生育与不育男性精子顶体反应释放的顶体酶活性比较

目的比较生育与不育男性精子顶体反应释放的顶体酶活性,以此来评价精子顶体反应。方法30例不育男性和10例健康生育男性精液,用Percoll密度梯度分离法优选精子,FITC-PSA荧光染色法分析AR;用分光光度比色法测定顶体酶活性。结果少精子症组、弱精子症组和畸形精子症组精子顶体反应率和顶体反应释放的顶体酶活性均明显低于生育组(P<0.05),前3组中顶体反应率和顶体反应释放的顶体酶活性以畸形精子症组最低,但不育各组间比较均没有明显差异。将顶体反应释放的顶体酶活性与其相应的顶体反应率进行相关分析,两者存在明显正相关关系(P<0.001)。结论用顶体反应释放的顶体酶活性可以评价精子顶体反应功能。     

人精子甘露糖受体表达与顶体反应的关系

目的 研究人精子的甘露糖受体(MR)表达与顶体反应的关系，以探讨体外获能培养精子的MR表达是否为人精子获能的标志。方法 将上游法收获的活精子在获能培养基BWW中进行体外获能培养后，加入小鼠透明带溶解液(mZPS)诱导人精子顶体反应，精子悬液用异硫氰酸荧光素标记的甘露糖基化牛血清白蛋白(FITCDMA)为探针标记MR，用豌豆凝集素(PSA)法检测顶体反应。结果 体外获能培养人精子的MR表达率与mZPS诱导的顶体反应率无相关性。结论 体外获能培养精子的MR表达可能不是人精子获能的标志。     

考马期亮蓝染色法检测人精子顶体反应的评价

为建立一种检测人检测人精子顶体反应的简便实用的新技术，用３．５％高氧酸水溶液本制０．０５％考马斯亮蓝染色液浸染人精子３０ｍｉｎ，顶体完整者顶体区染成紫蓝色，顶体反应者则不梁。对获能前后和钙离子载体Ａ２３１８７诱导顶体反应的精子进行染色并与经典的顶体反应检测技术ＰＳＡ法对照，两种方法检测顶体反应率无显著差异。     

甲氧滴滴涕对大鼠精子顶体反应的影响

目的:探讨甲氧滴滴涕(methoxychlor,MXC)对雄性大鼠精子顶体反应的影响。方法:在体外培养的精子悬液中加入不同浓度的MXC和孕酮处理,通过精子考马斯亮蓝染色,镜检计数顶体反应型精子的百分率。结果:与空白组比较,单纯经MXC处理组的顶体反应型精子百分率明显增加,另加孕酮的各处理组顶体反应型精子的百分率增加尤为明显,MXC终浓度≤6.25μg/ml的处理组顶体反应型精子百分率不增加。结论:MXC可促进大鼠精子发生顶体反应且与其剂量有关,当MXC终浓度≤6.25μg/ml不诱发顶体反应。MXC能加强孕酮诱发精子顶体反应的作用。     

宫颈粘液对人精子甘露糖受体表达的影响

目的：研究人宫颈粘液是否影响人精子甘露糖受体的表达。方法：正常人精子穿透宫颈粘液2h后．用金霉素（CTC）荧光染色法鉴定其获能及顶体状态,并用异硫氰酸荧光素标记的甘露糖化牛血清白蛋白（FITC-DMA）检测精子甘露糖受体的表达。对照组分别用获能培养基BWW培养0、2、6h后用相同方法检测。结果：人精子穿透宫颈粘液后“获能”型精子百分率较穿透前增高,而“顶体反应”型精子百分率及甘露糖受体的标记阳性率与穿透前无差别。结论：宫颈粘液促进精子获能,但不诱导顶体反应,且不影响其甘露糖受体的表达。     

考马斯亮蓝染色法检测人精子顶体反应的评价

为建立一种检测人精子顶体反应的简便实用的新技术，用３．５％高氯酸水溶液配制０．０５％考马斯亮蓝（Ｒ２５０）染色液浸染人精子３０ｍｉｎ，顶体完整者顶体区染成紫蓝色，顶体反应者则不染。对获能前后和钙离子载体Ａ２３１８７诱导顶体反应的精子进行染色并与经典的顶体反应检测技术ＰＳＡ法对照，两种方法检测顶体反应率无显著差异。方法学检验证明新技术结果可靠。本法操作简便、经济，普通显微镜即可检测，易于推广，克服了原有技术复杂昂贵的缺点，具有研究和临床诊断的良好价值。     

整合素系统对人精子顶体反应的作用

目的：观察整合素系统与人精子顶体反应的关系。方法：采用整合素VLA—5的特异性配体纤连蛋白(Fn)及整合素配体的活性片断RGD肽作用于人精子lh后，用三色法行顶体染色，采用了常规显微镜下肉眼计数和图像处理软件进行分析两种方法，观察Fn和RGD作用前后顶体反应(AR)率的变化。结果：常规显微镜下肉眼计数发现，100nM的Fn、200nM的Fn、250mg/L的RGD肽和500mg/L的RGD肽作用于人精子后AR率较作用前有明显增高；运用图像分析软件分析发现，100nW的Fn、200nM的Fn、250mg/L的RGD肽和500mg／L的RGD肽作用于人精子后AR率也有显性升高。以上P均＜0.05。结论：人精子膜上的整合素参与了人精子的顶体反应。     

不同功能状态精子表面复型的透射电镜观察

建立实用的实验程序，观察不同功能状态下的精子表面超微结构特点。方法用ＢＷＷ上游法分离活动的人精子：ＢＷＷ体外获，诱导顶体反应和低渗膨胀试验处理后，将精子贴附于阳离子膜玻片上，叔丁醇冷却干燥后真空铂－碳喷镀复型。     

精子顶体反应率对宫腔内人工授精临床妊娠率的影响

目的探讨精子顶体反应率与宫腔内人工授精临床妊娠率的关系。方法选取2013年3月-2014年5月我院生殖医学科宫腔内人工授精周期120例,均为促排卵周期且药物与方案一致,排除女方不育因素,术前2-7d取精模拟IUI精液优化,优化后的精液经钙离子载体A23187诱发顶体反应、PSA-FITC染色后观察并计数精子顶体反应率,比较妊娠组与未妊娠组患者精子顶体反应(Acrosome Reaction,AR)率,探讨诱发AR率与IUI临床妊娠率的关系,3次IUI未孕与妊娠组患者诱发顶体反应率的比较。结果 120例周期中,临床妊娠17例,总临床妊娠率14.17%(17/120)。其中3次IUI未孕患者8例。妊娠组与未妊娠组患者处理后正常形态率、前向运动精子总数、正常形态精子前向运动精子总数、自发AR率分别比较,均无统计学差异(P0.05),而妊娠组患者诱发AR率显著高于未妊娠组(P0.05)。诱发AR率小于15%、15%~30%、30%~50%、大于50%4组妊娠率分别为0%(0/12)、12.5%(3/24)、13.2%(5/38)、19.6%(9/46),诱发AR率小于15%患者临床妊娠率显著降低(P0.05);3次IUI未孕组与妊娠组诱发AR率分别15.35±17.62、63.75±14.33,3次IUI未孕组诱发AR率明显低于妊娠组(P0.05)。结论精子顶体反应率与宫腔内人工授精临床妊娠率有相关性,当诱发顶体反应率小于15%时妊娠率明显降低;反复IUI发生与顶体反应率降低密切相关。     

卵胞浆内单精子注射在受精基础研究中的应用

卵胞浆内单精子注射(ICSI)近十年来广泛应用于人类辅助生育；作为一种旁路受精方式，精子绕过了自然受精的多个环节，如精子与卵透明带的结合、顶体反应以及精子与卵胞膜融合等，丰富了经典的受精理论，也为研究受精机制提供了一个有效的实验模型。本文概述了ICSI受精后的精子变化、顶体反应、活化卵细胞的分子机制、不同发育阶段精子及生精细胞受精潜力等方面的进展。     

Regional differentiation in the heads of spermatozoa of rabbit,man and bull

The heads of the spermatozoa of rabbit, man, and bull are flattened in the dorsoventral plane and exhibit three distinct regions in the head coverings. The anterior one-half to two-thirds of the nucleus is covered by the acrosome, the middle of the nucleus by the thinned equatorial segment of the acrosome and the remainder of the head is covered by the postacrosomal segment. Species variation is evident in the head coverings; most prominent are the equatorial bands of the rabbit spermatozoon. The bands are subacrosomal bulges curving around the head; the arms of each band are displaced in the anteroposterior axis and do not cross the midline axis. The equatorial bands are visible in scanning electron micrographs, which also show a serrated junction between the equatorial segment and the postacrosomal region.     

An electron microscopic study of sperm penetration into the human egg investments

Summary The ultrastructure of human spermatozoa located in the cumulus cells and the zona pellucida of a pronuclear egg, and in the zona pellucida of a two-cell egg, both fertilized in-vivo, has been analysed in order to understand how the human spermatozoon penetrates the investing coats of the oocyte. Among the 36 spermatozoa found in the cumulus cells, 31 were phagocytosed by cumulus cells and 5 were wedged in the matrix between the cells. These spermatozoa were acrosome-reacted and their equatorial segment was intact. Six of the seven spermatozoa found in the zona pellucida (four spermatozoa in the pronuclear egg and three in the two-cell egg) had lost the equatorial segment, while the other one was partially reacted. The sperm heads were located in slits with sharp edges. From these findings it was concluded that in the human (1) only few and normal spermatozoa seem to reach the cumulus cells after natural insemination, (2) the acrosome reaction probably occurs sometime before the spermatozoa reach the vicinity of the corona cells, (3) the reaction of the equatorial segment seems to occur during or before the initial phase of zona penetration, since the spermatozoa located in the matrix of the zona pellucida had no equatorial segment. No evidence of the presence of spermatozoa with an intact acrosome in the matrix of cumulus cells or with an intact equatorial segment in the zona pellucida were found.     

Ultrastructure of Octodon degus spermatozoon with special reference to the acrosome

The ultrastructure of spermatozoa from the cauda epididymidis and vas deferens of Octodon degus — a Chilean hystricomorph rodent—is presented. The head of spermatozoa measured 7.7 μm long by 5.9 μm wide and the tail was 41 μm long. The head was flattened dorso-ventrally and ovate in outline. The acrosome was the most distinctive feature of O. degus spermatozoa. In a frontal view of the head, the rim of the acrosome surrounding the nucleus had the shape of an inverted U. The acrosomal region covering the plane of the flattened head exhibited dome-shaped protrusions. Transverse or sagittal sections of acrosomal protrusions showed that the plasma membrane and outer acrosomal membrane were evaginated, while the inner acrosomal membrane followed the contour of the nucleus. The protrusions were not distributed at random and they were absent in the equatorial segment and in the rim of the acrosome. In frontal views, near the boundary between the acrosome and post-acrosomal region, fine rods about 170 nm long ran obliquely on the caudal part of the equatorial segment. Behind the same boundary, the post-acrosomal region showed a serrated border. Phosphotungstic acid treatment at pH 0.3 produced staining at the surface of the sperm as well as within a superficial layer of the marginal thickening of the acrosome and on the acrosomal protuberances.     

Posttesticular development of spermatozoa of the tammar wallaby (Macropus eugenii)

Tammar wallaby spermatozoa undergo maturation during transit through the epididymis. This maturation differs from that seen in eutherian mammals because in addition to biochemical and functional maturation there are also major changes in morphology, in particular formation of the condensed acrosome and reorientation of the sperm head and tail. Of spermatozoa released from the testes, 83% had a large immature acrosome. By the time spermatozoa reached the proximal cauda epididymis 100% of sperm had condensed acrosomes. Similarly 86% of testicular spermatozoa had immature thumb tack or T shape head-tail orientation while only 2% retained this immature morphology in the corpus epididymis. This maturation is very similar to that reported for the common brush tail possum, Trichosurus vulpecula. However, morphological maturation occurred earlier in epididymal transit in the tammar wallaby. By the time spermatozoa had reached the proximal cauda epididymis no spermatozoa had an immature acrosome and thumbtack orientation. Associated with acrosomal maturation was an increase in acrosomal thiols and the formation of disulphides which presumably account for the unusual stability of the wallaby sperm acrosome. The development of motility and progressive motility of tammar wallaby spermatozoa is similar to that of other marsupials and eutherian mammals. Spermatozoa are immotile in the testes and the percentage of motile spermatozoa and the strength of their motility increases during epididymal transit. During passage through the caput and corpus epididymis, spermatozoa first became weakly motile in the proximal caput and then increasingly progressively motile through the corpus epididymis. Tammar wallaby spermatozoa collected from the proximal cauda epididymis had motility not different from ejaculated spermatozoa. Ultrastructural studies indicated that acrosomal condensation involved a complex infolding of the immature acrosome. At spermiation the acrosome of tammar wallaby spermatozoa was a relatively large flat or concave disc which projected laterally and anteriorly beyond the limits of the nucleus. During transit of the epididymal caput and proximal corpus the lateral projections folded inwards to form a cup like structure the sides of which eventually met and fused. The cavity produced by this fusion was lost as the acrosome condensed to its mature form as a small button-like structure contained within the depression on the anterior end of the nucleus. During this process the dorsal surface of the immature acrosome and its outer acrosomal membrane and overlying plasma membrane were engulfed into the acrosomal matrix. This means that the dorsal surface of the acrosomal region of the testicular tammar wallaby sperm head is a transient structure. The dorsal acrosomal surface of the mature spermatozoon appears ultrastructurally to be the relocated ventral surface of the acrosomal projections which previously extended out beyond the acrosomal depression on the dorsal surface of the nucleus of the immature spermatozoon.     

Identification and localization of G protein subunits in human spermatozoa

Antibodies to alpha and beta subunits of guanine nucleotide regulatory proteins (G proteins) were used to identify which G proteins are present in mature human spermatozoa and to determine their subcellular localization. Immunoblots of membranes from spermatozoa demonstrate the presence of Galphai2, Galphai3, Galphaq/11 and Gbeta35 and the absence of Galphai1, Galpha0, Galphas, Galpha12, Galpha13, Galpha16, Galpha and Gbeta36. Indirect immunofluorescence demonstrates the presence of Galphaq/11 in the acrosome, with the highest proportion in the equatorial segment. Galphai2 is present in the acrosome, midpiece and tailpiece and Galphai3 in the postnuclear cap, midpiece and tailpiece. The Gbeta35 subunit is found mostly in the midpiece, with marginal labelling of the head, tailpiece and the equatorial segment of the acrosome. The distinct pattern of distribution of G proteins suggests that they may couple to receptors or effectors which also have discrete regions of localization in spermatozoa. These highly localized signal transduction pathways may regulate discrete functions, such as activation of the acrosome reaction, fusion with the oocyte and motility.     

Progesterone promotes the acrosome reaction in capacitated human spermatozoa as judged by flow cytometry and CD46 staining.

The acrosome reaction is a necessary prerequisite for spermatozoa to acquire fertilizing ability. Several different moieties appear to promote the acrosome reaction through different pathways, including solubilized zona pellucidae, recombinant zona protein ZP3, follicular fluid, calcium ionophores, and mannosylated bovine serum albumin (BSA). Although many investigators have presented evidence that progesterone also promotes the acrosome reaction through the mediation of a non-genomic cell membrane receptor, this concept has been challenged. Other workers have suggested that progesterone does not promote an acrosome reaction in human spermatozoa, as judged by the detection of CD46, a complement regulatory protein present on the inner acrosome membrane, through flow cytometric analysis of large numbers of spermatozoa. Prior investigations were criticized by the limited numbers of spermatozoa enumerated visually, the use of non-specific staining techniques, and the failure to eliminate dead spermatozoa during the scoring of the acrosome reaction. We have repeated these experiments, using both a supravital dye to eliminate dead spermatozoa from flow cytometric analysis, and anti-CD46 monoclonal antibody to score acrosome-reacted spermatozoa. Care was taken to validate the adequacy of capacitation conditions, which were proven by the ability of spermatozoa to acrosome react in response to mannosylated BSA and to penetrate zona-free hamster eggs. Confocal microscopy was used to confirm that CD46 immunostaining was limited to the acrosomal region of the spermatozoon head. Our results indicate that progesterone does promote an acrosome reaction within capacitated spermatozoa.     

Is the oocyte a non-professional phagocyte?

Although fertilization has been described as a series of events during which the spermatozoon penetrates the oocyte, introducing its nuclear contents, there is strong evidence that either gamete can be the active partner at different stages of this process. Indeed, while sperm motility is essential for its penetration of the egg vestments, immotile spermatozoa are capable of entering the ooplasm once they adhere to the oolemma. Entry of the spermatozoon into the oocyte appears to require two distinct but perhaps related events, namely gamete cell membrane fusion, at the level of the equatorial segment of the sperm acrosome with the oolemma, and a quasi-phagocytic event involving the incorporation by the oocyte of the rostral portion of the acrosome-reacted spermatozoon head within an oolemmal-derived vesicle. This review explores the biology of phagocytosis by macrophages and non-professional phagocytes, and in particular the roles played by phagocytosis-promoting receptors (FcgR, complement receptors and integrins), in both signal transduction and their linkage with the cytoskeleton. It asks whether the oocyte might not utilize similar mechanisms during its incorporation of the spermatozoon.     

Development of membrane differentiations in the guinea pig spermatid during spermiogenesis

We have studied the development of membrane differentiations in guinea pig spermatids during spermiogenesis, using electron microscopy of thin sections, freeze-fracturing, and filipin labeling as an indicator of membrane cholesterol distribution. The development of the distinctive membrane specializations closely correlated with the developmental steps of spermatid differentiation. The annulus, the zipper, and the circumferential strands of particles overlying the mitochondrial gyres of the midpiece appeared in the plasma membrane over the tail during the last two steps of spermiogenesis. The outer acrosomal membrane showed trnsient crenations during the acrosome phase and serrations over the equatorial segment of the acrosome during the maturation phase. On the inner nuclear membrane, a depression appeared opposite the inner zone of the acrosome during the Golgi phase and persisted throughout spermiogenesis. The implantation fossa also appeared during this phase, as a bulging on the inner nuclear envelope covered with small membrane particles. By the cap phase, small and large 15–20-nm particles were present at the implantation-fossa site, and clusters of 9-nm particles were seen over the postacrosomal segment of the nuclear envelope. The migration of the nuclear pores began when close contacts were established between adjacent nuclear membranes. It started simultaneously at the anterior pole and the implantation fossa and later progressed over the segment of membrane between the caudal margin of the acrosome and the posterior ring. By the maturation phase, the nuclear pores had migrated to the redundant nuclear envelope. After filipin labeling, a gradient was visible in the distribution of the cholesterol-filipin complexes, decreasing from the plasma membrane to the nuclear membrane. Within the same membrane, cholesterol distribution varied from one pole of the cell to the other. On the differentiations of the plasma membrane over the tail, cholesterol-filipin complexes were absent, but they were profuse in the membrane over the head. In the acrosomal membrane, the complexes were gathered in clumps associated with the crenations. They became fewer in the rostral segment but remained moderate in the caudal equatorial segment as spermiogenesis proceeded. The nuclear membrane contained cholesterol-free regions opposite the inner acrosome and at the site of the implantation fossa. Otherwise, the acrosomal segment of the nuclear envelope demonstrated a homogeneous distribution of cholesterol-filipin complexes, while the postacrosomal segment showed small clumps of complexes adjacent to nuclear pores. We propose that each of the spermatid cell membranes is not an autonomous system, but is dependent upon its exchanges with the immediate external environment under the one side of the membrane and its close coupling to the internal environment (i.e., membrane-associated structures: annulus, zipper, etc.) under the otehr side of the membrane. We suggest that the cholesterol in spermatid cell membranes contributes to the establishment of their mosaicism and regulates the modeling of the spermatid by modulating the internal fluidity of individual membrane segments during spermiogenesis.     

Immunofluorescent studies on human spermatozoa.I. The detection of different spermatozoal antibodies and their occurrence in normal and infertile women

Sera from children, blood donors, pregnant women and women from infertile couples were tested by the indirect immunofluorescent technique on methanol-fixed human spermatozoa. Four different staining patterns were observed: (1) Staining of the acrosome: a homogeneous fluorescence usually covering the entire acrosome and thus including the equatorial segment, but with some sera only the front part of the acrosome was stained. (2) Staining of the equatorial segment: a narrow well-demarcated fluorescent band across the head at the equator. (3) Staining of the postnuclear area: two fluorescent bands on the posterior third of the head, forming a bowl-like figure closely corresponding to the localization of the postnuclear cap. (4) Staining of the tail: usually only involving the main tail piece. Combinations of these staining patterns were often seen.

Staining of the postnuclear area was found with only three out of the 330 sera tested, whereas the other antibodies were frequently present in low titres (titre ≤ 4) in all groups studied. On the other hand, titres ≥ 16 were found significantly more frequent among women from infertile couples than in any of the control groups, and in the further analysis of the infertile group the antibody levels appeared to be significantly higher among women with unexplained infertility than among those in whom the infertility could be explained.

     

Immunofluorescent studies on human spermatozoa. II. Characterization of spermatozoal antigens and their occurrence in spermatozoa from the male partners of infertile couples

The object of this study was to elucidate properties, organ specificity, and the occurrence in individual spermatozoa of the four antigens which can be identified in methanol-fixed human spermatozoa by the indirect immunofluorescence technique using human antisera, i.e. the antigen in the front part of the acrosome, in the equatorial segment, in the postnuclear region, and in the main tail piece.

With unfixed spermatozoa the reactions were weak and often uncharacteristic. After fixation with ethanol and acetone all four antigens were preserved, while after fixation with formalin the postnuclear antigen was not demonstrable. The antigens in the equatorial segment and in the postnuclear region were destroyed at 60°C and those in the front part of the acrosome and in the tail at 80°C. After the spermatozoa had been treated with trypsin the reaction with the three antigens in the head disappeared. However, with rabbit antiserum to human spermatozoa fluorescence could be induced even after heating the spermatozoa to 100°C or treating them with trypsin.

Absorption of the human antisera with seminal plasma, human milk, liver, kidney, and adrenal extract disclosed an antigenic relationship between the postnuclear antigen and seminal plasma as well as between the antigen in the front part of the acrosome and adrenal extract.

By means of known antisera the occurrence of acrosome antigens in the individual spermatozoa was investigated in ejaculates from seventy-six male partners of infertile couples. In ejaculates with a normal concentration of spermatozoa the antigens were generally demonstrable in at least 50% of the spermatozoa, whereas samples with a low concentration showed a marked variation, the staining percentages ranging from 0 to 90%.