N—Acety1 β—D—glucosaminidase is not attached to human sperm membranes through the glycosylphosphatidyl inositol（GPI）—anchor

AIM: The mode of anchorage of N-acetyl beta-D-glucosaminidase (NAGA) on human ejaculated sperm was investigated. METHODS: Sperm plasma membrane was prepared by discontinuous sucrose gradient centrifugation from human sperm. NAGA was solubilized from these membranes by two detergents: octyl-glycoside and triton X-100. In separate studies, the release of the enzyme from the sperm membrane preparation by phosphatidylinositol specific phospholipase C (PI-PLC) was also examined. RESULTS: NAGA activity was detected on sperm membranes isolated from human ejaculates. The pattern of the enzyme solubilization by detergents indicated that the enzyme was an integral protein of sperm membrane. NAGA was not released from the sperm membranes by PI-PLC treatment. CONCLUSION: The evidence presented strongly suggests that human sperm membrane bound NAGA is not attached via the GPI anchor.     

Immunocytochemical localization and biochemical characterization of two seminal plasma proteins that protect ram spermatozoa against cold shock

We have already shown that seminal plasma proteins in the ram can repair cold-shock sperm membrane damage and that the addition of seminal plasma proteins before cold-shock treatment prevents sperm membrane injury. In this study, we prove that 2 protein bands of approximately 14 (P14) and 20 (P20) kd isolated from seminal plasma are responsible for this protective effect. In vitro capacitation (CA) and acrosome reaction (AR) modified the content of both proteins on the sperm surface. P20 release began at the beginning of CA, and the induction of the AR accounted for an additional release of both proteins; not more than 35% of these proteins remained on acrosome-reacted sperm. Cytochemical analysis detected that there are several binding sites for P14 and P20 on the sperm surface and that membrane alterations induced by CA and the AR accounted for the loss and redistribution of both proteins to the equatorial and postequatorial regions. The P14-sequenced fragment showed a high homology with several seminal plasma proteins of different species and contained the FN 2 domain like bovine PDC-109. However, the sequence of the P20 fragment was not homologous with any reported protein. By immunochemical analysis, we obtained evidence that P14 is phosphorylated in serine and threonine residues and that P20 is a glycosylated protein. These results suggest that both proteins are involved in sperm CA and gamete interaction, first by stabilizing the sperm membrane and then by participating in CA in the female reproductive tract. The protective effect of P14 and P20 could be related to their decapacitating role.     

Characterization of human sperm N-acetylglucosaminidase

N-acetylglucosaminidase (NAG) is particularly active in mammalian spermatozoa and appears to be involved in fertilization. Although it is assumed that this enzyme is acrosomal, previous results from our laboratory suggest the presence of NAG at the sperm plasma membrane level. The present study attempted to analyse the subcellular distribution of this enzyme in human spermatozoa. Sperm were incubated under different conditions and NAG activity measured in the soluble extracts and cell pellets using a specific fluorometric substrate. A significant proportion of NAG activity was released when sperm were incubated in culture medium, suggesting a weak association with the plasma membrane. This location was confirmed by western blot analysis of plasma membrane fractions and immunofluorescence on non-permeabilized sperm, which showed a positive signal mainly on the acrosomal domain. The distribution of NAG activity between plasma membrane and acrosome was analysed after cell disruption by freezing and thawing. Triton X-100 stimulated sperm and epididymal NAG activity but not the enzyme obtained from other sources. In addition, biotinylated human recombinant NAG was able to bind to human sperm. Finally, after sperm incubation under capacitating conditions, NAG total activity increased and the sperm enzyme lost its ability to be stimulated by Triton X-100. The possible connection of these results with sperm maturation, capacitation and NAG participation in primary binding to the zona pellucida, was discussed.     

Interleukin 1 and the glomerular mesangium. III. IL-1-dependent stimulation of mesangial cell protein kinase activity

Interleukin 1 (IL-1) exerts a number of biologic actions upon cultured glomerular mesangial cells (MC). These include stimulation of cellular proliferation and induction of prostaglandin and type IV collagenase secretion. It was determined that this activity, as with other polypeptide growth factors, was associated with the activation of specific MC plasma membrane protein kinases. Plasma membranes from cycling MC were incubated with purified IL-1 and (32P) ATP in the absence of calcium and cyclic nucleotides. Macrophage IL-1 stimulated the rapid phosphorylation of several plasma membrane proteins, the most significant of which were 52-55 kd, 46 kd, and 20 kd in size. Macrophage IL-1 induced specific membrane phosphorylation in concentrations as low as 1.5 x 10(-12) M, an effect obtained with equivalent concentrations of purified MC IL-1. The 46 kd phosphoprotein, which was the most prominent, was alkali-resistant and contained phosphotyrosine when examined by phosphoamino acid analysis. The 52-55 kd and 20 kd phosphoproteins were alkali-labile and contained phosphoserine. The 46 kd phosphoprotein was the major phosphoprotein recovered from Con A-Sepharose and IL-1 affinity columns. Induction of plasma membrane-associated protein kinase activity may represent one mechanism whereby IL-1 initiates mesangial cellular activation.     

Calpain and calpastatin are located between the plasma membrane and outer acrosomal membrane of cynomolgus macaque spermatozoa

Mammalian sperm must undergo an acrosome reaction prior to penetration of the zona pellucida and subsequent fusion with an oocyte. Sperm gain the capability to acrosome react after a period of capacitation, which primarily involves biochemical changes in the sperm membranes. The morphological events of the acrosome reaction have been well-documented, but the underlying cellular mechanisms that regulate capacitation and the acrosome reaction remain unclear. Antibodies to the 2 ubiquitous calpains, mu and m, as well as the small subunit, which associates with both calpains, were localized at the ultrastructural level to the region between the plasma membrane and the outer acrosomal membrane of cynomolgus macaque sperm. After the acrosome reaction, all of the anti-calpain antibodies labeled the acrosomal shroud, suggesting that calpains are located throughout the cytoplasmic area between the 2 outer sperm membranes. Calpastatin is an endogenous modulator of calpain activity and is also localized within the same cytoplasmic region as calpains. The antibodies used for ultrastructural localization were also used to probe Western blots of sperm extracts. Antibodies to either the mu- or m-calpain recognized an 80-kd protein, which is similar to the molecular weights of other ubiquitous calpains described. The small subunit (30 kd) was also recognized with a specific monoclonal antibody. An antibody to calpastatin recognized a major band at 78 kd and a lighter band at 45 kd, while the antibody to the testis-specific isoform of calpastatin (TCAST) recognized a 110-kd protein. We hypothesize that this cysteine protease system may be functional in cynomolgus macaque sperm during capacitation, the acrosome reaction, or both.     

Capacitated and acrosome reacted spermatozoa of goat (Capra indicus): a fluorescence and electron microscopic study

Summary. Plasma membrane alterations accompanying in vitro capacitation and acrosome reaction of goat spermatozoa were studied using lectin labelling, scanning electron microscopy, and freeze-fracture methods. Fluorescein isothi-ocyanate linked lectins namely; Canavalia ensiformis (ConA), Maclura pomifera (MPA), Arachis hypogaea (PNA), Glycine max (SBA) and Triticum vulgaris (WGA) agglutinin were used to examine the distribution of surface carbohydrates during these two events. The head and the sperm tail reveal altered lectin labelling features after capacitation and acrosome reaction. After capacitation the surface coat components for MPA, SBA, and WGA are shed from the spermatozoon head. ConA receptors on the head are retained after capacitation but are partially shed in the acrosome reacted spermatozoa. SBA receptor sites appear on the sperm tail of the capacitated spermatozoa. Unusual morphological changes attending capacitation involve the sperm tail-end which develops a novel entity, which we have termed 'spatula'. The 'spatula' shows strong binding with ConA and WGA only. In the acrosome reacted spermatozoa the spatulated tail-end unwinds with a concomitant loss of lectin labelling. Highly ordered membrane particles, 'ladders' of the middle piece of the epididymal sperm tail, disappear and IMP clearings appear on the middle piece and in the spatulated ends of the capacitated spermatozoa. But in the acrosome reacted sperm IMPs reappear and are randomly disposed on the middle-piece and are clustered in small patches on the principal-piece. IMP free areas appear on the plasma membrane covering the acrosome and the outer acrosomal membrane (OAM) of the capacitated spermatozoa. The plasma membrane and OAM fuse at multiple foci and appear as acrosomal 'ghosts' which remain associated with the sperm head even after acrosome reaction.     

Interaction of PDC-109, the major secretory protein from bull seminal vesicles, with bovine sperm membrane Ca2+-ATPase

PDC-109 is the prevalent secretory protein from bovine seminal vesicles that binds to the midpiece of sperm once they pass the ampulla of the vas deferens during emission. Thereby, the protein changes biophysical membrane properties, eventually resulting in increased sperm motility. To elucidate the underlying biochemical mechanism, we have studied the ion-pumping activity (Ca(2+)-ATPase) in membrane preparations of bovine spermatozoa following in vitro incubation with the protein and analyzed whether PDC-109 influences sperm motility. PDC-109 was purified to homogeneity from bull seminal vesicle extracts using a newly described method. The effect of PDC-109 on sperm motility was analyzed using the CASA-method. These experiments clearly demonstrated that PDC-109 significantly increases sperm motility. Calcium-pumping mechanisms were analyzed by monitoring the effect of PDC-109 on various parameters of enzyme activity of Ca(2+)-ATPase in epididymal sperm plasma membranes and were compared with Ca(2+)-ATPase activities from other organs and from epididymal sperm of different species, respectively. Specificity studies were performed using different Ca(2+)-antagonists. Enzyme activities of both Mg(2+)-dependent and Mg(2+)-independent Ca(2+)-ATPases increased in a dose-dependent manner following the addition of the PDC-109 (range 5-20 microg). Preincubation of PDC-109 at temperatures above 37 degrees C and pHs ranging from below 6.5 and above 8.5 led to the loss of the stimulatory effect. An analysis of enzyme kinetics pointed to irreversible, cooperative interaction of PDC-109 with the enzyme. The effect was organ-specific, that is, restricted to sperm ATPases, but it was not species-specific, as it could be elicited also in rat sperm.     

微波辐射家兔阴囊后对精子透明质酸酶活力的影响

本文对用微波辐射阴囊后的新西兰种雄兔排出精子内透明质酸酶活力进行了测定,并与辐射前排出精子内透明质酸酶活力进行了比较。结果发现:辐射后第一次排出的精子(约辐射后2周左右),其所含透明质酸酶的活力比辐射前明显降低(P<0.01),以后各次采集到的精子的酶活力基本与辐射前一致。此结果提示:微波辐射动物阴囊能使贮存在附睾内精子透明质酸酶活力受到抑制。     

Identification of a role for a mouse sperm surface aldo‐keto reductase (AKR1B7) and its human analogue in the detoxification of the reactive aldehyde,acrolein

Mouse vas deferens protein (AKR1B7), a member of the aldo‐keto reductase family, was purified to homogeneity. Antibodies raised to AKR1B7 revealed an aldo‐keto reductase on the human sperm surface, while confocal microscopy experiments demonstrated that this enzyme covered the entire human sperm surface and was concentrated on the mid‐piece. Further functional characterisation of a recombinant form of AKR1B7 showed that the likely role of AKR1B7 is the reduction of the reactive aldehyde, acrolein, a by‐product of spermine catabolism in the reproductive tract. A similar acrolein detoxification activity was displayed by human sperm membrane extracts but was not present in seminal plasma. These results indicate that human sperm possess an aldo‐keto reductase on their membrane surface and are thus enzymatically protected against reactive aldehyde species both in the male and female reproductive tract.     

Binding of protein D/E to the surface of rat epididymal sperm before ejaculation and after deposition in the female reproductive tract

The objectives of the present investigation were to study the interaction of protein D/E with the surface of rat epididymal spermatozoa and to assess its topology on the spermatozoa surface before and after deposition in the female reproductive tract. Protein D/E, a member of the cysteine-rich secretory protein (CRISP-1) family, has been proposed to be involved in sperm-egg membrane fusion. In vitro competitive photoactivated cross-linking experiments followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis revealed that protein D/E molecules specifically interact with two surface proteins exhibiting an M(r) approximately 120.0 kd and approximately 130.0 kd, respectively, on the sperm surface. In vitro treatment of epididymal spermatozoa with phosphatidylinositol specific-phospholipase C revealed the release of protein D/E molecules over the head region but not the tail region of spermatozoa. Indirect immunofluorescence experiments using polyclonal antibodies generated against a highly purified protein D/E preparation demonstrated that protein D/E molecules were bound to the surface of spermatozoa recovered from the epididymal and female reproductive tracts, even after 7 hours. These results indicate that protein D/E molecules interact with specific membrane proteins, and is subsequently covalently bound to the surface of spermatozoa via a glycosyl-phosphatidyl inositol linkage. In addition, protein D/E molecules remain covalently bound to spermatozoa after deposition in the female reproductive tract, an observation that is consistent with the proposed physiological function of the protein in the fertilization process.     

自身免疫性睾丸炎对精子特异性酶和生育功能的影响

目的 :研究自身免疫性睾丸炎对精子特异性酶和生育功能的影响。　方法 :复制豚鼠实验性变态反应性睾丸炎 (EAO)模型 ,采用酶动力学分光光度法和明胶固定底物薄膜法 ,观察EAO状态下精子顶体蛋白酶、透明质酸酶、精子胞质乳酸脱氢酶、附睾尾部精子和睾丸组织形态的变化。　结果 :EAO造成附睾精子顶体酶系中顶体蛋白酶、透明质酸酶和乳酸脱氢酶活性下降、附睾尾部精子质量下降、睾丸生精细胞发生退行性病变。　结论 :EAO明显影响雄性豚鼠生育力 ,睾丸生精细胞和附睾精子可能是主要作用环节。     

人精子透明质酸酶活性测定的临床意义

目的 :测定人精子透明质酸酶 (HYD)活性并分析其与精液常规参数之间的相关性。　方法 :用改良Singer法测定 14 6例男性精子HYD活性 ,常规检测精子密度、活动率和正常形态百分率。　结果 :精子HYD活性与密度、活动率及与正常形态百分率存在显著相关性 (r值分别为 0 .65、0 .63和 0 .72 ,P均 <0 .0 1)。不育男性精子密度在 4 0× 10 6 /ml以上组的HYD活性明显高于精子密度少于 2 0× 10 6 /ml以下组 (P <0 .0 1)。不育男性精子活动率 >60 %组的HYD活性明显高于 <3 0 %组 (P <0 .0 1)。　结论 :精子HYD活性测定是评价精子功能的有效指标之一     

育精阴对精子特异性酶的影响

本实验通过复制实验性变态反应性睾丸炎（EAO）模型,采用酶动力学分光光度法以及明胶固定底物薄膜法,观察EAO状态下精子顶体蛋白酶、透明质酸酶和乳酸脱氢酶的变化,观察育精阴对精子特异性酶的影响。结果：EAO造成的附睾精子顶体酶系中顶体蛋白酶和透明质酸酶以及精子胞浆乳酸脱氢酶活性下降。在育精阴治疗后,顶体蛋白酶、透明质酸酶和乳酸胶氢酶活性显著恢复。揭示育精阴对EAO造成的免疫损伤有修复作用。     

Changes in the lectin binding sites on the testicular, epididymal, vas, and ejaculated spermatozoon surface of dog

Summary. Changes in the localization of sperm surface glycocomponents of testicular, epididymal, vas deferens, and ejaculated spermatozoa of dog ( Canis domesticus ) were studied employing fluorescein isothiocyanate conjugated lectins viz., Concanavalin A (ConA), Triticum vulgaris (WGA), Maclura pomifera (MPA), and Arachis hypogaea (PNA) agglutinins. The plasma membrane clothing the acrosome of the testicular, epididymal, and vas deferens spermatozoa shows reactivity with all the lectins used. However, in the ejaculated spermatozoa, the entire sperm surface shows reactive sites for ConA, WGA, and PNA. Variation in the labelling of the cytoplasmic droplet in different stages of spermatozoon transit in the epididymis has been discussed.     

精子膜抗原的研究新进展

精子表面的膜抗原是精子发生成熟过程中的重要标志分子 ,对其深入研究有助于揭示精子发生成熟及受精过程的生理机制和不育的病理改变。本文回顾了近年来随着分子生物学技术的广泛应用 ,传统的一些重要的精子膜抗原相继得到了克隆和测序 ,一些新的表达精子膜抗原的基因也陆续被发现 ,为从蛋白水平进一步深入研究其功能以及免疫避孕疫苗的筛选奠定了基础。     

Heme oxygenase enzyme activity in human seminal plasma of fertile and infertile males

This work aimed to assess heme oxygenase (HO) enzyme activity relationship with different human semen parameters. One hundred and twenty men were divided according to their sperm count and clinical examination into: obstructive azoospermia (n = 20), nonobstructive azoospermia (NOA) (n = 25), oligozoospermia (n = 35) and normozoospermia (n = 40). Semen analysis, western blot for HO-1 and HO-2, and estimation of seminal plasma HO enzyme activity chemically in the form of bilirubin concentration were carried out. Seminal plasma HO enzyme activity was very low in OA specimens, low in NOA, moderate in oligozoospermia while higher in normozoospermia (mean +/- SD; 6.26 +/- 2.2, 81.4 +/- 35.5, 283.8 +/- 90.1, 657.4 +/- 227.6 pmol ml(-1) min(-1)) with significant differences. Western blot analysis demonstrated HO-2 expression in all studied groups whereas HO-1 was highly expressed in fertile normozoospermic group compared with other groups. There was positive correlation between seminal plasma HO enzyme activity and sperm concentration, sperm motility percentage, motile spermatozoa ml(-1) and sperm normal morphology per cent. It is concluded that HO enzyme activity in the human seminal plasma is related to spermatogenesis and sperm-motility processes.     

Uteroglobin and transglutaminase modulate human sperm functions

During the process of capacitation, spermatozoa undergo significant changes in membrane composition, including removal of decapacitating factors (DFs), which are present in seminal plasma, that lead to increased sensitivity to physiological stimuli of the acrosome reaction. In the present study we investigated the presence, localization, and effects on human spermatozoa of 2 proteins of seminal plasma origin, uteroglobin (UG) and transglutaminase (TG). These 2 proteins interact with one another because TG promotes covalent links of UG to sperm surface proteins. We found that UG is localized around the entire surface of ejaculated human sperm, whereas TG is predominantly localized in the neck. FACScan analysis confirmed the surface localization of both antigens and demonstrated that swim-up selection of spermatozoa was associated with a significant reduction in the contents of the 2 substances when compared with unselected samples. Western blot analysis of UG in total sperm lysates confirmed the lower content of the protein in swim-up-selected sperm. Swim-up-selected sperm were characterized by their ability to undergo a spontaneous, time-dependent increase of capacitation-characteristic chlortetracycline pattern of fluorescence and increase in responsiveness to progesterone. Such changes were not observed in unselected sperm. Exogenous addition of TG, together with recombinant rabbit UG, prevented the spontaneous increase in responsiveness to progesterone (acrosome reaction and intracellular calcium) at 24 hours in swim-up-selected sperm, suggesting the occurrence of a capacitation-inhibiting activity of the 2 substances. In addition, we found that endogenous UG and TG contents, as determined by FACScan analysis, were negatively correlated (P < .0001) with sperm motility and that exogenous addition of the 2 substances resulted in a substantial reduction of progressive motility (P < .01). Collectively, these data indicate that TG and UG represent 2 DFs, and contribute to understanding the biochemical mechanisms that characterize the process of capacitation.     

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.