精子顶体反应及其相关调控因子研究进展

正受精是精子将所携带的单倍体遗传物质与卵子的单倍体遗传物质融合成双倍体合子的过程,此过程是一高度复杂且严格有序的生理过程。有学者[1]将人类受精过程概括为:1精子获能;2精子与卵丘细胞间相互作用;3精子活动力改变;4精子与透明带结合、透明带诱发精子顶体反应;5顶体内酶激活、释放;6精子穿透透明带;7精卵质膜融合;8卵子激活;9精子染色质解凝;10精卵核融合。其中,精卵     

精子与卵子的相互作用:人精子顶体反应与透明带结合动力学的研究

成功的精子和卵子相互作用取决于精子获能,而其获能状态则由顶体反应和精子活动力所确定。作者从三个不同方面评价了配子的相互作用:(1)结合到透明带的精子数量与精子浓度和孵化时间的关系。(2)精子与透明带结合过程中的获能状况和精子顶体反应的动力学。(3)人的卵泡液对不同人精子与透明带结合能力和精子顶体反应状态的影响。     

人及哺乳动物受精早期的糖类调节

人及哺乳动物受精早期的重要步骤包括精子获能、精卵识别(或结合)、顶体反应和精卵融合。越来越多的研究表明它们是由糖类调节的。输卵管特异糖蛋白(OGP)对精子获能起直接作用。蛋白多糖和氨基葡聚糖均可诱发精子获能。精卵识别中存在着糖类识别机制,即精子受体和卵透明带(ZP)配体相互作用。但它们的化学性质和特性还不清楚。     

精子获能(Sperm Capacitation)的含义

1951年 Austin 曾推测,耥子似乎需要在雌性生殖管道度过一些时间才能穿入卵的透明带。次年,该氏又指出,精子在具备穿入卵子的能力之前,必定要经过某种形式的生理学改变或“获能”,从而提出了精子获能这一概念。研究精子获能有许多方法。1955年张明觉把射出精子、附睾精子和在交配后不同时间从子宫内取得的精子输入排卵后不久的兔输卵管,结果发现只有从子宫内取出的精子能使兔卵受精;这表明获能可在子宫中完成。进一步的实验揭示,从兔子宫中取出的已获能精子,用5～20%的兔、牛或人的精浆液处理,可使之“去获能”,而去获能的精子在输卵管中还可以“再获能”。     

IVF和ICSI后的异常受精

IVF常会引起多精受精,其发生率超过30％。研究认为多精受精与夫妻的年龄和卵巢刺激无关。而认为与卵子的不成熟、卵子过熟、精子浓度、卵子募集过程中造成的透明带损伤或先天透明带缺损、卵泡液中孕激素水平、血清中高雌激素水     

哺乳动物精子顶体反应相关的蛋白及信号传导

哺乳动物精子顶体反应是自然受精起始过程中的重要环节,有多种精子膜蛋白和卵子透明带糖蛋白参与.这些蛋白一方面通过精子膜跨膜信号传导引起精子的顶体反应,另一方面,也为精子穿过透明带及精卵融合等提供必要的条件.精子顶体反应过程中涉及多种细胞信号传导的过程.如G蛋白耦联的信号传导、受体酪氨酸激酶(RTK)途径等.研究精子顶体反应过程中相关分子作用的信号传导通路,对进一步深入了解受精的分子机制具有重要的影响.     

四次跨膜蛋白CD9与精卵融合

当精子与卵子结合并穿透卵透明带后,迅速越过卵周隙,并到达卵膜.在该处精卵结合进而发生融合.精卵结合,使配子间有了分子间联系,而融合仅出现在卵子的特异区域,并有许多分子参与调节.四次跨膜蛋白CD9由卵细胞表达,是参与精卵融合调节的一个重要成员.四次跨膜蛋白家族成员与许多蛋白质结合形成四次跨膜蛋白网,在细胞膜生物学中发挥重要作用.CD9与精子受精素(ADAM2,fertilinβ)或精子免疫球蛋白/癌胚抗原(IgSF/CEA)超家族蛋白质相结合参与精卵融合及受精.     

透明带D-甘露糖残基在精子识别中的作用

目的:卵子透明带在受精中的作用,一般考虑为和特异性的精子识别,糖链结构的作用尤其受到注目。在这次研究中,为了弄清楚透明带糖链结构在精子识别中的作用,在下述条件下进行了透明带穿透试验。方法:用于透明带穿透试验的卵子是在体外培养成熟的人成熟卵子     

IVF和ICSI后的异常受精

IVF常会引起多精受精，其发生率超过30％。研究认为多精受精与夫妻的年龄和卵巢刺激无关。而认为与卵子的不成熟、卵子过熟、精子浓度、卵子募集过程中造成的透明带损伤或先天透明带缺损、卵泡液中孕激素水平、血清中高雌激素水平等相关。其中卵子的成熟度，特别是皮质颗粒的形成和皮质反应在多精受精的发生中起非常重要的作用。不成熟的卵子     

人类精子中存在的核糖核酸:正常精子与异常精子之间含量的区别

男性生育需要有足够数量的成熟精子而这些精子有足够的活力和获能的能力,发生顶体反应和结合以及穿透透明带受精的能力。这些必要因素中任何一点缺陷都会导致男性不育。     

哺乳动物精子顶体反应相关的蛋白及信号传导

哺乳动物精子顶体反应是自然受精起始过程中的重要环节,有多种精子膜蛋白和卵子透明带糖蛋白参与。这些蛋白一方面通过精子膜跨膜信号传导引起精子的顶体反应,另一方面,也为精子穿过透明带及精卵融合等提供必要的条件。精子顶体反应过程中涉及多种细胞信号传导的过程,如G蛋白耦联的信号传导、受体酪氨酸激酶(RTK)途径等。研究精子顶体反应过程中相关分子作用的信号传导通路,对进一步深入了解受精的分子机制具有重要的影响。     

Current Concepts of Molecular Events During Bovine and Porcine Spermatozoa Capacitation

Spermatozoa are required to undergo the processes of capacitation before they obtain fertilizing ability. The molecular changes of capacitation are still not fully understood. However, it is accepted that capacitation is a sequential process involving numerous physiological changes including destabilization of the plasma membrane, alterations of intracellular ion concentrations and membrane potential, and protein phosphorylation. There are no known morphological changes that occur to the spermatozoon during capacitation. The purpose of this review is to summarize current evidence on the molecular aspects of capacitation both in vivo and in vitro in bovine and porcine spermatozoa. For the purpose of this review, the process of sperm capacitation will encompass maturational events that occur following ejaculation up to binding to the zona pellucida, that triggers acrosomal exocytosis and initiates fertilization.     

Immunoelectronmicroscopic imaging of spermadhesin AWN epitopes on boar spermatozoa bound in vivo to the zona pellucida

Spermadhesin AWN is a major protein of boar seminal plasma and a sperm surface-associated lectin. AWN binds to beta-galactosides and to porcine zona pellucida glycoproteins, suggesting a role for this protein in primary gamete interaction. However, because capacitation induces remodelling of the sperm surface and AWN is peripherally bound to the plasma membrane, the present study sought to investigate whether AWN is present or absent in the subpopulation of spermatozoa that reaches the ovulated oocyte at the period of fertilization in vivo. Therefore, tubal tissues and oocytes from sows mated with a fertile boar were collected 6-8 h after ovulation. Tissues and oocyte sperm complexes were fixed, immunolabelled with anti-AWN monoclonal antibodies, and examined by means of light and scanning electron microscopy. The results show that spermadhesin AWN is present in spermatozoa seen along the genital tract of the natural mated sow as well as on plasmalemmal remnants of spermatozoa bound to the zona pellucida in vivo.     

Fucoidin inhibits the zona pellucida-induced acrosome reaction in human spermatozoa

We recently reported that fucoidin (a polymer of predominantly sulfated L-fucose) significantly inhibits tight binding of human sperm to the human zona pellucida in vitro and that several oligosaccharides obtained after acid hydrolysis possess sperm-zona pellucida binding inhibitory activity equal to the original fucoidin. This inhibition may be specific to sperm-zona interactions or may be the consequence of the interruption of capacitation, a series of biochemical and physiological events leading to final sperm maturation, that must occur for successful fertilization. Completion of capacitation is most often determined by assessing two end-points of the process: acquisition of hyperactivated motility and ability to complete the acrosome reaction. Here, we examined the effects of fucoidin on these two end-points of capacitation in vitro. Fucoidin did not affect the proportion of sperm with hyperstimulated motility. Neither did fucoidin cause an increase in sperm that had spontaneously acrosome-reacted at 4.5 hours compared to controls as evaluated by indirect immunofluorescence using the acrosomal marker, monoclonal antibody, T-6. Comparable percentages of sperm had completed the acrosome reaction when exogenously stimulated by calcium ionophore A23187 with and without the addition of fucoidin. However, in the presence of fucoidin, stimulation of the acrosome reaction by acid solubilized human zonae pellucidae was significantly inhibited. These data indicate that fucoidin does not impede the normal progression of capacitation. These results provide strong evidence to support the hypothesis is that the inhibitory effect of fucoidin is at the level of the sperm membrane since inhibition can be bypassed by increasing intracellular calcium directly with a calcium ionophore.     

Relationship between Sperm Concentration and Polyspermy in Intact and Zona-Free Mouse Eggs Inseminated in Vitro

Intact and zona-free mouse eggs were cultured with preincubated (capacitated) spermatozoa for 1 or 4 hr. High proportions of eggs (84%-100%), examined either 1 or 4 hr after insemination, were undergoing fertilization in the intact and zona-free eggs in sperm concentration from 25-800 × 10³ sperm/ml. The average number of spermatozoa attached to the zona pellucida and to the vitellus was only slightly increased as the sperm concentration increased. Polyspermy was increased from 25-200 × 10³ sperm/ml but there was no clear correlation between the incidence of polyspermy and further increase of sperm concentration in both the intact and zona-free eggs. Besides a functional zona reaction, there was a definite vitelline block to further sperm entry. It seems that due to the chance collision of sperm and egg with the subsequent formation of a block mechanism in the zona pellucida and in the vitelline membrane within a short time, polyspermy cannot be increased by further increase of sperm concentrations.     

Polyspermic penetration in porcine IVM-IVF systems

Although techniques for in vitro production of porcine embryos have proceeded very rapidly during the past decade, polyspermic penetration still remains a persistent obstacle to porcine in vitro fertilization (IVF) systems. Considerable research on in vitro polyspermic penetration in porcine in vitro-matured (IVM) oocytes has been undertaken to try to solve this problem. In the current paper, recent advancements in overcoming the problems of polyspermy in porcine IVF systems are reviewed. Partial induction of the acrosome reaction of boar spermatozoa in IVF media that contain caffeine is likely to be one of the major causes of polyspermy. A reduction in the number of incompletely acrosome-reacted spermatozoa, which can bind tightly to the zona pellucida and mask free sperm receptors of the zona pellucida, could reduce the incidence of polyspermic penetration; however, morphological differences in the reaction of the zona pellucida have been observed between IVM and ovulated oocytes, which suggests that altered zona morphology may be another cause of polyspermic penetration. It has been shown that the developmental ability of polyspermic porcine embryos to the blastocyst stage is similar to that of normal embryos but that developmental competence to term is much lower. To overcome the current problems of polyspermy, it is suggested that future efforts should be focused on controlling boar sperm function and/or sperm-zona binding to achieve the final maturation associated with normal zona modifications of porcine oocytes at fertilization.     

Immunological approaches to fertility regulation.

Strong evidence that specific immunogenic components of the reproductive system exist that are not represented in other body systems has led to efforts to develop an acceptable vaccine for fertility regulation. The aim is to create a vaccine administered infrequently by trained technicians outside the clinical environment. For safety and practical reasons, an approach using active immunization with a vaccine is preferred to passive immunization with antibodies. In current research with sperm antigens, a lactate dehydrogenase isoenzyme (LDH-X), an enzyme normally present on the sperm surface, reduced fertility in mice and rabbits. However, significant embryo mortality occurred. Other sperm antigens have been tested and rejected. Most of the research on ovum antigens is directed toward the zona pellucida, and work is in progress to isolate experimental quantities of specific zona pellucida antigens. Antibodies to human zona are reported to react with pig zona and vice versa, providing a model system. Antibodies to whole-placenta homogenates reportedly disrupt pregnancy in several laboratory animal species, and 2 placenta-specific proteins are potential antigens since antibodies to them do not react with any other tissue so far tested. Of 3 protein hormones isolated from placental tissue, 2 are potential antigens. The possible hazards of antifertility vaccines can be divided into 2 categories: problems related to immunization and problems caused by antibodies produced.     

Baboon spermatozoa-zona pellucida binding assay

This study developed a baboon in vitro system that allows transport of sperm from a treatment facility to an off-site location for subsequent evaluation of sperm functional capacity. We further described a sperm functional assay that evaluates baboon sperm binding to homologous zona pellucida, a baboon hemizona assay (HZA). Semen samples were collected from baboons via electroejaculation directly into refrigeration transport buffer. Postshipment semen characteristics were analyzed and each specimen prepared for assessment of sperm-zona pellucida interaction. Optimization of the baboon HZA included determination of the relationship between motile sperm concentration and zona pellucida binding. The effect of the sperm activators, caffeine and dbcAMP, on computerized sperm motion characteristics and HZ binding was also determined. A significant motile sperm concentration dependent increase was observed in sperm-zona pellucida binding. Maximal binding was observed at approximately 1-2 million motile sperm/mL. Treatment with the sperm activators, caffeine and dbcAMP, resulted in a significant increase in sperm progressive motility, straightline velocity (VSL), and amplitude of lateral head displacement (ALH), p <0.05 and a highly significant increase in curvilinear velocity (VCL), p <0.01. Treatment with caffeine and dbcAMP was not an absolute requirement for sperm-zona pellucida binding, inasmuch as binding did occur in the absence of activators. However, treatment with the two activators, caffeine and dbcAMP, resulted in a highly significant increase in HZ binding, p <0.0001. This system allows for the short-term maintenance of baboon sperm in a semiquiescent state until stimulation with the activators, caffeine and dbcAMP. It further provides a novel approach to delineating a contraceptive regimen's or agent's (ie, sperm vaccine) impact on specific cellular events occurring in the male gamete during fertilization.     

Factors Regulating Sperm Capacitation

Capacitation is broadly defined as the functional modifications rendering sperm competent to fertilize, encompassing the ability of the sperm to bind the zona pellucida and subsequently undergo the acrosome reaction, hyperactivated motility, and the capacity to fuse with the oocyte. Although discovered in 1951, research over the past 15 years has considerably clarified the mechanisms leading to capacitation. The purpose of this review is to discuss the challenges of studying capacitation and to summarize recent notions regarding its regulation. Of particular interest is an atypical soluble adenylyl cyclase that is stimulated by bicarbonate to activate protein kinase A and drive sperm protein tyrosine phosphorylation. The identities of the phosphorylated sperm-protein substrates and the kinase(s) responsible for their tyrosine phosphorylation have fostered major questions regarding this pathway. Recent investigations, however, have made exciting advances toward resolving these queries. Advanced proteomic approaches have revealed the tyrosine phosphorylated substrates to be implicated in a diverse range of cellular activities. SRC tyrosine kinase is a particularly interesting candidate as the mediator of the protein kinase A-driven sperm protein tyrosine phosphorylation. Future studies are merited to fully characterize additional signaling mediators such as phosphatases and other kinases that may be involved, to elucidate the functional importance of the tyrosine phosphorylation on those particular substrates and to appreciate the differences that may exist among species.     

Abnormal fluid transport by the epididymis as a cause of obstructive azoospermia

It has been known for more than a decade that in many mammalian species including man, spermatozoa once shed from the testis are immature, immotile and incapable of fertilizing the ovum. During their transit through the epididymis, they undergo various morphological and functional changes that confer on them the ability to ascend the female tract, to undergo an acrosome reaction, to penetrate the zona pellucida and to effect a successful fertilization. By the time spermatozoa have reached the cauda epididymidis, they are held in a quiescent state by factors in the epididymal fluid. The epididymis plays a vital role by creating a favourable fluid environment for sperm maturation and storage. The exact mechanisms underlying sperm maturation and storage are unclear and it appears that no single epididymal factor is held entirely responsible. In contrast, spermatozoa are directly bathed in the epididymal fluid; the fluidity of the microenvironmental has a direct effect on epididymal spermatozoa. The epididymal epithelium has been shown to transport electrolytes and water by processes involving ion pumps, ion carriers and ion channels. These components are under nervous, hormonal and paracrine control and are susceptible to interference by pharmacological agents. This paper reviews the physiology of electrolytes and fluid transport in the epididymis and describes how abnormal fluid transport across the epididymal duct could predispose towards epididymal obstruction, a condition that may occur in cystic fibrosis, Young's syndrome or other unexplained cases of male infertility.