recognized by 1B5 McAb on the other hand was initially distributed over the OAM of the entire acrosomal domain (cap + equatorial segment), but during maturation it became progressively more restricted in area until in cauda spermatozoa only the anterior tip of the OAM bound the McAb. McAb 2D6 also bound to the entire OAM and acrosomal contents of caput spermatozoa, but, unlike 5B1 and 1B5 McAbs, reactivity was transient. That is, staining was first detected in caput spermatozoa but then disappeared in corpus and cauda spermatozoa. In contrast to all of the above, 1B6 McAb bound to the surface membrane overlying the entire head domain of caput spermatozoa, but during maturation it became restricted to the postacrosomal domain. These results indicate that, in addition to remodeling of the surface membrane during epididymal maturation, extensive processing of intracellular membrane antigens also takes place and that it is very active within the acrosome. The nature of these intracellular processing events remains to be elucidated, but they may have important consequences for membrane fusion and cell recognition phenomena during fertilization.
Mol Reprod Dev 1991 Aug
PMID:Alterations in distribution of surface and intracellular antigens during epididymal maturation of rat spermatozoa. 188 15

Because the exceptionally high incidence of polyspermic fertilisation has been emphasised as a major defect in systems of in vitro fertilisation in pigs, the aetiology of the condition has been analysed in a series of experiments in vivo in the search for a common underlying cause and possible means of mitigation. Whereas the defense mechanism against polyspermy in pig oocytes is classically viewed as zona reaction, more recent evidence suggests a secondary block at the vitelline surface. Both blocks may be compromised in situations leading to polyspermy, although deleterious influences seem to be expressed principally in an inadequate zona block, as judged by the presence of perivitelline spermatozoa. Postovulatory aging of mammalian oocytes prior to sperm penetration leads to polyspermy, as can be demonstrated in pig eggs. The primary lesion may concern the cortical reaction, owing to a delayed and incomplete exocytosis of the vesicular contents. Eggs ovulated after gonadotrophin treatment during the luteal phase of the cycle show a high incidence of polyspermic penetration (60.6%), as do those shed at estrus in animals treated with progesterone systemically (40%) or by local microinjections in the oviduct wall (32.3%). Whereas progesterone may be modifying interactions of the gametes and responses of the egg organelles in all four above experimental situations, enhanced numbers of spermatozoa ascending a more patent isthmus appear to be the principal cause of polyspermy.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Reprod Dev 1991 Aug
PMID:Oviduct function in pigs, with particular reference to the pathological condition of polyspermy. 188 18


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