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.     

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     

精子顶体发育相关蛋白的研究进展

受精是新生命诞生的第一步,在此过程中,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。哺乳动物的受精过程涉及到一系列复杂而精细的活动,如精子的超活化与获能、顶体反应以及精卵结合等。精子顶体是一种特殊的膜细胞器,具有帽状结构并覆盖在精子的细胞核前部,在受精过程中发挥着关键作用。顶体的形成主要包括囊泡形成、囊泡运输、囊泡融合和顶体与细胞核结合等过程,其独特的蛋白运输机制需要内质网、高尔基体等细胞器以及某些特殊结构之间的相互配合。顶体形成是精子细胞分化为精子的关键步骤之一,受多基因精细调控,其发育缺陷与包括圆头精子症在内的多种男性不育症有关。综述近年精子顶体发育过程中主要生物学事件的发生顺序中相关蛋白的研究,并介绍了自噬相关蛋白对顶体发育过程作用的新近研究,以期为男性不育症的诊疗提供新思路。     

哺乳动物精子膜与精卵识别相关的研究进展

哺乳动物的精卵识别是受精中一个复杂的、关键性的步骤,它涉及生殖的普遍规律性和种属特异性,其中精子膜成分可能与卵细胞透明带蛋白或糖基交替进行连续性识别。详细阐述精子膜与识别相关成分如:sp56、β-1,4-半乳糖转移酶、p95、顶体素原、PH-20、受精素β、精子粘连素、甘露糖等糖基受体,RSA及其作用机制,对受精秘密的揭示、避孕疫苗的研制及对生育率的控制等都具有十分重要的现实意义。     

哺乳动物精子膜与精卵识别相关的研究进展

哺乳动物的精卵识别是受精中一个复杂的、关键性的步骤,它涉及生殖的普遍规律性和种属特异性,其中精子膜成分可能与卵细胞透明带蛋白或糖基交替进行连续性识别.详细阐述精子膜与识别相关成分如:sp56、β-1,4-半乳糖转移酶、p95、顶体素原、PH-20、受精素β、精子粘连素、甘露糖等糖基受体,RSA及其作用机制,对受精秘密的揭示、避孕疫苗的研制及对生育率的控制等都具有十分重要的现实意义.     

哺乳动物精子膜与精卵识别相关的研究进展

哺乳动物的精卵识别是受精中一个复杂的、关键性的步骤,它涉及生殖的普遍规律性和种属特异性,其中精子膜成分可能与卵细胞透明带蛋白或糖基交替进行连续性识别,详细阐述了精子膜与识别相关成分如：sp56、β-1,4-半乳糖转移酶、p95、顶体素原、PH－20、受精素β、精子粘连素、甘露糖等糖其受体,RSA及其作用机制,对受精秘密的揭示、避孕疫苗的研制及对生育率的控制等都具有十分重要的现实意义。     

哺乳动物精子膜离子通道的研究进展

哺乳类动物精子膜上存在着2+,Na+,K+和Cl-等诸多的离子通道,这些离子通道在精子成熟、获能、顶体反应等一系列受精过程中发挥了极其重要的作用.不同的离子通道在精子膜上的分布不同,并且在精子的各种生理活动中起的作用也不同.其中Ca2+及其通道更被认为是触发受精过程中不可缺少的关键性因素之一.因此,对精子膜离子通道研究的进一步深入,能对受精机理有更深的了解,而且在避孕药物研发的思路上还可能有一个新的突破.     

精子功能检测在男性不育诊治中的应用

<正>在临床实践中,对男性生育功能的评估主要依靠精液分析,然而常规精液分析只检测精子浓度、活力及形态学等参数,仅反映精子的质量,而只有精子的功能才能反映精子的授精能力。正常授精精子要经历获能、顶体反应、与透明带结合及穿透等一系列过程[1]。精子功能试验可检测精子执行这些功能的情况,包括精子膜完整性、顶体反应、精子核成熟度和DNA完整性、活性氧(ROS)、精子与透明带相互作用、精子-宫颈黏液相互作用检测等[2],本文主要     

抗精子抗体对受精及受精前后各阶段的影响

原因不明性不孕夫妇中10％～30％可能与AsAb有关。AsAb可影响受精前的各个环节及受精和受精后的过程。如干扰精子获能和顶体反应、抑制精子穿透宫颈粘液、促进巨噬细胞和白细胞吞噬杀伤精子、阻碍精子接触和穿过透明带、影响精卵融合、引起补体介导的受精卵的融解、损害胚泡植入及前期胚胎发育引起早期流产等过程,从而引起免疫不孕。     

男性不育时顶体素活性测定法

顶体素(acrosin)是存在于精子顶体内膜上的胰蛋白酶样蛋白水解酶,具有溶解卵透明带使精子通过等作用,在受精过程中极为重要。此酶自本世纪60年代后半期才逐渐了解。据报道,不孕症占已婚夫妇的1/10,其中约半数原因在男性。因此,了解男性的生育能力十分重要。既往对男性生育力的评     

受精调控机制的研究进展

人类和哺乳动物的受精机制是近年来的研究热点.通过生物化学、转基因和基因敲除等研究得出的受精机制主要分为4个部分：①精子获能;②精子结合至卵子透明带;③精子与透明带结合诱发顶体反应（AR）;④精子与卵子的结合和膜融合.笔者拟就近期研究阐明的精子获能、AR及精、卵融合的新信息、新观点,进行综述如下.     

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.     

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.     

Spermatozoa from a marsupial, the brushtail possum, contain beta1,4-galactosyltransferase

Beta1,4-galactosyltransferase-I (GalTase-I) is one of the key molecules on the sperm surface of eutherian mammals that is likely to be involved in binding to the egg coat, the zona pellucida, to mediate sperm-egg interaction. In laboratory mice, the species for which most data are available, this protein functions as a receptor for the zona pellucida protein ZP3 of the oocyte and, upon binding, triggers the sperm acrosome reaction. In the present study, we investigated the presence and abundance of GalTase-I in epididymal sperm extracts of a marsupial, the brushtail possum, Trichosurus vulpecula. For this, spermatozoa were collected from cauda epididymides and the amount of beta1,4-galactosyltransferase activity in washed sperm extracts was compared with that of porcine spermatozoa. Overall beta1,4-galactosyltransferase enzyme activity was found to be more abundant in possum sperm extracts than those from porcine spermatozoa (P<0.05). Immunoblots with an antibody to mouse GalTase-I revealed that the molecular weight of possum spermatozoa GalTase-I was 66 kDa, which is similar to the molecular weight of GalTase-I in spermatozoa from eutherian mammals. The molecular weight of GalTase-I was the same in sperm extracts collected from the caput and cauda epididymides. These results demonstrate that GalTase-I is indeed present in possum spermatozoa and thus it may be a gamete receptor molecule on the sperm surface of marsupials as well as those of eutherian mammals.     

DETECTION AND BIOIMAGING OF NITRIC OXIDE IN BOVINE OOCYTES AND SPERM CELLS

Mammalian gametes contain constitutive nitric oxide synthases (NOS) to synthesize nitric oxide (NO). The detection and bioimaging of NO in bovine gametes is important to determine the regulatory roles of NO during the different events of fertilization. Diaminofluoresceins, new fluorescence indicators for NO, were applied to detect the release of NO from bovine gametes. These compounds yield green fluorescent triazolofluoresceins, which provide the advantages of high specificity, sensitivity, and simplicity for the detection of NO. In this study, we mapped the expression of NOS in the bovine sperm and ova. NOS activity in sperm first appeared in the acrosome, then 60?min later in the head, middle piece, cytoplasmic droplet, and tail. Cow ova had high NO activity in the cytoplasm and in the surrounding corona cells, but not in the zona pellucida. These results show that for bovine gametes, the synthesis NO by the NOS system presents clear patterns of time and spatial distribution that may be important for the different events of 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.     

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.     

精子质量和功能参数与IVF受精率和优胚率的关系探讨

目的:对优化处理后前向运动精子总数、精子正常形态百分率和精子顶体反应阳性率,这三项能体现精子质量和功能的参数与IVF受精率和优胚率之间的关系进行探讨。方法:分析226对接受IVF治疗的不育夫妇优化处理后前向运动精子总数、精子正常形态百分率、精子顶体反应阳性率、IVF受精率和优胚率等数据,观察精子质量和功能参数对IVF受精率和优胚率的影响。结果:当处理后前向运动精子总数<5×106时,IVF受精率极显著低于其他组(P<0.01);当处理后前向运动精子总数≥5×106时,IVF受精率不随处理后前向运动精子总数的增加而上升(P>0.05);处理后前向运动精子总数对优胚率无显著影响(P>0.05);精子正常形态百分率和顶体反应阳性率对IVF受精率和优胚率均无显著影响(P>0.05)。结论:优化处理后前向运动精子总数与IVF受精率相关,与优胚率无关;正常精子形态和顶体反应与IVF受精率和优胚率无关。