Binding of epididymal proteins to rat spermatozoa in vivo.

The secretion of epididymal proteins and their binding to spermatozoa in rats were examined after retrograde perfusion of the superior and inferior epididymal arteries with [35S]methionine. PAGE revealed that the pattern of radioactive proteins in the luminal fluid was markedly different from the well-characterized pattern of secretory proteins obtained by in vitro incubation of epididymal minces with labeled methionine. Of the proteins secreted into the lumen, about 1% were associated with Percoll-purified spermatozoa. More proteins were associated with the spermatozoa in the corpus epididymidis than in the caput. Sequential extraction of spermatozoa with an isotonic buffer, a high-salt buffer, Triton X-100, and SDS revealed that almost half of the radiolabeled proteins could be extracted with the isotonic buffer. The firmly bound radioactive proteins remaining, which were extracted with Triton X-100 or SDS, consisted of one major band of 25 kDa and two minor bands of 30 kDa and 32 kDa. Analysis of the sperm-associated proteins at various times after the isotope was administered indicated that tight binding of proteins to spermatozoa occurs within 3 h after isotope injection.     

Biosynthetic precursors and in vitro translation products of the glucose transporter of human hepatocarcinoma cells, human fibroblasts, and murine preadipocytes

Antisera to the human erythrocyte Glc transporter immunoblotted a polypeptide of Mr 55,000 in membranes from human hepatocarcinoma cells, Hep G2, human fibroblasts, W138, and murine preadipocytes, 3T3-L1. This antisera immunoprecipitated the erythrocyte protein which had been photoaffinity labeled with [3H]cytochalasin B, immunoblotted its tryptic fragment of Mr 19,000, and immunoblotted the deglycosylated protein as a doublet of Mr 46,000 and 38,000. This doublet reduced to a single polypeptide of Mr 38,000 after boiling. When Hep G2, W138, and 3T3-L1 cells were metabolically labeled with L-[35S]methionine for 6 h, a broad band of Mr 55,000 was immunoprecipitated from membrane extracts. In pulse-chase experiments, two bands of Mr 49,000 and 42,000 were identified as putative precursors of the mature transporter. The t1/2 for mature Glc transporter was 90 min for Hep G2 cells that had been starved for methionine (2 h) and pulsed for 15 min with L-[35S]methionine. Polypeptides of Mr 46,000 and 38,000 were immunoprecipitated from Hep G2 cells that had been metabolically labeled with L-[35S]methionine in the presence of tunicamycin. This doublet reduced to the single polypeptide of Mr 38,000 after boiling. In the absence of tunicamycin, but not in its presence, mature polypeptide of Mr 55,000 was immunoprecipitated from Hep G2 cells metabolically labeled with D-[3H]GlcN. A polypeptide of Mr 38,000 was observed in boiled immune complexes from the in vitro translation products of Hep G2, W138, and 3T3-L1 cell RNA. Dog pancreatic microsomes cotranslationally, but not posttranslationally, converted this to a polypeptide of Mr 35,000. A model for Glc transporter biogenesis is proposed in which the primary translation product of Mr 38,000 is converted by glycosylations to a polypeptide of Mr 42,000. The latter is then processed via heterogeneous complex N-linked glycosylations to form the mature Glc transporter, Mr 55,000.     

Surface changes in chimpanzee sperm during epididymal transit

Intact chimpanzee caput and cauda epididymal sperm, sperm cell lysates, and caput and cauda epididymal fluid were radiolabeled by enzymatic iodination with lactoperoxidase and Na125 I and were compared by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Caput epididymal sperm showed nine labeled macromolecular components of 90, 64, 56, 48, 38, 31, 20, 18 and 16 Kd and cauda epididymal sperm showed eleven macromolecular components of 90, 64, 55, 47, 42, 33, 27, 18, 17, 15 and 11 Kd. Six of the components labeled on caput sperm (90, 64, 56, 48, 18 and 16 Kd) were detected in equal amounts of cauda sperm and two (38 and 20 Kd) were detected at greatly reduced labeling intensities. In the cauda epididymidis, four new components (33, 27, 17 and 11 Kd) became prominent features of the sperm surface. Analysis of labeled caput and cauda sperm cell lysates resolved components distinct from those detected on sperm surfaces. Electrophoresis of caput epididymal fluid showed five labeled components of 66, 56, 47, 41 and 37 Kd, while electrophoresis of cauda epididymal fluid showed eight labeled components of 92, 66, 56, 48, 31, 27, 24 and 11 Kd. Three components (66, 56 and 47 Kd) were present in both caput and cauda fluid, two (41 and 37 Kd) in caput fluid only, and five (92, 31, 27, 24 and 11 Kd) in cauda fluid only. Components of 37 Kd were labeled in caput fluid and on caput sperm but not on cauda sperm, whereas components of 27 Kd and 11 Kd were labeled in cauda fluid and on cauda sperm but not on caput sperm. These data show that chimpanzee sperm undergo extensive surface modifications during epididymal maturation and that some of these modifications may be related to exogenous proteins/glycoproteins in epididymal fluids.     

Newly synthesized proteins in seminiferous intertubular and intratubular compartments of the rat testis

Two-dimensional gel electrophoresis combined with autoradiography and Western blot procedures have been used to characterize newly synthesized proteins in testicular intertubular fluid (TIF) and seminiferous tubular fluid (SNF). Fluids were collected following in vivo and in vitro intratesticular injection of [35S]methionine into control and hypophysectomized adult rats. A discrete number of [35S]methionine-labeled proteins were detected within TIF and SNF. Their presence and relative abundance varied according to in vivo and in vitro labeling conditions. While two major blood plasma proteins, albumin and transferrin, were radioactively labeled after in vivo labeling, these two proteins were insignificantly labeled in samples collected after in vitro labeling. Three acidic proteins, possibly secreted by Sertoli cells (Mr = 72,000, 45,000 and 35,000), were more abundant in TIF samples collected after in vitro [35S]methionine labeling than after in vivo labeling. Incubated seminiferous tubules and TIF of hypophysectomized rats showed a decrease in [35S]methionine-labeling intensity of the Mr = 72,000 acidic protein, possibly reflecting changes in the seminiferous epithelium caused by pituitary hormonal deprivation. Autoradiographs of TIF and most remarkably, of SNF, showed many protein spots that suggested cell breakage and leakage during sample collection. Results of this study suggest that most albumin and transferrin found in TIF and SNF have an extratesticular origin and that proteins secreted by the Sertoli cell can gain access to both TIF and SNF.     

Identification of epididymal proteins associated with hamster sperm.

The electrophoretic analysis of the proteins that were extracted from immature caput and mature cauda sperm showed evidence of accumulation of several proteins during the epididymal transit of the sperm. An antiserum, raised against detergent-extracted proteins from mature spermatozoa, immunostained six epididymal proteins with apparent molecular masses of 16, 22.5, 26, 37, 60, and 80 kDa on Western blots of epididymal fluid. Of these proteins, only the 26 kDa protein was significantly immunodetected in proximal caput epididymal fluid. Its biosynthesis by caput epididymis was confirmed by immunoprecipitation of an in vitro translated product of caput poly (A) RNA. The homology of the 26 kDa epididymal protein with the 26 kDa sperm protein was verified by epitope mapping. The other epididymal proteins were found in the fluid of the more distal portions of the organ. Their presence in the epididymal fluid coincided with their detection on the sperm. These epididymal proteins were considered to be sperm-coating proteins.     

The cell surface receptor for immunoglobulin E. Effect of tunicamycin on molecular properties of receptor from rat basophilic leukemia cells

Cell surface receptors for immunoglobulin E were isolated by repetitive affinity chromatography from rat basophilic leukemia cells biosynthetically labeled with L-[35S]methionine and D-[3H]mannose. Native immunoglobulin E receptor appeared as a very broad band in the 45,000 to 62,000 Mr region in sodium dodecyl sulfate polyacrylamide gels. However, from cells cultured in the presence of tunicamycin, a relatively narrow band with an apparent Mr of 38,000 was isolated. The 38,000 Mr band rebound to immunoglobulin E-Sepharose, was immunoprecipitated with antibodies to immunoglobulin E receptor, shared tryptic peptides with native receptor, and was labeled with L-[35S]methionine but not D-[3H]mannose, and thus appears to be immunoglobulin E receptor lacking N-linked oligosaccharides. It is demonstrated that N-linked oligosaccharides account for much of the apparent heterogeneity of native receptor in sodium dodecyl sulfate polyacrylamide gels and in two-dimensional gel electrophoresis. A receptor-associated protein with apparent Mr = 30,000, prominently labeled with L-[35S]methionine but not with D-[3H]mannose, did not have altered molecular properties when isolated from tunicamycin-cultured cells, and did not share tryptic peptides with receptor.     

Effects of caput ligation on rat sperm and epididymis: protein thiols and fertilizing ability.

Mammalian spermatozoa mature while passing through the epididymis. Maturation is accompanied by thiol oxidation to disulfides. In rats, sperm become motile and fertile in the cauda. We have previously demonstrated that rat caput sperm contain mostly thiols and that upon passage from the corpus to the cauda epididymidis, sperm protein thiols are oxidized. The present work was undertaken to study the role of the regions of the epididymis in sperm maturation as reflected in the thiol status, fertility, and motility of the spermatozoa. The distal caput epididymidis of mature albino rats was ligated on one side. After 5 days, sperm were isolated from the ligated caput and from caput and cauda of the control side. Thiol groups in sperm, epididymal luminal fluid (EF), and epididymal tissue were labeled using the fluorescent thiol-labeling agent monobromobimane. After ligation, changes were observed in a) sperm proteins, sperm nuclear proteins, and epididymal fluid by electrophoresis; b) epididymal tissues by histochemistry; c) progressive motility by phase microscopy; and d) fertilizing ability after insemination into uteri of immature females. We found that after ligation, caput sperm thiols, especially protamine thiols, are oxidized, rendering them similar to mature sperm isolated from the cauda epididymidis. Spermatozoa from ligated caput epididymidis gain progressive motility and partial fertilizing ability. Morphology of epithelial cells of ligated caput is similar to that of cauda cells. However, other changes in caput EF and epithelium induced by ligation render the ligated caput epididymidis different from either control caput or cauda. Hence, sperm thiol oxidation, along with the development of fertilizing ability, can occur in sperm without necessity for sperm transit through the corpus and cauda epididymidis.     

Interactions of labeled epididymal secretory proteins with spermatozoa after injection of 35S-methionine in the mouse

The sequential interactions of epididymal secretory proteins with spermatozoa during epididymal transit were examined. Mice received injections of 35S-methionine, and the radiolabeled luminal fluid and sperm-associated proteins were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis at various times after injection. The majority of the luminal fluid and sperm-associated proteins were found in the caput epididymidis at 8 h; by 7 days, many of these proteins had been transported to the cauda epididymidis. Two classes of epididymal protein-sperm interactions were distinguished on the basis of regional synthesis and secretion. The major class consisted of proteins that were synthesized, secreted, and bound to spermatozoa in the caput epididymidis. In this class, however, the binding of proteins to the spermatozoa was variable. For example, a protein of 25 kDa remained associated with spermatozoa in substantial amounts during epididymal transit, while proteins of 40 and 35 kDa decreased in amount. Other proteins such as a protein of 18 kDa did not remain associated with spermatozoa. Another class of proteins (54, 44, 29 kDa) were synthesized and secreted from all epididymal regions but bound only to caput spermatozoa. Most of the epididymal proteins appeared to be tightly bound to the spermatozoa since spermatozoa already saturated with the unlabeled protein in the distal epididymis remained so even though the spermatozoa were surrounded by labeled proteins in the luminal fluid. These studies demonstrate that a variety of specific interactions occur between epididymal secretory proteins and spermatozoa as they migrate and mature in the epididymis.     

Turnover of the surface proteins and the receptor for serum asialoglycoproteins in primary cultures of rat hepatocytes

The turnover of plasma membrane proteins in primary rat hepatocyte cultures was examined by following the loss of polypeptides labeled in situ by lactoperoxidase-catalyzed iodination using 125I and 131I. Most plasma membrane proteins had similar rates of degradation, having a half-life of approximately 85 h. By in situ labeling via lactoperoxidase-catalyzed iodination, as well as metabolically labeling cells with L-[35S]methionine, the asialoglycoprotein receptor, a plasma membrane constituent, was identified and shown to exist in three forms which were structurally related. The turnover of receptor on the cell surface was examined by following the loss of iodinated cell surface receptor, while the turnover of total cellular receptor, including both surface and internally localized receptor was assayed by following the loss of receptor labeled metabolically with [35S]methionine. The turnover rate in both cases was approximately 20 h. Receptor-mediated endocytosis of asialoglycoproteins had no effect on the turnover of the plasma membrane proteins or receptor. Based on estimates of the rate of metabolism of the asialoglycoprotein ligand relative to the turnover rate of the receptor, we conclude each molecule of receptor can deliver about 1,000 molecules of ligand to the lysosome to be degraded.     

In vivo secretion and association of clusterin (SGP-2) in luminal fluid with spermatozoa in the rat testis and epididymis.

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.     

Biosynthesis and turnover of a Mr = 110,000 glycoprotein localized to the hepatocyte bile canaliculus

Antiserum was raised in rabbits against a bile canalicular glycoprotein of Mr = 110,000 purified to homogeneity from of rat liver. The antisera specifically immunoprecipitated a Mr = 110,000 polypeptide from hepatocytes metabolically labeled with [35S]methionine. When hepatocytes in primary culture were incubated with tunicamycin before labeling with [35S]methionine in the presence of tunicamycin, the major polypeptide immunoprecipitated by the specific antiserum from Triton X-100 extracts of cells had a molecular weight of 59,000. Enzymatic removal of N-linked carbohydrates from the Mr = 110,000 glycoprotein by N-glycanase digestion also yielded a polypeptide with minimum Mr = 59,000. In pulse-chase experiments using [35S]methionine, the Mr = 110,000 protein detected by the specific antisera first appears as Mr = 85,000 and 75,000 intermediate species which are endoglycosidase H sensitive. The Mr = 85,000 intermediate form is lost first with time followed by the Mr = 75,000 form giving rise to the Mr = 110,000 form that is endoglycosidase H resistant. Neuraminidase digestion of the Mr = 110,000 form generated an Mr 85,000 form but with a different carbohydrate structure than the intermediate Mr 85,000 form detected in the pulse-chase experiments. The time required to accomplish the processing of the Mr = 85,000 and 75,000 forms is relatively slow. Finally, the terminal sugars are added and the mature Mr = 110,000 glycoprotein is rapidly transported to the cell surface. A minimum time of 90 min is required for the Mr = 110,000 bile canalicular glycoprotein to be synthesized, processed, and reach the cell surface which is long relative to the time required (10 min) for another domain-specific protein, the receptor for asialoglycoproteins, to reach the sinusoidal surface. The Mr = 110,000 bile canalicular glycoprotein turns over in the bile canalicular domain with a half-life of 43 h while the asialoglycoprotein receptor turns over in the sinusoidal domain with a half-life of 23 h.     

Identification and validation of mouse sperm proteins correlated with epididymal maturation

Sperm need to mature in the epididymis to become capable of fertilization. To understand the molecular mechanisms of mouse sperm maturation, we conducted a proteomic analysis using saturation dye labeling to identify proteins of caput and cauda epididymal sperm that exhibited differences in amounts or positions on two-dimensional gels. Of eight caput epididymal sperm-differential proteins, three were molecular chaperones and three were structural proteins. Of nine cauda epididymal sperm-differential proteins, six were enzymes of energy metabolism. To validate these proteins as markers of epididymal maturation, immunoblotting and immunofluorescence analyses were performed. During epididymal transit, heat shock protein 2 was eliminated with the cytoplasmic droplet and smooth muscle γ-actin exhibited reduced fluorescence from the anterior acrosome while the signal intensity of aldolase A increased, especially in the principal piece. Besides these changes, we observed protein spots, such as glutathione S-transferase mu 5 and the E2 component of pyruvate dehydrogenase complex, shifting to more basic isoelectric points, suggesting post-translational changes such dephosphorylation occur during epididymal maturation. We conclude that most caput epididymal sperm-differential proteins contribute to the functional modification of sperm structures and that many cauda epididymal sperm-differential proteins are involved in ATP production that promotes sperm functions such as motility.     

Secretion and transport of mouse epididymal proteins after injection of 35S-methionine

The presence of epididymal secretory proteins in crude luminal fluids of the epididymis of mice was investigated at varying times after i.v. injection of 35S-methionine or after incubation of epididymal minces with 35S-methionine. The amount of label incorporated into luminal proteins after in vivo injection was not significantly different at 4, 8, 12, and 16 h. The quantity of labeled proteins in the crude luminal fluids of Regions 1-3 (caput) was about two and four times higher than in Regions 4 (corpus) and 5 (cauda), respectively. Increasing the dosage of 35S-methionine strongly increased the amount of labeled protein present. Approximately half of the labeled protein present in the epididymis was found in the luminal fluid. Polyacrylamide gel electrophoresis revealed comparable patterns of proteins at 8 h after injection of isotope or after a 5-h in vitro incubation of minced epididymal tissues with isotope. The protein patterns from the five regions, however, were markedly different from each other and highly characteristic. Two proteins (25 kDa and 18 kDa) were found in crude luminal fluids of Regions 2 and 3 eight hours after in vivo injection, but not in Regions 4 and 5. One protein (29 kDa) was found in high amounts in Regions 4 and 5 eight hours after injection. Five days after injection, the three proteins were found in Regions 4 and 5. However, the 25-kDa protein was present in reduced amount, whereas the 18- and 29-kDa proteins accumulated in caudal fluid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two-dimensional electrophoresis of proteins in principal cells, spermatozoa, and fluid associated with the rat epididymis

Spermatozoa, fluids, and principal cells from different regions of the epididymis were characterized by two-dimensional electrophoresis. Rete testis fluid was collected after 36-h efferent duct ligation, and cauda epididymal fluid was collected by retrograde perfusion through the vas deferens. Spermatozoa were collected after their exudation from minced caput and corpus epididymal tissue. Principal cells were recovered after enzymatic disaggregation and centrifugal elutriation of epididymides. Two-dimensional polyacrylamide gel electrophoresis was used to prepare protein profiles of all samples. Comparison of the proteins found in rete testis fluid versus those found in cauda epididymal fluid revealed a dramatic change in composition, including the loss, addition, or retention of specific proteins as well as changes in the relative concentrations of certain proteins. Prominent cauda epididymal fluid proteins, possibly contributed by the epididymal epithelium, were detected at 16, 23, and 34 kDa. After epididymal transit, a considerable decrease was observed in the number of aqueous-soluble sperm proteins. Differences in the protein composition of epididymal epithelial principal cells from the caput versus corpus epididymidis were also noted, suggesting that functional differences exist for these epididymal regions. Of particular interest was the occurrence of a prominent protein of approximately 20-23 kDa found in all sperm samples, in fluids, and in caput and corpus principal cells. However, this protein was absent in cauda epididymal sperm after 36-h efferent duct ligation. The rapid loss of this protein from sperm after efferent duct ligation suggests that this surgical intervention may affect spermatozoa residing within the epididymis.     

Phosphorylation of lens intrinsic membrane proteins by protein kinase C

Two intrinsic proteins of bovine lens membranes with apparent relative molecular masses (Mr, app) of 26,000 and 18,000 were phosphorylated in intact membranes by protein kinase C prepared from either bovine brain or lens. The kinase preparations exhibited histone H1 phosphorylation dependent on calcium and phospholipid but not on cAMP. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the lens membranes showed a major band at Mr, app = 26,000 (identified as MP26, the main intrinsic protein of lens fiber cells), an intermediate band at Mr, app = 18,000 and several minor bands. Autoradiography of complete assay mixture containing protein kinase C, calcium, magnesium and [gamma-32P]ATP showed major bands at Mr, app = 18,000 and 26,000. Several lines of evidence indicated that the label at Mr, app = 26,000 was associated with MP26, a protein which has been found in lens junctions and which may form cell-cell channels. Treatment of the phosphorylated membranes with chymotrypsin and V8 protease cleaved the major band at Mr, app = 26,000 to fragments of Mr, app .= 22,000 and 24,000. Label was not detected in the resulting Mr, app = 22,000 peptide, but the Mr, app = 24,000 peptide was found to be labeled. Phosphoamino acid analysis of MP26 indicated that approximately 75% of the label was on phosphoserine and 25% was on phosphothreonine. No label was found on phosphotyrosine. These results differ from those reported for cAMP-dependent phosphorylation of lens proteins. Phosphorylation by protein kinase C may account for some of the labeling of MP26 detected in vivo.     

Lectin-binding sites on the plasma membranes of rabbit spermatozoa: Changes in surface receptors during epididymal maturation and after ejaculation

Modifications in rabbit sperm plasma membranes during epididymal passage and after ejaculation were investigated by used of three lectins: concanavalin A (Con A); Ricinus communis I (RCA(I)); and wheat germ agglutinin (WGA). During sperm passage from caput to cauda epididymis, agglutination by WGA drastically decreased, and agglutination by RCA(I) slightly decreased, although agglutination by Con A remained approximately unchanged. After ejaculation, spermatozoa were agglutinated to a similar degree or slightly less by Con A, WGA, and RCA(I), compared to cauda epididymal spermatozoa. Ultrastructural examination of sperm lectin-binding sites with ferritin- lectin conjugates revealed differences in the densities of lectin receptors in various sperm regions, and changes in the same regions during epididymal passage and after ejaculation. Ferritin-RCA(I) showed abrupt changes in lectin site densities between acrosomal and postacrosomal regions of sperm heads. The relative amounts of ferritin-RCA(I) bound to heads of caput epididymal or ejaculated spermatozoa. Tail regions were labeled by ferritin RCA(I) almost equally on caput and cauda epididymal spermatozoa, but the middle-piece region of ejaculated spermatozoa was slightly more densely labeled than the principal-piece region, and these two regions on ejaculated spermatozoa were labeled less than on caput and cuada epididymal spermatozoa. Ferritin-WGA densely labeled the acrosomal region of caput epididymal spermatozoa, although labeling of cauda epidiymal spermatozoa was relatively sparse except in the apical area of the acrosomal region. Ejaculated spermatozoa bound only a few molecules of ferritin-WGA, even at the highest conjugate concentrations used. Caput epididymal, but not cauda epididymal or ejaculated spermatozoa, bound ferritin-WGA in the tail regions. Dramatic differences in labeling densities during epididymal passage and after ejaculation were not found with ferritin-Con A.     

Expression and activity of gamma-glutamyl transpeptidase in the rat epididymis.

Following Northern analysis, GGT mRNA was found predominantly within the caput epididymides and kidney. The size of mRNAs for kidney, caput, corpus, and ductus deferens were 2.2, 2.3, 2.2, and 2.3 kb, respectively, whereas cauda showed a doublet of 2.2 and 2.3 kb. GGT transpeptidation and hydrolytic activity within epididymal luminal fluids collected by micropuncture showed caput = corpus greater than cauda and corpus greater than caput greater than cauda, respectively. Caput luminal GGT transpeptidation activity was significantly inhibited by serine-borate and was optimal at pH 8.0. The calculated Km and Vmax values for hydrolysis of GSH by caput luminal GGT were 0.06 microM and 2.19 nmoles/min/microliters luminal fluid at pH 8.5 compared to 0.49 microM and 0.49 nmoles/min/microliters luminal fluid, respectively, at the physiological pH 6.5 of caput fluid. These studies would suggest that the epididymis can control the activity of luminal GGT by pH. Lower Km (0.12 microM) and higher Vmax (1.13 nmoles/min/microliters luminal fluid) values were also calculated when GSSG was used compared to GSH. Results from Triton X-114 partitioning experiments suggest that luminal GGT probably exists in both membrane bound and nonmembrane bound forms. Western blot analysis of proteins within epididymal luminal fluids revealed both subunits of GGT in all epididymal regions studied. However, two lower molecular bands, approximately 22 kDa and 21 kDa, were also observed in cauda fluid. It is suggested that as GGT is transported along the epididymal duct it undergoes degradation, which accounts for its loss of activity in the distal epididymal regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of shRNA-mediated gene silencing of transcription factor SNAI1 on the biological phenotypes of breast cancer cell line MCF-7

Developing spermatozoa require a series of posttesticular modifications within the luminal environment of the epididymis to achieve maturation; this involves several surface modifications including changes in plasma membrane lipids, proteins, carbohydrates, and alterations in the outer acrosomal membrane. Epididymal maturation can therefore allow sperm to gain forward motility and fertilization capabilities. The objective of this study was to identify maturation-dependent protein(s) and to investigate their role with the production of functionally competent spermatozoa. Lectin blot analyses of caput and cauda sperm plasma membrane fractions identified a 17.5 kDa wheat germ agglutinin (WGA)-binding polypeptide present in the cauda sperm plasma membrane not in the caput sperm plasma membrane. Among the several WGA-stained bands, the presence of a 17.5 kDa WGA-binding polypeptide band was detected only in cauda epididymal fluid not in caput epididymal fluid suggesting that the 17.5 kDa WGA-binding polypeptide is secreted from the cauda epididymis and binds to the cauda sperm plasma membrane during epididymal transit. Proteomic identification of the 17.5 kDa polypeptide yielded 13 peptides that matched the sequence of peroxiredoxin-5 (PRDX5) protein (Bos Taurus). We propose that bovine cauda sperm PRDX5 acts as an antioxidant enzyme in the epididymal environment, which is crucial in protecting the viable sperm population against the damage caused by endogeneous or exogeneous peroxide.     

Immunochemical localization and association with spermatozoa of androgen-regulated proteins of MR 24000 secreted by the mouse epididymis

A polyclonal monospecific immune serum was raised against androgen-regulated proteins with Mr 24000 secreted by the mouse caput epididymidis. Sections of frozen tissues from the different regions of the epididymis have been studied by indirect immuno- fluorescence. Results indicate that the antigens are secretory proteins produced by the epithelial cells of the caput epididymidis, essentially in the medial and distal segments. Accumulation of the antigens was observed in the lumen of the caput and the corpus epididymal duct. Subsequently, their association with the sperm surface occurred and persisted down to the cauda epididymidis.