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

An adaptation of the Feulgen procedure to visualise DNA at the ultrastructural level, using osmium ammine instead of Schiff reagent, was applied to ultrathin sections of rabbit epididymal spermatozoa, known to display increasing chromatin compaction as they progress through the epididymis. In contrast with the somatic cell nuclei of the epididymal epithelium, which display classical staining, the chromatin of spermatozoa is partly or fully destroyed during the hydrolysis step of the technique. The sperm chromatin resistance towards destruction is a function of the initial sperm nuclear compaction and of the duration of hydrolysis prior to Feulgen-like staining. For a given nucleus, the maximum staining intensity is only obtained after an optimal duration of hydrolysis. However, because of this duration and local differences in chromatin compaction, the total DNA of a section is never completely visualized. The most compact parts of the nuclei are not yet stained, when the less compact parts have already been destroyed. This gives rise to a sperm-specific pattern which corresponds to the enhancement of microheterogeneities present in all sperm nuclei and to the local depolymerisation of the nuclear material during hydrolysis. The depolymerised parts of nuclei sit over the sections, and they also bind uranyl acetate, ethidium bromide and anti-protamine antibodies. Therefore, in sperm nuclei, the Feulgen-like staining at the ultrastructural level does not reveal the true DNA distribution. However, the amount of staining can be quantified to evaluate sperm chromatin compaction.
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PMID:Distribution of DNA, nuclear micro-heterogeneities and compaction of the chromatin in rabbit epididymal spermatozoa. Ultrastructural evaluation of the Feulgen-like technique using osmium ammine. 751 30

Single rat epididymal cell studied under whole cell patch-clamp condition responded to 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP) (500 microM) and to ionomycin (1 microM) by an increase in whole cell conductance. A major part of the stimulated current was carried by Cl-, although a small part was due to nonselective cation current. After elimination of the cation current component by using impermeant cation, the cells revealed different Cl- conductance properties in response to adenosine 3',5'-cyclic monophosphate (cAMP) and ionomycin. The cAMP-stimulated Cl- conductance was independent of time and voltage and showed a linear current-voltage relationship. The anion permselectivity was NO3- > Br- > Cl- approximately I- >> SO(4)2-. The ionomycin-stimulated Cl- conductance showed marked time and voltage dependency. In contrast to the cAMP-induced anion permselectivity, the ionomycin-induced anion permselectivity was I- > Br- approximately NO3- > Cl- >> SO(4)2-. These results indicate that the epididymal epithelial cells exhibit different Cl- conductances sensitive to cAMP and Ca2+. The cAMP-activated conductance has properties resembling the type associated with the cystic fibrosis transmembrane conductance regulator found in cystic fibrosis-affected epithelia. This finding supports the notion that the epididymis is a cystic fibrosis epithelium.
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PMID:Properties of cAMP-dependent and Ca(2+)-dependent whole cell Cl- conductances in rat epididymal cells. 768 72

Swelling-induced Cl- conductance in cultured rat epididymal cells was characterized using whole cell patch-clamp techniques. Activation of whole cell current with an outwardly rectifying current-potential relationship was observed in cells exposed to hyposmotic solutions. This current was determined, from the observed current-reversal potentials at different Cl- concentrations, to be Cl- selective. The anion selectivity sequence of the swelling-induced Cl- conductance was I- approximately NO3- approximately Br- > Cl- > 2-(N-morpholino)ethanesulfonic acid. The swelling-induced Cl- conductance was reversibly inhibited by different Cl- channel blockers. Unlike diphenylamine-2-carboxylate or 5-nitro-2-(3-phenylpropylamino)-benzoate, which showed voltage-independent blockade, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid showed a marked voltage-dependent blockade of the volume-sensitive Cl- current, with a greater effect at depolarizing voltages. The swelling-induced Cl- conductance appeared to be different from the Ca(2+)- or adenosine 3',5'-cyclic monophosphate-activated Cl- conductances on the basis of the following observations: 1) swelling-induced current activation was seen even in the presence of kinase inhibitor (H-8) or absence of external free Ca2+, and 2) further increase in current activation could be produced by swelling after Ca(2+)- or adenosine 3',5'-cyclic monophosphate-induced current activation. The swelling-induced Cl- conductance may be involved in regulating epithelial cell volume as well as serving other important epididymal functions such as facilitating transepithelial secretion of organic compounds.
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PMID:Characterization of a swelling-induced chloride conductance in cultured rat epididymal cells. 769 76

A trypsin-like protease was extracted with 1% cetyltrimethylammonium bromide (CTAB) at pH 7.0 from boar cauda epididymal sperm nuclei whose acrosin had previously been removed by acid extraction. The CTAB-extracted sperm protease (CSP) was purified by ion-exchange chromatography on CM-23, gel filtration on Sephadex G-100, affinity chromatography on benzamidine-CH-Sepharose 4B, and HPLC on CM-5PW. CSP is a two chain protein composed of M(r) 2.6K and M(r) 37K chains, which are covalently cross-linked by disulfide bonds. CSP exhibited a pH optimum between pH 8.0 and 9.0, and was inhibited by diisopropyl phosphorofluoridate, antipain, leupeptin, and 1-chloro-3-tosylamide-7-amino-L-2-heptanone. The activity of CSP was enhanced about 1.2-fold with 50 mM CaCl2, with which acrosin is enhanced 2.0-fold. The catalytic efficiency (kcat/Km) of CSP toward Bz-L-Arg-OEt, Tos-L-Arg-OMe, and Tos-L-Lys-OMe in the presence of 50 mM CaCl2 differed from that of acrosin by factors of 0.53, 1.2, and 0.80, respectively. Amino acid sequencing of V8-digested peptides of CSP, and its L- and H-chains showed that the amino acid sequence of CSP was closely related to, but different from, that of acrosin. These results suggest that CSP is a novel acrosin-like enzyme that differs from acrosin in its location in the sperm head, the effect of calcium ions on its activity, and its substrate specificity.
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PMID:Purification and characterization of a novel acrosin-like enzyme from boar cauda epididymal sperm. 782 68

A fraction from bovine seminal vesicle fluid that initiated acrosome reaction of bovine epididymal spermatozoa in vitro in the presence of heparin was prepared by sequential chromatographies on heparin-Sepharose, gel filtration (Superose 12) and reversed phase chromatography (ProRPC). Sequence analysis of the separated fraction showed that it contained the major protein (PDC-109) with 100% homology. This fraction contained also Ca(2+)-dependent phospholipase A2-like activity which hydrolysed phosphatidylethanolamine and phosphatidylcholine with 14C-labelled linoleic (lino-PE, lino-PC) or arachidonic acid (ara-PE, ara-PC) at sn-2 position. This protein was not detected in N-terminal sequence analysis. Lysophosphatidylcholine, lysophosphatidylethanolamine, and p-bromophenacyl bromide (p-BPB) inhibited this lipolytic activity. Sulfoglycolipid (Seminolipid) had inhibitory effect at concentrations above 0.1 mM but activated slightly the enzyme at lower concentrations. Boiling destroyed acrosome initiating activity in the separated fraction.
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PMID:Partial characterization of a fraction from bull seminal vesicle fluid that potentiates the bull sperm acrosome reaction in vitro. 804 72

Phospholipase A2 and lysophospholipids have been implicated in the mammalian sperm acrosome reaction. In this study we further investigated the role of this enzyme and lysophospholipids on the acrosome reaction of hamster spermatozoa. Hamster epididymal spermatozoa were incubated under capacitation and acrosome reaction-inducing conditions. After 3.0 and 3.5 h, the spermatozoa were treated with different doses of lysophosphatidylcholine for 12 min. Then the percentage of motility, hyperactivation, and acrosome reaction was evaluated by light microscopy. Lysophosphatidylcholine, 10 micrograms/ml, was the highest acrosome reaction-inducing dose without an effect on sperm motility. Lysophosphatidylcholine induced the acrosome reaction only when added to spermatozoa capacitated for a minimum of 2 h. This effect was apparent after 1 min of its addition and reached a plateau after 5 min. Lysophosphatidylethanolamine and lysophosphatidylinositol were also effective in inducing the acrosome reaction. Lysophosphatidylserine did not have any effect on the reaction, but caused an increase in sperm hyperactivation. Sperm treated with the phospholipase A2 inhibitors quinacrine dihydrochloride and p-bromophenacyl-bromide showed an inhibition of the spontaneous occurrence of the acrosome reaction. These inhibitors, however, did not block the acrosome reaction induced by lysophosphatidylcholine. The time course of the lysophosphatidylcholine-induced acrosome reaction was the same whether control or inhibitor treated spermatozoa were used. These results suggest that the membrane events of the acrosome reaction initiate with the activation of the phospholipase A2, thus producing the fusogen agents necessary for this exocytotic event.
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PMID:Studies of lysophospholipids related to the hamster sperm acrosome reaction in vitro. 840 1

The extent to which chromatin of rat caput (CAP), corpus (COR), cauda (CAU) spermatozoa undergo condensation and compaction is known to be a function of progressive increase in the formation of inter- as well as intra-protamine disulphide bridges during their transit through the epididymis. Relative compaction undergone by the nuclear chromatin of these sperm populations was studied by monitoring their susceptibility to in vitro decondensation induced by varying concentrations (0, 0.01, 1, 5, 10, 50 mM) of disulphide reducing agent, dithiothreitol (DTT) after an initial exposure to 0.01% papain. Following this treatment and staining with the nucleic acid specific fluorochrome, ethidium bromide (EB), it was observed that irrespective of the epididymal region from which they were collected, spermatozoa exhibited DTT dose-dependent (a) increase in nuclear size as seen under fluorescence microscopic examination, (b) decrease in flow cytometrically quantifiable light scatter parameters--forward scatter (FSc, 'nuclear size') and side scatter (SSc, nuclear 'granularity'), (c) increase in individual cell EB binding when analyzed by DNA flow cytometry, and (d) increase in thiol specific 14C-iodoacetamide (14C-IA) uptake. The decrease in both FSc and SSc occurring in spite of actual increase in nuclear size has been attributed to increase in translucency of spermatozoan nuclei consequent to decondensation. The FSc, SSc and EB bindability were studied by monitoring both the channels of maximal cell concentration detected in the flow cytograms as well as by digitally quantitating the numbers of cells within specific channels (1-64, 65-128, 129-192 and 193-256) of the flow cytogram. The latter indicated a measure of the variability in the response of populations of sperm within each sample to DTT induced decondensation. At any given concentration of DTT, especially between 5-10 mM, the differences observed between sperms of different regions were consistent and significant (P < 0.01-P < 0.001), maximal changes being shown by CAP and minimal by CAU sperm, COR sperm appearing in between. The effective concentration of DTT required to elicit 50% of maximal (i.e. that exhibited by CAP sperm when taken as 100%) effect (ED50) varied significantly among CAP, COR and CAU sperms for each of the parameters studied (P < 0.01-P < 0.001). It is concluded that the differences observed among the three epididymal sperm populations are due to differences in the extent of susceptibility to decondensation in vitro and that this is dependent upon the variation in the -S-S-content of their chromatin during different stages of epididymal transit. All the parameters used (with the exception of fluorescence microscopy) can be quantified and as all of them show a similar dose dependency to DTT treatment, any one of these parameters can be conveniently used to determine the mature/immature status of the sperms voided. Application of such a method to determine the quality of sperms voided by man appears feasible.
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PMID:Rat epididymal sperm exhibit on dithiothreitol treatment in vitro quantifiable differences in patterns of light scatter, uptake of 14C-iodoacetamide and binding of ethidium bromide to DNA. 871 72

The outer membranes of mitochondria of mammalian sperm are encased in a keratinous structure known as the mitochondrial capsule. The experiments in the present study were designed to resolve a controversy surrounding the intracellular localization, developmental expression, and selenium-content of a cysteine-rich 17-20 kD protein that has been reported to constitute the major structural protein in the mitochondrial capsule of mammals. An antibody to a synthetic oligopeptide based on the predicted sequence of mouse cysteinerich protein recognizes a 24 kD protein in epididymal sperm tails of mice. The 24 kD protein does not appear to be a selenoprotein because: (1) it is not labeled with 75Se-selenite in seminiferous tubule culture; (2) cleavage with cyanogen bromide and translation of T7 RNA polymerase transcripts in vitro indicate that the translation start site is located downstream of potential UGA selenocysteine codons in the mouse cysteine-rich mRNA; (3) the reading frame encoding the cysteine-rich protein in rat lacks inphase UGA selenocysteine codons. Light and electron microscopy immunocytochemistry detects the cysteine-rich protein first during step 11 of spermiogenesis in the mouse demonstrating that the cysteine-rich protein mRNA is under temporal translational control. Electron microscope immunocytochemistry reveals that the cysteine-rich protein is evenly distributed in the cytoplasm in spermatids in steps 11 through early step 16 in mouse, and that it is associated with the outer mitochondrial membranes of spermatids in late step 16 and epididymal spermatozoa.
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PMID:Developmental expression, intracellular localization, and selenium content of the cysteine-rich protein associated with the mitochondrial capsules of mouse sperm. 891 43

Immunoneutralization of endogenous follicle-stimulating hormone (FSH) of adult male monkeys leads to oligospermia and infertility despite unchanged testosterone levels. The inability of these monkeys to impregnate despite repeated exposures to cycling females appeared to be due to abnormal alterations in the kinetics of germ cell transformations and deficient spermiogenesis. Here we investigated the stability of sperm chromatin in oFSH-immunized monkeys as a marker for spermiogenesis. The susceptibility of spermatozoa to in vitro decondensation induced by dithiothreitol (DTT, 0.05-50 mM) was studied by measuring the nuclear fluorescence of DTT-treated, ethidium bromide (EB)-stained sperm using flow cytometry. Changes in sperm morphology and binding of thiol-specific 14C-iodoacetamide (14C-IA) were also monitored under the same conditions. Sperm from the immunized monkeys decondensed at a lower concentration of DTT, bound more EB, and decondensed more extensively than those from control animals. The difference was apparent in sperm from all regions of the epididymis. Immunized monkey sperm also bound significantly more 14C-IA at all concentrations of DTT. Overall, the effective concentration of DTT required to elicit 50% of maximal decondensation (ED50) of epididymal and ejaculated sperm was significantly lower for the immunized monkeys than even the caput sperm of controls. These results suggest that FSH deprivation in monkeys results in production of sperm with limited potential for disulfide formation and reduced chromatin stability.
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PMID:Enhanced susceptibility of follicle-stimulating-hormone-deprived infertile bonnet monkey (Macaca radiata) spermatozoa to dithiothreitol-induced DNA decondensation in situ. 943 42

We have previously shown that the in vitro development of deer embryos differed according to the IVF conditions. The aim of the study was to use heterologous IVF with zona-free matured bovine oocytes to assess the in vitro fertility of 3 samples of deer semen (2 ejaculates from sika deer (Cervus nippon) and 1 pool of epididymal spermatozoa from red deer (Cervus elaphus)). The frozen/thawed semen samples were selected on Percoll gradient and resuspended in Tyrode modified medium supplemented with estrus sheep serum (0, 2, 20% v/v) or heparin (10 microg/mL). During 8 h of culture, the sperm motility index according to the post-insemination time (hpi) did not differ either between samples or between supplemented IVF media. In vitro matured zona-free bovine oocytes were inseminated in different IVF media with the semen samples. Penetration rates assessed at 15 hpi were optimal with 20% estrus sheep serum for sika deer ejaculates whereas 2% were sufficient to reach the maximum functionality of epididymal spermatozoa from red deer. The mean time of pronuclear formation was similar regardless of the semen sample. The precocity of the onset of the first S-phase in both pronuclei was characterized by Bromo-deoxy-Uridine exposures between 5 and 15 hpi in order to assess the developmental potential conferred by the semen sample (intrinsic value). As we previously observed in homologous IVF, this value seemed to be higher for the epididymal sperm sample.
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PMID:Assessment of in vitro fertility of deer spermatozoa by heterologous IVF with zona-free bovine oocytes. 1148 Jun 18


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