补救性ICSI后受精失败的原因探讨

目的:研究受精失败卵母细胞内精子解浓缩,卵子激活情况及纺锤体染色体分析,探讨补救性ICSI后受精失败的原因.方法:收集生殖中心行补救性ICSI后受精失败(受精率为0)的MII期卵母细胞,并分为早救性ICSI组和晚救性ICSI组.于取卵后48小时用免疫荧光法在激光共聚焦显微镜下观察.结果:收集95个受精失败的卵母细胞(其中早救性ICSI组46个.晚救性ICSI组49个),两组卵子激活障碍的发生率分别为60.9%,51%,没有统计学差异(P＞0.05),卵子激活障碍的发生率远高于其他影响卵母细胞受精的因素的发生率.结论:卵子激活障碍是补救性ICSI后受精失败的最主要原因.     

哺乳动物精子超激活运动研究进展

哺乳动物精子经过雌性生殖道与卵子发生融合完成受精过程,需要正常的精子运动能力作为保障。超激活运动可以帮助精子穿透卵子透明带,与卵子发生结合形成受精卵。精子超激活运动的激发和维持是通过自身生理和雌性生殖道内生化环境共同调节完成的。"超激活运动"自发现起到现在已成为生殖学研究的重要部分,现对精子超激活运动与受精的关系、调节因子、鞭毛结构、技术手段及未来研究方向进行综述,为男性不育及避孕等相关临床研究提供理论依据。     

昆虫单性生殖及其性别决定

通常昆虫必须通过精子、卵子结合,即受精才能繁育后代。有一些昆虫的卵没有受精也能发育成新个体。这种精子、卵子不发生核融合,卵子单独发育的繁殖方式称单性生殖,亦有人称之为无融合生殖。单性生殖是有性生殖的一种特殊方式或变态。在植物界中较为普遍,有许多昆虫也行单性生殖。单性生殖是某些昆虫长期的自然适应性的表现。利用单性生殖可以     

浅谈受精机制

多数动、植物,通过精子和卵子相互结合,产生出了新一代的个体。这个过程的内在机制非常复杂。交配时,千百万精子蜂涌而上,都要进入卵子,可是最终只有一个精子和卵子结合,受精之后,又立即产生一种封锁机制,别的精子都再也无法进入。有意思的是受精卵在生化本质上发生了根本性变化,由休眠状态转变为强有力的活化状态,开始了活跃的合成和分裂过程。不少学者对受精的机制进行多年的研究,探求其中的奥秘。现就下面三个问题作简要的介绍。     

鲸类繁殖生物学的研究概况

生殖是生物繁殖自身的能力,生殖过程包括生殖细胞(精子与卵子)的生成与成熟、受精过程、妊娠、胎儿的发育、分娩等环节。       

PNAS：钙离子有助卵子激活

近期来自美国国立卫生研究院环境健康研究所（NIH／NIEHS）的研究人员报道了钙离子流动在卵子受精过程中的最新调控机制。该报道揭示钙离子流动在受精过程中,不仅单单为卵子的钙库提供钙离子来源。而且其在跨入卵子质膜的过程中为卵子的激活提供了必须的信号。这一研究发现为人类辅助生殖提供了重要的理论依据并且对于推动卵子保存具有重要的意义。相关研究论文发表在美国《国家科学院院刊》（PNAs）杂志上。     

我国的蕨类植物杂交种

蕨类植物是植物界中的一大类群,是靠孢子进行繁殖的孢子植物。它的生活史中具有一个独立生活的配子体世代和一个独立生活的孢子体世代。配子体极小,其上发育着精子器、颈卵器。蕨类植物的精子具鞭毛,受精过程离不开水,通过水精子才能达到颈卵器与卵子结合受精。在自然界潮湿的环境中许多不同种的蕨类植物的配子体生活在一起时,就有可能发生不同种的精子和卵子结合成受精卵而发育出杂交后代的可能性。但杂交后代能否正常发育并存     

丝裂原活化蛋白激酶信号通路与受精

受精是雌雄生殖细胞相互融合形成合子的过程 ,是生命的开端。有性生殖的生命体通过受精 ,使基因组由生殖细胞的单倍体恢复为体细胞的二倍体 ;同时 ,精子与卵子的结合激活了以母源信息形式贮存在卵子中的发育程序 ,使卵子由受精前的生命活动抑制状态转变为活跃状态 ,即活化卵子。受精涉及到两个不同个体间异源细胞的识别、融合、信号转导和分裂周期调节 ,因此久已成为发育生物学、细胞生物学和生物化学的重要研究课题。多种蛋白激酶参与对受精过程的调节。作者和其他人的研究均发现丝裂原活化蛋白激酶 (ｍｉｔｏｇｅｎａｃｔｉｖａｔｅｄｐｒ…     

唇鲳精子早期入卵观察

用扫描电镜对唇鳃成熟卵子及早期精子人卵过程进行观察。结果显示,唇鲋成熟卵子在动物极中央有一深凹陷的表面光滑的精孔器,其外径2．512μm,内径2．330μm,精子直径1．567μm。混匀的精卵刚遇水时,没有精子进入精孔器。受精后1s,精孔器内出现精子。受精后5S,组织切片显示,精子已经进入卵子内,并形成具有强烈抑制多精人卵作用的受精锥。受精后10S,精子在精孔器前庭集结,尚未形成受精塞。受精后20S,在精孔器内形成受精塞。受精塞没有阻塞精孔管,经分析它不是来源于皮层反应产物。受精塞形成后,可以吸附人卵的精子,这对多精入卵有积极的抑制作用;精子尾部在入卵过程中相互缠绕,这也是减少多精入卵的重要机制。受精后30s,受精塞和吸附的精子向精孔器外移动。受精后50S,受精塞和吸附的精子堵塞精孔器。受精后60s,受精塞吸附的精子开始解体,但是由于精孔管未封闭,还有精子通过精孔管进入到质膜。在人工受精过程中,卵子的单精受精屏障会因其周围精子密度大、精子与卵子距离短、精子运动速度快而被打破,从而导致这些卵子出现多精入卵的现象。受精后80s,精孔管仍然没有封闭,精孔器附近的精子明显出现活动能力的差异：精孔器外面的精子活动能力最强,精孔管旁边的精子活动能力较弱;精孔管外堆积的精子活性消失,受精塞吸附的精子已开始解体,经初步分析,这可能是进入其内的精子耗能有所差异的结果。受精后100S,受精塞吸附的精子解体。     

唇精子早期入卵观察

用扫描电镜对唇成熟卵子及早期精子入卵过程进行观察.结果 显示,唇成熟卵子在动物极中央有一深凹陷的表面光滑的精孔器,其外径2.512 μm,内径2.330 μm,精子直径1.567 μm.混匀的精卵刚遇水时,没有精子进入精孔器.受精后1 s,精孔器内出现精子.受精后5 s,组织切片显示,精子已经进入卵子内,并形成具有强烈抑制多精入卵作用的受精锥.受精后10 s,精子在精孔器前庭集结,尚未形成受精塞.受精后20 s,在精孔器内形成受精塞.受精塞没有阻塞精孔管,经分析它不是来源于皮层反应产物.受精塞形成后,可以吸附入卵的精子,这对多精入卵有积极的抑制作用;精子尾部在入卵过程中相互缠绕,这也是减少多精入卵的重要机制.受精后30 s, 受精塞和吸附的精子向精孔器外移动.受精后50 s, 受精塞和吸附的精子堵塞精孔器.受精后60 s, 受精塞吸附的精子开始解体,但是由于精孔管未封闭,还有精子通过精孔管进入到质膜.在人工受精过程中,卵子的单精受精屏障会因其周围精子密度大、精子与卵子距离短、精子运动速度快而被打破,从而导致这些卵子出现多精入卵的现象.受精后80 s, 精孔管仍然没有封闭,精孔器附近的精子明显出现活动能力的差异:精孔器外面的精子活动能力最强,精孔管旁边的精子活动能力较弱;精孔管外堆积的精子活性消失,受精塞吸附的精子已开始解体,经初步分析,这可能是进入其内的精子耗能有所差异的结果.受精后100 s,受精塞吸附的精子解体.     

Current trends in evaluation of sperm function: in vitro selection and manipulation of male gametes for assisted conception

With increasing medical utilization of assisted reproductive technology (ART), scientists and clinicians have been able to study extensively multiple cell functions operating synchronously and flawlessly during the events preceding, before and after fertilization. Critical evaluation of the functional status of spermatozoa for in vitro techniques such as sperm-mucus interaction, acrosome reaction status, sperm-zona pellucida binding and penetration tests, hyaluronic acid binding assay, and computer assisted semen analysis etc. can direct a male partner of an infertile couple to more aggressive forms of treatments. In vitro selection of functionally competent sperm cells is a pre-requisite for successful outcome in in vitro fertilization or in intracytoplasmic sperm injection (ICSI). Direct injections of acrosome-intact spermatozoa into oocyte during ICSI bypassing the normal events of sperm oocyte interaction and fusion events have raised concerns with regard to fertilization abnormalities and genetic issues. The present communication briefly reviews the sperm function tests with emphasis on its correlation with fertility outcome, and the currently employed sperm selection and manipulation procedures which may have implications in assisted conception programs.     

Complete fertilization failure in ICSI

Abstract  Fertilization failure is one of the causes of infertility that becomes evident only after in vitro fertilization (TVF) and intracytoplasmic sperm injection (ICSI) have been attempted. Although the frequency of incidence of fertilization failure is low, if fertilization failure is encountered, medical treatment is usually stopped and serious psychological damage may occur to the patient.
While fertilization failure in IVF can be dealt with using ICSI, there is no treatment for fertilization failure in ICSI. At present, clinical investigations are being conducted to evaluate oocyte activation in combination with ICSI to cope with fertilization failure of ICSI.     

Essential role of the nonreducing terminal alpha-mannosyl residues of the N-linked carbohydrate chain of bovine zona pellucida glycoproteins in sperm-egg binding

It has been proposed that mammalian sperm bind species-specifically to carbohydrate chains of zona pellucida glycoproteins at fertilization. Although the sperm ligand carbohydrate chains have been characterized in mice and pigs, the existence of the ligands of other mammals remains unclear. In order to explore the bovine sperm ligand, two in vitro competition assay methods were applied. As a result, a high-mannose-type carbohydrate chain, Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAcbeta1-4GlcNAc, which is the major neutral chain in bovine egg zona glycoproteins, was shown to possess bovine sperm ligand activity. When nonreducing terminal alpha-mannosyl residues were eliminated from the zona glycoproteins by alpha-mannosidase digestion, the ligand activity was reduced, indicating that the alpha-mannosyl residues play an essential role in bovine sperm-egg binding. The number of sperm binding to eggs was reduced to about one-half after fertilization. The ligand-active high-mannose-type chain may be buried after fertilization, since its amount remains unchanged. Pretreatment of bovine sperm with the sperm ligand-carbohydrate chain significantly inhibited penetration of the sperm into oocyte and the male pronucleus formation. Thus, a correlation between the sperm ligand activity and in vitro fertilization rate was observed.     

Investigation of assisted fertilization and biology of reproduction by sperm microinjection

Recent success in assisted fertilization mainly depended on the development of sperm microinjection methods: intracytoplasmic sperm injection and subzonal insemination. Some basic mechanisms that under-lie fertilization were revealed by using intracytoplasmic sperm injection. In respect to this, problems of fertility, oocyte activation, formation of pronuclei and practical aspects of intracytoplasmic sperm injection are discussed.     

Selection of nonapoptotic spermatozoa as a new tool for enhancing assisted reproduction outcomes: an in vitro model

Magnetic cell sorting (MACS) using annexin V-conjugated microbeads eliminates apoptotic spermatozoa based on the externalization of phosphatidylserine residues. The procedure delivers two sperm fractions: annexin V-negative (nonapoptotic) and annexin V-positive (apoptotic). Our aim was to determine whether the sperm fertilizing potential can be improved by selecting a nonapoptotic fraction using MACS. Semen samples (n = 35) were subjected to separation on a density gradient followed by MACS. Extent of apoptosis was assessed by measuring levels of activated caspase 3 using fluorescein-labeled inhibitors of caspase, alterations in mitochondrial membrane potential (MMP) using a lipophilic cationic dye, and DNA fragmentation using terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling assay. The sperm fertilization potential was assessed using hamster oocyte penetration assay and hamster oocyte-intracytoplasmic sperm injection (ICSI). Annexin V-negative sperm displayed superior quality in terms of high motility, low caspase 3 activation, MMP integrity, and small extent of DNA fragmentation. Annexin V-negative sperm demonstrated higher oocyte penetration capacity but comparable sperm chromatin decondensation (SCD) following ICSI. Conversely, the annexin V-positive sperm presented with poor quality and fertilization potential. The oocyte penetration rate was negatively correlated with apoptotic marker expression, whereas SCD following ICSI was only associated with apoptosis on sperm-damaged membranes. We conclude that apoptosis appears to impact sperm-oocyte penetration rate; however, it does not seem to affect early stages of fertilization such as SCD in spermatozoa of healthy donors. The selection of nonapoptotic sperm by MACS may be used to enhance results of in vitro fertilization by increasing sperm-oocyte penetration.     

Ultrastructural study of cat zona pellucida during oocyte maturation and fertilization

The mammalian zona pellucida (ZP) is an extracellular glycoprotein structure formed around growing oocytes, ovulated eggs and preimplantation embryos. The specific functions of ZP are highly determined by its morphological structure. Studies on cat oocytes during maturation and after fertilization were undertaken, using routine transmission (TEM) and scanning electron microscopy (SEM). Two basic ZP layers – outer with rough spongy appearance and inner with smaller fenestrations and smooth fibrous network – were visible. Deposits, secreted by oviductal cells formed new layer, the so called oviductal ZP. After fertilization outer ZP showed rougher meshed network due to fusion between filaments as a consequence from sperm penetration while the inner was smoother with melted appearance. The presented data on the SEM and TEM characteristics of cat oocytes, together with our previous studies on carbohydrate distribution suggest that during oocyte maturation and fertilization ZP undergoes structural and functional rearrangements related to sperm binding and penetration.     

Inhibition of bovine sperm-oocyte fusion by the p-aminophenyl derivative of D-mannose

Several steps in the process of mammalian fertilization are mediated by carbohydrates. This study investigated the role of the p-aminophenyl derivative of d-mannose (APMP) during bovine fertilization. Inseminating cumulus-oocyte complexes (COCs) in the presence of increasing APMP concentrations resulted in a significant dose-dependent decrease of the fertilization rate (P < 0.05). No negative effect of 50 mM APMP on total sperm motility and progressive motility was found. Subsequently, the fertilization steps at which this blocking effect could be exerted were investigated, i.e., sperm penetration of the cumulus oophorus, sperm-zona binding, acrosome reaction, sperm-oolemma binding, and/or sperm-oocyte fusion. Inseminating cumulus-enclosed and cumulus-denuded oocytes in the presence of 50 mM APMP significantly decreased the fertilization rate to a comparable minimum level (P < 0.05). There was no significant relationship between the number of spermatozoa bound to the zona pellucida and the APMP concentration, and APMP nor d-mannosylated bovine serum albumin (BSA) suppressed or stimulated sperm acrosomal status. Inseminating zona-free oocytes in the presence of 50 mM APMP did not influence sperm-oolemma binding, but significantly inhibited sperm-oocyte fusion (P < 0.05). Preincubating zona-free oocytes with 200 microg/ml Con A but not with 50 mM APMP inhibited the sperm-oocyte fusion rate to the same extent as when the gametes were simultaneously exposed to 50 mM APMP. These data indicate that the blocking effect of APMP on bovine fertilization is mainly due to an inhibition of sperm-oocyte fusion, probably by specific obstruction of the sperm receptor sites that are responsible for the fusion process.     

Oocyte diameter in relation to meiotic competence and sperm penetration

This study was conducted to determine the diameter of canine oocytes that are able to attain full meiotic competence and sperm penetration. Oocytes were collected from ovaries of bitches at various stages of the estrous cycle. Only healthy-looking cumulus-oocyte complexes were used for in vitro maturation, and were divided into four groups based on diameter: <100, 100 to <110, 110 to <120 and >120 microm. Following in vitro maturation or fertilization, oocytes were stained to assess nuclear maturation and penetration rates. The mean oocyte diameter was 108.5 +/- 0.4 microm. The oocytes displayed size-related ability to undergo meiotic maturation. After culture for 72 h, the rates of oocytes that remained at the germinal vesicle stage in the <110 microm groups were significantly higher (P<0.01) than in the > or = 110 microm groups. None of the oocytes <110 microm reached metaphase II (MU), but 4.9 and 21.5% of the oocytes that were greater than 110 and 120 microm, respectively, progressed to MII. After in vitro fertilization for 20 h, 10 to 25% of oocytes were penetrated by spermatozoa, but there were no clear relationships between oocyte diameter and penetration rates of the oocyte by sperm. In the <120 microm groups, sperm penetration was mostly found in oocytes arrested at the germinal vesicle stage. However, a total of eight oocytes > or = 120 microm in diameter were penetrated by spermatozoa, of which five oocytes reached MII. These results suggest that there is a clear relationship between oocyte diameter and meiotic competence, but no relationship between oocyte diameter and sperm penetration. Canine oocytes may have acquired meiotic competence once they reach at a diameter of 120 microm, but the oocytes may allow the entry of spermatozoa into the ooplasm irrespective of oocyte diameter.     

The Elusive Acrosome of Chiton Sperm

Summary

In our study of spermiogenesis in the lined chiton Tonicella lineata, we traced the formation and migration of small Golgi vesicles to the apex of the sperm, where they fused to form an apical granule. This apical granule and other Golgi secretions tested positively for acid phosphatase. In preliminary experiments on fertilization, sperm swam inside open hull (chorion) cupules down to the surface of the egg and penetrated it. No micropyle was observed. Serial 1μm sections of eggs fixed during fertilization demonstrated that the sperm nucleus had penetrated not only the hull but also the vitelline and oocyte membranes. Serial thin sections showed that the tip of the anterior filament of the sperm had fused with a single microvillus of the oocyte membrane, creating a membranous tube through which the nucleus had entered the egg cortex. We suggest that the apical granule of chiton sperm is an acrosome that enables the nucleus to penetrate the egg membranes.     

Glycobiology of fertilization in the pig

By adopting internal fertilization, the meeting of both gametes - the sperm and the egg - and thus the highly coordinated sequence of interactions leading to fertilization, occur in the female reproductive tract. In mammals, the oviduct has been shown to translate the requirements of the female, coordinating sperm activation (capacitation) and sperm transport with the arrival of the ovulated egg. A hierarchy of carbohydrate-based interactions accompanies these events ranging from the binding of uncapacitated sperm to the oviductal epithelium (establishment of the female sperm reservoir), to the primary and secondary binding processes contributing to gamete recognition and sperm penetration of the oocyte zona pellucida. The current perspective will focus on the carbohydrate-recognition systems in the binding events during fertilization in the pig. The roles of the major carbohydrate-binding proteins, the spermadhesins and the acrosomal serine proteinase, pro/acrosin are discussed under consideration of recent structural data. The glycans and the glycoproteins of the porcine oviduct with a focus on the candidate sperm receptors as well as the zona pellucida N-glycans of prepuberal pigs have been characterized by a mass spectrometric approach. Furthermore, some preliminary data supporting the hypothesis that the zona pellucida has to undergo a maturation process during oocyte development are presented.