Calcium-dependent actin filament-severing protein scinderin levels and localization in bovine testis, epididymis, and spermatozoa

We assessed the levels and localization of the actin filament-severing protein scinderin, in fetal and adult bovine testes, and in spermatozoa during and following the epididymal transit. We performed immunoblots on seminiferous tubules and interstitial cells isolated by enzymatic digestion, and on bovine chromaffin cells, spermatozoa, aorta, and vena cava. Immunoperoxidase labeling was done on Bouin's perfusion-fixed testes and epididymis tissue sections, and on spermatozoa. In addition, immunofluorescence labeling was done on spermatozoa. Immunoblots showed one 80-kDa band in chromaffin cells, fetal and adult tubules, interstitial cells, spermatozoa, aorta, and vena cava. Scinderin levels were higher in fetal than in adult seminiferous tubules but showed no difference between fetal and adult interstitial cells. Scinderin levels were higher in epididymal than in ejaculated spermatozoa. Scinderin was detected in a region corresponding with the subacrosomal space in the round spermatids and with the acrosome in the elongated spermatids. In epididymal spermatozoa, scinderin was localized to the anterior acrosome and the equatorial segment, but in ejaculated spermatozoa, the protein appeared in the acrosome and the post-equatorial segment of the head. In Sertoli cells, scinderin was detected near the cell surface and within the cytoplasm, where it accumulated near the base in a stage-specific manner. In the epididymis, scinderin was localized next to the surface of the cells; in the tail, it collected near the base of the principal cells. In Sertoli cells and epididymal cells, scinderin may contribute to the regulation of tight junctional permeability and to the release of the elongated spermatids by controlling the state of perijunctional actin. In germ cells, scinderin may assist in the shaping of the developing acrosome and influence the fertility of the spermatozoa.     

Seminal plasma proteins and the reaction of spermatozoa from intact boars and from boars without seminal vesicles to cooling.

Spermatozoa from intact boars and from boars without seminal vesicles were resuspected in diluent and cooled at different rates to 0 degrees C. Glutamic oxaloacetic transaminase and lactate dehydrogenase activities were greater in the diluents which had contained spermatozoa from intact boars than in those which contained spermatozoa from animals without seminal vesicles. The incubation of seminal plasma from an intact boar with spermatozoa from a vesiculectomized animal before cooling also increased the enzyme activity in the diluent. The factors responsible for this effect were associated with the basic protein fractions of boar seminal plasma, in particular the proteins with haemagglutinating activity which may have been adsorbed onto the spermatozoa. Spermatozoa were exposed to colloidal Fe(OH)2+ to determine by electron microscopy the charge on the surface of the plasma membrane of washed epididymal spermatozoa and ejaculated spermatozoa from intact and vesiculectomized boars. Epididymal spermatozoa bound the positively charged particles more readily than the ejaculated spermatozoa from the intact boars, due to the absence of membrane-bound protein.     

Calcium-binding phosphoprotein: the principal acidic protein of mammalian sperm

A calcium-binding phosphoprotein previously found only in brain and adrenal medulla has been isolated from the adult bovine testis. The testicular protein is electrophoretically indistinguishable from the protein of adult bovine brain and adrenal medulla in 15% polyacrylamide gels at pH 8.3 and 4.7. Crude homogenates and acidic protein fractions of adult testis, fetal testis and epididymal spermatozoa were examined electrophoretically for the presence of this calcium-binding protein. The protein was present in homogenates of adult testis and epididymal spermatozoa but only to limited extent in the homogenate of fetal testis. It was the major acidic protein of spermatozoa. It appears likely that the calcium-binding protein evident in adult testicular tissue is contributed largely by the developing spermatozoa.     

Transformation of sperm histone during formation and maturation of rat spermatozoa.

Changes of chromosomal basic proteins of rats have been followed during transformation of spermatids into spermatozoa in the testis and during maturation of spermatozoa in the epididymis. Rat testis chromatin has been fractionated on the basis of differing sensitivity to shearing, yielding a soluble fraction and a condensed fraction. The sperm histone is found in the condense fraction. Somatic-type histones are found in both fractions. The somatic-type histones in the condensed fraction contains much more lysine-rich histone I, than does the somatic-type histones in the soluble fraction. This may suggest that the lysine-rich histone I is the last histone to be displaced during the replacement of somatic-type histones by sperm histone. After extensive shearing followed by sucrose centrifugation, the condensed portion of testis chromatin can be further fractionated into two morphologically distinctive fractions. One is a heavy fraction possessing an elongated shape typical of the head of late spermatids. The other is a light fraction which is presumably derived from spermatids at earlier stages of chromatin condensation and which is seen as a beaded structure in the light microscope. Sperm histone of testis chromatin can be extractable completely by guanidinium chloride without a thiol, wheras 2-mercaptoethanol is required for extraction of sperm histone from caput and cauda epididymal spermatozoa. The light fraction of the condensed testis chromatin contains unmodified and monophospho-sperm histone. The sperm histones of the heavy fraction is mainly of monophospho and diphospho species, whereas unmodified and monophosphosperm histones are found in caput and cauda epididymal spermatozoa. Labeling of cysteine sulfhydryl groups of sperm histone releases by 2-mercaptoethanol treatment shows that essentially all of the cysteine residues of sperm histone in testis chromatin are present as sulfhydryl groups, while those of sperm histone isolated from mature (cauda epididymal) spermatozoa are present as disulfide forms and approximately 50% of the cysteine residues of sperm histone obtained from caput epididymal spermatozoa are in disulfide forms. These results suggest that phosphorylation of sperm histone is involved in the process of chromatin condensation during transformation of spermatozoa in the epididymis.     

Biochemical and binding characteristics of boar epididymal fluid proteins

During the passage through the epididymis, testicular spermatozoa are directly exposed to epididymal fluid and undergo maturation. Proteins and glycoproteins of epididymal fluid may be adsorbed on the sperm surface and participate in the sperm maturation process, potentially in sperm capacitation, gamete recognition, binding and fusion. In present study, we separated proteins from boar epididymal fluid and tested their binding abilities. Boar epididymal fluid proteins were separated by size exclusion chromatography and by high-performance liquid chromatography with reverse phase (RP HPLC). The protein fractions were characterized by SDS-electrophoresis and the electrophoretic separated proteins after transfer to nitrocellulose membranes were tested for the interaction with biotin-labeled ligands: glycoproteins of zona pellucida (ZP), hyaluronic acid and heparin. Simultaneously, changes in the interaction of epididymal spermatozoa with biotin-labeled ligands after pre-incubation with epididymal fluid fractions were studied on microtiter plates by the ELBA (enzyme-linked binding assay) test. The affinity of some low-molecular-mass epididymal proteins (12-17 kDa and 23 kDa) to heparin and hyaluronic acid suggests their binding ability to oviductal proteoglycans of the porcine oviduct and a possible role during sperm capacitation. Epididymal proteins of 12-18 kDa interacted with ZP glycoproteins. One of them was identified as Crisp3-like protein. The method using microtiter plates showed the ability of epididymal fluid fractions to change the interaction of the epididymal sperm surface with biotin-labeled ligands (ZP glycoproteins, hyaluronic acid and heparin). These findings indicate that some epididymal fluid proteins are bound to the sperm surface during epididymal maturation and might play a role in the sperm capacitation or the sperm-zona pellucida binding.     

Rat sperm enzymes during epididymal transit

The activities of cAMP and cGMP phosphodiesterases (EC 3.1.4.1), adenylate cyclase (EC 4.6.1.1) and protein carboxyl-methylase (EC 2.1.1.24) were measured in the particulate and soluble (105 000 g supernatant) fractions of washed spermatozoa isolated from five segments of the adult rat epididymis. The activities of both phosphodiesterases decreased during epididymal transit, whereas adenylate cyclase and protein carboxyl-methylase underwent a progressive increase, the latter showing the most marked alteration. Both cAMP and cGMP phosphodiesterases as well as the adenylate cyclase were all associated primarily with the particulate fraction, and the extent to which these enzymes were associated with the membranes increased as the spermatozoa passed through the epididymis. Sperm protein carboxyl-methylase activity was, on the other hand, predominantly soluble in all segments of the epididymis. Adenylate cyclase, cAMP phosphodiesterase and protein carboxyl-methylase activities were found predominantly in the sperm tails, whereas cGMP phosphodiesterase was equally distributed between heads and tails. These observations imply that the acknowledged increase in intracellular cAMP levels which occurs in spermatozoa during epididymal transit may be a consequence of both increased synthesis (adenylate cyclase) and reduced hydrolysis (phosphodiesterase).     

Antibodies against epididymal glycoproteins block fertilizing ability in rat

Antiserum against rat androgen-dependent secretory epididymal protein DE (raised in rabbit) was added to suspensions of rat spermatozoa from the cauda epididymidis which were used for artificial insemination. While control spermatozoa fertilized 41.6% of oocytes, those exposed to antiserum to protein DE fertilized only 6.6% (P less than 0.01). An equal amount of normal rabbit serum (NRS) did not cause inhibition (33.1%). To study the entry of antibodies into the epididymis, caudal tubules were cultured for 24 h and the fertility of the contained spermatozoa was assessed by artificial insemination. Culture in Medium 199 alone or with NRS resulted in spermatozoa which fertilized 52% of oocytes while the presence of antiserum to protein DE in the culture medium yielded spermatozoa which fertilized only 16.6% of oocytes (P less than 0.01). These results suggest (1) that the epididymal protein DE might be part of a sperm structure involved in the fertilization process, and (2) that, at least under the present culture conditions, immunoglobulins penetrate the epididymal epithelium in sufficient numbers to reduce fertility significantly.     

Immunochemical localization of secretory antigens in the human epididymis and their association with spermatozoa

Ejaculated spermatozoa were washed and extracted with 0.6 M NaCl (2 h at 0 degree C) and the extract used to immunize rabbits. The crude antibody reacted with epididymal fluid and cytosol and with prostatic cytosol but did not recognize blood serum and testicular cytosol. After adsorption with prostatic proteins, the serum was specific for epididymis. Using immunoelectrophoresis and affinity chromatography, it was found that the antibody reacted with antigens which co-electrophoresed with androgen-dependent proteins (mobility relative to albumin, Ra) 0.3, 0.43 and 1.0, previously identified in human epididymis. Weak immunofluorescence in the epithelium of proximal caput tubules was detected on tissue sections. In contrast, distal caput and corpus tubules displayed a strong fluorescence in the cytoplasm of basal and principal cells as well as in spermatozoa present in lumen. Intense fluorescence was limited to the luminal content and the apical border and sterociliae of principal cells in caudal tubules. When applied to isolated spermatozoa, the reaction was negative for testicular sperm, while 49%, 82% and 100% of spermatozoa from caput, corpus and cauda, respectively, had a fluorescent acrosomal cap. An apparent gradient of increasing fluorescent intensities was also observed in this sequence. The reaction was strongest over the acrosomal cap, apparently absent in the postacrosomal region and weaker over the midpiece and principal piece. These results are interpreted as suggestive of the progressive coating of human spermatozoa with androgen-dependent epididymal proteins during epididymal transit.     

Lactate dehydrogenase activity of rat epididymis and spermatozoa: effect of constant light

During its passage through the epididymis, the gamete undergoes a process of "maturation" leading to the acquisition of its fertilizing ability. The epididymis displays regional variations in the morphology and metabolic properties of its epithelium which are relevant for the progressive development of mature sperm characteristics. The epididymis has spontaneous peristaltic contractions and receives sympathetic innervation that is modulated by melatonin, a hormone synthesized and released by the pineal gland. Constant lighting disrupts melatonin synthesis and secretion. We have studied the effect of constant light on lactate dehydrogenase (LDH; EC 1.1.1.27) and its isozyme C4 activities and protein content in whole epididymis, epididymal tissue and in spermatozoa from caput and cauda segments. Animals were exposed from birth to an illumination schedule of 14 h light:10 h dark (group L:D). At 60 days of age one group of animals was submitted to constant light over 50 days (group L:L). In order to test the fertilizing ability, the rats of each group were mated with soliciting estrous females. The percentage of pregnancies in females mated with males maintained in L:L was remarkably lower than those in females mated with males maintained in the L:D photoperiod (44% and 88% respectively). Constant light increased protein concentration and LDH activity in caput as well as in cauda of total epididymis. On the contrary, in epididymal tissue, the protein content decreased in both epididymal sections compared with controls. When enzymatic activity was expressed in Units per spermatozoa, constant light induced a significant reduction of total LDH and LDHC4 in caput and cauda spermatozoa while LDH activity of epididymal tissue was not affected. In spite of the decrease in LDH per sperm cell when rats were exposed to constant light, in total epididymis (epididymis tissue plus sperm cells content) and in spermatozoa, values of enzyme activities expressed per weight unit were higher than those of controls. This is explained by the increase in the amount of stored spermatozoa, both in caput and cauda, produced by exposure of animals to constant light. Our results confirm that in rats, chronic exposure to constant light promotes a reduction of fertilizing ability and indicates that continuous lighting reduces the total LDH and LDHC4 activities, possibly due to moderate aging of spermatozoa within the duct by lengthening of the sperm transit through the epididymis.     

Effects of glucose and insulin on the activation of lipoprotin lipase and on protein-synthesis in rat adipose tissue.

Glucose, and certain sugars that can readily be converted to glucose 6-phosphate, bring about an activation of adipose-tissue lipoprotein lipase when epididymal fat-bodies from starved rats are incubated in the presence of cycloheximide. Other substrates do not support the activation. If the tissue is preincubated in the presence of cycloheximide for longer than 2h, the ability of added glucose to activate the enzyme is lost. On the other hand, the addition of glucose still brings about an increase in lipoprotein lipase activity after preincubation in the absence of cycloheximide for as long as 4h. The magnitude of the increase in enzyme activity brought about by the addition of glucose is increased when protein synthesis is stimulated during the preincubation period by insulin. The results are interpreted in terms of the existence in adipose tissue of a proenzyme pool of lipoprotein lipase that is normally maintained by protein synthesis and that is converted to complete enzyme of higher specific activity by a process that specifically requires glucose.     

Expression, activity, and subcellular localization of testicular hormone-sensitive lipase during postnatal development in the guinea pig

The present work reports on testicular hormone-sensitive lipase (HSL), the biological significance of which has been documented in male fertility. The HSL protein levels and enzymatic activity were measured, respectively, by densitometry of immunoreactive bands in Western blots, performed with antibodies against recombinant rat HSL, and by spectrophotometry in seminiferous tubules (STf) and interstitial tissue (ITf) enriched fractions generated from neonatal, pubertal, and adult guinea pig testes. In addition, HSL was studied in subcellular fractions obtained from STf isolated from adult testes and in epididymal spermatozoa (Spz). A 104-kDa HSL protein was detected in STf and ITf, the expression and activity of which increased with testicular development. Three immunoreactive bands of 104, 110, and 120 kDa were detected in the lysosomal subfraction, and two bands of 104 and 120 kDa were detected in Spz. The HSL activity was positively correlated with free (FC) and esterified (EC) cholesterol ratios in STf and ITf, but not with triglyceride (TG) levels, during testicular development. Immunolabeling localized HSL to elongated spermatids and Sertoli cells, where its distribution was stage-dependent, and within the cells lining the excurrent ducts of the testis. The findings of the 104- and 120-kDa HSL immunoreactive bands and of HSL activity in Spz as well, as the detection of the 104-, 110-, and 120-kDa immunoreactive bands in lysosomes, suggest that part of HSL may originate from germ cells and be imported in Sertoli cells. The HSL protein levels and enzymatic activity in ITf and STf were positively correlated with serum testosterone levels during development. To the best of our knowledge, this study is the first to contribute insights regarding the impact of HSL on FC:EC cholesterol ratios and TG levels in the interstitial tissue and tubules in relation to serum testosterone levels during postnatal development, and regarding the immunolocalization of the enzyme in regions of the male gamete consistent with spermatozoa-oocyte interaction.     

Binding of a 15 kDa glycoprotein from spermatozoa of boars to surface of zona pellucida and cumulus oophorus cells.

A highly purified 15 kDa glycoprotein isolated from ejaculated spermatozoa was used to raise antisera in female rabbits. An indirect immunofluorescence technique was used to detect the antigen in the seminal vesicle tissue and on the acrosomes of ejaculated, native and capacitated, boar spermatozoa. No immunoreactivity was detected on cells of the seminiferous tubules (spermatogonia, spermatocytes, and spermatids), on spermatozoa in the ductus epididymis and in cells of the epididymal and testicular tissues. These observations support the view that the 15 kDa protein is produced in the seminal vesicle secretory epithelium, and is attached to the sperm plasma membrane during the exposure of spermatozoa to seminal vesicle compounds. The observations that the antigen remained on the acrosome of ejaculated spermatozoa after capacitation and blocked sperm-oocyte binding in vitro suggest that the antigen plays a role in sperm-egg interactions. The strong immunoreactivity exhibited by cumulus cells after incubation of antisera with the porcine egg surrounded by cumulus cells shows the possible importance of the 15 kDa glycoprotein for contact of spermatozoa with cells of the cumulus oophorus surrounding the egg.     

The effect of insulin on the synthesis of lipoprotein lipase in rat adipose tissue

A novel method is described for measuring the incorporation of radiolabelled amino acids into rat adipose tissue lipoprotein lipase (LPL) in vitro. Following the incubation of epididymal fat-bodies in the presence of [3H]leucine, the radiolabelled enzyme was isolated from extracts of the delipidated tissue, in a single step, by affinity chromatography on heparin-Sepharose, SDS-PAGE of such purified enzyme preparations revealed the presence of a single radiolabelled polypeptide of molecular weight 56 000, corresponding to LPL. In the presence of insulin, the rates of incorporation into LPL and into total tissue protein were increased respectively by 2.3 fold and 1.7 fold, compared to controls. It is concluded that part of the increase in incorporation into LPL is due to the general stimulus of protein synthesis in the tissue by insulin. Additionally insulin may either specifically increase the rate of synthesis or decrease the rate of degradation of the enzyme.     

Proteins of demembraned mouse sperm heads. Characterization of a major sperm-unique component

A 15-kilodalton protein has been identified as a major component of the residual protein fraction of mouse epididymal/vas spermatozoal heads, demembraned by treatment with Triton X-100 and sequentially extracted with 1 M NaCl/2-mercaptoethanol/DNase I. Two-dimensional electrophoresis of that protein before and after treatment with alkaline phosphatase indicated that it is present in epididymal/vas spermatozoa as a series of five differentially phosphorylated molecules with pI 6.0-7.0. Cyto-immunofluorescence with an affinity-purified antibody to the 15-kDa protein localized that protein to a circumscribed region of the demembraned mouse sperm head mediad from the dorsal margin. By radioimmunoassay, the 15-kDa protein was shown to be sperm-unique and species-specific. The antibody was nonreactive with homogenates of meiotic spermatogenic cells and round spermatids (stages 1-11) but was reactive with a non-phosphorylated 15.5-kDa protein of elongating spermatids (stages 12-16) and testicular spermatozoa. Following alkaline phosphatase treatment, the spermatozoal 15-kDa protein migrated to the position of the spermatidal 15.5-kDa protein on a sodium dodecyl sulfate gel. Thus, we conclude that the 15-kDa protein of mouse spermatozoa is synthesized during the elongation phase of spermiogenesis (stages 12-16) and is phosphorylated in the terminal period of that phase and/or after excursion of spermatozoa from the seminiferous tubules.     

Immunolocalization of CRES (Cystatin-related epididymal spermatogenic) protein in the acrosomes of mouse spermatozoa.

The CRES (cystatin-related epididymal spermatogenic) protein is a member of the cystatin superfamily of cysteine protease inhibitors and exhibits highly restricted expression in the reproductive tract. We have previously shown that CRES protein is present in elongating spermatids in the testis and is synthesized and secreted by the proximal caput epididymal epithelium. The presence of CRES protein in developing germ cells and in the luminal fluid surrounding maturing spermatozoa prompted us to examine whether CRES protein is associated with spermatozoa. In the studies presented, indirect immunofluorescence, immunogold electron microscopy, and Western blot analysis demonstrated that CRES protein is localized in sperm acrosomes and is released during the acrosome reaction. Interestingly, while the 19- and 14-kDa CRES proteins were present in testicular and proximal caput epididymal spermatozoa, the 14-kDa CRES protein was the predominant form present in mid-caput to cauda epididymal spermatozoa. Furthermore, following the ionophore-induced acrosome reaction, CRES protein localization was similar to that of proacrosin/acrosin in that it was detected in the soluble fraction as well as associated with the acrosome-reacted spermatozoa. The presence of CRES protein in the sperm acrosome, a site of high hydrolytic and proteolytic activity, suggests that CRES may play a role in the regulation of intraacrosomal protein processing or may be involved in fertilization.     

Protein thiols in spermatozoa and epididymal fluid of rats.

Thiol (SH) oxidation to disulphides (SS) is thought to be involved in sperm chromatin condensation and tail structure stabilization, which occur during maturation of spermatozoa. Previously developed procedures, using the fluorescent labelling agent monobromobimane (mBBr), enabled us to study the thiol-disulphide status of spermatozoa. Electrophoretic separation of labelled sperm proteins from the caput and cauda regions showed that during maturation thiol oxidation occurs in many protein fractions from the tail and that the magnitude of oxidation differs between proteins. Among the protein bands, one major band (MPB), probably a dense fibre constituent, is quantitatively prominent. N-Ethylmaleimide (NEM) or mBBr alkylation (of intact spermatozoa) changes the mobility of the caput MPB, but not that of the cauda MPB. The results indicated that the altered mobility of MPB is mainly due to a change in its shape, possibly resulting from the alkylation of a few critical SH groups. Epididymal fluid proteins contain both SH and SS. The thiol and disulphide content of the various epididymal proteins appears similar, although some diminution in fluorescence is seen in epididymal fluid proteins from the cauda region as compared with those from the caput region. The prominent changes in thiol status occur in the spermatozoa.     

Effects of insulin, glucocorticoids and adrenaline on the activity of rat adipose-tissue lipoprotein lipids.

The lipoprotein lipase activity of epididymal fat-bodies from starved rats was measured during incubations at 37 degrees C in vitro. Protein synthesis independent activation of the enzyme, previously observed during incubations at 25 decrease C, also occurs at 37 degrees C. Protein-synthesis-dependent increases in the activity of the enzyme occur in the presence of insulin and are markedly potentiated by glucocorticoids. The effects on the activity of the enzyme of insulin alone, or in the presence of glucocorticoids, are correlated with its effects on total protein synthesis in the tissue. Adrenaline antagonizes the increase in activity of the enzyme brought about by insulin and abolishes the potentiation of insulin action by glucocorticoids. These changes may be due, at least in part, to its stimulation of inactivation of the enzyme in the tissue. It is suggested that changes in adipose-tissue lipoprotein lipase activity that occur with changes in nutritional status in vivo result from the combined effects of changes in plasma insulin and glucocorticoid concentrations.     

Altered molecular dynamics and antioxidant status in the spermatozoa in testosterone-induced oligospermia in mouse

Though supraphysiological doses testosterone (T) and its derivatives are known to suppress spermatogenesis in mammals by interfering with the hypothalamus-pituitary axis leading to oligozoospermia, no study has been performed to evaluate the integrity of the sperm cells produced by such individuals. In T-induced oligozoospermia in the mouse, the spermatozoa showed suppressed zona-binding ability though the motility and viability remained unchanged. In order to assess whether this decreased zona-binding ability is due to perturbations in the mechanical properties of the sperm membranes, we attempted to examine the molecular dynamics employing a lipophilic spin label (16-doxyl stearate) and a protein-binding label (Mal-Net) in two sets of independent experiments. The results showed that the rotational freedom of lipophilic molecules reduced significantly within the first week of T-treatment. During weeks 1 through 4, the protein rotation was found to be retarded significantly. We observed a sharp increase in the ascorbyl radical associated with the cauda epididymal spermatozoa and epididymal fluid of testosterone-treated mice. Moreover, the glutathione (GSH) content in the spermatozoa and the epididymal fluid increased significantly after testosterone-treatment. Further, there was a elevation in the superoxide dismutase (SOD) activity and suppression in the superoxide anion radical generated by the cauda epididymal spermatozoa of testosterone-treated animals. A change in the mechanical properties of a bilayer could modify both the mechanical properties and the function of incorporated proteins. In many instances, a liquid-crystalline bilayer is necessary for protein function. It is likely that the change in the physical properties of sperm membranes might cause the inhibition of enzymes associated with spermatozoa after T-treatment. The alterations in the sperm membrane structure and the antioxidant potentials of both the spermatozoa and the cauda epididymal fluid could also account for the decrease in the zona-binding index of the spermatozoa in T-treated animals. Thus, this study demonstrates for the first time that supraphysiological doses of testosterone could modify the mechano-dynamic properties of sperm membranes and could perturb the redox status of both spermatozoa and the epididymal fluid.     

Spermatozoa modulate epididymal cell proliferation and protein secretion in vitro

Normal epididymal function, such as protein expression and secretion, is primarily regulated by testicular androgens and temperature. However, the role of spermatozoa in this critical process has never been studied. In order to determine whether sperm itself could regulate epididymal function, we have developed a cell culture system of bovine epididymal cells to study the interactions between spermatozoa and the epididymal epithelium. Primary cells from caput, corpus, and cauda epididymal tissues were cultured in the presence of androgens at 32 degrees C (scrotal) and 37 degrees C (abdominal). Newly synthesized proteins were metabolically labeled with (35)S-methionine after sperm co-incubation and the pattern of secreted proteins was analyzed by two-dimensional polyacrylamide gel electrophoresis. Proliferation rate, protein secretion rate and electrophoretic patterns of secreted proteins were evaluated 48 hr post-co-incubation. Incubation at 32 degrees C indicated that spermatozoa stimulation increases the level of protein secretion of cultured cells from all epididymal sections while it slightly decreases proliferation of corpus cells. At 37 degrees C, spermatozoa co-incubation significantly decreases the protein secretion rate of cultured cells from all epididymal sections. Independently of cell incubation temperature, spermatozoa stimulation induces both an increase in the intensity of radiolabeled proteins and the appearance of new secreted proteins of caput cells without affecting the protein pattern of corpus or cauda cells. Incubation at 37 degrees C, however, greatly modifies the pattern of proteins expressed at 32 degrees C by cauda cells. Taken together, these results support the hypothesis that spermatozoa themselves affect epididymal cell function, most importantly for caput epididymides.