Localization of epididymal secretory proteins on rat spermatozoa

Spermatozoa from the testis and cauda epididymidis of the rat were surface labelled with radioactive iodide. Detergent extracts of radioiodinated spermatozoa immunoprecipitated with antisera against specific epididymal proteins, followed by polyacrylamide gel electrophoresis, revealed two proteins (D and E of Mr 27 000 and 28 000, respectively) which became associated with spermatozoa during epididymal transit. These proteins were observed by immunofluorescence microscopy to be located over a restricted area of the head surface. Proteins with similar molecular weight were labelled on spermatozoa from the cauda epididymidis, but not from the testis, by reaction with sodium boro[3H]hydride in the presence of galactose oxidase. However, failure to immunoprecipitate with antibodies to Proteins D and E and non-coincident migration on two-dimensional gel electrophoresis established the non-identity of these proteins. Compared with Proteins D and E, two other major epididymal secretory proteins (Proteins B and C of Mr 16 000) associated with spermatozoa to a relatively minor extent during epididymal transit.     

Selective binding of specific rat epididymal secretory proteins to spermatozoa and erythrocytes

Tissue pieces from the caput epididymidis of the rat were incubated in vitro with (³⁵S) methionine to produce radioactive secretory proteins. The radioactive secretory proteins so formed were tested for their ability to bind to washed rat spermatozoa collected from the rete testis and cauda epididymidis, and to rat erythrocytes. The sperm and erythrocytes bound approximately 5% of the total radioactive protein. Binding was protein-specific in that only selected proteins became associated with the cells. Binding was not cell-specific, however, since testicular spermatozoa, caudal spermatozoa, and erythrocytes all bound the same proteins to a similar degree.     

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.     

Ultrastructural localization of epididymal secretory proteins associated with the surface of spermatozoa from rabbit cauda epididymis

Summary Antibodies against whole rabbit epididymal fluid as well as against three purified proteins from this fluid (namely EP21, EP35 and uteroglobin) were prepared and characterized by Western immunoblot. These antibodies were used to study the association of those proteins to the spermatozoon by means of immunoelectron microscopy using a colloidal gold-labelling technique. Antibodies against whole fluid intensely stained the spermatozoon surface at the acrosomal and postacrosomal regions of the head and at the middle piece of the tail. The equatorial region and the principal piece were much less labelled. The EP21 antigen associated with the whole surface of the head and the middle piece but not with the principal piece of the tail. EP35 was distributed over the acrosomal but not the postacrosomal region. The principal piece also contained this antigen in considerable amounts. The antibody against uteroglobin did not stain the head surface but intensely labelled both the middle and principal pieces.     

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.     

Development changes occurring in the lipids of ram epididymal spermatozoa plasma membrane

Ram spermatozoa were obtained from different regions (caput, corpus, and cauda) of the epididymis and their plasma membrane was removed using a nitrogen cavitation treatment (750 psi, 10 min equilibration at 4 degrees C). Membrane was recovered after sucrose gradient centrifugation and identified using 125I-succinylated concanavalin A (125I-succConA) as a surface marker. Based on fluorescein isothiocyanate-succConA (FITC-succConA) labeling and electron microscopy, cavitation removed plasma membrane from the anterior sperm head in the area overlying the acrosome. Cholesterol was the major sterol in plasma membrane, with desmosterol present in sperm entering the epididymis (caput sperm) but negligible in sperm after epididymal transit (cauda sperm). Ethanolamine and choline phosphoglycerides represented 70-80% of membrane phospholipids, with the ethanolamine fraction decreasing relative to choline phosphoglycerides during epididymal transit. The molar ratio of cholesterol to phospholipid increased in the plasma membrane during maturation. The bulk phospholipid-bound fatty acids consisted primarily of palmitoyl acyl groups (16:0) in caput sperm and docosahexaenoyl acyl groups (22:6) in cauda sperm. The choline phosphoglyceride fraction was purified and analyzed. It consisted of a mixture of ether acyl glycero-3-phosphocholine and diacyl phosphoglyceride, with the dominant acyl residue, at all stages of epididymal maturation, being 22:6 throughout epididymal transit. The significance of these findings relative to acquisition of fertilization capacity by sperm during epididymal maturation is discussed.     

Lipid phase transition in the plasma membrane of the goat epididymal maturing spermatozoa

Microviscosity of the highly purified plasma membranes isolated from the maturing goat caput, corpus and cauda epididymal sperm, was measured using l,6-diphenyl-l,3,5-hexatriene as the lipophilic probe at varying temperatures (12–42°C). As shown by the Arrhenius plot of the data each of the maturing sperm membranes had two distinct lipid phase transitions in the temperature zones 19–25°C and 34–37°C. The low-temperature transitions for the immature caput- and mature cauda-sperm membranes were noted at 19–20°C, and 24–25°C, respectively, whereas both these membranes showed high temperature transition at 36–37°C. The maturing corpus-sperm membrane had phase transitions at 21–22°C and 35–36°C that were significantly different from those of the immature/mature sperm membranes. The data implicate significant alteration of the sperm membrane structure during epididymal maturation. The phase transition of the mature male gametes at 36–37°C may have a great impact on the subsequent events of the sperm life cycle since the mature spermatozoa that are stored in the epididymis a few degrees below the body temperature, experience higher temperature when ejaculated into the female reproductive tract.     

A maturation-related differential phosphorylation of the plasma membrane proteins of the epididymal spermatozoa of the hamster by endogenous protein kinases

When the plasma membranes of caput and cauda epididymal spermatozoa of hamster were evaluated for their ability to undergo phosphorylation, a differential phosphorylation of the membrane proteins was observed. In the plasma membranes of the caput epididymal spermatozoa (immature), twelve proteins were phosphorylated (100, 76, 67, 65, 55, 52, 47, 42, 38, 32, 30, and 20 kD), whereas in the plasma membranes of cauda epididymal spermatozoa (mature), a differential phosphorylation pattern was observed with respect to the 94, 67, 52, and 47 kD proteins. The 94 kD protein was found to be phosphorylated and the 67 kD protein was found to be not phosphorylated in cauda spermatozoal plasma membrane (Cd SPM) in contrast to this protein in caput spermatozoal plasma membrane (Cpt SPM). The 52 and 47 kD proteins were also more intensely phosphorylated in Cd SPM than Cpt SPM. The 100 kilodalton protein, although present in both Cpt and Cd sperm plasma membranes, was found to be phosphorylated at the tyrosine residues only in the Cd SPM, as indicated by the Western blot using antiphosphotyrosine antibody. Further, a differential phosphorylation of the substrate proteins present in the Cpt and Cd SPM was seen when Mg²⁺ in the assay buffer was replaced by other divalent cations. For instance, Zn²⁺ stimulated the phosphorylation of an 85 kD protein in cauda SPM and not in the caput SPM and Ca²⁺ stimulated the phosphorylation of a 76 kD protein only in the cauda SPM. The phosphoproteins in both the plasma membranes were found to be phosphorylated predominantly at the tyrosine residue. The differential phosphorylation of a 100 kD protein at tyrosine in the Cd SPM (Western blot), which is absent in the immature Cpt SPM, also indicated that certain proteins in the hamster spermatozoa are phosphorylated in a maturation-specific manner. Mol. Reprod. Dev. 47:341–350, 1997. © 1997 Wiley-Liss, Inc.     

Coincident localization of secretory and plasma membrane proteins in organelles of the yeast secretory pathway.

Immunoelectron microscopy of Saccharomyces cerevisiae cells embedded in Lowicryl K4M has been used to localize invertase and plasma membrane (PM) ATPase in secretory organelles. sec mutant cells incubated at 37 degrees C were prepared for electron microscopy, and thin sections were incubated with polyclonal antibodies, followed by decoration with protein A-gold. Specific labeling of invertase was seen in the lumen of the endoplasmic reticulum, Golgi apparatus, and secretory vesicles in mutant cells that exaggerate these organelles. PM ATPase accumulated within the same organelles. Double-immune labeling revealed that invertase and PM ATPase colocalized in secretory vesicles. These results strengthen the view that secretion and plasma membrane assembly are biosynthetically coupled in yeast.     

Seminal plasma proteins reduce protein tyrosine phosphorylation in the plasma membrane of cold-shocked ram spermatozoa.

Capacitation of spermatozoa, a complex process occurring after sperm ejaculation, is required to produce fertilization of the oocyte in vivo and in vitro. Although this process results from a poorly understood series of morphological and molecular events, protein tyrosine phosphorylation has been associated with sperm capacitation in several mammalian species, but it still remains to be demonstrated in ram spermatozoa. Studies of capacitation in ram spermatozoa are of great interest, since several reports have suggested that the reduced fertility of cryopreserved spermatozoa is due to their premature capacitation. In this work, we report for the first time, to our knowledge, that tyrosine phosphorylation of ram sperm membrane proteins is related to the capacitation state of these cells. Capacitation induced tyrosine phosphorylation of some plasma membrane proteins of ram spermatozoa freed from seminal plasma by a dextran/swim-up procedure. It has also been proved that cold-shock induces protein tyrosine phosphorylation as well as a decrease in plasma membrane integrity. Addition of seminal plasma proteins prior to cold-shock not only improved sperm survival but also promoted a decrease in protein tyrosine phosphorylation.     

Modification of the metabolism of rat epididymal spermatozoa by spermine.

Spermine (1 to 10 mM) markedly enhanced (2- to 7-fold) the formation of labelled lactate from radioactive fructose in rat epididymal spermatozoa $\text{in vitro}$. The combination of spermine and calcium (1 mM), the latter ion inconsistently stimulating the formation of carbon dioxide and lactate from fructose, resulted in a striking synergistic stimulation of the fructolysis in epididymal spermatozoa. The production of lactate from fructose in the presence of spermine and calcium increased 5- to 30-fold over that in normal Ringer solution. Spermidine, but not putrescine, also stimulated the formation of lactate from fructose in epididymal spermatozoa. While stimulating the aerobic fructolysis the polyamines inhibited the formation of radioactive CO₂ from (2-¹⁴C)pyruvate thus apparently interfering with the reactions of the tricarboxylic acid cycle. In this respect, however, oxidized spermine {N,N′-bis(3-propion-aldehyde)-1,4-diaminobutane} was far more effective.Spermine (and spermidine) being normal constituents of the secretions of male accessory sexual glands in most mammals could conceivably belong to the factors responsible for the “intensely glycolytic” nature of ejaculated spermatozoa.     

Identification of membrane antigens of goat epididymal spermatozoa

Purified goat sperm plasma membrane was used as antigen to raise the antibody in rabbit. Using this antisera four groups of antigenic membrane polypeptides are determined in caput and cauda epididymal sperm. The immunoresponsiveness of the polypeptides in caput and cauda sperm differs significantly. In case of cauda epididymal sperm, the polypeptides of region A (96KDa, 82KDa, 78KDa, 68KDa) and region D (24KDa, 20KDa, 18KDa) are highly immunoresponsive whereas in case of caput epididymal sperm the same antisera recognized the polypeptides of region B, C and D. By surface labelling with lactoperoxidase iodination and subsequent immunoprecipitation in the iodinated cell extract we demonstrate eight of these above polypeptides (96KDa, 82KDa, 68KDa, 50KDa, 29KDa, 24KDa, 20KDa and 18KDa) as surface antigen. The 96KDa, 82KDa and 68KDa surface polypeptides are highly immunoresponsive than the other lower molecular weight surface antigens in cauda epididymal goat spermatozoa.     

Distribution of filipin-sterol complexes in the plasma membrane of stallion spermatozoa during the epididymal maturation process

The presence and distribution of cholesterol in mature and immature epididymal spermatozoa was analyzed using filipin as a cytochemical tool in freeze-fracture replicas and thin section preparations. The polyenic-antibiotic filipin formed complexes with 3, beta -OH sterols, producing characteristic protrusions, or pits, that were heterogeneously distributed in the plasma membrane of stallion spermatozoa, revealing a specific organization in a functionally specialized area of the gamete. The acrosomal region of the sperm head presented a significantly higher density of filipin sterol complexes than the postacrosomal region, which was usually free of these complexes. The plasma membrane of the flagellum also showed filipin sterol complexes randomly distributed in freeze-fracture replicas. The strong filipin labeling observed in the membrane of spermatozoa obtained from the caput region of the epididymis decreased significantly during epididymal passage. The significance of these changes is not completely understood, but they might contribute to establishing the molecular organization necessary for sperm transit and storage in the epididymis as well as to development of motile spermatozoa that are able to fertilize the oocyte and induce normal embryonic development.     

Rabbit epididymal secretory proteins. I. Characterization and hormonal regulation

Analyses of samples of luminal fluid from the rete testis, distal efferent ducts, and epididymal regions 2-5 and 8 revealed that 91% of the fluid leaving the testis is reabsorbed by the efferent ducts, 79% of the remainder is reabsorbed proximal to epididymal regions 4 and 5, and there is a net secretion of fluid into the duct caudally. There is a net reabsorption by the efferent ducts of 73% of the protein leaving the testis and then a net secretion along the epididymis. SDS-PAGE of the luminal fluids indicated that four new protein bands that were not present in blood appeared in the efferent ducts, 5 in epididymal regions 1-5, 6 in regions 6 and 7, and one in region 8. Two bands in samples from the efferent ducts were absent caudally, and one band present in region 7 was absent in region 8. The rates of incorporation of (35)S-methionine into minced duct in vitro varied among regions when expressed per milligram of wet weight of tissue (region 2-5 > region 7 > region 6 > region 1 > region 8 > ductuli efferentes), and orchidectomy had little effect on the rates. Incorporation into four proteins that were secreted in vitro (M(r) 38 000, 20 000, 15 000, and 13 000) was reduced or abolished by orchidectomy and restored by testosterone therapy. The secretion of three proteins (M(r) 52 000, 23 000, and 22 000) was reduced or abolished by orchidectomy and not restored by testosterone therapy. SDS-PAGE of detergent extracts of sperm indicated that five proteins were lost and nine were gained during epididymal transit. Seven of the proteins gained were about the same molecular weight as proteins secreted by the epididymis (M(r) 94 000, 52 000, 38 000, 36 000, 22 000, 20 000, and 13 000) and were analyzed using N-terminal amino acid microsequencing.     

Studies on the binding of a 32K rat epididymal protein to rat epididymal spermatozoa

A glycoprotein of molecular weight 32K has been isolated and purified from the rat caudal epididymal fluid by gel filtration, ion-exchange and affinity chromatography. The highly purified protein was labeled with radioactive iodine and the binding of the 125I-labeled 32K rat epididymal protein (REP) to washed rat caudal epididymal sperm was studied under various conditions. Scatchard plots of the binding data revealed two binding kinetics. One bound with high affinity (KD = 2.6 X 10(-10) ) but low capacity. The other bound with lower affinity (KD = 2.2 X 10(-9)M) but high capacity. The rate of binding of the labeled protein to sperm was dependent on the temperature of the incubation medium. At the scrotal temperature of 33 degrees C, maximal binding was obtained after 40 min. However, at 22 degrees C equilibrium state was reached after 90 min and at 0 degrees C, the equilibrium rate was not reached even after 120 min of incubation. Binding showed dependence on extracellular pH (optimal pH at 4) and ionic strength of the incubation medium. High ionic strength was found to inhibit binding of the 125I-labeled 32K REP to rat caudal epididymal sperm. Specific binding was abolished by 100-fold molar excess unlabeled 32K REP or by native rat caudal epididymal fluid proteins, but not by albumin or ovalbumin. This indicates high specificity of binding. This study has provided direct evidence for the interaction of an epididymal protein with epididymal spermatozoa.     

Loss of plasma membrane proteins of bull spermatozoa through the freezing-thawing process

The widespread application of A. I. and realization of its full potential depends largely on the use of frozen semen. However, fertility resulting from A. I. is poorer than that from fresh semen in most species. The objective of this study was to compare the protein composition of fresh and frozen-thawed bull sperm plasma membrane surface. The effect of Tween 20 on protein removal from fresh and frozen sperm plasma membrane surface was studied and compared. The effect of incubation with different detergent concentrations on sperm motility and viability was examined. Approximately 2 x 10(8) frozen-thawed bull spermatozoa washed through a discontinuous Percoll gradient were incubated for 15 min at 20 degrees C with 0.01, 0.03 and 0.05% Tween 20. Sperm motility was completely eliminated at all 3 assayed detergent concentrations, while the initial sperm viability of 52% was decreased to 26, 10 and 5%, respectively, at the 3 concentrations. The removal of sperm plasma membrane proteins also increased from 0.72 mg to 2 mg with 0.05% Tween 20. Similar results were found with fresh semen samples. Although the amount of extracted proteins was significantly lower than that obtained with frozen spermatozoa, fresh sperm motility was likewise eliminated by the detergent treatment, and sperm viability was decreased. A semen sample with an initial sperm viability of 59% had a value of only 8% after treatment with 0.05% Tween 20. Comparative SDS-PAGE analysis of the extracted fractions from fresh and frozen-thawed semen treated with Tween 20 showed that the higher amount of extracted proteins in the frozen semen samples corresponded to the egg yolk lipoproteins in the cryoprotectant medium. However, it is worth noting that 4 more bands were found in the sample obtained from fresh semen than from frozen semen. These results indicate that some cell membrane proteins are lost through the freezing-thawing process.     

Gossypol-induced modifications in the microenvironment of rat epididymal spermatozoa

Gossypol acetic acid (20, 25 or 30 mg/kg/day orally for 5 weeks) decreased epididymal weight in adult Sprague-Dawley rats but the epididymal concentrations of proteins, lactate dehydrogenase and acid phosphatase were unchanged. The concentrations of carnitine, inositol and potassium in epididymal fluid were decreased in a dose-related manner. These modifications were not due to disturbances of Leydig and Sertoli cell functions which were normal. We suggest that the reduction in epididymal secretion results from a decrease in the number of spermatozoa rather than from a direct action of gossypol on the epididymal epithelium.     

Semen plasma proteins prevent cold-shock membrane damage to ram spermatozoa

Although the effect of semen plasma on the function of spermatozoa has been widely studied, results are contradictory. We showed that semen plasma proteins are adsorbed onto the cold-shocked ram sperm surface, and that this adsorption is able to reverse the membrane alterations induced by cold-shock. In the present study we evaluate whether the addition of semen plasma proteins before the cold-shock would prevent membrane damage and maintain ram sperm viability. Ram spermatozoa freed from semen plasma by a dextran/swim-up procedure were strongly affected by the cold-shock treatment, lowering cell viability (membrane integrity by fluorescence markers) from 72.2+/-3.4% to 24.6+/-2.1%. Adding semen plasma proteins (> 3 kDa) to the medium before the cold treatment had an immediate beneficial effect on sperm survival in all samples. This effect was concentration-dependent, since the percentage of membrane-intact spermatozoa increased significantly with increased protein concentration in the incubation medium. The highest concentration of proteins (2.1 mg) continued to protect the membranes after 1 h of incubation at 20 degrees C while lower concentrations (0.7 and 1.4 mg) showed a slight decline. Inclusion of linoleic-oleic acids had a beneficial effect on preserving sperm viability when 25, 37 or 75 microM linoleic-oleic acids were added. There was a positive interaction between fatty acids and semen plasma proteins. Thus, the addition of 25 microM oleic-linoleic acid in the presence of 2.1 mg semen plasma proteins accounted for an increase in viability up to 50.7% significance (P < 0.001) relative to the control sample (25%). Likewise, semen plasma proteins significantly promoted the ability of Vitamin E (alpha-tocopherol phosphate) to improve sperm survival. A 26% viability value obtained after cold-shock in the control sample significantly increased (P < 0.001) up to 57% in the sample with 1.6 mM Vitamin E phosphate and 2.1 mg semen plasma proteins (0 h). This study demonstrates that impaired function of cold-shocked ram spermatozoa freed from semen plasma could be prevented by addition of semen plasma proteins, resulting in higher maintained viability values. Inclusion of either linoleic-oleic acids or vitamin E together with semen plasma proteins would increase the improvement in ram spermatozoa survival.