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

A study was undertaken to estimate the activities of the key enzymes of glycolysis, the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle in purified rat spermatocytes and spermatids, which have been shown to die in glucose-containing medium and require lactate/pyruvate for maintaining normal ATP concentrations. The aim was to elucidate the changes in the glycolytic and oxidative potential of germ cells undergoing meiosis. Pachytene spermatocytes and round spermatids from adult rat testis were purified to approximately 90% purity by trypsin digestion followed by a combination of centrifugal elutriation and Percoll density gradient centrifugation. After the purity and viability of these cells had been established, their contents of hexokinase, phosphofructokinase, lactate dehydrogenase (LDH) and LDH-X of glycolysis, glucose 6-phosphate dehydrogenase of the pentose phosphate pathway and citrate synthase, aconitase, malate dehydrogenase and 2-oxoglutarate dehydrogenase of the TCA cycle were estimated. These enzymes were also estimated in epididymal spermatozoa for comparison with the testicular germ cells. The results indicate greater activity of glycolytic and pentose phosphate pathway enzymes in spermatocytes than in spermatids, which exhibited greater activity of TCA cycle enzymes than the former. The difference in activity was statistically significant for most of the enzymes studied. In contrast, spermatozoa exhibited markedly greater activity of glycolytic enzymes and significantly lower activity of pentose phosphate pathway and TCA cycle enzymes than did the testicular germ cells. We conclude that the unusual dependence of spermatids exclusively on lactate may be due to their lower glycolytic potential, whereas spermatocytes with comparatively greater glycolytic activity have an intermediate dependence on lactate and are therefore able to utilise lactate, pyruvate, or both, while retaining a better ability to utilise glucose. Spermatozoa with the greatest glycolytic potential and the lowest TCA cycle activity appear to be 'programmed' to utilise exclusively glucose/fructose for energy.
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PMID:Changes in carbohydrate metabolism of testicular germ cells during meiosis in the rat. 953 8

To identify a novel candidate(s) for acrosomal proteins that act on the sperm/egg interaction, a DNA fragment was PCR-amplified from a cDNA library of acrosin-deficient mouse testis and then used as a probe to screen a mouse testis cDNA library. Complementary DNA clones encoding each of two similar but different serine proteases, TESP1 and TESP2, have been identified. The nucleotide sequences of these clones indicate that mouse TESP1 and TESP2 are initially synthesized as preproproteins of 367 and 366 amino acids, respectively. Comparison of the two TESP sequences with those of typical serine proteases suggests that each TESP zymogen is probably converted into a two-chain mature enzyme consisting of light and heavy chains covalently linked by a single pre-existing disulfide bond. The conversion may be accomplished by another protease(s) with a trypsin-like cleavage specificity, since it is unlikely that the mature TESP1 and TESP2 are capable of splitting the Lys-Ile bond between the light and heavy chains. Northern blot analysis of total cellular RNA demonstrates that the TESP1 and TESP2 genes are expressed only in the testis, and the transcripts are abundantly present in the haploid round spermatids. Moreover, immunocytochemical analysis of mouse cauda epididymal sperm using affinity-purified antibodies reveals that these two TESPs are both localized in the sperm acrosome and are released during the acrosome reaction induced by calcium ionophore A23187. These findings provide additional clues for elucidating the mechanisms involved in the sperm/egg interactions, including penetration of the zona pellucida by sperm.
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PMID:Two novel testicular serine proteases, TESP1 and TESP2, are present in the mouse sperm acrosome. 958 71

Guinea pig intestinal phospholipase B is a calcium-independent phospholipase hydrolyzing sequentially the acyl ester bonds at sn-2 and sn-1 positions of glycerophospholipids, promoting the formation of sn-glycero-3-phosphocholine from phosphatidylcholine. This 140-kDa glycoprotein from the brush border membrane of differentiated enterocytes contributes to lipid digestion as an ectoenzyme. The cDNA coding for guinea pig phospholipase B was revealed to be the homologue of AdRab-B, an mRNA appearing in rabbit upon intestine development. The sequence predicts a polypeptide of 1463 amino acids displaying four homologous repeats, two of them containing the lipase consensus sequence GXSXG. A 5-kilobase transcript was particularly abundant in mature ileal and jejunal enterocytes but was also detected in epididymis, where phospholipase B displayed a higher molecular mass (170 kDa versus 140 kDa in intestine), with no obvious evidence for enzyme activity. Trypsin treatment of phospholipase B immunoprecipitated from epididymal membranes reduced its size to 140 kDa, coinciding with the appearance of a significant phospholipase A2 activity. The same results were obtained in COS cells transfected with phospholipase B cDNA. Since sn-glycero-3-phosphocholine present at high concentrations in seminal plasma mainly stems from epididymis, this suggests a possible role of phospholipase B in male reproduction. This novel localization also unravels a mechanism of phospholipase B activation by limited proteolysis involving either trypsin in the intestinal lumen or a trypsin-like endopeptidase in the male reproductive tract.
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PMID:Ectopic epididymal expression of guinea pig intestinal phospholipase B. Possible role in sperm maturation and activation by limited proteolytic digestion. 959 72

To identify peptide-specific antibodies which define sperm surface antigens, hybridomas were derived from the splenocytes of mice immunized with swollen human spermatozoa which had been subjected to limited proteolytic cleavage under reducing conditions prior to immunization. A total of 13.7% of the hybrid clones secreted antibodies which reacted with deglycosylated human seminal plasma glycoproteins when screened by an ELISA. A monoclonal antibody, designated mAb 4A8 sp., specifying a peptide epitope of human epididymal and a sperm surface glycoprotein was selected which inhibited human sperm-egg binding in a dose-dependent manner, and totally blocked sperm penetration in vitro. This inhibition did not result from an effect of the antibody on the motility of spermatozoa, nor was it due to premature induction of the acrosome reaction. Exclusion of oligosaccharide chains by chemical hydrolysis with trifluoromethane sulphonic acid (TFMS), enzymatic degradation and binding of lectins, did not abrogate the reactivity of mAb 4A8 to the cognate epitope whereas antibody binding was precluded upon digestion with proteolytic enzymes. In Western immunoblots of human seminal plasma glycoproteins, the antigen presented as a set of immunoreactive polypeptides, a major glycoprotein of M(r) 78 kDa and less prominent bands of M(r) 56 and 44 kDa. Immunocytochemical staining of a number of human reproductive and somatic tissues revealed strong immunostaining of the luminal epithelium of the epididymis as well as of spermatozoa in the lumen. Immunolocalization to the plasma membrane of ejaculated human spermatozoa was demonstrated by immunofluorescence, although on undigested spermatozoa the antigen epitope was less accessible. Upon capacitation the antigen persisted on the sperm surface and was present on the head of capacitated acrosome-intact spermatozoa. The pronounced peripheral immunostaining of the sperm head was accentuated after DTT/trypsin treatment, implicating the dynamic accessibility of the epitope on the plasma membrane of capacitated spermatozoa. It is suggested that the protein in question appears on the sperm membrane as a consequence of its modification in the epididymis (insertion and processing), and may be involved in the processes leading to sperm attachment and interaction with the human zona pellucida.
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PMID:Human sperm surface glycoprotein involved in sperm-zona pellucida interaction. 966 98

The effects of insulin and rapamycin on the phosphorylation of the translation regulator, initiation factor 4E-binding protein 1 (4E-BP1) have been studied in rat fat cells by following changes in the incorporation of 32P from [32P]Pi under steady-state conditions. Both unbound 4E-BP1 and 4E-BP1 bound to eukaryotic initiation factor 4E (eIF4E) were isolated from the cells and then digested with trypsin and other proteases; the radiolabelled phosphopeptides were then separated by two-dimensional thin- layer analysis and HPLC. The results provide confirmation of the conclusion of Fadden, Haystead and Lawrence [J. Biol. Chem. (1997) 272, 10240-10247] that insulin increases the phosphorylation of four sites that fit a Ser/Thr-Pro motif (Thr-36, Thr-45, Ser-64 and Thr-69) and that taken together these phosphorylations result in the dissociation of 4E-BP1 from eIF4E. The effects of insulin on the phosphorylation of these sites, and hence dissociation from eIF4E, are blocked by rapamycin. However, the present study also provides evidence that insulin increases the phosphorylation of 4E-BP1 bound to eIF4E on a further site (Ser-111) and that this is by a rapamycin-insensitive mechanism. Extraction of rat epididymal fat cells followed by chromatography on Mono-S and Superose 12 columns resulted in the separation of both an insulin-stimulated eIF4E kinase and an apparently novel kinase that is highly specific for Ser-111 of 4E-BP1. The 4E-BP1 kinase was activated more than 10-fold by incubation of the cells with insulin and was markedly more active towards 4E-BP1 bound to eIF4E than towards unbound 4E-BP1. The effects of insulin were blocked by wortmannin, but not by rapamycin. A 14-mer peptide based on the sequence surrounding Ser-111 of 4E-BP1 was also a substrate for the kinase, but peptide substrates for other known protein kinases were not. The kinase is quite distinct from casein kinase 2, which also phosphorylates Ser-111 of 4E-BP1. The possible importance of these kinases in the phosphorylation of 4E-BP1 in fat cells is discussed. It is suggested that the phosphorylation of Ser-111 might be a priming event that facilitates the subsequent phosphorylation of Thr-36, Thr-45, Ser-64 and Thr69 by a rapamycin-sensitive process that initiates the dissociation of 4E-BP1 from eIF4E and hence the formation of the eIF4F complex.
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PMID:Insulin-stimulated kinase from rat fat cells that phosphorylates initiation factor 4E-binding protein 1 on the rapamycin-insensitive site (serine-111). 980 82

We have previously identified a hamster sperm protein, P26h, proposed to be involved in the interaction between spermatozoa and the egg's zona pellucida. In this study we investigated the mechanism of P26h accumulation on hamster spermatozoa during epididymal maturation. Immunocytochemical studies showed an accumulation of P26h on the acrosomal cap of hamster spermatozoa during epididymal transit. To document the anchoring mechanism of P26h, cauda epididymal spermatozoa were exposed to different treatments. High-salt buffered solutions were unable to remove P26h from the surface of intact spermatozoa. P26h was released in a dose-dependent manner when live spermatozoa were treated with a solution of phospholipase C specific to phosphatidylinositol. In contrast, the P26h remained associated to the sperm surface following treatment with trypsin. To document the transfer mechanisms of P26h on the maturing spermatozoa, prostasomes were isolated from the epididymal fluid and subjected to immunodetection. Western blots and immunogold studies showed that P26h was associated to epididymal prostasomes. Phospholipase C treatment performed on epididymal prostasomes, indicated that P26h also is anchored to these vesicles via a phosphatidylinositol. These results suggest that epididymal sperm maturation involves a cell to cell transfer of a phosphaditylinositol-anchored protein and that prostasomes may be implicated in this process.
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PMID:Hamster sperm antigen P26h is a phosphatidylinositol-anchored protein. 989 Jul 54

A fraction of acrosomal proteins dispersed during calcium ionophore A23187-induced acrosome reaction was prepared from cauda epididymal sperm of wild-type and acrosin-deficient mice, rat, and hamster. The acrosome-reacted sperm were further extracted by Nonidet P-40 to obtain the detergent-soluble protein fraction. Activities of serine proteases in the two protein fractions were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of gelatin. A mixture of 42- and 41-kDa gelatin-hydrolyzing proteases was found in both fractions of the wild-type mouse sperm, whereas the acrosin-deficient mouse sperm contained the active 42-kDa protease and apparently lacked the activity of the 41-kDa protease. However, exogenous bovine pancreatic trypsin compensated for the absence of acrosin in the protein fractions of the mutant mouse sperm; the gelatin-hydrolyzing activity of the 41-kDa protease appeared when the sperm proteins of the mutant mice were treated with pancreatic trypsin. Two-dimensional polyacrylamide gel electrophoresis revealed that the 42- and 41-kDa proteases were distinguished from acrosin by the isoelectric point and immunoreactivity with affinity-purified antibody against an oligopeptide corresponding to the N-terminal amino acid sequence of mouse proacrosin. Moreover, the gelatin-hydrolyzing proteins corresponding to these two proteases were not detected in rat and hamster sperm, in spite of the treatment of the sperm extracts with pancreatic trypsin, and the total amount of gelatin-hydrolyzing activities in mouse was much smaller than those in rat and hamster. These results may reflect the difference of the serine protease system for the sperm penetration through the egg zona pellucida between mouse and other rodent animals, possibly explaining why the acrosin-deficient mouse sperm are capable of penetrating the zona pellucida.
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PMID:Difference of acrosomal serine protease system between mouse and other rodent sperm. 1044 Aug 45

We have identified cDNA and genomic clones encoding a homologue of pancreatic trypsin, termed TESP4, as a candidate protein involved in the sperm penetration of the egg zona pellucida in mouse. The deduced amino acid sequence indicates that TESP4 is 90% identical to pancreatic trypsin. Analysis of Northern blotting and reverse transcriptase-polymerase chain reaction reveals that the mouse TESP4 gene is ubiquitously expressed in all tissues tested, including the pancreas and testis, and the transcript is present in the haploid stages of male germ cells. Moreover, immunochemical analysis of mouse cauda epididymal sperm using an affinity-purified antibody against bovine pancreatic trypsinogen shows that TESP4 is localized only in the sperm acrosome and is released during the acrosome reaction induced by calcium ionophore A23187. These findings may open a new point of view regarding the molecular mechanisms of the sperm/egg interactions, including the sperm penetration of the egg zona pellucida.
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PMID:A homologue of pancreatic trypsin is localized in the acrosome of mammalian sperm and is released during acrosome reaction. 1050 5

Dramatic inhibition of trypsin activity by rat caltrin and guinea pig caltrin I was spectrophotometrically demonstrated using the artificial substrate benzoylarginyl ethyl ester. Approximately 6% and 21% of residual proteolytic activity was recorded after preincubating the enzyme with 0.22 and 0.27 microM rat caltrin and guinea pig caltrin I, respectively. Reduction and carboxymethylation of the cysteine residues abolished the inhibitor activity of both caltrin proteins. Rat caltrin and guinea pig caltrin I show structural homology with secretory trypsin/acrosin inhibitor proteins isolated from boar and human seminal plasma and mouse seminal vesicle secretion and share a fragment of 13 amino acids of almost identical sequence (DPVCGTDGH/K/ITYG/AN), which is also present in the structure of Kazal-type trypsin inhibitor proteins from different mammalian tissues. Bovine, mouse, and guinea pig caltrin II, three caltrin proteins that have no structural homology with rat caltrin or guinea pig caltrin I, lack trypsin inhibitor activity. Rat caltrin, guinea pig caltrin I, and the mouse seminal vesicle trypsin inhibitor protein P12, which also inhibits Ca(2+) uptake into epididymal spermatozoa (mouse caltrin I), bound specifically to the sperm head, on the acrosomal region, as detected by indirect immunofluorescence. They also inhibited the acrosin activity in the gelatin film assay. Caltrin I may play an important role in the control of sperm functions such as Ca(2+) influx in the acrosome reaction and activation of acrosin and other serine-proteases at the proper site and proper time to ensure successful fertilization.
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PMID:Trypsin/acrosin inhibitor activity of rat and guinea pig caltrin proteins. Structural and functional studies. 1085 40

Trypsinogen is a serine proteinase produced mainly by the pancreas, but it has recently been found to be expressed also in several cancers such as ovarian and colon cancer and in vascular endothelial cells. In this study, we found that trypsinogen-1 and -2 are present at high concentrations (median levels, 0.4 and 0.5 mg/L, respectively) in human seminal fluid and purified them to homogeneity by immunoaffinity and anion exchange chromatography. Purified trypsinogen isoenzymes displayed a M(r) of 25 to 28 kd in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Most of the trypsinogen-1 purified from seminal fluid was enzymatically active whereas trypsinogen-2 occurred as the proform, which could be activated by enteropeptidase in vitro. Immunohistochemically, trypsinogen protein was detected in the human prostate, urethra, utriculus, ejaculatory duct, seminal vesicles, deferent duct, epididymal glands, and testis. Expression of trypsinogen mRNA in the same organs was demonstrated by in situ hybridization. Trypsinogen mRNA was also detected in the prostate and seminal vesicles by reverse transcriptase-polymerase chain reaction and Northern blotting. Isolated trypsin was shown to activate the proenzyme form of prostate-specific antigen. These results suggest that trypsinogen isoenzymes found in seminal fluid are produced locally in the male genital tract and that they may play a physiological role in the semen.
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PMID:Expression and characterization of trypsinogen produced in the human male genital tract. 1110 74


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