Monoclonal antibody c262 counteracts the stimulatory effect of progesterone on sperm-oocyte fusion

Some evidence suggests that the non-genomic effects exerted by progesterone (P) on human spermatozoa are mediated by membrane receptor(s) displaying the C-terminal domain, but not the N-terminal domain of the genomic P receptor (PR). This study aimed at determining whether the monoclonal antibody (mAb) c-262, directed against the C-terminal domain of the genomic PR, counteracts the stimulatory effect of P on the human sperm ability to fuse with oocytes. Sperm/oocyte fusion was evaluated by means of the hamster egg penetration test. The brief exposure of capacitated spermatozoa to P produced a stimulatory effect on sperm/oocyte fusion. mAb c262 counteracted this stimulatory effect in a dose-dependent manner. No counteraction was observed when capacitated spermatozoa were pre-exposed to PGR-312, a mAb directed against the N-terminal domain of the genomic PR. These results reinforce the hypothesis that the non-genomic effects exerted by P on human spermatozoa are mediated by membrane receptor(s) displaying the C-terminal domain, but not the N-terminal domain of the genomic PR.     

人精子质膜孕激素受体研究

目的 :观察人精子表面孕激素受体 (PR)的定位及阳性表达率。　方法 :精子体外获能后 ,应用异硫氰酸荧光素标记的牛血清白蛋白 孕酮复合物 (P BSA FITC)染色 ,荧光显微镜观察及流式细胞术 (FCM )定量分析法 ,分别观察与孕酮 (P)结合精子形态及标记精子所占比例。　结果 :P BSA FITC染色精子的形态主要表现为 2种类型 :整个顶体区域或仅赤道区呈绿色荧光 ,顶体后区及尾部不着色。与P结合精子的百分率为 (30 2± 2 4 ) %。　结论 :人精子顶体表面有PR表达 ,且这种表达呈选择性     

Migration and ultrastructural localization of the c-kit receptor protein in spermatogenic cells and spermatozoa of the mouse

PURPOSE: The c-kit receptor is a proto-oncogene important in germ cell migration and maturation and has also been demonstrated on the acrosomal region of mature sperm. The purpose of the present study was to examine the ultrastructural location of the c-kit receptor in mouse testis and sperm. MATERIALS AND METHODS: Testis and sperm from mature male mice were examined for the c-kit receptor utilizing electron microscopy and Western blot analysis techniques. Thin sections of mouse testis and sperm were stained with immunogold-labeled anti-c-kit antibodies. The protein from these testes and sperm was also utilized for Western blot analysis. RESULTS: The c-kit protein was localized within the mouse testes to the type A spermatogonia, the round spermatids, and the mature testicular spermatozoa. The c-kit receptor was noted to migrate from the lumen of the acrosomal vesicles in the early spermatids to the plasma membrane of the late spermatids. It was also noted in the acrosomal region of the testicular spermatozoa, as well as the sperm from the epididymis. Sperm undergoing the acrosome reaction demonstrated association of the c-kit receptor with the plasma membrane of the acrosome, but not on the acrosomal membrane itself. Western blot analysis demonstrated protein bands of 150 kDa in testis and intact sperm. CONCLUSIONS: The present study confirms the presence of the c-kit receptor in mouse testis and sperm. It also demonstrates that this receptor is localized to the region of the developing acrosome.     

精子表面蛋白受精素β在精卵结合和融合中的作用研究

为探讨精子表面蛋白受精素β（Fβ）在精－卵结合和融合中的作用。西文应用鼠β单抗,通过体外受精试验观察该单抗预卿育不同浓度精子后,对精－卵结合和融合功能的影响;应用免疫细胞化学方法确定Fβ在精子表面的特异性定位。结果在不同精子浓度下,Fβ单抗均可显著降低体外精－卵的结合和融合能力;可使精－卵结合指数降低34．43％－75．46％,平均56．05％;使精－卵融合的受精指数降低47．68％－56．32％,平均52．18％;受精率下降28．06％－54．32％,平均41．56％。在精子顶体反应前后,Fβ在精子表面均特异性定位于精子头部赤道区域和顶体内膜区域。结论：Fβ在精－卵的结合和融合中均具有重要作用,支持将Fβ蛋白作为精子靶抗原之一用于避孕疫苗的开发研究。     

Progesterone Receptors in Mammary Gland Development and Tumorigenesis

The steroid hormone, progesterone (P), is a central coordinator of all aspects of female reproductive activity and plays a key role in pregnancy-associated mammary gland morphogenesis and mammary tumorigenesis. The effects of P on the mammary gland are mediated by two structurally and functionally distinct nuclear receptors PR-A and PR-B that arise from a single gene. Null mutation of both receptors in PR knockout (PRKO) mice has demonstrated a critical role for PRs in mediating pregnancy-associated mammary ductal branching and lobuloalveolar differentiation and in initiation of mammary tumors in response to carcinogen. Analysis of the molecular genetic pathways disrupted in PRKO mice has recently yielded important insights into the molecular mechanisms of regulation of mammary gland morphogenesis by PRs. In addition to its essential role in regulating proliferative and differentiative responses of the adult mammary gland during pregnancy, P plays a critical role in the protection against mammary tumorigenesis afforded by early parity. Thus, the effects of P on postnatal developmental plasticity of the mammary gland differ between young and adult glands. This review will summarize recent advances in our understanding of 1) the molecular mechanisms by which PRs mediate pregnancy-associated mammary gland morphogenesis, 2) the role of PRs in mediating tumorigenic responses of the adult mammary gland to carcinogen, and 3) the role of P in long-term protection of the juvenile mammary gland against tumorigenesis. In addition, we will summarize recent insights into the isoform selective contributions to some of these activities of PRs obtained from comparative analysis of P-dependent mammary gland development in PR isoform specific knockout mice lacking either the PR-A (PRAKO) or PR-B (PRBKO).     

正常生育男性精子膜孕激素受体的放射性配基结合分析

目的:研究体外获能2h后正常生育男性精子膜上的孕酮结合位点。方法:用上游法分离活力良好的精子并于体外获能2h,然后进行多点结合饱和实验,在7个总结合管内分别加入精子悬液和浓度递增的孕酮-11α-葡萄糖醛酸甙-[125I]碘化酪胺(125I-孕酮),在相应的非特异管内除加入与总结合管等量的精子悬液和125I-孕酮外,另加入10μmol/L孕酮,于4℃孵育1h,终止孵育后分别测定各管以及离心后沉淀的放射性,运用Scatchard函数的单位点多点饱和法数学模型,用最小二乘回归法计算平衡解离常数(Kd)和最大结合容量(Bmax)。结果:Kd为(0.61±0.04)nmol/L,Bmax为(830±344)结合位点数/细胞,对回归方程进行的显著性检验表明r=-0.980,P<0.01。结论:正常生育男性精子膜上存在一种高亲和力和低容量性孕酮结合位点即孕激素受体。     

Con A-binding protein Zn-α2-glycoprotein on human sperm membrane is related to acrosome reaction and sperm fertility

Fertilization, the recognition and fusion between spermatozoa and oocyte, involves various molecules on the spermatozoa and oocyte membranes. Concanavalin A (ConA)-binding proteins may be one of the molecules involved in mammal spermatozoa fertilization; however, their structure and function remain largely unknown. Here, we initially identified a ConA-binding protein, Zn-α2-glycoprotein (ZAG), involved in regulating the acrosome reaction (AR) of human spermatozoa. ZAG is localized on the pre-equatorial region covering the acrosome, neck and tail (some parts of middle piece and principal piece respectively) regions of the acrosome intact human spermatozoa, and disappears in the acrosomal region of the acrosome-reacted spermatozoa. Polyclonal antibodies against human recombinant ZAG significantly reduced the AR and sperm capability binding to human zona pellucida or penetration into zona-free hamster oocytes. Furthermore, assessment of the signaling pathways regulated by ZAG revealed that ZAG affects sperm AR through both the cAMP/PKA and PKC pathways. These results indicate that ZAG, which is present on the human sperm membrane, plays a critical role in the AR and subsequently, may be involved in sperm fertility.     

An acrosomal antigen of human spermatozoa and spermatogenic cells characterized with a monoclonal antibody

Monoclonal antibodies were raised against acrosomal antigens of human sperm by immunizing BALB/CA mice with purified ejaculated human spermatozoa. An ELISA-assay, employing glutaraldehyde-fixed spermatozoa as antigen, was used to screen the hybridomas producing anti-human sperm antibodies. Two hybridoma cell-lines produced antibodies which bound to the acrosomal region of spermatozoa. Both gave identical results in preliminary tests and therefore only one was chosen for further experiments. This antibody stained the acrosomal region of fixed but not living spermatozoa by indirect immunofluorescence, indicating an intra-acrosomal localization of the antigen. In acetone-fixed frozen sections of human testis this antigen was expressed only in germ cells in the adluminal compartment of seminiferous tubules. The antigen was clearly visible in round spermatids from the beginning of the cap phase of acrosome development and was also present in premature germ cells which were present in ejaculates and which were in the early stages of acrosome development. By immunochemical analysis this antibody recognized a molecule of 50 K MW as well as other components of 24 to 34 K. The pattern of staining for the antigen was similar in the presence or absence of beta-mercaptoethanol in the sample buffer. The species specificity of the antigen was studied by indirect immunofluorescence using acetone-fixed spermatozoa and the antigen was found to be present in mouse, bovine, ram and boar spermatozoa. This antibody may be useful as an acrosomal marker.     

Detection of progesterone receptors in human spermatozoa and their correlation with morphological and functional properties

In previous reports, it has been demonstrated that progesterone (P) stimulates capacitation, hyperactivation of human sperm motility and initiates the acrosome reaction (AR). This last effect has been related to the presence of non-genomic receptors for the steroid, localized on the sperm head plasma membrane. These receptors can be detected after treating spermatozoa with the non-permeable conjugate Progesterone - 3-(O-carboxymethyl) oxime: bovine serum albumin-fluorescein isothiocyanate (P-BSA-FITC). In the present study, the presence of progesterone receptors was determined in a selected sperm population with normal morphology and high progressive motility. In addition, other parameters such as the AR, hypo-osmotic swelling test, stability of chromatin and capacitating effect of P were evaluated. The percentage of P-BSA-FITC positive-spermatozoa present in the selected sperm population was higher than in total seminal spermatozoa. Furthermore, spermatozoa incubated with P showed a higher percentage motility and AR than did control spermatozoa. The HOS test indicated that membrane integrity of P-treated spermatozoa was not different to that found in the control sperm suspensions. Unexpectedly, the total sperm population treated with P showed a marked susceptibility to nuclear decondensation with reducing agents. According to these results, the selected sperm population of this study, able to respond to P, may be similar to that with good motility and normal morphology selected in the female reproductive tract, before fertilization.     

Immunolocalization and possible functional role of PSP-I/PSP-II heterodimer in highly extended boar spermatozoa

PSP-I/PSP-II heterodimer is a major protein of boar seminal plasma which is able to preserve, in vitro, the viability, motility, and mitochondrial activity of highly extended boar spermatozoa for at least 5 hours. However, little is known about the binding pattern of the heterodimer to the sperm plasma membrane and its eventual relation with the maintenance of the sperm functionality. The present study investigated the effect of exposing highly extended boar spermatozoa (1 million/mL) to 1.5 mg/mL of PSP-I/PSP-II for 0.5, 5, and 10 hours at 38 degrees C on sperm characteristics and the changes in PSP-I/PSP-II localization as a result of both the addition of PSP-I/PSP-II to the extender and the incubation time. Exposure of the spermatozoa to PSP-I/PSP-II preserved sperm viability, motility, and mitochondrial activity when compared to nonexposed spermatozoa. This protective effect lasted for 10 hours (P < .05). After immunolabeling of highly extended semen with rabbit monospecific polyclonal antibody against PSP-I/PSP-II, the percentage of immunopositive spermatozoa declines over time from 71% (0.5 hours) to 49% (10 hours). However, more than 80% of spermatozoa remained labeled during the 10-hour incubation period if PSP-I/PSP-II was added. Scanning electron microscopy revealed 4 different binding patterns. The heterodimer was mainly localized to the acrosomal area, being redistributed to the postacrosomal area or lost during in vitro incubation. In conclusion, the protective effect of the heterodimer appears to be related to its adhesion to the acrosomal area, and the loss of this protective effect coincides with a stepwise redistribution of PSP-I/PSP-II during incubation.     

Protease activated receptor 2 and epidermal growth factor receptor are involved in the regulation of human sperm motility

Aim： To investigate mechanisms of tryptase-induced reduction of sperm motility and explore whether epidermal growth factor receptor （EGF-R） and protease activated receptor 2 （PAR-2）- associated pathways are involved. Methods： Fresh semen was collected from healthy donors （n = 15）. Semen parameters and quality were assessed in accordance with the World Health Organization （WHO） criteria. Swim-up sperm were fixed and subjected to immunocytochemistry and immunoelectronmicroscopy with specific antibodies directed against PAR-2 and EGF-R. Protein extractions from swim-up spermatozoa were analyzed by Western blotting with antibodies for both receptors. Motility of spermatozoa was evaluated by computer-assisted semen analysis. Results： Immunocytochemistry found PAR-2 and EGF-R in approximately 30% of examined human ejaculated spermatozoa. Both receptors were localized in the plasma membrane. Like tryptase, the PAR-2 synthetic agonist SLIGKV reduced sperm motility, and this effect was inhibited by application of two specific EGF-R pathway blockers （AG1478 and PD168393）. Conclusion： The observed reduction of sperm motility by tryptase through the PAR-2 receptor involves EGF-R pathways.     

The fate of acrosomal staining during the acrosome reaction of human spermatozoa as revealed by a monoclonal antibody and PNA-lectin

A monoclonal anti-human sperm antibody, raised against an acrosomal antigen and indicated to recognize in boar sperm the serine protease, acrosin, stained in human spermatozoa a 50 Kd antigen and several others in the region 24-34 Kd by immunoblotting. The 50 Kd band and the region of 30-34 Kd showed proteolytic activity by zymographic enzyme detection. The fate of the antigen was studied in the acrosome reaction induced by the calcium ionophore A23187. In control incubations 69.5 +/- 14.2% (mean +/- SD) of the spermatozoa had intact acrosomal staining according to indirect immunofluorescence using this antibody whereas in acrosome-reacted samples only 21.0 +/- 2.0% of the sperm were stained. Another marker for the acrosome, peanut agglutinin-lectin (PNA), was used to detect the acrosome with similar results. Acrosome reactions were verified by electron microscopy. The present results indicate that the corresponding antigen, evidently acrosin, and PNA-positive material are liberated during the acrosome reaction which suggests that they are not bound to the inner acrosomal membrane but are components of the acrosomal matrix.     

Secretory Phospholipase A2 Group IID Is Involved in Progesterone-Induced Acrosomal Exocytosis of Human Spermatozoa

Phospholipase A2 (PLA(2)) plays a major role during acrosomal exocytosis (AE) in mammalian spermatozoa, but the identity of PLA(2) subtypes present in spermatozoa remains elusive. This study explored whether secretory PLA(2) Group IID (sPLA(2)-IID) isoform is present in human spermatozoa and whether it is involved in AE. Localization and expression of sPLA(2)-IID in human spermatozoa were explored by immunofluorescence staining and Western blot analysis. Occurrence of AE was evaluated by triple staining, and arachidonic acid (AA) levels were quantified by gas chromatography-mass spectrometry. Sperm motion parameters and hyperactivation were analyzed by computer-assisted sperm analysis. sPLA(2)-IID was localized in the postacrosomal region of the head and the midpiece of tail in human sperm. A 16-kd protein band was detected by Western blotting in sperm extracts. Progesterone-induced AE was significantly inhibited in a concentration-dependent manner using a sPLA(2)-IID neutralizing antibody. The increase in AA levels seen during progesterone-stimulated exocytosis was significantly abrogated by the antibody. The sPLA(2)-IID antibody significantly inhibited hyperactivation, sperm curvilinear velocity, and amplitude of lateral head displacement, but it did not affect the proportion of motile sperm. In conclusion, sPLA(2)-IID is present at the head and midpiece in the human sperm, and activation of such sPLA(2)-IID seems to be involved in AE. Therefore, sPLA(2)-IID isoform plays a functional role during the AE in human sperm.     

Paramagnetic beads coated with Pisum sativum agglutinin bind to human spermatozoa undergoing the acrosome reaction

Summary. For the evaluation of sperm functions it is important to assess the acrosome reaction after induction with various stimuli. Acrosome reaction tests normally include the capacitation of spermatozoa, treatment with an inducer, and detection of acrosomal loss by dyes, lectins or antibodies. Since most of these methods are time-consuming or require expensive equipment, paramagnetic beads coated with Pisum sativum agglutinin (PSA) were investigated for their usefulness in facilitating the detection of human sperm acrosome reaction.
Binding of PSA beads to the acrosomal region increased significantly after incubation of capacitated spermatozoa with 10 μM A23187 (20.3±6.7% [mean±SD, absolute binding], n = 21), 1 mM dibutyryladenosine cyclic monophosphate (17.1±8.5%, n = 25) and 10 mM phorbol myristate acetate (21.1±12.5%, n = 10). Bead binding was significantly reduced by preincubation with a protein kinase inhibitor. Beads bound to Concanavalin A (ConA) were also attached to the acrosomal region after induction of the acrosome reaction by A23187 or dbcAMP, but a lower number of spermatozoa were bound to ConA-beads than to PSA beads. Pre-treatment of spermatozoa with α-methyl-D-mannoside before addition of the PSA beads markedly decreased bead binding, which indicates its mannose-specificity. Electron microscopic examinations demonstrated that PSA beads mainly bound to membrane structures of spermatozoa that were undergoing, but had not completed the acrosome reaction.     

透明质酸受体精子的质量特性研究

目的:分析具有透明质酸受体精子的质量特征,寻找精子质量评估新指标。方法:用透明质酸包被载玻片检测活动精子-透明质酸结合率,分析其与精液常规、精子膜功能、精子受精功能和精子成熟障碍指标的关系。结果:头部具有透明质酸结合位点的精子,顶体完整性[(95.4±3.9)%]、线粒体膜高电位率[(97.8±2.1)%]明显高于原始活动精子[(68.8±6.2)%和(72.8±7.4)%,P均0.01];精子-透明质酸结合率与多项精液常规参数呈弱相关性(r=0.195~0.268,P均0.05),与诱发顶体反应率和精子正常形态率呈正相关(r=0.666、0.417,P均0.01),与精子核蛋白不成熟度、DNA碎片率和过量残留胞质率呈负相关(r=-0.266、-0.308、-0.218,P0.01、0.01、0.05)。结论:头部存在透明质酸受体的精子,其质膜结构、受精潜能、成熟度俱佳;用于检测精子透明质酸受体的精子-透明质酸结合试验,是独立的精子多重质量评价新指标。     

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.     

Presence and tyrosine phosphorylation of c-met receptor in human sperm.

The c-met receptor is a p190MET tyrosine kinase proto-oncoprotein that through its binding to its ligand, designated hepatocyte growth factor (HGF), induces mitogenic, motogenic, and morphogenic activities in a variety of cell types. The present study was conducted to examine whether or not the c-met receptor is expressed and tyrosine phosphorylated in the human sperm cell. The Western blot analysis, using a monoclonal antibody (MAb2) directed against the extracellular domain of the c-met receptor, showed a specific band of 195 kDa corresponding to the intact c-met receptor in the detergent-solubilized human sperm preparation (HSP). This protein band was not recognized by the control myeloma lg (immunoglobin). In the immunoprecipitation procedure, a similar specific band of 195 kDa and a 145-kDa band corresponding to the beta-subunit of c-met receptor were seen. In the indirect immunofluorescence technique, the c-met receptor was localized predominantly in the acrosomal region of the sperm cell. The c-met receptor was tyrosine phosphorylated/autophosphorylated during capacitation and in the cell-free in vitro kinase assay. Incubation of human sperm with hepatocyte growth factor (HGF) or MAb2 to c-met receptor enhanced the degree of tyrosine phosphorylation/autophosphorylation of the c-met receptor up to 5.1-fold. These findings indicate that the c-met receptor is present in the acrosomal region of human sperm cell and is tyrosine phosphorylated, which is enhanced by HGF and the receptor antibody. The c-met system may have an important role in sperm function.     

孕酮及其受体与男性生殖

孕酮是一种女性激素,它对女性生理功能及妊娠有着很重要的作用。近年来发现孕酮及其受体与男性生殖也有关。在男性生殖腺中存在孕酮受体mRNA。孕酮通过与其受体结合调节精子的产生,促进精子受精,而在少精、弱精、畸形精子症和不明原因不育男性精子中,孕酮受体的表达缺陷及其对孕酮的反应性降低导致受精能力下降。本文综述了孕酮及其受体与男性生殖的研究进展。     

Physiological action of oestradiol on the acrosome reaction in human spermatozoa

The acrosome is a secretory vesicle located in the sperm head. The acrosome reaction consists in the fusion of the sperm plasma membrane with the external acrosomal membrane. It has been observed that this reaction does not take place in spermatozoa incubated in cervical mucus, hydrogel that contains high concentrations of oestradiol in the peri-ovulatory period. The objective of the present study was to analyse the influence of oestradiol on the acrosome reaction in human spermatozoa to evaluate the possible inhibitory effect of this hormone. Spermatozoa were incubated in progesterone (10.1 nmol l⁻¹); oestradiol plus progesterone (oestradiol at 840 pmol l⁻¹ and progesterone at 10.1 nmol l⁻¹), oestradiol (840 pmol l⁻¹) and control (without steroidal hormones) for 30 min, 60 min, 240 min and 24 h. The acrosome reaction was evaluated by stain with Hoechst 33258 and fluorescein isothiocyanate-conjugated Pisum sativum agglutinin lectin. Progesterone-incubated spermatozoa showed the highest percentage of acrosome reaction ( P  <   0.05). Spermatozoa incubated with oestradiol and oestradiol plus progesterone showed the lowest percentage of acrosome reaction. The present study demonstrates the inhibitory role of oestradiol on the acrosome reaction, stimulated by progesterone in human spermatozoa under physiological conditions.