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.     

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.     

大鼠精子在附睾成熟过程中肉毒碱量变化的研究

肉毒碱被认为是与精子在附睾中成熟有关的成熟因子。本文报告测定大鼠附睾头部,体部及尾部精子中的肉毒碱。结果表明精子在循附睾头、体、尾运行过程中,精子中肉毒碱含量逐步增加,在附睾体一头部,附睾尾一头部的精子内的肉毒碱量呈现十分显著性差异。结果提示附睾体部或体一头部交界部位可能是精子在附睾中成熟的关键部位。     

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.     

Erratum

During passage of hamster spermatozoa through the epididymis their maturation is shown to involve changes in the sperm head, midpiece (mitochondria) and tail. The sum of these changes results in a dramatic increase in the fertilizing potential of the spermatozoa. When comparable numbers of spermatozoa from the caput or corpus epididymis were injected into one uterine horn of mature females, following ovulation induction, and spermatozoa from the cauda epididymis were injected into the contralateral horn, no fertilization was observed with caput epididymal spermatozoa, 1.7% of oocytes were fertilized by corpus epididymal spermatozoa, whereas 79.5% fertilization was obtained with cauda epididymal spermatozoa. Total sperm numbers increased from caput to corpus to cauda [28.3 ± 12.2, 40.6 ± 20.8, 144 ± 62 million, respectively]. The percentage of progressively motile spermatozoa increased from 27.9 ± 6.4 to 33.8 ± 4.8 to 70 ± 10.7 during this passage. Viability, measured by exclusion of the dye, propidium iodide, was significantly less in spermatozoa from the cauda than from the proximal or mid-caput epididymis. The percentage of the live cells that were stained intensely by rhodamine-123 (a measure of mitochondria1 membrane potential) increased during epididymal passage from 22.8 ± 7.8% in the proximal caput epididymis to 57.2 ± 16.5% in the cauda epididymis. Staining with acridine orange (a measure of DNA packaging in the sperm head) indicated an increase in chromatin condensation in cauda epididymal spermatozoa, when compared to those obtained from the caput or corpus.     

Investigation of epididymal sperm maturation in the golden hamster

During passage of hamster spermatozoa through the epididymis their maturation is shown to involve changes in the sperm head, midpiece (mitochondria) and tail. The sum of these changes results in a dramatic increase in the fertilizing potential of the spermatozoa. When comparable numbers of spermatozoa from the caput or corpus epididymis were injected into one uterine horn of mature females, following ovulation induction, and spermatozoa from the cauda epididymis were injected into the contralateral horn, no fertilization was observed with caput epididymal spermatozoa, 1.7% of oocytes were fertilized by corpus epiddymal spermatozoa, whereas 79.5% fertilization was obtained with cauda epididymal spermatozoa. Total sperm numbers increased from caput to corpus to cauda [28.3 ± 12.2, 40.6 ±20.8, 1434 ±62 mihon, respectively]. The percentage of progressively motile spermatozoa increased from 27.9 ±6.4 to 33.8 ± 4.8 to 70 ± 10.7 during this passage. Viability, measured by exclusion of the dye, propidium iodide, was significantly less in spermatozoa from the cauda than from the proximal or mid-caput epididymis. The percentage of the live cells that were stained intensely by rhodamine-123 (a measure of mitochondrial membrane potential) increased during epididymal passage from 22.8 ±7.8% in the proximal caput epididymis to 57.2 ± 16.5% in the cauda epididymis. Staining with acridine orange (a measure of DNA packaging in the sperm head) indicated an increase in chromatin condensation in cauda epididymal spermatozoa, when compared to those obtained from the caput or corpus.     

非阻塞性输精管滤过装置节育术的精浆中性α-糖苷酶的检测

本文采用ＷＨＯ推荐的改良精浆中性α－糖苷酶的测定方法测定输精管滤过装置节育术（ＩＶＤ组）和输精管结扎术（结扎组）的精浆中性α－糖苷酶活性。术前两组精浆均测出中性α－糖苷酶活性：ＩＶＤ组为４３．５０±２９．０１ｍＵ／每次射精（ｘ±ｓ）；结扎组为４７．８１±３１．２０（ｘ±ｓ）ｍＵ／每次射精；两组间无显著差异（Ｐ＞０．０５）。术后６、１２个月ＩＶＤ组分别有９１．５７％和７９．１７％的精浆测出中性α－糖苷酶活性；而结扎组仅有４．４８％和４．２４％的精浆测出中性α－糖苷酶活性；两组间均有非常显著的差异（Ｐ＜０．００１）。结果提示：ＩＶＤ组术后一年内其大部分受试对象的精浆中仍有附睾液存在。     

Biochemical alterations in the proacrosin-acrosin system during epididymal maturation of the rat spermatozoa.

Acrosin, an acrosomal serine protease, is believed to have a role in fertilization. The enzyme is synthesized in an enzymatically inactive precursor form, proacrosin, and is processed to enzymatically active form(s). In the studies presented here, maturation-associated changes in the proacrosin-acrosin system of rat spermatozoa are reported. Acid-solubilized components of spermatozoa from caput, corpus, and cauda epididymidis were resolved on gelatin-sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and the proteolytic bands visualized by enzymography. These studies reveal the presence of one form (52 kDa), two forms (52 and 41 kDa), and four forms (52, 41, 34, and 31 kDa) in the spermatozoa from caput, corpus, and cauda, respectively. The findings suggest that the enzymatically inactive high molecular weight component (proacrosin) present in the caput spermatozoa is partially converted to the low molecular weight components (acrosin) during epididymal transit. The sensitivity of these molecular forms to an inhibitor of acrosin, p-nitrophenyl p'-guanidino benzoate (NPGB), and the fact that all four forms cross-reacted with the antibody against guinea pig testis proacrosin, suggest that these molecular forms are proacrosin-acrosin components. To understand the mechanism of the changes in molecular forms, spermatozoa from caput, corpus, and cauda regions were subjected to in vitro activation, and the acid-solubilized components resolved on gelatin-SDS-polyacrylamide gel. A smaller component of 34 kDa was generated from both the caput and corpus spermatozoa. No changes in the molecular form(s) of cauda spermatozoa were observed, even after in vitro activation for 4 hours. Inclusion of NPGB during in vitro activation blocked generation of the new molecular form from the caput spermatozoa. These studies indicate that intra-acrosomal events during epididymal transit may be important in the production of functionally mature spermatozoa.     

Characterization of sulfhydryl proteins involved in the maintenance of flagellar straightness in hamster spermatozoa

Hamster caput epididymal spermatozoa exhibit a marked 90-180 degree bend when induced to acquire progressive motility in vitro (Cornwall et al, 1988). Flagellar bending is prevented by oxidizing sperm sulfhydryl (SH) groups with diamide. In the present study, the authors examined the SH proteins involved in sperm flagellar bending using two-dimensional polyacrylamide gel electrophoresis and monobromobimane, an SH-specific fluorescent dye. Proteins extracted from samples containing bent caput spermatozoa contained more reduced SH than the same proteins extracted from straight caput spermatozoa. To further characterize these sperm SH proteins, caudal epididymal spermatozoa that exhibited straight flagella were induced to undergo flagellar bending by treatment with the SH reductant dithiothreitol. Specific proteins of 85-94 kDa, 50-55 kDa and 35-40 kDa, which were rich in SH groups when extracted from samples containing bent caput spermatozoa, were also SH-rich in samples containing bent cauda spermatozoa. These studies suggest that oxidation of specific SH proteins may be important for maintaining sperm flagellar morphology.     

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.     

葡萄糖调节蛋白前体GRP78和肿瘤排斥抗原GP96为仓鼠附睾头部精子特有

哺乳动物睾丸中的精子经过“附睾成熟”期,由静止状态转变成为运动状态。该过程中精子从附睾头部向附睾尾部移动,同时精子发生了一系列的形态、生理和生化改变,如蛋白组成和蛋白修饰的改变可能会影响精子获能的潜能。本实验使用基质辅助激光解吸／电离串联质谱（MALDI-MS／MS）法分析仓鼠睾丸头部和尾部精子的蛋白组学,成功发现了113个蛋白质点。对113个蛋白质点进一步对照比较发现30个蛋白质点（对应20个蛋白）的密度发生了显著改变,其中附睾尾部精子5个蛋白的密度增加,11个蛋白密度减少;此外,葡萄糖调节蛋白前体GRP78和肿瘤排斥抗原GP96为仓鼠附睾头部精子特有,而纤维蛋白原样蛋白1为附睾尾部精子所特有。几个蛋白密度增加可能与附睾成熟过程中精子代谢和ATP产生相关。一些蛋白如ERp57,GRP78,GP96,Hsp60,Hsp70和二氢硫辛酰胺S-乙酰转移酶的密度改变通过免疫印迹法得到验证。本研究首次报道了仓鼠精子的蛋白质组学研究,全面展示了仓鼠精子附睾成熟过程中的蛋白结构改变。     

Effects of putative epididymal osmolytes on sperm volume regulation of fertile and infertile c-ros transgenic Mice

Volume regulation by spermatozoa has been demonstrated to be crucial in both mice and men for transport in the female tract. In order to determine the nature of osmolytes used by spermatozoa, they were released from the cauda epididymis of fertile c-ros heterozygous mice into incubation medium of uterine osmolality (representing an osmotic challenge), containing increasing concentrations of compounds that are major epididymal fluid components and known osmolytes in somatic cells. This should nullify the concentration gradients for osmolytes that mediate volume regulation, prevent osmolyte efflux, and lead to swelling. Of the osmolytes tested, K(+) caused the most rapid and extensive volume increases; glutamate, taurine, L-carnitine, and myo-inositol also were effective, but glycerophosphocholine was not. Such effects were not observed in cauda sperm from the infertile knockout mice, demonstrating a defect in normal volume regulation. K(+) concentrations in cauda epididymal fluid were 21 mM higher in the knockout than the heterozygous mice, but no differences were found in caudal fluid glutamate, carnitine, or myo-inositol. The carnitine content of cauda sperm from knockout males was not different from that of fertile males, but lower amounts of glutamate and inositol were found that could explain the poor volume regulation. In heterozygous mice, cauda but not caput sperm responded to the K(+) channel blocker quinine by swelling, demonstrating development of volume regulation during epididymal transit, whereas knockout cauda sperm showed no response, as with the osmolytes. Major epididymal secretions could serve as osmolytes in murine spermatozoa for volume regulation in response to physiological osmotic challenge in the normal fertile mice; the reduced sperm content of inositol and glutamate in the c-ros knockout mice might reflect maturational abnormalities in volume regulation.     

Nonprotein thiols and disulfides in rat epididymal spermatozoa and epididymal fluid: role of gamma-glutamyl-transpeptidase in sperm maturation

Sperm thiol oxidation during sperm maturation is important for sperm component stabilization, the acquisition of sperm motility, and fertilizing ability. A correct degree of oxidation is required, since spermatozoa are very susceptible to oxidative damage. The pathways involved in physiologic sperm thiol oxidation in the epididymis are not completely understood. The nonprotein thiol glutathione (GSH), in addition to playing a major role as an antioxidant and in eliminating toxic compounds, has been implicated in prooxidation processes in various cells, via gamma-glutamyl-transpeptidase (gamma-GT)-dependent catabolism. Little information is available on the dynamics of nonprotein thiols (NPSHs) and disulfides (NPSSNPs) in spermatozoa and epididymal fluid (EF) during sperm passage in the epididymis. It is not clear whether NPSHs and NPSSNPs are involved in sperm protein thiol (PSH) oxidation or whether GSH catabolism in the epididymis can serve as a pathway for sperm PSH oxidation. In the present study, we used the thiol fluorescence labeling agent monobromobimane to analyze NPSHs and nonprotein disulfides (NPSSRs) (R, nonprotein or protein) in spermatozoa and EF in the rat caput and cauda epididymis. NPSH levels are shown to be significantly higher in the caput than in the cauda (spermatozoa and fluid). GSH in the caput lumen is subject to high gamma-GT activity. A marked loss of sperm GSH and a shift to an oxidized state (resulting in a significantly higher concentration of glutathione disulfides [GSSRs] than GSH) occur during the passage of spermatozoa from the caput to the cauda epididymis. Caput EF and extracellular NPSSNPs induce sperm thiol oxidation. The results suggest that epididymal NPSH/NPSSNP participates in sperm PSH oxidation and that some reactions of GSH in the gamma-GT pathway (in the epididymis) provide oxidizing power, leading to physiologic sperm thiol oxidation.     

An assessment of the fertilizing ability of spermatozoa in the epididymis of the marmoset monkey (Callithrix jacchus)

The fertilizing ability of spermatozoa from different regions of the epididymis of the marmoset monkey, Callithrix jacchus , was assessed by determining their rate of fusion with zonaless hamster ova in vitro. This technique tests for functional competence and was validated using epididymal spermatozoa of the hamster whose fertility have been measured previously by in vivo fertilization experiments. The results suggest that some marmoset spermatozoa first acquire the ability to fertilize in the distal caput and proximal corpus epididymidis although the majority become fertile on reaching the proximal cauda region. Acquisition of fertilizing capacity was associated with a change in the degree and character of sperm motility. However, it is considered that modifications to the sperm plasmalemma which allow capacitation and the acrosome reaction to occur are of primary importance for sperm fertility. The development of sperm fertilizing ability in the marmoset is discussed in relation to that in other primates.     

The appearance of basal cells in the developing murine epididymis and their temporal expression of macrophage antigens

This work demonstrates similarities between epididymal basal cells and macrophages in the mouse. Light microscopic studies of the postnatal development of the murine epididymis showed that basal cells were not present before days 12, 14 and 16 in the cauda, caput and corpus epididymis, respectively. An increase in cell number per unit length of tubule perimeter was demonstrated in all segments between days 20 and 27, when testicular fluid and spermatozoa start entering the epididymis. In the adult, there were more basal cells per unit perimeter in the cauda than caput or corpus epididymis. Conspicuous and consistent expression by basal cells of antigens detected by antibodies against tissue-fixed macrophages (F4/80) and mature macrophages (Mac-1) occurred only after they became established within the epithelium. Basal cells in the cauda epididymis did not display either antigen in the adult, although they persisted in the caput region. Such developmental patterns are compatible with the hypothesis that basal cells play a role in immune defence against sperm autoantigens.     

Forward motility protein (FMP): localization in hamster epididymal spermatozoa

The presence of FMP was investigated by immunocytochemistry in spermatozoa from the hamster caput and cauda epididymis. Spermatozoa from the caput showed no staining whereas spermatozoa pre-incubated with FMP were stained on the acrosome. Pre-treatment of the same sperm with epididymal plasma induced staining on the principal piece of the flagellum. Spermatozoa from the cauda were stained without previous incubation both on the acrosome and on the principal piece of the flagellum. These results suggest that the action of FMP, which prevents head-to-head agglutination of motile spermatozoa and allows acquisition of forward motility, may be due to at least two proteins. The first localizes to the acrosome during epididymal transit (anti-sticking factor), whilst the second localizes to the principal piece of the flagellum (forward motility initiation factor).     

Epididymal epithelial cells cultured in vitro prolong the motility of bovine sperm

It is well known that the epididymis is an excellent environment to maintain sperm viability. Therefore, we used different sections of bovine epididymis (caput, corpus, and cauda) to develop epithelial cell culture monolayers to identify factors that will increase sperm survival in the freezing-thawing process. Each epididymal section was dissected and treated with collagenase to obtain epithelial cell clusters. The cells were cultured in RPMI-1640 medium with 10% serum at 38.5 degrees C. A confluent monolayer was obtained after 5-7 days in culture and preliminary characterization using cytokeratin antibody indicated that the cell culture contained 85%-95% of epithelial cells. These cellular cultures were tested for their ability to maintain motility of epididymal and frozen-thawed spermatozoa. Washed spermatozoa were added to obtain a final dilution of 1 x 10(6) spermatozoa/mL. The motility of frozen-thawed spermatozoa was also recorded after incubation in conditioned media. Our results show that cocultures of spermatozoa and epididymal cell monolayers for 24 and 48 hours were beneficial for maintaining epididymal and frozen-thawed sperm motility (36.0% and 20.4%) compared with spermatozoa cultured with fibroblast cells or in the absence of a cell monolayer (0%; P < .01). The conditioned medium provides favorable conditions for sperm motility. Results with conditioned medium on maintenance of frozen-thawed sperm motility suggest that epididymal cells in vitro secrete beneficial factors that prolong the sperm survival.     

Glutathione, L-glutamic acid and γ-glutamyl transpeptidase in the bull reproductive tissues

The distribution of glutathione (GSH), L-glutamic acid (Glu) and gamma-glutamyl transpeptidase (gamma-GT) was studied in bull reproductive organs and fluids. Glutathione, the physiological substrate of gamma-GT, was localized specifically by a fluorescence method in the testis, epididymis and spermatozoa. Of the reproductive tissues, the testis, caput epididymis and ampulla had the highest levels of GSH, but it was also present in seminal fluid. Washed caput epididymal sperm had three times the GSH content of cauda epididymal or ejaculated sperm. In spermatozoa, GSH displayed maximal staining in the midpiece and tail regions. The highest levels of gamma-GT were encountered in the epididymis. The concentration of Glu was also high in the epididymis. Its formation may be due to the hydrolytic activity of gamma-GT, which, in addition, may have an important role in the transfer of Glu residues to reactive groups on the sperm surface.     

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.     

The ultrastructure of dog epididymis

Summary The ultrastructure of the epididymal duct and ductuli efferentes in the dog has been studied by electron microscopy. The epididymidis can be separated into the classical divisions of caput, corpus and cauda epididymidis on the basis of general morphology and ultrastructure. The ductuli efferentes have a low epithelium with pronounced cilia at the apices of cells and appear to provide primarily a transport role for spermatozoa. In the epididymis proper the caput region is characterized by an extremely large Golgi apparatus with large numbers of lysosomes and nuclear inclusions. Secretory activity appears to be most common in the corpus region. Absorption and secretion are most active in the first two segments while in the cauda epideidymidis the long-term storage of spermatozoa in the lumen is associated with many dense crystalline bodies formed in the epithelial cells within the Golgi apparatus and possibly deriving from absorbed macromolecular material from the lumen. The theory of whole sperm cell resorption by the epididymal duct is not supported by this study.