Further characterization of a secreted epididymal glycoprotein in mice that binds to sperm tails

Sperm maturation antigen 4 (SMA-4) is a surface component of the mouse sperm tail. Previously, immunofluorescence studies indicated that SMA-4 may be secreted by principal cells of the distal caput epididymidis and bound to spermatozoa as they pass through that region of the duct. In the present study, detergent extracts of spermatozoa from the cauda epididymidis were subjected to polyacrylamide gel electrophoresis under reducing and denaturing conditions, transferred to nitrocellulose, and immunostained with a monoclonal antibody against SMA-4. A band of approximately 54,000 molecular weight was revealed. The band was also stained by the periodic acid-Schiff (PAS) procedure. This glycoprotein was not detected in extracts of spermatozoa from the proximal caput epididymidis or of spermatozoa from the cauda epididymidis that were preincubated for 4 hours in an in vitro fertilization environment. Blots of sperm-free fluid from the corpus and cauda epididymidis displayed an immunoreactive and PAS-positive band of about 85,000 molecular weight that was not observed in fluid from the caput epididymidis. The difference in the molecular weights of the antigen in the fluid and that in extracts of cauda spermatozoa suggests that SMA-4 may be modified chemically upon association with the sperm surface.     

Two-dimensional gel electrophoretic analysis of rat sperm membrane interaction with cauda epididymal fluid

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to analyze the polypeptide composition of rat cauda epididymal fluid, blood serum and membrane-enriched fractions of caput, corpus, and cauda epididymal spermatozoa. Several polypeptides were found in both cauda fluid and blood serum, and in both cauda fluid and epididymal spermatozoa. Prominent cauda epididymal fluid polypeptides that were associated with caput, corpus, and cauda sperm membranes were 32 and 33 kDa. Passage of spermatozoa from the caput to the cauda epididymidis was characterized by the loss of three glycopolypeptides of 32, 30 and 29 kDa, and by the addition of a 37-kDa glycopolypeptide. Incubation of intact caput, corpus and cauda spermatozoa with cauda epididymal fluid revealed major changes in the polypeptide maps of the incubation fluid and the membrane-enriched fractions of caput and corpus, but not cauda spermatozoa. The incubation of cauda fluid with caput and corpus sperm cells was characterized by a loss of several polypeptides and the addition of a 24-kDa glycopolypeptide. The most striking change in spermatozoa incubated with cauda epididymal fluid was the addition of two glycopolypeptides of 32 and 33 kDa to the polypeptide maps of caput sperm cells. These data demonstrate that rat spermatozoa undergo surface modifications during epididymal maturation and that these modifications can be influenced by epididymal fluid.     

Modification of the rat sperm flagellar plasma membrane during maturation in the epididymis

Previously, we demonstrated that surface radiolabeling of rat epididymal spermatozoa by lactoperoxidase-catalyzed iodination reveals a major component with an apparent molecular weight of 26,000 to 28,000 daltons (26 kDa) on spermatozoa from the cauda but not the caput epididymidis. To characterize this surface component further, sperm surface constituents radiolabeled by lactoperoxidase-catalyzed iodination were separated by 2-D PAGE. The 26 kDa component was localized by autoradiography and appeared as the major labeled acidic spot on cauda spermatozoa, but neither a radiolabeled spot nor a corresponding stained spot was present on caput spermatozoa. The 26 kDa spot was excised from 2-D gels of plasma membranes from cauda spermatozoa and utilized for immunization. The monospecific antiserum stained a single band of 26 kDa on Western blots of SDS-PAGE-separated plasma membranes from cauda spermatozoa and in a 100,000 X g supernatant fluid of the luminal contents of the cauda epididymidis. Immunohistochemical staining of cauda spermatozoa revealed antigen exclusively on the flagellar domain; the antigen was not seen on caput spermatozoa but first appeared in spermatozoa from the proximal corpus epididymidis. Immunoelectron microscopy confirmed the 26 kDa component was localized to the external face of the flagellar plasma membrane. Immunohistochemical staining of caput spermatozoa incubated in vitro with cauda epididymal luminal fluid revealed the 26 kDa component specifically bound the flagellar domain of immature spermatozoa.     

Localization of antigenic determinants recognized by a monoclonal antibody in rat epididymal spermatozoa, with particular reference to sperm maturation

Summary.  This study localized antigenic determinants recognized by a mouse anti-human sperm monoclonal antibody TüS10 immunocytochemically and immunoelectron microscopically in the rat sperm recovered from the caput and cauda epididymidis. Immunocytochemistry showed that the antibody bound specifically to the plasma membrane overlying the principal piece of membrane-intact sperm from the caput and cauda epididymidis. Demembranation by Triton X-100 significantly decreased the affinity of the monoclonal antibody TüS10 to the caput sperm but did not obviously change that to the cauda sperm. Immunoelectron microscopy with biotinstreptavidin peroxidase complex pre-embedding method confirmed the localization of the antigenic determinants over the cell surface of the principal piece of the membrane-intact spermatozoa from the caput and cauda epididymidis. The demembranated sperm from the caput epididymidis showed no intracellular labelling, while those from the cauda displayed labelling on their external surface of the fibrous sheath. Using monoclonal antibody TüS10 as a probe, we detected different distribution patterns of the antigenic determinants between the spermatozoa in the caput and cauda epididymidis. These results suggest that spermatozoa mature with immunologically detectable changes in the fibrous sheath during their epididymal transit.     

The Effect of Caffeine on Rat Epididymal Spermatozoa: Motility, Metabolism and Fertilizing Capacity

The metabolism, motility and fertilizing capacity of spermatozoa from the caput, corpus and cauda epididymidis were examined. Motility increased from 3% to almost 50% from caput to cauda.
Caput sperm were almost infertile, when inseminated into the uterus. Corpus and cauda sperm fertilized ova in 54 and 60% (respectively) of females, but the number of offspring was below our average colony litter size. Caffeine, 10 mM, had no effect on any of the sperm characteristics measured. No teratogenic effects or long-term effects on fertility of the offspring were seen.     

Origin of the luminal fluid proteins of the rat epididymis

Using a combined microperfusion and high resolution gel electrophoresis technique, the origin of the epididymal fluid proteins of the rat has been investigated. Some proteins originate from the testis, others are secreted by the epididymis or are released by spermatozoa. Of particular interest is a 32 000 dalton protein found to be actively secreted by the caput epithelium in situ and concenrated in the lumen. The cauda epididymidis contained the highest concentration of this protein. Radioactive labelling of the sperm surface proteins revealed that this protein was present on the surface of the mature cauda but not on the immature caput or corpus sperm, suggesting its acquisition by the sperm surface during epididymal transit. Another sperm surface protein of interest (MW 40 000) is present only on the plasma membrane of the cauda but not on that of the caput or corpus sperm. Since this protein was not identified in the epididymal perfusates or luminal fluids, its presence may result from some modification events taking place in the sperm membrane during maturation.     

Electrolyte and Water Transport in Rat Epididymis; Its Possible Role in Sperm Maturation

Electrolyte and water transport in different regions of the rat epididymis has been studied using a microperfusion technique. The caput and proximal corpus epididymides were found to absorb NaCl and water and secrete K⁺ at a lower rate than the cauda epididymidis. The secretion rate of protein was the same in both regions. In the caput and proximal corpus, reabsorption of chloride was hypertonic. Reabsorption of sodium could not account for water reabsorption. In contrast, water reabsorption in the cauda epididymidis was dependent upon the intraluminal sodium ions. Amiloride inhibited both the Na⁺ and water reabsorption in this region. It was concluded that in the proximal regions of the rat epididymidis, water reabsorption may be secondary to an active transport of chloride, whereas in the cauda, a net transepithelial transport of sodium ions is the driving force for water reabsorption.
Transport of electrolytes and water across the perfused rat cauda epididymidis has also been studied under various experimental conditions. Treatment of rats with alpha-chlorohydrin (9 mg/kg/day) for 7 days inhibited the rate of sodium and water reabsorption without affecting the secretion of proteins. Ligation of the testicular efferent duct or the corpus epididymidis had no significant effect on the transport functions of the cauda epididymidis. When cyproterone acetate (10 mg/rat/day) was injected into male rats, the rate of sodium and water reabsorption was reduced. This effect was accompanied by a loss of sperm motility. It is concluded that the transport functions of the cauda do not require the normal flow of testicular fluid, but may depend on the supply of circulating androgen in the blood. Alpha-chlorohydrin and cyproterone acetate may affect sperm maturation by disrupting the normal milieu of the epididymal duct.     

Alterations in protein profile of rat spermatozoa during maturation

Alterations in the polypeptide pattern of rat spermatozoa during epididymal transit were studied by SDS-PAGE and compared with that of epididymal cytosol and luminal fluid. The total number of cytosol and luminal fluid polypeptides increase from caput to cauda epididymidis but sperm associated polypeptides decrease during epididymal transit. Changes in polypeptide pattern of spermatozoa are due to their acquisition, loss or modification. Spermatozoa acquire seven polypeptides, of which six are acquired in corpus (MW 16.5, 38, 41, 72, 75 and 100 Kdal) and one (MW 28.5 Kdal) in cauda epididymidis. Spermatozoa lose one polypeptide of MW 72.5 Kdal in caput and two polypeptides of MW 70 and 115 Kdal in cauda epididymidis. Four polypeptides of MW 18.5, 19.5, 64 and 67.5 Kdal disappear from cauda spermatozoa without appearing in the luminal fluid. Polypeptide of MW 62.5 Kdal is observed only in spermatozoa and luminal fluid from cauda epididymidis.     

Changes in Porcine Sperm Lactate Dehydrogenase Isoenzymes During Sperm Maturation

The changes in sperm lactate dehydrogenase (LDH-lactate: NAD oxidoreductase, EC 1.1.1.27) activity and the relative occurrence of LDH isoenzymes during sperm maturation were studied using pubertal German improved Landrace boars. The LDH of spermatozoa liberated from the testis, caput epididymis, proximal and distal corpus epididymidis and cauda epididymidis was spectrophotometrically quantified while the LDH isoenzymes were separated on fine cellulose acetate membrane strips with the Sartorius Sartophor system. The LDH content of sperm dropped drastically as they moved from the testis to the caput epididymidis. Thereafter, only little and insignificant changes were observed. Testicular sperm was composed more of the fastest anodically-migrating isoenzyme (LDH1) while with sperm maturation, the least or slowest migrating isoenzymes (LDH4 and 5) became progressively more dominant. This loss in LDH content in sperm and the shifts in the LDH isoenzyme patterns indicate that the development of sperm during maturation is dependent on a delicate balance between lactate and pyruvate, such that the cathodic isoenzymes involved in the anaerobic energy-supplying metabolic processes are sufficiently available for sperm activity and survival.     

Effect of gossypol on the concentration of sodium and potassium in the rat epididymis

The effects of administration of gossypol acetic acid (7.5 mg/kg daily for 4 weeks) on the concentration of Na+ and K+ in the rat epididymis was assessed. Epididymal fluid samples, collected by micropuncture, from the caput, corpus, proximal cauda and distal cauda epididymis from gossypol-treated and control animals were analysed for Na+ and K+ concentrations. Gossypol-treated males failed to impregnate healthy females, presumably because their sperm were immotile. In gossypol-treated rats, Na+ levels decreased significantly (P less than 0.01) in the caput, corpus, proximal and distal cauda epididymis. In contrast, the K+ concentration was increased significantly (P less than 0.05) only in the caput and corpus epididymis. This altered electrolyte milieu may be responsible, to some extent, for immotility and hence infertility.     

Regulation of rat caput epididymidis contractility by prostaglandins

Mechanical activity of the rat caput epididymidis in vitro was recorded using a videomicrography system. The effects of prostaglandin (PG)F2 alpha, PGE2, and aspirin on caput epididymidis contractility were determined by measuring the frequency of contraction, luminal diameter, and amplitude of contraction at various concentrations of each test compound in vitro. PGF2 alpha stimulated contractility of the tubules at physiological concentrations, while PGE2 reduced contractility. Aspirin strongly inhibited contractility at concentrations of 10(-3) and 10(-2)M. Endogenous levels of PGF2 alpha and PGE were determined for rat testes, caput, corpus, and cauda epididymidis and vas deferens. While the concentrations of PGE were consistently higher than those of PGF2 alpha, both compounds were relatively low in the testes, high in the vas deferens, and intermediate throughout the epididymis. Results from these experiments strongly suggest that PGs are important regulators of proximal epididymidis contractions and thus may regulate sperm transport through that organ.     

Use of neonatal tolerization and chemical immunosuppression for the production of monoclonal antibodies to maturation-specific sperm surface molecules.

Mammalian sperm acquire functional maturity as they move from the caput to the cauda epididymidis. Changes occur in the protein/glycoprotein composition of the sperm plasma membrane during this time, and may be essential to the maturation process. The production of monoclonal antibody (Mab) probes to the maturation-specific molecules has been difficult since new proteins comprise a minor portion of total membrane proteins. This report describes a protocol for enhancing the production of Mabs to maturation specific molecules. By injecting neonatal mice with caput epididymal sperm plasma membranes, in combination with chemical immunosuppression at adulthood, the mice were made tolerant to the antigens expressed on the caput sperm membranes. Subsequent immunization with cauda epididymal sperm plasma membranes allowed the production of Mabs to the maturation-specific moieties without the necessity for extensive antigen purification procedures. The majority of the resulting Mabs recognize cauda, not caput, epididymal sperm plasma membranes as determined by enzyme-linked immunosorbent assay (ELISA), immunocytochemistry on unfixed cells, and Western blot analyses, even though the protein profile from caput epididymal sperm plasma membranes is very similar to that from cauda membranes. The five Mabs described also recognize cauda fluid antigens, suggesting that the maturational changes on the sperm plasma membranes arise from interactions with the epididymal fluid. Use of the tolerization/immunosuppression protocol has provided Mab tools to assist in the study of sperm maturation during epididymal transit.     

Sperm maturation in dogs: sperm profile and enzymatic antioxidant status in ejaculated and epididymal spermatozoa

Spermatozoa become more susceptible to the attack of reactive oxygen species during maturation. To avoid oxidative damage, the epididymis must provide the necessary antioxidant protection. The aim of this study was to compare the canine sperm profile and the enzymatic antioxidant status of the ejaculated fractions and samples collected from the different segments of the epididymis (caput, corpus and cauda). Five adult dogs were used, and after 1–3 weeks, subsequently to bilateral orchiectomy and epididymal storage, sperm samples were collected from the different segments of the epididymis. Samples were evaluated for conventional microscopy and computer‐assisted motility analysis: sperm plasma membrane permeability and the activity of the antioxidant enzymes catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD). Samples collected from the caput and corpus showed lower values for most of the motility variables evaluated, indicating different levels of immaturity. Catalase activity was observed only in ejaculated samples. Conversely, GPx activity was higher in the cauda epididymidis. Correlations were found between SOD and GPx and SOD and sperm motility in the epididymal cauda and corpus, highlighting the importance of the enzymes for the protection of spermatozoa during the transit along the epididymis.     

Effect of a nonsteroidal antiandrogen, flutamide on intraluminal acidification in rat testis and epididymis

Summary. Flutamide, a pure antiandrogen was administered to intact adult male rats to study the effect of altered availability of hormones on in situ pH, PCO₂ and bicarbonate concentration ([HCO⁻₃]) of seminiferous tubules, proximal caput, middle caput, middle corpus, and proximal cauda epididymidis. The weights of the epididymis and ventral prostate as well as the plasma testosterone level showed antiandrogenic effects of flutamide. Relative to controls, flutamide elevated significantly in situ pH in proximal caput, middle caput, middle corpus and proximal cauda epididymidis but not in seminiferous tubules. In situ PCO₂ values in the above segments, after flutamide, were indistinguishable from controls and from each other but all values remained significantly higher than systemic arterial blood PCO₂. Flutamide treatment did not change the [HCO⁻₃] in systemic arterial blood or seminiferous tubules but increased markedly the values in proximal caput and middle caput. The results of the present studies support the view that luminal acidification in the rat epididymis is under androgen control and may be important for sperm maturation and storage.     

Apparent fertility of human spermatozoa from the caput epididymidis

Fifty-one patients with obstructive azoospermia caused by blockage at the caput epididymidis have been followed for 4 years after undergoing "specific tubule" vasoepididymostomy, bypassing the corpus and cauda. The patency rate was 73%, and the pregnancy rate was 31%. There was no correlation between sperm count and pregnancy rate, but there was a strong correlation with sperm motility. With less than 20% motility, only 15% of the patients became pregnant, but with greater than 20% motility postoperatively, 58% became pregnant. If the wife was over 30 years old, only 21% got pregnant. If the wife was under 30, 67% got pregnant. "Redo" cases were just as likely to succeed as "first-time" attempts. In the "patent" cases, 43% of patients with spermatozoa that never reached or traversed the corpus or cauda epididymidis produced a pregnancy. Spermatozoa from the proximal caput produced a 33% pregnancy rate, whereas spermatozoa from the distal caput produced a 50% pregnancy rate. One-half of the pregnancies occurred more than 2 years postoperatively.     

大鼠附睾上皮细胞的培养及其功能测定

用酶消化法分离成年雄性ＳＤ大鼠附睾头、体、尾的上皮细胞，贴壁培养。４～１１天后取贴壁细胞进行ＨＥ染色及扫描电镜观察，发现附睾头部和体部大部分为带有微绒毛的主细胞，而尾部细胞较小。并以ＰＮＰＧ为底物测定上清液内α－１．４糖苷酶的活性，结果为体部最高，尾部次之，头部最低。用硫代巴比妥酸法检测上清液中唾液酸的含量，仍以体部最高，但尾部最低。统计学处理均有显著差异。提示大鼠附睾体部可能是精子成熟的关键部位。     

Alteration of epididymal function and its relation to maturation of spermatozoa

Alteration of epididymal function and its relation to maturation of spermatozoa was studied in 54 adult male albino rats. Levels of free and bound sialic acid in the spermatozoa and luminal contents of the epididymis and vas deferens were determined. A group of 10 received rabbit antiserum to ovine luteinizing hormone (LHAS) sc .2 ml/day for 5 days. 2 groups of 8 animals each received 2.5 mg cyproterone acetate twice daily for either 15 or 30 days. 16 animals served as intact controls and 12 animals served as castrate controls. Epididymis and vas deferens sperm counts were not affected by LHAS for 5 days or by cyproterone acetate for 15 days; however, sperm counts were decreased in the corpus (p less than .02), cauda (p less than .05), epididymidis and vas deferens (p less than .01) when rats were treated with cyproterone acetate for 30 days. Castration resulted in a marked reduction in all regions within 5 days. In the intact rats spermatozoa sialic acid decreased in the cauda epididymidis (p less than .01) and increased in the vas deferens (p less than .001). Sialic acid concentration was similar in those treated with either LHAS or cyproterone acetate for 30 days. Bound sialic acid in the epididymal fluid increased (p less than .02) to a maximum in the corpus and cauda and decreased in the vas deferens (p less than .05). LHAS or cyproterone acetate caused a reduction in bound sialic acid in the fluid of the epididymis and vas deferens.     

Appearance of specific proteins in rat epididymis during postnatal development

In the immature 30-day-old caput and cauda epididymides of the rat, only a single prealbumin protein was visible. Additional epididymis-specific proteins appeared in the caput on day 45, coinciding with the entry of testicular fluid containing first wave of spermatozoa. In the cauda epididymides, additional proteins appeared only on day 50. These results indicate that the functional differentiation of caput precedes that of the cauda epididymidis.     

The presence of α-glucosidase, glycerophosphocholine and carnitine in the epididymis and ejaculate of the vervet monkey (Cercopithecus aethiops) and the Chacma baboon (Papio ursinus)

Summary.  The epididymis is the site of post-testicular sperm maturation in the male genital tract. Studies on human epididymides are hampered by the practical inaccessibility of epididymides of healthy men in their reproductive years. The limited use of laboratory animals therefore seems unavoidable. The objective was to establish baseline values of the epididymal markers α-glucosidase, glycerophosphocholine (GPC) and carnitine in the lumen of the caput, corpus and cauda epididymidis and in the ejaculate of adult male Chacma baboons and vervet monkeys. In both primates, α-glucosidase was found throughout the epididymis and in the ejaculate; values did not vary significantly. In monkeys, the highest concentration of GPC was found in the cauda epididymidis, but smaller amounts were found in the other regions and the ejaculate. In baboons, GPC was absent from the caput, but present in the other regions, including the ejaculate. Carnitine concentrations increased significantly from the caput to the cauda in monkeys and from the caput to the corpus in baboons. With this study, the relative concentration ranges in which these markers are present in the epididymides of these primates have been established. In future studies, changes in concentrations of these substances would probably indicate changes in epididymal function.     

Gamma-glutamyl transpeptidase activity in the epididymis during fetal and postnatal development in rats.

The histochemical and biochemical distributions of gamma-glutamyl transpeptidase (gamma-GT) were investigated in the epididymis of rats during fetal and postnatal development. In the epididymal homogenates, gamma-GT activity was detected on the fifth day after birth. A sharp increase was observed after 30 days of life in the caput homogenates. Moderate levels of the enzyme were found in the cauda epididymis. Gamma-GT is histochemically detected from the 15th day of gestation in Wolffian ducts and in 17- to 18-day-old fetuses in newly differentiated epididymal tubules. Enzyme activity, was associated with the plasma membranes (apical, lateral, and basal), was preponderant on the apical part of the epithelial cells. During the first 15 days of the postnatal life, the histochemical reaction intensities were identical from the caput to the cauda epididymidis. From the 18th day onwards, enzyme activity decreased in the corpus and in the cauda, while gamma-GT increased in the caput epididymidis, and a strong activity was found on the apical surface of epithelial cells. Weak or moderate gamma-GT activity of spermatozoa in the caput tubules, increasing steadily from caput to cauda epididymidis, suggests that gamma-GT may be related to the functional maturation of spermatozoa.