Carbohydrates mediate sperm-ovum adhesion and triggering of the acrosome reaction

The fertilization process is the net result of a complex sequence of events that collectively result in the fusion of theopposite gametes. The male gamete undergoes continuous morphological and biochemical modifications during spermdevelopment in the testis (spermatogenesis), maturation in the epididymis, and capacitation in the female reproductivetract. Only the capacitated spermatozoa are able to recognize and bind to the bioactive glycan residue(s) on the ovum'sextracellular coat, the zona pellucida (ZP). Sperm-zona binding in the mouse and several other species is believed totake place in two stages. First, capacitated (acrosome-intact) spermatozoa loosely and reversibly adhere to the zona-in-tact ovum. In the second stage tight irreversible binding occurs. Both types of bindings are attributed to the presence ofglycan- binding proteins (receptors) on the sperm plasma membrane and their complementary bioactive glycan units(ligands) on the surface of the ZP. The carbohydrate-mediated adhesion event initiates a signal transduction cascade re-sulting in the exocytosis of acrosomal contents. This step is believed to be prerequisite which allows the hyperactivatedacrosome-reacted spermatozoa to penetrate the ZP and fertilize the ovum. This review focuses on the role of carbohy-drate residues in sperm-ovum interaction, and triggering of the acrosome reaction. I have attempted to discuss extensiveprogress that has been made to enhance our understanding of the well programmed multiple molecular events necessaryfor successful fertilization. This review will identify these events, and discuss the functional significance of carbohy-drates in these events.

卵胞浆内单精子注射后人精子激活鼠卵母细胞能力的研究

目的 :研究各种质量精子的鼠卵母细胞激活能力。方法 :选用 2 0～ 2 5 g昆明种雌性白鼠 ,超排卵取卵细胞 ,用 Percoll密度梯度离心法处理正常精液、少精、弱精、畸精以及冷冻后的正常精液 ,选择优质的精子进行显微注射 ,计算卵母细胞激活率、卵细胞形态正常率。结果 :各种质量精液中优选的精子激活鼠卵母细胞能力差异无显著性 ( P>0 .0 5 )。结论 :人精子激活鼠卵母细胞能力与最初精液状态没有直接的相关性 ,它不受精子数量、活力。

人精子甘露糖受体对地鼠卵母细胞超微结构的影响

目的研究人精子甘露糖受体(MR)在精卵融合中的作用。方法去透明带金黄地鼠卵经不同浓度的纯化人精子MR作用后,用透射电子显微镜观察卵母细胞超微结构的变化。结果MR作用卵母细胞后,卵母细胞内的皮质颗粒(CG)减少,并伴随有CG胞吐现象,同时卵母细胞表面的微绒毛数量减少。结论MR作用于卵母细胞表面后,可触发CG胞吐,并促使卵母细胞表面微绒毛减少,提示MR在介导精子与卵母细胞识别与融合中起作用。

腺苷脱氨酶在人精子膜上的定位及其单克隆抗体对人精子穿卵能力的影响

利用本实验室制备的抗人精子膜结合腺苷脱氨酶（ＡＤＡ）单克隆抗体（ＭｃＡｂ）和间接荧光免疫技术，分析了该酶在正常人精子膜上的位置。同时，利用该酶单抗分析了该酶活性对人精子穿透去透明带卵的影响。上述两个实验的结果显示：（１）ＡＤＡ结合于人精子中段、尾部膜的外侧；（２）利用抗ＡＤＡＭｃＡｂ抑制ＡＤＡ活性可明显抑制正常人精子穿透去透明带卵的能力。这一研究结果提示，ＡＤＡ作为精子的膜蛋白组分，可能参与对精子生理功能的调节过程。

The human acrosome reaction

We developed tests of sperm-oocyte interaction: sperm-zona binding, zona-induced acrosome reaction, sperm-zona penetration and sperm-oolemma binding, using oocytes which failed to fertilise in clinical in vitro fertilization(IVF). Although oocyte defects contribute to failure of sperm oocyte interaction, rarely are all oocytes from one wom-an affected. Low or zero fertilization in standard IVFwas usually caused by sperm abnormalities. Poor sperm-zona pel-lucida binding was frequently associated with failure of standard IVF and obvious defects of sperm motility or morpholo-gy. The size and shape of the acrosome is particularly important for sperm binding to the oocyte. The proportion ofacrosome intact sperm in the insemination medium was related to the IVF rote. Inducing the acrosome reaction with acalcium ionophore reduced sperm-zona binding. Blocking acrosome dispersal with an acrosin inhibitor prevented sperm-zona penetration. Sperm-zona penetration was even more highly related to IVF rates than was sperm-zona binding.Some patients had low or zero fertilization rates with standard IVF but normal sperm by conventional tests and normalsperm-zona binding. Few of their sperm underwent the acrosome reaction on the surface of the zona and none penetrat-ed the zona. In contrast, fertilization and pregnancy rates were high with intmcytoplasmic sperm injection. We call thiscondition defective zona pellucida induced acrosome reaction. Discovery of the nature of the abnormalities in the signaltmnsduction and effector pathways of the human zona pellucida induced acrosome reaction should result in simpler testsand treatments for the patients and also provide new leads for contraceptive development.

Molecular aspects of mammalian fertilization

Mammalian fertilization is a highly regulated process, much of which are not clearly understood. Here we presentsome information in order to elaborate a working hypothesis for this process, beginning with the sperm modifications inthe epidydimis up to sperm and egg plasmalemma interaction and fusion. We also discuss the still poorly understood ca-pacitation process, the phenomenon of sperm chemo-attraction that brings the capacitated sperm to interact with theoocyte vestments and certain aspects of the acrosome reaction. (Asian J Androl 2001 Dec; 3 : 243 - 249)

CRISP2蛋白研究进展

CRISPs(cysteine rich secretory proteins)蛋白家族有4个成员,CRISP2是唯一在睾丸中表达的CRISPs成员,组织特异性较高。精母细胞表达和分泌CRISP2,不受雄激素的影响。CRISP2分布于生精上皮内,定位于精母细胞和支持细胞间的连接处,可以增强精母细胞连接到支持细胞上的能力;继而存在于圆形精子细胞及延长型精子细胞,可结合到精子尾部和头部顶体,并最终定位于精子尾部以及顶体。CRISP2可能与精子顶体RyR3受体结合调节Ca^(2+)内流;此外,CRISP2可能还具有调节RyR受体之外的其他离子通道(如抑制K^(+)通道)的功能。研究表明,CRISP2参与了精子成熟和获能的全程,也在精卵结合的过程中起一定作用。综述CRISP2蛋白在睾丸的表达和分布、调控以及作用。

RNase9蛋白在男性生殖系统中的表达及功能

目的：研究核糖核酸酶9（RNase9）在男性生殖系统中的表达、定位及功能，为其异常所引起的不育症诊断和治疗提供理论基础。方法采用生物信息学分析人RNase9的结构，预测其功能；利用基因重组技术构建了原核表达载体制备重组RNase9蛋白。通过RT-PCR检测RNase9蛋白在不同组织中的表达，比较老年人（76.3±8.1岁）、成年人（31.2±5.6岁）和胎儿（0.8±0.2岁）附睾组织RNase9蛋白表达量的差异。应用免疫荧光实验观察RNase9蛋白在睾丸、附睾、精子上的定位。应用酵母tRNA底物实验检测RNase9核糖核酸酶活性。分别向精子悬液中加入免疫前血清（A组）和鼠抗RNase9多抗血清（B组），应用抗体封闭精子运动实验检测RNase9蛋白对精子运动能力[平均曲线运动速度（VCL）、平均直线运动速度（VSL）、平均路径速度（VAP）]的影响。8～12周龄金黄地鼠肌注孕激素和人绒毛膜促性腺激素，取卵子与两组精子孵育后（A组精子用免疫前血清，B组精子用RNase9抗血清封闭5 h；实验中所用卵子的数目分别是A组60个和B组62个），应用抗体封闭精子穿卵实验检测RNase9蛋白对精子穿卵能力的影响。结果经过生物信息学分析，RNase9基因属于核糖核酸酶A超家族一新成员，N末端含23个氨基酸的信号肽序列，相对分子质量约为20000，等电点PI 5.92；含5个N-糖基化位点、2个酪蛋白激酶Ⅱ磷酸化位点、1个酪氨酸激酶磷酸化位点、1个NDUFC2位点和2个胰-核糖核酸酶功能域标签位点。RNase9蛋白在人附睾、睾丸、心脏，肺脏、肝脏、脾脏、肾脏、胃多种组织广泛表达，在附睾组织中表达量较高；在成年人附睾组织中的表达量明显高于胎儿和老年人附睾组织（均P〈0.05）。RNase9蛋白特异结合在精子的赤道后颈部，原核表达获得高纯度具有生物活性的重组蛋白和相应的抗体。核糖核酸酶实验显示RNase9蛋白缺乏核糖核酸酶活性，为0.111±0.007。抗体封闭精子后不影响精子的运动及穿卵能力，两组VAP、VSL、VCL和受精率差异没有统计学意义（均P〉0.05）。结论 RNase9蛋白在附睾及精子上的表达及定位表明该蛋白可能在男性精子成熟及发育过程中起重要作用，但不影响成熟精子的运动及穿卵能力。