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Query: UNIPROT:P56851 (epididymal)
 11,273 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The fertilization in vitro of mouse with intact zonae pellucidae by mouse cauda epididymal spermatozoa was inhibited in a concentration- dependent fashion by 3-quinuclidinyl benzilate (QNB), normally used as a specific antagonist for the muscarinic class of cholinergic receptors. Inhibition was observed with both cumulus-intact and cumulus-free preparations. QNB at 50 microM inhibited fertilization of cumulus-free eggs by greater than 90% but had no effect on the fertilization of zona-free eggs. At this concentrations, QNB had no adverse effect on sperm motility, nor did it prevent binding of spermatozoa to the zona pellucida. The inhibitory effects of QNB were fully reversible. QNB is therefore a useful specific inhibitor of zona penetration. Spermatozoa in the in vitro fertilization medium bound QNB with a concentration dependence which matched that of the inhibition of fertilization. This binding was saturable and corresponded to 700 pmole/10(7) cells with KD = 10 microM. The in vitro fertilization medium contains 2% (w/v) bovine serum albumin (BSA) which also binds QNB according to the mass action law. The large amount of QNB bound to sperm in this medium appears to be QNB binding to BSA adsorbed on the sperm cell surface: these spermatozoa bind QNB specifically in the absence of BSA with a saturable capacity of only 70 fmole/10(7) cells with KD = 5 nM. Calculation of the distribution of QNB between BSA binding sites and sperm surface binding sites in the in vitro fertilization medium indicates that the specific sperm sites become saturated with the same concentration dependence as inhibition of fertilization. However, the dissociation rate of QNB from sperm in both the presence and absence of BSA is too rapid to permit confirmation of these sites as the locus of the inhibitory effect; this locus remains to be clarified.
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PMID:Inhibition of in vitro fertilization of mouse eggs: 3-quinuclidinyl benzilate specifically blocks penetration of zonae pellucidae by mouse spermatozoa. 728 85

The inhibitory action of cholesterol-containing suspensions on fertilizing capacity in uterine-capacitated rabbit sperm cells showed a direct dependence on the concentration of sterol. Dispersion with synthetic phosphatidylcholine as a nonsonicated suspension or as liposomes did not alter this antifertilization effect. Esterification of the sterol, however, caused a complete loss of inhibitory activity. Cholesterol inhibited induction of the acrosome reaction among epididymal rat spermatozoa incubated under chemically defined conditions. Other agents with a negative effect on the acrosome reaction were seminal plasma membrane vesicles and palmitic acid. Egg lecithin-liposomes and bovine serum albumin, especially after being delipidated, facilitated it. These results corroborate the viewpoint that changes in the lipid bilayer of sperm plasma membrane significantly influence fertilizing capacity among mammalian spermatozoa.
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PMID:Interaction of lipids with the plasma membrane of sperm cells. I. The antifertilization action of cholesterol. 743 24

In the accompanying report (Visconti, P.E., Bailey, J.L., Moore, G.D., Pan, D., Olds-Clarke, P. and Kopf, G.S. (1995) Development, 121, 1129-1137) we demonstrated that the tyrosine phosphorylation of a subset of mouse sperm proteins of M(r) 40,000-120,000 was correlated with the capacitation state of the sperm. The mechanism by which protein tyrosine phosphorylation is regulated in sperm during this process is the subject of this report. Cauda epididymal sperm, when incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin do not display the capacitation-associated increases in protein tyrosine phosphorylation of this subset of proteins. This NaHCO3, CaCl2 or bovine serum albumin requirement for protein tyrosine phosphorylation can be completely overcome by the addition of biologically active, but not inactive, cAMP analogues. Addition of the active cAMP analogues to sperm incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin overcomes the inability of these media to support capacitation, as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. The effects of the cAMP analogues to enhance protein tyrosine phosphorylation and to promote capacitation appears to be at the level of the cAMP-dependent protein kinase (PKA), since two specific inhibitors of this enzyme (H-89 and Rp-cAMPS) block the capacitation-dependent increases in protein tyrosine phosphorylation in sperm incubated in media supporting capacitation. Capacitation, as assessed by the aforementioned endpoints, also appears to be inhibited by H-89 in a concentration-dependent manner. These results provide further evidence for the interrelationship between protein tyrosine phosphorylation and the appearance of the capacitated state in mouse sperm. They also demonstrate that both protein tyrosine phosphorylation and capacitation appear to be regulated by cAMP/PKA. Up-regulation of protein tyrosine phosphorylation by cAMP/PKA in sperm is, to our knowledge, the first demonstration of such an interrelationship between tyrosine kinase/phosphatase and PKA signaling pathways.
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PMID:Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. 753 69

The molecular basis of mammalian sperm capacitation, defined functionally as those processes that confer on the sperm the acquisition of fertilization-competence either in vivo in the female reproductive tract or in vitro, is poorly understood. We demonstrate here that capacitation of caudal epididymal mouse sperm in vitro is accompanied by a time-dependent increase in the protein tyrosine phosphorylation of a subset of proteins of M(r) 40,000-120,000. Incubation of sperm in media devoid of bovine serum albumin, CaCl2 or NaHCO3, components which individually are required for capacitation, prevent the sperm from undergoing capacitation as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. In each of these cases the protein tyrosine phosphorylation of the subset of capacitation-associated proteins does not occur. Protein tyrosine phosphorylation of these particular proteins, as well as sperm capacitation, can be recovered in media devoid of each of these three constituents (bovine serum albumin, CaCl2 or NaHCO3) by adding back the appropriate component in a concentration-dependent manner. The requirement of NaHCO3 for these phosphorylations is not due to an alkalinization of intracellular sperm pH or to an increase in media pH. Caput epididymal sperm, which lack the ability to undergo capacitation in vitro, do not display this capacitation-dependent subset of tyrosine phosphorylated proteins in complete media even after extended incubation periods, and do not fertilize metaphase II-arrested eggs in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation. 774 26

The aim of the present study is to analyse the relative and combined effects of ethanol and protein deficiency on serum testosterone and LH, and on gonadal histology, in ethanol fed rats. The study was performed in 32 animals divided into four groups, fed with the Lieber & DeCarli control, 36% ethanol, 2% protein, and 36% ethanol 2% protein containing diets, respectively. Two months later, rats were anaesthetized with pentobarbital and sacrificed, and the right testes and epididymus were carefully removed. Both ethanol and protein deficiency independently lead to a decrease in serum testosterone levels, and to testicular atrophy, lowest testosterone levels and highest degrees of atrophy being observed in the rats receiving the 36% ethanol, 2% protein containing diet. Both serum testosterone and testicular size and weight significantly correlated with final weight and serum albumin. Hypospermia, atrophy of the seminiferous tubules, and reduced epididymal diameter were also observed in this last group of animals. Thus, protein deficiency may contribute to hypogonadism of alcoholics.
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PMID:Relative and combined effects of ethanol and protein deficiency on gonadal function and histology. 781 91

The fluorescent calcium indicator, fluo-3, was loaded as the membrane permeant tetraacetoxymethyl (AM) ester into cauda epididymal mouse sperm at 25 degrees C for 20 min in the absence of bovine serum albumin (BSA) and presence of the dispersant, Pluronic F-127. Excess indicator was removed by two centrifugation washes at 100g for 10 min, a procedure that did not impair sperm motility. Upon resuspension in medium containing 20 mg/ml BSA to promote capacitation, the sperm cells exhibited readily detectable fluorescence uniformly distributed in the cytoplasm. Cell fluorescence was stable over the time of the experiments and was responsive to changes in intracellular calcium concentration, [Ca2+]i. Initial [Ca2+]i was 231 +/- 58 nM (+/- SE, n = 43). Addition of heat-solubilized mouse zonae pellucidae to capacitated sperm increased [Ca2+]i by 106 +/- 19 nM (+/- SE, n = 18), the higher steady-state concentration being reached after 30 min. Subsequent addition of the non-fluorescent calcium ionophore Br-A23187 resulted in a further increase of 114 +/- 18 nM (+/- SE, n = 18), the higher steady-state concentration being reached after 6 min. The increase in [Ca2+]i induced by solubilized zonae pellucidae was largely blocked by 3-quinuclidinyl benzilate (QNB), an antagonist of muscarinic receptors that was earlier shown to block the zona pellucida induced acrosome reaction in mouse sperm (Florman and Storey, 1982: Dev Biol 91:121-130). This [Ca2+]i increase was completely blocked by the tyrosine kinase inhibitor, tyrphostin A48, and by the inactivator of G1 proteins, pertussis toxin. At the concentrations at which they blocked the zona pellucida-induced increase in [Ca2+]i, all three inhibitors also blocked the zona pellucida-induced acrosome reaction. These results indicate that [Ca2+]i increase in is an early, if not the initial, reaction in the sequence leading to zona pellucida induced acrosomal exocytosis in mouse sperm. The observation that the three inhibitors, each having a different mode of action, all block the zona pellucida induced [Ca2+]i suggests that the sperm plasma membrane receptors mediating the zona pellucida induced acrosome reaction may function as a complex, whose formation is activated by zona pellucida ligand binding.
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PMID:Calcium influx into mouse spermatozoa activated by solubilized mouse zona pellucida, monitored with the calcium fluorescent indicator, fluo-3. Inhibition of the influx by three inhibitors of the zona pellucida induced acrosome reaction: tyrphostin A48, pertussis toxin, and 3-quinuclidinyl benzilate. 788 69

Corpus epididymal and efferent duct epithelial cells on permeable supports formed confluent monolayers that resisted hydrodynamic equilibrium and created electrical resistance. Monolayers were formed sooner and were of better quality when fetal bovine serum (FBS), rather than bovine serum albumin (BSA), was present in glucose-free, rather than glucose-containing, media. Testosterone was converted to androstenedione by both cell types and conversion of both steroids to 5 alpha-reduced metabolites was higher in cells from the corpus epididymidis than from efferent ducts. Addition of heat-treated human spermatocoele fluid (similar to rete testis fluid) to the apical aspects of the cells increased cell heights when they were initially low, but some cytoplasmic damage was observed. New serum-free media (especially those designed for keratinocytes and mammary epithelial cells) could maintain cultured cells at heights found in situ.
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PMID:Epithelial monolayers from human epididymal and efferent duct tubules; testosterone metabolism and effects of culture conditions on cell height and confluence. 789 70

To establish the mode of fertilization in a marsupial, a morphological investigation was made of the gametes of the South American grey short-tailed opossum. Monodelphis domestica, at the time of fertilization in vivo and in vitro. Oestrus was induced in females by the introduction of an unfamiliar male. To obtain oocytes recently fertilized in vivo, females were killed 18-24 hours after the first mating and the region of the oviduct containing eggs excised and fixed. Unfertilized mature oocytes were recovered from ovarian follicles 15-18 hours after first mating and fertilized in vitro with cauda epididymal spermatozoa in a modified MEM medium supplemented with bovine serum albumin at 37 degrees C in 5% CO2 in air. Following sperm-egg binding and fertilization, oocytes were fixed and prepared for light and electron microscopy. Spermatozoa unpaired prior to fertilization in vivo and in vitro and single spermatozoa bound to the zona surface by their plasmalemma overlying the acrosome on the dorsal face of the sperm head. The acrosome reaction was only observed at the zona surface (suggesting that it may be induced by zona components) and involved a vesiculation of sperm plasma and acrosomal membranes over the main body of the acrosome but not over the narrow, marginal region which persisted after the acrosome reaction was complete. Sperm penetration of the zona pellucida caused a large breach in the zona and the dispersal of perivitelline material. The fusion of the spermatozoon with the oolemma occurred first over the marginal acrosomal region and was accompanied by a fertilization cone which protruded through the zona penetration hole. Activation of the egg was characterized by the release of material from vesicles in the peripheral cytoplasm and extrusion of the second polar body. The mode of fertilization in Monodelphis was compared with what is known in other marsupials (New World and Australian) and eutherian (placental) mammals. It was concluded that the general features of the acrosome reaction and sperm-egg fusion may be essentially similar in both groups and that an evolutionary schism did not occur following the development of the eutherian mode of fertilization.
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PMID:Ultrastructural characteristics of in vivo and in vitro fertilization in the grey short-tailed opossum, Monodelphis domestica. 821 40

The same low M(r) protein fraction of epididymal luminal fluid as that studied previously by Hamilton in 1981 was assayed for alpha-lactalbumin (alpha-lac) activity with bovine milk galactosyltransferase (GalTase) as the enzyme source and bovine serum albumin (BSA) to make the total protein concentrations of all the samples in each assay constant. No alpha-lac activity could be detected in this fraction. When glucose was used as an acceptor, radioactivity passing through the ion exchange column used to retain the remaining UDP-galactose did increase with the amount of the proteins of the low M(r) fraction, but this increase was observed not only for samples with an acceptor but also for samples without. The increased radioactive product co-migrated in high-voltage electrophoresis with galactose, not lactose. When GlcNAc was used as an acceptor, the situation was the same, and the increased radioactive product co-migrated with galactose, not LacNAc. No inhibition of bovine milk GalTase activity by the low M(r) proteins was observed. Addition of protein, either BSA or the low M(r) fraction of epididymal luminal fluid, to assay medium that contained no proteins other than GalTase enhanced the GalTase activity both in forming lactose in the presence of glucose and also LacNAc in the presence of GlcNAc. It appears that the earlier reports of alpha-lac-like activity in epididymal fluids and extracts may have been due to the presence of enzymes liberating free galactose from UDP-galactose and/or a stimulatory non-specific effect of the protein in the solutions on the lactose synthesis activity of the GalTase.
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PMID:No alpha-lactalbumin-like activity detected in a low molecular mass protein fraction of rat epididymal extract. 826 6

The activity of adipose tissue hormone-sensitive lipase in animals with hyperinsulinemia has been reported to be increased compared with that in control animals. We examined whether this results from a direct effect of insulin on the tissue and whether it is accompanied by alteration in the regulation of lipolysis. When rat epididymal fat pads are incubated in culture medium with bovine serum albumin for 2-4 h with 2 ng/ml or 50 microU/ml of insulin, hormone-sensitive lipase activity in the postmicrosomal supernatant fraction after acid precipitation and activation with ATP-Mg2+ increases significantly compared with preparations from tissues incubated with the vehicle. The specific activities of hormone-sensitive lipase in sonicates of adipocytes after primary culture with insulin at concentrations from 10 to 4000 ng/ml (250 microU to 100 mU/ml) increase in an insulin-dose-related manner. Lipolysis in response to 10(-7) M isoproterenol also increases in an insulin-dose-dependent manner. Enhancement of isoproterenol-mediated lipolysis is not attributable to a difference in the triglyceride content of the cells. Lipolysis caused by the beta-agonist could be completely blocked by the simultaneous presence of insulin in both control and insulin-treated cells reflecting normal responsiveness of both types of cells to the acute effect of insulin. Although an increase in lipolysis is seen with norepinephrine and growth hormone after insulin treatment, other lipolytic agents such as ACTH, thyrotropin, and glucagon evoke similar responses in insulin-treated and control cells. The simultaneous presence of growth hormone and insulin during the 16-h culture results in additive effects on the subsequent response of the cells to 10(-7) M isoproterenol compared with the responses of the cells cultured with each hormone alone. beta-Agonist-mediated cAMP accumulation in the presence of Ro-20.1724, a specific phosphodiesterase inhibitor, is significantly higher in cells cultured in the presence of insulin than in control cells. Forskolin (1-25 microM) increases the lipolytic responses of insulin-treated cells compared with control cells, but the maximal response of the insulin-treated cells to forskolin is lower than that to isoproterenol. We conclude that changes produced by chronic insulin treatment involve more than one site along the lipolytic cascade.
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PMID:Chronic exposure of rat fat cells to insulin enhances lipolysis and activation of partially purified hormone-sensitive lipase. 839 27


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