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.     

Effects of antifertility drugs on epididymal protein secretion, acquisition of sperm surface proteins and fertility in male rats

The possibility that α-chlorohydrin, 6-chloro-6-deoxyglucose (6CDG) and cyproterone acetate (CPA) might affect epididymal protein secretion or acquisition of sperm surface proteins as the cause of their antifertility action in male rats was investigated. Daily administration of 9 mg/kg α-chlorohydrin for 7–14 days and 24 mg/kg 6CDG for 14–21 days induced sterility in male rats and impaired the capacity of the cauda epididymal spermatozoa to initiate motility. Treatment with CPA (30 mg/kg/day) for 21–28 days, however, was found to have no effect on fertility and initiation of sperm motility, although the epididymis of the treated animals underwent a loss in weight. The antifertility effects of α-chlorohydrin or 6CDG did not seem to be attributed to an interference with epididymal protein secretion. The cauda epididymal fluids of the α-chlorohydrin, 6CDG and CPA treated animals have similar protein patterns compared to those of the control animals. However, when the surface proteins of the spermatozoa were labelled with radioactive iodine, the sperm surface proteins of the α-chlorohydrin and 6CDG treated animals were found to differ from those of the control animals. Two peaks (MW 32 000 and 70 000) and one peak (70 000) were significantly reduced in the α-chlorohydrin treated and 6CDG treated animals, respectively. Additional bands appeared on the surface of the treated (infertile) animals. In contrast, CPA treatment did not affect the surface protein pattern of the epididymal spermatozoa. It was concluded that the antifertility affects of α-chlorolydrin and 6CDG are not due to an interference with epididymal secretion of specific proteins but to an intervention of the subsequent acquisition of these proteins by epididymal spermatozoa. This results in a decrease in the capacity of the epididymal sperm to initiate motility and hence a loss of fertilizing capacity.     

Correlation between changes in rat sperm membrane lipids, protein, and the membrane physical state during epididymal maturation.

An enriched plasma membrane fraction was isolated from caput, corpus, and cauda rat spermatozoa and analyzed for lipid and protein content, thermal phase transition temperature using electron paramagnetic resonance spectroscopy (EPR), and enzymatic assays of calcium-dependent ATPase activity. Based on sperm concentration, total membrane phospholipid, cholesterol, and protein content declined as sperm passed through the epididymis. A more refined analysis of the bulk plasma membrane phospholipid revealed that approximately 56% of the phospholipid consisted of choline (PC) and ethanolamine (PE) phosphoglycerides; the remainder consisted of sphingomyelin (SM), phosphatidylserine (PS), and diphosphatidylglycerol (DPG). The mole percent of PE increased in sperm proceeding from the caput to the corpus epididymis and then declined from the corpus to the cauda epididymis. The phospholipid-bound fatty acids consisted primarily of palmitate (C16:0) and stearate (C18:0), with a significant increase in the mole percent of the docosapentenoyl acyl group (C22:5) in cauda sperm. Arrhenius' plots of the EPR peak height signals using the lipid soluble spin label, 5-doxyldecane, and the calcium-dependent ATPase activity as a function of temperature demonstrated a change in the apparent fluidity of the membrane and energy of activation of the calcium-dependent ATPase associated with the three sperm membrane preparations. These data suggest that the apparent fluidity and biochemical composition of the sperm membrane change during epididymal maturation.     

Beneficial effects of vitamin E in sperm functions in the rat after spinal cord injury

Male infertility as a result of spinal cord injury (SCI) is associated with abnormal semen qualities including low sperm counts and poor sperm motility and morphology. Clinical studies suggest that reactive oxygen species (ROS)-related events might contribute to abnormal sperm functions after SCI. The current study examined whether impaired sperm functions after SCI can be ameliorated by an antioxidant, vitamin E. Vitamin E feeding of spinal cord transected (SCX) rats during the acute (maintenance) and chronic (restoration) phases of the injury partially preserved sperm viability and mitochondrial potential; similar effects were only seen in spinal cord contused (SCC) rats during the chronic phase. A beneficial effect of vitamin E on sperm motility, however, was only observed in SCX rats during the chronic phase of the injury. These results suggest that ROS-related events might account for some of the effects of cord injury on sperm functions, depending on the extent of injury and time postinjury. Furthermore, we found that sperm heads from SCC and SCX rats were less condensed compared to those from sham control rats. Such effects were attenuated by vitamin E, suggesting that ROS-related events may also contribute to abnormal sperm morphology after SCI. Partial restoration of male accessory gland weights in those rats fed vitamin E further suggests its beneficial effects on the functions of these glands. Conclusion: Vitamin E feeding attenuated some of the effects of spinal cord injury on sperm functions and male accessory glands in the rat. These results support a role of ROS-related events in deterioration of semen quality after cord injury. Further understanding of the underlying mechanisms for effects of vitamin E on sperm functions and male accessory glands will provide scientific rationale for the use of vitamin E or other antioxidant as therapeutic means to preserve sperm functions and semen quality in SCI men.     

人精子体外获能前后计算机辅助分析参数的比较

目的为临床使用计算机辅助精子分析(CASA)系统的精液分析参数提供参考。方法对48例人类冷冻复温精液和116例临床进行丈夫人工授精(AIH)的新鲜精液体外获能前后CASA检测结果,分组进行配对资料t-检验和独立样本t-检验。结果所有实验组精子体外获能后的曲线运动速度(VCL)、直线运动速度(VSL)、平均路径速度(VAP)、精子头侧摆幅度(ALH)和鞭打频率(BCF)显著提高(P<0．01);除冻存精液精子体外获能后的运动前向性(STR)和头部椭圆度外,其余各组精液精子获能后的STR、直线性(LIN)和头部椭圆度显著下降(P<0,01)。行AIH治疗的精液标本,无论获能前后,已孕组精子VAP、LIN和头部椭圆度显著高于未孕组(P<0．05),而BCF和头部面积低于未孕组(P<0.05);获能前两组的(a b)级精子百分率无显著性差异,获能后差异显著(P<0．05)。结论体外获能处理可显著提高精子的各种运动速度,但精子运动的前向性和直线性降低;精液常规分析结果中的精子运动等级和速度不直接反映精子的受精能力。     

Conjugation of maturation-related wheat-germ-lectin-binding proteins to caput epididymal sperm in co-cultures with corpus epididymal epithelial cells of BALB/c mouse

In BALB/c mice, two maturation-related wheat-germ-binding glycoproteins (GP-49 and GP-83) are synthesized and secreted by corpus and cauda epididymis. A co-culture technique was used to investigate these glycoproteins in principal cells of corpus epididymis and the conjugation of these molecules on caput sperm. The principal cells were recovered from corpus epididymides of 4-week-old mice and cultured in RPMI 1640 medium supplemented with 10% fetal calf serum. After culturing for 3-4 days, most cells revealed epithelial cell-specific keratins in immunofluorescent localization with monoclonal antibody. By electron microscopy, a prominent nucleolus with well-extended euchromatin was revealed in the nucleus and the cytoplasm contained multivesicular bodies, and a well-developed Golgi apparatus with endoplasmic reticulum. By SDS-PAGE, GP-83 and GP-49 were revealed in the cell extracts and cell culture supernatants after incubation with 35S-methionine. Radiolabeled binding sites were also found on the surface of caput sperm co-cultured with the principal cells for 4 h in the presence of 35S-methionine. WGA-binding glycoproteins may be synthesized and secreted by the principal cells of corpus epididymis and conjugated to caput sperm during the epididymal transit.     

计算机辅助分析人、家兔、大鼠和小鼠附睾精子运动能力

本研究应用计算机辅助精子分析（ＣＡＳＡ）定量分析了人、家兔、大鼠和小鼠精子附睾成熟过程中，精子运动能力的发生和发展。同时对这几种动物和人进行了系统分析和比较。结果表明：在不同种属之间，其运动的发生和发展具有一定的差异；各种不同种属动物精子在各自附睾成熟过程中，其运动能力的两个方面参数，运动速度和运动方式的发展是不平行的；附睾尾部精子的运动能力（包括运动速度和直线程度）最强。     

Cathepsin D in human reproductive tissues: cellular localization in testis and epididymis and surface distribution in different sperm conditions

Mammalian sperm surface antigens are acquired either during spermatogenesis or sperm maturation in the epididymis. These antigens, many of which are hydrolytic enzymes, are actively synthesized and secreted by the resident epithelial cells and adsorbed to the sperm membrane as part of posttesticular sperm modification. In this study, we aimed to investigate the expression of cathepsin-D (CAT-D) in human reproductive tissues and its distribution on the sperm surface in different sperm conditions. Immunohistochemical results revealed the expression of CAT-D in the somatic Sertoli and Leydig cells without showing any immunoreactivity in any germ cells, despite their engagement of the acrosomal system. A strong immunoreactivity of anti-CAT-D was also detected in the epididymal epithelium, chiefly in the principal cells, which are known to actively synthesize and secrete proteins into the epididymal lumen. The absence of CAT-D in the clear cells was unexpected because these cells are known to engage the endosomal machinery. We further showed that CAT-D was anchored on the sperm surface confined to the postacrosomal region without any lateral redistribution within the membrane during sperm capacitation. However, the enzyme underwent changes to be an active form of a 29/30-kd doublet during sperm capacitation. Using CAT-D as a marker, we were able to demonstrate here localization of the enzyme in human reproductive tissues, as well as reveal membrane modification in human sperm during maturation and capacitation processes.     

Acidification of testicular and epididymal fluids in the rat after surgically-induced varicocele

Experimental left varicocele (ELV) in adult rats produces a bilateral increase in testicular blood-flow and temperature which may alter the intraluminal environment in which sperm mature. The purpose of the present study was to evaluate the effect of ELV on the in-situ pH, PCO2 and bicarbonate concentration ([HCO3-]) in seminiferous tubules (ST), initial segment (IS), proximal caput (PCP), middle caput (MCP), middle corpus (MCR), and proximal cauda epididymidis (PCD) of the rat employing pH and PCO2 microelectrodes. Adult male rats with ELV or sham-surgeries (control) were studied after 30 days. Relative to controls, ELV significantly reduced the in-situ PCO2 in the testicular artery, ST, IS, PCP, MCP, MCR and PCD. In spite of this reduction, all values remained significantly higher than the systemic arterial blood PCO2. Values for in-situ pH in ST, IS, PCP, MCP, MCR, and PCD of control rats were significantly more acidic than systemic arterial blood. Furthermore, ELV decreased the pH in ST, IS, PCP, and MCP when compared to control values. The calculated [HCO3-] in ST from control animals was less than half that in systemic arterial blood and was reduced further in IS, PCP, and MCP. Varicocele did not change the [HCO3-] in systemic arterial blood but reduced markedly the values in ST, IS, PCP, and MCP. These alterations in the in-situ pH, PCO2, and [HCO3-] in structures of the rat testis and epididymis may play a role in the anti-spermatic effect of ELV.     

Expression and phosphorylation of mitogen-activated protein kinases during spermatogenesis and epididymal sperm maturation in mice.

The expression and phosphorylation/dephosphorylation of mitogen-activated protein (MAP) kinases during mouse spermatogenesis and epididymal sperm maturation have been investigated by immunoblotting and immunohistochemical staining with commercially available anti-ERK2 and anti-Active MAPK antibodies. Two forms of MAP kinases, p42ERK2 and p44ERK1, were expressed in a similar amount in spermatogenic cells at different stages. ERK1 and ERK2 were phosphorylated (activated) in early spermatogenic cells from primitive spermatogonia to zygotene primary spermatocytes, while only a small quantity of phosphorylated MAP kinases could be detected in pachytene primary spermatocytes and spermatids. MAP kinase activity in primative spermatogonia and preleptotene primary spermatocytes was the highest among spermatogenic cells. ERK1 and ERK2 were also present in epididymal spermatozoa, and their phosphorylation was increased while spermatozoa pass through epididymis and vas deferens for maturation. It would appear that MAP kinase activation may contribute to the mitotic proliferation of primative spermatogonia, an early phase of spermatogenic meiosis, and, later, sperm motility acquirement.     

禽流感病毒感染小鼠前后表面蛋白编码基因变异情况分析

目的了解禽流感病毒（AIV）在感染小鼠前后表面蛋白编码基因的特点及变异情况。方法在A／Goose／Guangdong／NH／2003（H5N1）感染小鼠肺组织的第12小时和第9天各分离1株AIV病毒，通过鸡胚增殖后提取RNA，反转录合成cDNA，经PCR扩增和产物纯化构建重组质粒，用双脱氧链终止法进行核苷酸序列测定并进行基因特性分析。结果3株病毒HA基因的核苷酸序列同源性为99．6％～99．8％，推导氨基酸序列的同源性为99．3％～99．6％。3株病毒NA基因的核苷酸序列同源性为99．8％-99．9％，均为同义突变。M基因未发生任何变异。结论AIV感染小鼠后HA基因出现变异，NA和肘基因未出现有意义的突变。     

The secretory activity of the seminal vesicles and its relationship to sperm motility: effects of infection in the male reproductive tract

In 146 males aged between 20 years and 40 years attending an infertility service, the secretory activity of the seminal vesicles was assessed by measurement of corrected seminal fructose concentration. This value was related to the presence of a positive semen culture, other evidence of inflammatory processes in the reproductive tract and sperm motility. Only 48% of subjects with a positive semen culture showed evidence of inflammation in the reproductive tract, as assessed by the presence of more than 20 white blood cells per high power field, and greater than 10% spermagglutination in the ejaculate. There was a relationship between the inflammatory process, hypofunction of the seminal vesicles and poor sperm motility. When the semen culture was positive but there was no evidence of inflammation neither seminal vesicle function nor sperm motility was affected. When the semen culture was negative, i.e. no evidence of inflammation and the subjects were asthenozoospermic, the corrected fructose levels were normal. It is proposed that in these conditions the cause of asthenozoospermia may be factors other than accessory sex organ dysfunction. In conclusion, there was no close relationship between the bacteriological results and evidence of inflammation of the accessory glands. A positive semen culture was related to lower levels of corrected fructose (hypofunction of the seminal vesicles) when the positive sperm culture was associated with inflammation of the reproductive tract and asthenozoospermia.     

大鼠睾丸扭转复位后附睾上皮细胞凋亡及唾液酸变化

目的:探讨大鼠睾丸扭转复位后,附睾上皮细胞凋亡与唾液酸分泌的关系。方法:24只雄性SD大鼠建立左侧睾丸扭转复位模型,分为对照组、扭转2h和4h共3组,每组8只。TUNEL法检测附睾上皮细胞凋亡,5-甲基苯二酚法检测扭转侧附睾唾液酸的含量。结果:睾丸扭转2h复位后24h,扭转侧附睾上皮细胞凋亡指数与对照组相比上升不明显(P>0.05),唾液酸含量改变不明显(P>0.05);扭转4h复位后24h,扭转侧附睾上皮细胞凋亡指数与对照组相比上升极显著(P<0.001),唾液酸含量下降明显(P<0.05)。结论:睾丸扭转2h复位后24h,附睾上皮细胞未发生凋亡,其分泌唾液酸功能不受影响;扭转4h复位后24h,附睾上皮细胞凋亡严重,其分泌唾液酸功能下降。     

尿毒症患者肾移植前后精子形态学变化的研究

目的 了解尿毒症患者肾移植前后精子形态及多重精子缺陷指数的变化。方法 对15例尿毒症患者、10例肾移植术后患者的精液进行精子形态学及MAI和SDI的检测并与12例正常男子进行对照。结果 尿毒症组的精子正常形态率明显低于肾移植组和正常男子组(P<0.01),肾移植组和正常男子组间无差异(P>0.05)。MAI及精子畸形指数(SDI)3组均处于正常范围,其中MAI 3组间无差异(P>0.05),而SDI3组间有明显差异(P<0.01)。结论 尿毒症组的精子正常形态率明显下降,肾移植组虽然精子正常形态率处于正常范围,但与尿毒症组一样缺陷精子总数仍明显多于正常男子。     

Motility potential of macaque epididymal sperm: the role of protein phosphatase and glycogen synthase kinase-3 activities

Human and monkey ejaculated sperm contain protein phosphatase-1 (PP1), PP1 inhibitor 2 (12), and glycogen synthase kinase-3 (GSK-3). Inhibition of ejaculated human sperm protein phosphatase (PP) activity with calyculin-a (CL-A) significantly stimulates motility, implicating protein dephosphorylation in motility regulation. The present experiments were conducted to characterize and compare PP and GSK-3 activity in monkey caput and caudal epididymal sperm, to determine the cellular distribution of these enzymes, and to test the thesis that epididymal sperm PP activity is inversely related to motility. Caput epididymal sperm populations, (8.8% motile) contained levels of PP activity that were >3 times as high as those of caudal spermatozoa. This PP activity was further identified by inhibitor response profiles as PP1. In both caput and caudal sperm, the majority of this PP1 activity was localized in 100,000 x g soluble fractions. Western blot analysis indicated that a portion of this difference was the result of elevated amounts of PP1 in caput compared with caudal epididymal sperm. The presence of GSK-3 activity was undetectable in 100,000 x g insoluble fractions of epididymal sperm, whereas both caput and caudal sperm soluble fractions contained GSK-3 activity, which was approximately threefold higher in caput sperm compared with caudal populations. Treatment of caput epididymal sperm from the rhesus macaque with the PP inhibitor CL-A resulted in a significant, dose-dependent increase from 8 to 38% motile cells (without any effect on their path velocity). In contrast, CL-A had no significant influence on either percent motility or path velocity of caudal epididymal sperm. Cytosolic PP1 and GSK-3 activities appear to be inversely related to the motility of monkey epididymal sperm and may have a regulatory role in the development of the potential for motility in epididymal sperm.     

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.     

Increase in fluid viscosity during epididymal transit and the immobilization of rat epididymal spermatozoa

A microcapillary method was developed to measure the viscosity of small volumes of undiluted epididymal fluid. Fluid from the cauda epididymis registered 82 +/- 17 centipoise which was much more viscous than fluid from the caput region (8 +/- 2 centipoise). Initiation of sperm motility was strongly suppressed in the viscosity range of 7 to 150 centipoise. A significant increase in the viscosity of fluid from the caput region was observed when immobilin in the fluid binds with a lectin from Jack fruit (Artocarpus heterophyllus). Thus, it is postulated that aggregation of the immobilin induced by a lectin-like material produced by the cauda epididymis may be a mechanism by which fluid viscosity is increased during epididymal transit.