Concentration of acrosome stabilizing factor (ASF) in rabbit epididymal fluid and species specificity of anti-ASF antibodies

Rabbit Acrosome Stabilizing Factor (ASF) concentrations were measured by immunoradiometric assay (IRA) in lumenal fluids obtained by micropuncture from the caput epididymidis, corpus epididymidis, cauda epididymidis, and the vas deferens of the rabbit. ASF was below the limit of detection in caput epididymidal fluids. Average ASF concentrations (3 bucks) in the corpus epididymidis, cauda epididymidis, and vas deferens were 880, 3363, and 3236 micrograms/ml, respectively. The average level of ASF in the cauda epididymidal fluid (CEF) represents from 10 to 23% of the total protein and is at least tenfold more than the amount previously determined to effect complete decapacitation of rabbit sperm by an in vivo assay. The average ASF concentration in seminal plasma from two vasectomized males was 0.155 micrograms/ml, approximately 100,000-fold less than is present in CEF and 2000-fold less than is present in normal seminal plasma. CEFs or seminal plasma from 11 different species were screened by Western blotting using high titer anti-ASF polyclonal antibodies to detect ASF-like molecules in other species. Only rabbit ASF was recognized.     

Heparin-binding proteins from seminal plasma bind to bovine spermatozoa and modulate capacitation by heparin

Bovine spermatozoa that have been exposed to seminal plasma possess more binding sites for heparin than sperm from the cauda epididymis that have not been exposed to accessory sex gland secretions. Seminal plasma exposure enables sperm, following incubation with heparin, to undergo zonae pellucidae-induced exocytosis of the acrosome. In this study, the regulatory role of seminal plasma heparin-binding proteins in capacitation of bovine spermatozoa by heparin was investigated. Plasma membranes from sperm exposed to seminal plasma in vivo or in vitro contained a series of acidic 15-17 kDa proteins not found in cauda epididymal sperm. Western blots of membrane proteins indicated that these 15-17 kDa proteins bound [125I]-heparin. Heparin-binding proteins were isolated by heparin affinity chromatography from seminal plasma from vasectomized bulls. Gel electrophoresis indicated that the heparin-binding peaks contained 14-18 kDa proteins with isoelectric variation, a basic 24 kDa protein, and a 31 kDa protein. Western blots probed with [125I]-heparin confirmed the ability of each of these proteins to bind heparin. Each of these proteins, as well as control proteins, bound to epididymal sperm. The seminal plasma proteins were peripherally associated with sperm since they were removed by hypertonic medium and did not segregate into the detergent phase of Triton X-114. Seminal plasma heparin-binding proteins potentiated zonae pellucidae-induced acrosome reactions in epididymal sperm. However, seminal plasma proteins that did not bind to the heparin affinity column were unable to stimulate zonae-sensitivity. Control proteins, including lysozyme--which binds to both heparin and sperm, were ineffective at enhancing zonae-induced acrosome reactions. These data provide evidence for a positive regulatory role of seminal plasma heparin-binding proteins in capacitation of bovine spermatozoa.     

Major contribution of epididymis to alpha-glucosidase content of ram seminal plasma

Acid alpha-glucosidase and L-carnitine (a well-known epididymal marker) were measured in rete testis and epididymal fluids of adult male rams. These fluids were collected by selective catheterization or by a micropuncture technique, respectively. Both parameters remained at a low and constant level in rete testis and all along caput and corpus epididymidis. Then they increased at equivalent rates in cauda epididymidis to much higher levels than those in seminal plasma (5 mU/mg protein and 10 mM, respectively). An optimum pH study of alpha-glucosidase activity in these fluids showed two well-separated peaks in rete testis and caput epididymal fluids around pH 4 and 7, respectively, but only a single peak at pH 4 in cauda epididymidis that was comparable to the one in seminal plasma. Sucrose density gradient fractions analyzed for their enzyme content in the absence or presence of sodium dodecyl sulfate (1% w/v), a selective inhibitor of acid alpha-glucosidase activity, allowed the demonstration of two enzyme forms at pH 6.8 in rete testis fluid sedimenting in the 7S and 4S regions of the gradient, while a unique 4S form was encountered in cauda epididymidis and in seminal plasma. Although the fate of the minor 7S component of the rete testis fluid in its epididymal transit is presently unknown, similarities between the enzyme in cauda epididymidis and seminal plasma are strong enough to support the hypothesis that epididymis contributes primarily to the acid alpha-glucosidase content of ram seminal plasma.     

Sodium,potassium, and protein concentrations and 2D-SDS-page of epididymal luminal and ejaculated seminal fluids of the adult chimpanzee (Pan troglodytes)

The concentration of soluble protein and of sodium and potassium ions was estimated in chimpanzee caput epididymal luminal fluid, cauda epididymal luminal fluid, and ejaculated seminal fluid. Protein concentration was 48.5 ± 1.5 μg/μl in caput fluid, 26.8 ± 2.0 μg/μl in cauda fluid, and 53.0 ± 7.9 μg/μl in seminal fluid. Sodium concentration was 127.0 ± 7.0 mM in caput fluid, 34.5 ± 1.8 mM in cauda fluid, and 18.8 ± 1.8 mM in seminal fluid. Potassium concentration was 58.0 ± 0.0 mM in caput fluid, 56.8 ± 5.2 mM in cauda fluid, and 77.0 ± 1.7 mM in seminal fluid. Proteins in caput epididymal, cauda epididymal, and ejaculated seminal fluids, with approximate molecular weights (kDa) between <14.4 and 45.0 kDa and apparent isoelectric points (pIs) between 4.5 and 7.5, were resolved by two-dimensional SDS-polyacrylamide gel electrophoresis (2D-SDS-PAGE) and silver stained. In caput fluid, the most intensely stained polypeptides resolved between 14.4 and 21.5 kDa (pI 5.4–7.4). In cauda fluid, the number and intensity of stained components increased markedly, and the most intensely stained polypeptides resolved between 21.5 and 31.0 kDa (pI 5.7–7.3). In seminal fluid, polypeptides between <14.4 and 45.0 kDa (pI 5.7–7.4) appeared characteristically diffuse and distributed. These results demonstrate that the ions and the polypeptides in the luminal microenvironment change significantly along the epididymal duct of the male chimpanzee. © 1994 Wiley-Liss, Inc.     

A Comparative Study on the Chemical Composition of Plasma from the Cauda Epididymidis,Semen Fractions,and Whole Semen in Boars

A comparative study was carried out on the chemical composition of plasma from the cauda epididymidis, semen fractions, and whole semen of boars. A total of 22 boars were used in this study. The boars, which ranged in age from 8 to 14 months, were of Swedish Landrace and Swedish Yorkshire breed. All boars used presented a normal semen picture. A dummy sow and an artificial vagina were employed for semen collection. The semen was collected as whole semen and as semen fractions in 10 nil volumes. The contents of the cauda epididymidis was removed post mortem. The following parameters were investigated: sperm concentration, dry weight of spermatozoa and of seminal plasma, osmotic pressure, sodium, potassium, chloride, inorganic phosphorus, calcium, magnesium, total protein, GOT, GPT, and alkaline phosphatase in seminal plasma. Paper electrophoresis was carried out on seminal plasma. Tlxe results of the analysis are summarized in Tables 1–6. The sperm concentration was approximately 3.2 mill./mm3 in the cauda epididymidis, 1 mill./mm3 in the sperm-richest fraction (II) and 0.25 mill./mm3 in whole semen. The dry weight (expressed in per cent dry matter) of spermatozoa was highest in the cauda epididymidis (25.47 %), showing a tendency to decreasing in semen fractions I—IV and was lowest in whole semen (15.29 %). The per cent dry weight in plasma was higher in the cauda epididymidis (4.56 %) than in semen fraction I (2.20 %). In semen fractions I—IV the per cent dry weight rose from 2.20 (U to 4.51 % and reached the level of approximately 3.80 % in the sperm-free fractions V—VII. The osmotic pressure was significantly higher in the cauda epidi-dymal plasma than in the whole seminal plasma or the seminal plasma fractions. The same phenomenon was observed in a boar where the cauda epididymal content was collected in vivo from a patent established fistula. There appears to be a connection between the per cent dry weight of spermatozoa and the osmotic pressure, which means that the per cent dry weight of the cauda epididymal spermatozoa decreases when mixed with the accessory gland secretions, which have a lower osmotic pressure. The fall in per cent dry weights is thought to be caused by an intake of water. The amount of sodium, chloride and magnesium was higher in ejaculated seminal plasma than in cauda epididymal plasma. The reverse was true for inorganic phosphorus and potassium. Moreover the sperm-free fractions contained more sodium, chlorides and magnesium than the sperm-containing fractions, while the concentration of potassium and inorganic phosphorus was comparatively higher in the sperm-containing fractions. A connection is apparent between sperm concentration and the potassium, inorganic phosphorus and magnesium levels. Statistical analysis of the values of chloride and magnesium revealed significant differences between individual boars for most of the semen fractions. The concentration of plasma proteins in the cauda epididymidis was approximately the same as in whole semen and in the semen fractions except for fraction I, which contained a relatively low concentration. As regards total protein there were significant differences between individual boars in most of the semen fractions as well. The paper electrophoretic pattern of epididymal plasma was different from that of semen plasma. Thus there were three or four distinct components in the cauda epididymidis numbered 1, 2, 3, and 4, and three distinct components in whole seminal plasma numbered 3, 4, and 5, while the sperm-richest semen fractions contained four components (2, 3, 4, and 5) and the others three components, namely 3, 4, and 5. The level of GOT was high in the cautlu cpiflidymill contents (99.1 i. u./ml) compared with that for whole seminal plasma (99.1 i.u/ml). In semen fractions there was a clear positive correlation between the level of GOT and the sperm concentration. The GPT concentration wis as a whole low and. in contrast to GOT. somewhat higher in the sperm-free fractions than in the sperm-containing fractions. The concentration of alkaline phosphatase was very high in cauda epididymal plasma (31,463 i. u./ml) as well as in the sperm-rich fractions (e.g. 7,096 i. u./ml in fraction II). Preliminary investigation has moreover revealed a very low alkaline phosphatase concentration in seminal plasma of vasectomized boars, which condition suggests thai the main origin for alkaline phosphatase in boars is the testis and epididymis.     

Identification and partial characterization of alpha-1,4-glucosidase activity in equine epididymal fluid

The expression of alpha-1,4-glucosidase activity was fluorometrically and electrophoretically assessed in the epididymal fluid and seminal plasma of stallions. alpha-Glucosidase specific activity in the epididymis increased significantly from the proximal caput to the cauda. Stallion epididymal glucosidase maintained activity in a wide range of pH, with two distinct peaks (around pH 4.0 and 6.0, respectively). Enzyme activities at different pH, inhibition assays with sodium dodecyl sulfate (SDS) and maltotriose (MTT, selective inhibitors of alpha-glucosidases "acidic" and "neutral" isoforms, described in other tissues) and the electrophoretic analysis in native and native/SDS-PAGE conditions, indicated that stallion epididymal glucosidase was due to two catalytically active forms. These forms, analyzed by non-denaturing electrophoresis, exhibited different electrophoretic mobility and molecular weight. Samples from the proximal caput of the epididymis were rich in Form II or "neutral" form, whereas the "acid" or Form I seemed to be predominate in the cauda epididymal region. At physiological pH, Form II was predominant in the seminal plasma. The physiological role(s) of these forms is uncertain, but based on their ability to hydrolyze glucosidic linkage, they probably are involved in degradation/modifications of epididymal fluid and/or spermatozoa glycoconjugates, thereby participating in plasma membrane remodeling associated with sperm maturation.     

Identification, proteomic profiling, and origin of ram epididymal fluid exosome-like vesicles

Small membranous vesicles, between 25- and 75-nm diameter, were collected by high-speed centrifugation from the ram cauda epididymal fluid and were found to be normal constituents of this fluid and of the seminal plasma. The SDS-PAGE protein pattern of these vesicles was specific and very different from that of the caudal fluid, seminal plasma, sperm extract, and cytoplasmic droplets. After two-dimensional electrophoresis separation and mass spectrometry analysis, several proteins were identified and grouped into i) membrane-linked enzymes, such as dipeptidyl peptidase IV (DPP-IV), neprilysin (NEP), phosphodiesterase-I (E-NPP3), and protein G-beta; ii) vesicle-associated proteins, such as lactadherin (MFEG8-PAS6/7) and vacuolar ATPase; iii) several cytoskeleton-associated proteins, such as actin, ezrin and annexin; and iv) metabolic enzymes. The presence of some of these proteins as well as several different hydrophobic proteins secreted by the epididymis was further confirmed by immunoblotting. These markers showed that the majority of the vesicles originated from the cauda epididymal region. The physical and biochemical characteristics of these vesicles suggest they are the equivalent of the exosomes secreted by several cell types and epithelium. The main membrane-linked proteins of the vesicles were not retrieved in the extract from cauda or ejaculated sperm, suggesting that these vesicles did not fuse with sperm in vivo.     

Biochemical characterization of two ram cauda epididymal maturation-dependent sperm glycoproteins

Rabbit polyclonal antibodies were raised against ram cauda epididymal sperm proteins solubilized by N-octyl-beta-D-glucopy-ranoside (anti-CESP) and against proteins of the fluid obtained from the cauda epididymidis (anti-CEF). The anti-CESP polyclonal antibody reacted with several bands from 17 to 111 kDa with different regionalization throughout the epididymis. The strongest epitopes at 17 kDa and 23 kDa were restricted to the cauda epididymidis. The anti-CEF polyclonal antibody reacted mainly with a 17-kDa and a 23-kDa compound in the cauda sperm extract. These cauda epididymal 17- and 23-kDa proteins disappeared after orchidectomy, but they reappeared in the same regions after testosterone supplementation, indicating that they were secreted by the epithelium. The fluid and membrane 17- and 23-kDa antigens had a low isoelectric point and were glycosylated. The fluid 17- and 23-kDa proteins had hydrophobic properties: they were highly enriched in the Triton X-114 detergent phase and could be extracted from the cauda epididymal fluid by a chloroform-methanol mixture. These proteins were further purified, and their N-terminal sequences did not match any protein in current databases. A polyclonal antibody against the fluid 17-kDa protein recognized the protein in the cauda epididymal sperm extract and immunolocalized it on the sperm flagellum membrane and at the luminal border of all cells in the cauda epididymal epithelium. These results indicated that secreted glycoproteins with hydrophobic properties could be directly integrated in a specific domain of the sperm plasma membrane.     

An acrosin inhibitor in ram spermatozoa that does not originate from the seminal plasma.

Ram seminal plasma, and ejaculated ram spermatozoa that have been washed with 0.25M sucrose, both contain acrosin inhibitor. The aim of this work was to determine whether the intracellular inhibitor originates from the seminal plasma. The amounts of inhibitor in ejaculated and epididymal spermatozoa were measured and compared with the amounts present in the seminal plasma of normal and vasectomized rams. One ejaculated ram spermatozoon contained 2.1 amol (2.1 X 10(-18) mol) of inhibitor and one epididymal spermatozoon contained 3.3 amol of inhibitor. (All molarities are mean values based on pooled ram semen or on single ejaculates from three vasectomized rams.) Calculations from results in earlier publications indicated that one ejaculated ram spermatozoon contains about 3 amol of acrosin; thus the inhibitor: acrosin ratio in washed ram spermatozoa is approximately 1. One ml of ram semen contains, on average, 3 X 10(9) spermatozoa and not more than 0.8 ml of seminal plasma. This number of ejaculated spermatozoa would contain 6.3 nmol of inhibitor, while the same number of epididymal spermatozoa would contain 9.9 nmol of inhibitor. These values exceed the quantities of inhibitor present in 0.8 ml of normal seminal plasma (approximately 1.6 nmol) or in 0.8 ml of seminal plasma from vasectomized rams (approximately 2.3 nmol). We conclude that seminal plasma is not a major source of the acrosin inhibitor that can be recovered from washed ejaculated ram spermatozoa.     

Beta-galactosidase in the seminal plasma and reproductive organs of the bull

The distribution of beta-galactosidase activity was studied in different reproductive organs, seminal plasma and spermatozoa of the bull. The highest specific activity of beta-galactosidase was found in testis and in different parts of the epididymis, where the activity seemed to be partly in secretory (cauda secretion) and partly in non-secretory, bound form (caput to cauda epididymidis). Gel filtration on Sepharose 6B at pH 7.0 revealed two beta-galactosidase forms (GF-1, Mr approximately 500,000-600,000 and GF-2, Mr approximately 190,000-220,000) in reproductive organs and seminal plasma. The pH-optimum of both beta-galactosidase forms was about 3.75-4.75. Hg2+ and p-chloromercuribenzoate inhibited strongly these activities. Further, form GF-2 seemed to be slightly more sensitive to the thermal inactivation at 50-70 degrees C than form GF-1. In chromatofocusing beta-galactosidase activities in bull seminal plasma coeluted with those of the cauda epididymidis (pI-values 7.5-6.4). On the contrary, prostate, Cowper's gland, testis, ampulla and seminal vesicles had enzyme activities eluting at lower pI-values (6.3-4.2). Thus, the seminal plasma activity is mainly an indicator for the function of the epididymal cauda.     

Heterogenous turnover of sperm and seminal vesicle proteins in the mouse revealed by dynamic metabolic labeling

Plasticity in ejaculate composition is predicted as an adaptive response to the evolutionary selective pressure of sperm competition. However, to respond rapidly to local competitive conditions requires dynamic modulation in the production of functionally relevant ejaculate proteins. Here we combine metabolic labeling of proteins with proteomics to explore the opportunity for such modulation within mammalian ejaculates. We assessed the rate at which proteins are synthesized and incorporated in the seminal vesicles of male house mice (Mus musculus domesticus), where major seminal fluid proteins with potential roles in sperm competition are produced. We compared rates of protein turnover in the seminal vesicle with those during spermatogenesis, the timing of which is well known in mice. The subjects were fed a diet containing deuterated valine ([(2)H(8)]valine) for up to 35 days, and the incorporation of dietary-labeled amino acid into seminal vesicle- or sperm-specific proteins was assessed by liquid chromatography-mass spectrometry of samples recovered from the seminal vesicle lumen and cauda epididymis, respectively. Analyses of epididymal contents were consistent with the known duration of spermatogenesis and sperm maturation in this species and in addition revealed evidence for a subset of epididymal proteins subject to rapid turnover. For seminal vesicle proteins, incorporation of the stable isotope was evident from day 2 of labeling, reaching a plateau of labeling by day 24. Hence, even in the absence of copulation, the seminal vesicle proteins and certain epididymal proteins demonstrate considerable turnover, a response that is consonant with the capacity to rapidly modulate protein production. These techniques can now be used to assess the extent of phenotypic plasticity in mammalian ejaculate production and allocation according to social and environmental cues of sperm competition.     

Immunolocalization of anti-agglutinin for spermatozoa in boars

A boar "anti-agglutinin," which inhibits head-to-head agglutination of spermatozoa, has been identified as a 25-kDa sialoprotein contained in epididymal and seminal plasma. This study was conducted to determine the location of the anti-agglutinin on spermatozoa and in various organs, including epididymides, by indirect immunofluorescence and Western blotting techniques. Ejaculated boar spermatozoa were washed and subjected to immunocytochemical observation. Epididymal plasma was recovered from three different regions of epididymides and subjected to sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting. Twelve kinds of organs (testis, epididymis, seminal vesicle, prostate, heart, liver, kidney, spleen, stomach, small intestine, lung, and muscle) were recovered from boars. The unilateral epididymides were fixed, cut into 10-microm frozen sections, and subjected to immunohistochemical observation. The other organs were homogenized and used for SDS-PAGE and Western blotting. Immunocytochemical observations revealed that the antiserum strongly recognized the acrosomal region and equatorial segment on unfixed and methanol-fixed spermatozoa. Immunohistochemical observations revealed that the epithelia of the epididymal ducts were recognized by the antiserum mainly in the corpus epididymides. Moreover, the antiserum reacted with the luminal contents of the corpus and cauda epididymides. However, no specific reaction was detected in the caput epididymides. Western blotting showed that the antiserum selectively recognized a band of the anti-agglutinin in the corpus and cauda epididymal plasma, although no band was detected in the caput epididymal plasma. In the extracts from various organs, the single band was detected in the corpus and cauda epididymides at the same mobility as the anti-agglutinin, but not in the other organs. Based on these results, the following matters concerning the anti-agglutinin are discussed: (1) the importance of its association with the acrosome of spermatozoa in inhibiting sperm head-to-head agglutination; (2) its origin in the epididymis; and (3) its tissue specificity.     

Seasonal changes in antioxidant defence systems in seminal plasma and fluids of the boar reproductive tract

This study aimed to analyze seasonal variations in the antioxidant defence systems of the seminal plasma and fluids of the cauda epididymis and vesicular glands of the boar. The analyzed antioxidants included superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total L-glutathione (GSH+GSSG). Seasonal changes in total protein content and total antioxidant status (TAS) of the seminal plasma and reproductive fluids were also analyzed. Compared with the spring-summer period, total protein content in the seminal plasma was significantly higher during the autumn-winter period. Among the antioxidants analyzed, only SOD activity showed marked seasonal variations, being significantly higher during the spring-summer period. Likewise, the fluid of the cauda epididymis exhibited greater SOD and CAT activity during the spring-summer period, whereas TAS levels were markedly higher during the autumn-winter period. Neither GPx activity nor total GSH+GSSG content in the cauda epididymal fluid was significantly affected by the seasonal periods. The vesicular gland fluid exhibited an approximately 4-fold greater level of SOD activity during the autumn-winter period, as compared with the spring-summer period. By contrast, greater CAT and GPx activity, and a higher level of total GSH+GSSG were observed in the vesicular gland fluid during the spring-summer period. In conclusion, the findings of this study indicate that seasonal variations could have varying effects on the antioxidant defence systems in the seminal plasma and fluids of the boar reproductive tract.     

Glycoprotein changes in the rat sperm plasma membrane during maturation in the epididymis.

To investigate surface glycoprotein changes during post-testicular maturation, plasma membranes were isolated from proximal caput, distal caput, and cauda epididymal rat spermatozoa. Membrane glycoproteins were identified on Western blots of SDS-PAGE fractionated samples using biotinylated lectins and Vecta-stain reagents; these were compared to glycoproteins present in cauda epididymal luminal fluid. Lens culinaris agglutinin, Pisum sativum agglutinin, peanut agglutinin, wheat germ agglutinin, Ricinus communis agglutinin, Ulaex europaeus agglutinin, and Dolichol biflorus agglutinin each bound a specific subset of the polypeptides present. Several types of glycoprotein changes were noted including their appearance, loss, alteration of staining intensity, and alteration of electrophoretic mobility. Some maturation-dependent sperm surface glycoproteins co-migrated with glycoproteins present in epididymal fluid. This approach of direct analysis of the glycoproteins in purified plasma membranes identifies a broader spectrum of maturation-related surface changes occurring within the epididymis than are noted with surface labeling procedures.     

Immunochemical detection, physicochemical characterization and levels of pregnancy-associated endometrial alpha 2-globulin (alpha 2-PEG) in seminal plasma of men

Pregnancy-associated endometrial alpha 2-globulin (alpha 2-PEG), the major secretory protein of the human uterine endometrium during the luteal phase of the menstrual cycle and early first trimester of pregnancy, has been detected by immunochemical techniques in seminal plasma. Biochemical analysis and immunoblotting has verified that immunoreactive alpha 2-PEG in seminal plasma exhibits properties identical to those of endometrial alpha 2-PEG, i.e. Concanavalin A-binding dimeric glycoprotein of native Mr 56,000, subunit Mr 28,000, average pI 4.7 and of alpha 2-mobility. Concentration of alpha 2-PEG in seminal plasma was 22.13 +/- 2.82 micrograms/ml (mean +/- s.e.m., n = 110) which compared to 12.02 +/- 1.65 micrograms/ml (mean +/- s.e.m., n = 48) found in amniotic fluid at 11-20 weeks of pregnancy, to 4.29 +/- 1.66 micrograms/ml (mean +/- s.e.m., n = 15) in uterine luminal fluid in women during the luteal phase and to 0.245 +/- 0.025 micrograms/ml (mean +/- s.e.m., n = 10) in sera at 10 weeks of pregnancy. This distribution is very different from that observed for pregnancy-associated placentally-derived serum proteins detected in seminal plasma. The source of alpha 2-PEG in seminal plasma is unknown but is unlikely to be the testis because of the normal levels observed in vasectomized men. In the endometrium alpha 2-PEG synthesis and secretion appears to be related to progesterone-dependent differentiation of the glandular epithelium. Therefore these observations suggest that a different mechanism of regulation of the gene for alpha 2-PEG operates in the male reproductive tract.     

The maintenance of motility and the surface properties of epididymal spermatozoa from bull, rabbit and ram in homologous seminal and epididymal plasma.

Epididymal spermatozoa from bull, rabbit and ram were incubated in homologous epididymal plasma or seminal plasma in a buffered saline-based medium with or without serum albumin. The spermatozoa were either diluted directly into the medium or were washed first. No effect of washing was observed on the subsequent reaction of the cells to the different media. A considerable proportion of the populations of epididymal spermatozoa survived (i.e. continued to exhibit motility) for up to 22 h at 30 degrees C in the simple saline-based medium. Initially epididymal plasma had a slight stimulatory effect on sperm motility in ram and bull but it had no effect on sperm survival in any of the 3 species. Seminal plasma stimulated motility markedly in ram initially, but in all 3 species seminal plasma was detrimental to survival: in ram even a 15-min exposure to the fluid reduced survival. Serum albumin also stimulated motility; it delayed, but did not prevent, the detrimental effect of seminal plasma, although it had no effect itself on survival. The effects of epididymal plasma, seminal plasma and serum albumin on surface properties of epididymal spermatozoa, i.e. agglutination, sticking-to-glass and eosinophilia, were also noted. These varied between species and there was no correlation between these effects and the effects on motility and survival.     

ATP-induced reactivation of ram testicular, cauda epididymal, and ejaculated spermatozoa extracted with Triton X-100

It was possible to demembrante and reactivate not only freshly collected testicular, cauda epididymal, and ejaculated ram sperm but also sperm that had been stored for several days at 0 degrees C and for several months at -196 degrees C in rete testis fluid or egg yolk citrate media. Sperm were usually washed free of seminal plasma before demembranation, but this was not essential for reactivation. Bovine serum albumin (1.0%) in the wash medium increased the survival of sperm, but more than 0.25% in the extraction medium decreased reactivation. A macro-molecular component of cauda epididymal fluid also inhibited the reactivation of testicular sperm. Triton X-100 concentrations between 0.01% and 1.00% in the extraction medium were satisfactory for demembranating the sperm. Rapid cooling (i.e., cold shock) mimicked the effect of detergent in making the sperm responsive to added ATP and demonstrated that damage to ram sperm in cold shock does not involve the axoneme. Ejaculated and cauda sperm were reactivated immediately on addition of ATP and activity persisted for up to 10 min. Testicular sperm, on the other hand, required about 4 min to become fully reactivated. The optimal ATP concentration for activation of sperm was 0.1-1.0 mM. Magnesium ions (0.1-1.0 mM) were important for reactivation, and testicular sperm required a higher magnesium concentration than did cauda or ejaculated sperm. Manganese ions were almost as effective as magnesium for reactivating cauda epididymal and ejaculated sperm. Cobalt and cadmium ions were much less active for cauda and ejaculated sperm and none of these ions were effective for testicular sperm. Fluoride (25-50 mM) inhibited reactivation. The presence of 50 microM cAMP in the extraction medium or preincubation of testicular sperm with theophylline or caffeine increased low levels of activation, but this was not evident with ejaculated or cauda sperm. We conclude that the motor apparatus is already functionally assembled in spermatozoa on leaving the testis, but some fine adjustment must take place during maturation in the epididymis.     

Identification and expression of boar sperm proteins in the reproductive tract that interact with antibodies in serum from an infertile woman

Serum designated as IS obtained from a young healthy infertile woman induced a head-to-head agglutination of ejaculated boar sperm. The immunoglobulin G (IgG) prepared from IS localized to the acrosomal region of the sperm head obtained from the corpus and cauda epididymis as determined by an indirect immunofluorescent method. The IgG interacted with a boar sperm protein with an estimated molecular weight of 45-kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) immunoblotting technique. However, the IgG did not interact with proteins extracted from sperm obtained from the testis and caput epididymis or from non-gonadal tissues including liver, kidney, spleen, muscle and serum. The IgG interacted with additional proteins of about 75- and 38-kDa present in the corpus and cauda epididymal fluids but not those in the caput epididymal fluid. The staining intensity of the 75-kDa band was reduced and that of the 38-kDa was nullified with ejaculated seminal plasma proteins. The interacting proteins were adsorbed when chromatographed on Concanavalin A Sepharose column, suggesting that they are glycoproteins.     

A simple and sensitive solid-phase radioimmunoassay for measuring the transferrin content of human biological fluids: its application to seminal plasma

A highly sensitive and reproducible radioimmunoassay was established to detect transferrin in human fluids. By this technique, applied to seminal fluid, transferrin levels (micrograms/ml) were found in normozoospermic individuals (64.49 +/- 25.41) at level higher than in oligozoospermic (38.93 +/- 21.35), azoospermic (19.49 +/- 10.23), or vasectomized (19.61 +/- 8.95) subjects. A relationship between transferrin and spermatozoid concentration in sperm was shown. These results reinforce previous findings that seminal transferrin can be used as a reliable clinical marker of Sertoli Cell function.     

Changes in binding of a 27-kilodalton chimpanzee cauda epididymal protein glycoprotein component to chimpanzee sperm

Motility patterns of caput epididymal chimpanzee sperm, caput epididymal chimpanzee sperm incubated in vitro with chimpanzee cauda epididymal fluid, and cauda epididymal chimpanzee sperm were assessed quantitatively. Sperm recovered from the caput epididymis showed no motility, whereas sperm recovered from cauda epididymis showed progressive forward motility. After incubation in cauda fluid, approximately 25% of caput epididymal sperm showed some motile activity. Electrophoretic analysis of ¹²⁵I-labeled sperm plasma membrane preparations revealed that the surface of caput epididymal sperm, incubated in cauda fluid, was modified by the appearance of a major protein-glycoprotein surface component with an apparent molecular weight of 27 kilodaltons (kD). THis 27-kD component was not detected on caput epididymal sperm incubated in buffer or in caput fluid. However, it was present in cauda fluid and on cauda epididymal sperm. Binding to caput epididymal sperm was cell specific in that chimpanzee erythrocytes incubated in cauda fluid did not bind this 27-kD cauda fluid component. Motility patterns of ejaculated chimpanzee sperm and of ejaculated chimpanzee sperm incubated in the uterus of adult female chimpanzees also were assessed quantitatively. Ejaculated sperm showed progressive forward motility, whereas in utero incubated ejaculated sperm showed hyperactivated motility typical of capacitated sperm. Electrophoretic analysis of ¹²⁵I-labeled sperm plasma membrane preparations revealed the loss of a 27-kD component from the surface of ejaculated sperm after in utero incubation. No significant change in the ¹²⁵I-distribution pattern was detectable when ejaculated sperm were incubated in buffer. These results suggest that the lumenal fluid component, which becomes adsorbed to the surface of chimpanzee sperm during maturation in the epididymis and which is removed from the surface of mature chimpanzee sperm in the female reproductive tract, affects sperm motility.