NO is necessary and sufficient for egg activation at fertilization

The early steps that lead to the rise in calcium and egg activation at fertilization are unknown but of great interest--particularly with the advent of in vitro fertilization techniques for treating male infertility and whole-animal cloning by nuclear transfer. This calcium rise is required for egg activation and the subsequent events of development in eggs of all species. Injection of intact sperm or sperm extracts can activate eggs, suggesting that sperm-derived factors may be involved. Here we show that nitric oxide synthase is present at high concentration and active in sperm after activation by the acrosome reaction. An increase in nitrosation within eggs is evident seconds after insemination and precedes the calcium pulse of fertilization. Microinjection of nitric oxide donors or recombinant nitric oxide synthase recapitulates events of egg activation, whereas prior injection of oxyhaemoglobin, a physiological nitric oxide scavenger, prevents egg activation after fertilization. We conclude that nitric oxide synthase and nitric-oxide-related bioactivity satisfy the primary criteria of an egg activator: they are present in an appropriate place, active at an appropriate time, and are necessary and sufficient for successful fertilization.     

The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs

Representing the 60 trillion cells that build a human body, a sperm and an egg meet, recognize each other, and fuse to form a new generation of life. The factors involved in this important membrane fusion event, fertilization, have been sought for a long time. Recently, CD9 on the egg membrane was found to be essential for fusion, but sperm-related fusion factors remain unknown. Here, by using a fusion-inhibiting monoclonal antibody and gene cloning, we identify a mouse sperm fusion-related antigen and show that the antigen is a novel immunoglobulin superfamily protein. We have termed the gene Izumo and produced a gene-disrupted mouse line. Izumo-/- mice were healthy but males were sterile. They produced normal-looking sperm that bound to and penetrated the zona pellucida but were incapable of fusing with eggs. Human sperm also contain Izumo and addition of the antibody against human Izumo left the sperm unable to fuse with zona-free hamster eggs.     

Transient activation of calcineurin is essential to initiate embryonic development in Xenopus laevis

At fertilization, an increase of cytosolic calcium ions (Ca2+) triggers various activation responses in animal eggs. In vertebrates, these responses include exit from metaphase arrest in meiosis II (MII exit) and cortical remodelling initiated by cortical granule exocytosis. Although the essential requirement of Ca2+/calmodulin-dependent protein kinase II for inducing MII exit has been documented, a role of the Ca2+/calmodulin-dependent protein phosphatase calcineurin in egg activation has not been investigated. Here we show, using cell-free extracts from unfertilized eggs of Xenopus laevis, that calcineurin is transiently activated immediately after Ca2+ addition to a concentration that induces MII exit. When calcineurin activation is inhibited, cyclin-dependent kinase 1 (Cdk1) inactivation by means of cyclin B degradation is prevented and sperm chromatin incubated in the extracts remains condensed. Similarly, if calcineurin is inhibited in intact eggs, MII exit on egg activation is prevented. In addition, the activation contraction in the cortex is suppressed whereas cortical granule exocytosis occurs. We further demonstrate that, when a high level of calcineurin activity is maintained after activation, growth of sperm asters is prevented in egg extracts and, consistently, migration of male and female pronuclei towards each other is hindered in fertilized eggs. Thus, both activation and the subsequent inactivation of calcineurin in fertilized eggs are crucial for the commencement of vertebrate embryonic development.     

Outbred embryos rescue inbred half-siblings in mixed-paternity broods of live-bearing females

Females commonly mate with more than one male, and polyandry has been shown to increase reproductive success in many species. Insemination by multiple males shifts the arena for sexual selection from the external environment to the female reproductive tract, where sperm competition or female choice of sperm could bias fertilization against sperm from genetically inferior or genetically incompatible males. Evidence that polyandry can be a strategy for avoiding incompatibility comes from studies showing that inbreeding cost is reduced in some egg-laying species by postcopulatory mechanisms that favour fertilization by sperm from unrelated males. In viviparous (live-bearing) species, inbreeding not only reduces offspring genetic quality but might also disrupt feto-maternal interactions that are crucial for normal embryonic development. Here we show that polyandry in viviparous pseudoscorpions reduces inbreeding cost not through paternity-biasing mechanisms favouring outbred offspring, but rather because outbred embryos exert a rescuing effect on inbred half-siblings in mixed-paternity broods. The benefits of polyandry may thus be more complex for live-bearing females than for females that lay eggs.     

Bidirectional incompatibility between conspecific populations of Drosophila simulans

Cytoplasmic incompatibility (CI) describes the phenomenon whereby eggs fertilized by sperm from insects infected with a rickettsial endosymbiont fail to hatch. Unidirectional CI between conspecific populations of insects is a well documented phenomenon. Bidirectional CI has, however, only been described in mosquito populations, and recently between closely related species of parasitic wasps, where it is of interest as both an unusual form of reproductive isolation and as a potential means of insect population suppression. Here we report on the first known example of bidirectional CI between conspecific populations of Drosophila simulans. Further, we show that defects as early as the first cleavage division are associated with CI. This observation suggests that the cellular basis of CI involves disruption of processes before or during zygote formation and that CI arises from defects in the structure and/or function of the sperm during fertilization.     

An extrachromosomal factor causing loss of paternal chromosomes

Extrachromosomal inheritance is ubiquitous among plants and animals; however, most extrachromosomal factors are uniparentally inherited through females, but not through males. Examples include chloroplasts, mitochondria and a variety of intracellular symbionts. The only known exception to maternal extrachromosomal inheritance in an animal is a paternally transmitted sex ratio factor (psr) which causes all-male families in the parasitic wasp, Nasonia vitripennis. Normally in this wasp, male offspring are haploid and develop from unfertilized eggs whereas females are diploid and develop from fertilized eggs. The psr factor is either a venereally transmitted infection which prevents egg fertilization (and therefore causes all-male families), or a factor transmitted to eggs by the sperm of males carrying psr, which somehow prevents incorporation of the paternal chromosomes. Here we report that sperm from psr males fertilizes eggs, but that the paternal chromosomes are subsequently condensed into a chromatin mass before the first mitotic division of the egg and do not participate in further divisions. Resulting haploid offspring are male, but have inherited the paternal factor. This extrachromosomal factor promotes its own transmission at the expense of the paternal chromosomes, and therefore can be considered a 'selfish' genetic element.     

Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis

It has been suggested that the failure of parthenogenetic mouse embryos to develop to term is primarily due to their aberrant cytoplasm and homozygosity leading to the expression of recessive lethal genes. The reported birth of homozygous gynogenetic (male pronucleus removed from egg after fertilization) mice and of animals following transplantation of nuclei from parthenogenetic embryos to enucleated fertilized eggs, is indicative of abnormal cytoplasm and not an abnormal genotype of the activated eggs. However, we and others have been unable to obtain such homozygous mice. We investigated this problem further by using reconstituted heterozygous eggs, with haploid parthenogenetic eggs as recipients for a male or female pronucleus. We report here that the eggs which receive a male pronucleus develop to term but those with two female pronuclei develop only poorly after implantation. Therefore, the cytoplasm of activated eggs is fully competent to support development to term but not if the genome is entirely of maternal origin. We propose that specific imprinting of the genome occurs during gametogenesis so that the presence of both a male and a female pronucleus is essential in an egg for full-term development. The paternal imprinting of the genome appears necessary for the normal development of the extraembryonic membranes and the trophoblast.     

五唇兰受精过程中卵细胞的响应

五唇兰Doritis pulcherrima Lindl．受精过程中超微结构观察结果显示：精子到达胚囊前。卵细胞呈梨形；精子到达胚囊时，卵细胞呈典型的倒梨形；精核和卵核融合时，卵细胞坐长圆型。卯核凹陷，整个精核被卵核包围着。由此可见：五唇兰受精过程的顺利进行，不仅需要精细胞的主动参与，还要卵细胞的积极响应。     

Rapid evolution of reproductive barriers driven by sexual conflict

A growing amount of experimental data indicates extremely rapid evolution of traits and proteins related to fertilization in many diverging taxa. These data come from studies of sperm or pollen competition between closely related species, and from molecular studies of fertilization proteins. The positive selection for evolutionary novelty that appears to be acting on fertilization systems seems paradoxical because successful reproduction requires the close matching of female and male traits. It has been suggested that perpetual coevolution between the sexes can result from sexual conflict in mating. Sexual conflict occurs when characteristics that enhance the reproductive success of one sex reduce the fitness of the other sex. Numerous examples of sexual conflict resulting from sensory exploitation, polyspermy and the cost of mating have been discussed in detail. The potential for coevolution due to such conflict has been evaluated experimentally. Here I develop a simple mathematical model describing coevolutionary dynamics of male and female traits involved in reproduction. The model shows that continual change in such traits at a constant speed is expected whenever females (or eggs) experience fitness loss from having too many compatible males (or sperms). The plausibility of runaway coevolution increases with increasing population size. Rapid evolution of reproductive barriers driven by sexual conflict may explain increased speciation rates after colonization of new habitats ('adaptive radiation') and high species richness in resource-rich environments.     

龟足（Pollicipes mitella）的胚胎发育

本文详细描述了龟足（Ｐｏｌｌｉｃｉｐｅｓｍｉｔｅｌｌａｌｉｎｎａｅｕｓ１７５８）从受精卵至孵化成无节幼虫的发育过程。卵细胞的两次成熟分裂是在受精后才先后完成，卵裂为螺旋形卵裂，若水温为２９～３１℃，从受精至孵化约需８～１５天。     

濒危植物-半日花(Helianthemum songaricum)的双受精作用及胚和胚乳发育

运用石蜡切片法,对半日花Ｈ．ｓｏｎｇａｒｉｃｕｍ的受精过程,胚及胚乳发育进行了初步观察．受精前,一个助细胞解体,另一助细胞宿存．精核与极核的融合早于精核与卵的融合．受精极核不经过休眠过程,经多次分裂形成核型胚乳．胚乳游离核于球胚晚期开始细胞化．种子成熟后无胚乳．胚胎发育属柳叶菜型．胚柄由一纵列细胞组成并于球胚晚期开始退化．胚的发育经历球形原胚、心形胚、鱼雷形胚及成熟胚各时期．     

Complementarity between sperm surface beta-1,4-galactosyltransferase and egg-coat ZP3 mediates sperm-egg binding.

Despite its importance, the molecular basis of mammalian gamete recognition has remained unclear. The enzyme beta-1,4-galactosyltransferase (Gal-transferase) has been viewed traditionally as a biosynthetic component of the Golgi complex, but is also found on the surface of many cells where it can bind its specific glycoside substrate on adjacent cell surfaces or in the extracellular matrix. In mouse it has been suggested that Gal-transferase on the sperm head mediates fertilization by binding oligosaccharide residues in the egg coat, or zona pellucida, and that the ability of the zona pellucida to bind sperm is conferred by oligosaccharides of the ZP3 glycoprotein. However, it has not been confirmed that Gal-transferase and ZP3 are in fact complementary gamete receptors whose interaction mediates sperm-egg binding. Here we show that mouse sperm Gal-transferase specifically recognizes those oligosaccharides on ZP3 that have sperm-binding activity, but does not interact with other zona pellucida glycoproteins. In contrast, all zona pellucida glycoproteins are recognized by non-sperm Gal-transferase, demonstrating a more stringent substrate specificity for the sperm enzyme. This interaction is required for sperm-egg binding because blocking or removing the binding site for Gal-transferase on ZP3 inhibits its ability to bind sperm. After the release of the sperm acrosome, the transferase relocalizes to a new membrane domain where it can no longer bind to ZP3, which is consistent with the inability of acrosome-reacted sperm to bind ZP3 or to initiate binding to the zona pellucida. Following fertilization, ZP3 is modified by egg cortical granule secretions so that it loses sperm receptor activity, which can be accounted for by a selective loss of its binding site for sperm Gal-transferase. These results show that sperm surface beta-1,4-galactosyltransferase and the egg-coat glycoprotein ZP3 are complementary adhesion molecules that mediate primary gamete binding in the mouse.     

Release of mature starfish oocytes from interphase arrest by microinjection of human centrosomes

Mature oocytes (unfertilized eggs) are arrested at definite cell-cycle stages which vary from species to species. In frogs and mammals, the oocytes are arrested at the second metaphase of meiosis whereas in echinoderms they are blocked later, at the pronucleus stage. What causes the maturing oocytes to stop at some point in the cell cycle is not entirely clear. In frogs, the metaphase arrest seems to be maintained by a cytostatic factor. In echinoderms, which stop at interphase, no such a factor has so far been found. The fertilization process, beyond the introduction of paternal chromosomes, releases the oocyte from cell-cycle arrest and provides a functional centrosome to replace the endogenous centrosome which is apparently lost during oogenesis in most species. Several lines of evidence suggest that release from cell-cycle arrest is mediated by a Ca2+ burst which is associated with fertilization, and it is known that the functional centrosome provided by the sperm is necessary for mitotic spindle formation and cleavages. We report here that microinjection of purified human centrosomes into mature starfish oocytes is sufficient to release them from arrest at interphase and to support many cleavages leading to the occasional formation of normal embryos. In this species centrosome induced re-entry into the cell cycle does not require a transient calcium burst nor does it require intact microtubules.     

Crucial function of histone lysine methylation in plant reproduction

Unlike animals, plants do not set aside germ cells early in development. In angiosperm species, reproduction occurs in the adult plant upon flowering. The multicellular male and female gametophytes differentiate from meiotic products within reproductive floral organs. Double fertilization is another remarkable feature of most angiosperm species. The zygote derived from fertilization of the egg cell by one of the sperm cells and the endosperm from fertilization of the central cell by the second sperm cell develop in a coordinated manner together and enclosed in the sporophytic maternal integuments, forming the seed. Understanding plant reproduction is biologically pertinent and agronomically and ecologically important. Here, we describe the known functions of histone lysine methylations in various steps of reproduction in the reference plant Arabidopsis thaliana. It is emerging that histone lysine methylation is key for understanding epigenetic regulation networks of genome function.     

Effects of ubiquitin-protea-some pathway on mouse sperm capacitation, acrosome reaction and in vitro fertilization

Chlortetracycline (CTC) fluorescence patterns were used to study changes in the patterns B and AR of mouse sperm after incubation with reagents that would block the UPP. They were the monoclonal antibody against ubiquitinated proteins-UCP1; the polyclonal antibody against ubiquitin anti-Ub, and a special inhibitor against proteasome ALLN. Furthermore, we treated the capacitated sperm or the eggs with these reagents separately and tested whether the normal in vitro fertilization was blocked or not. Results illustrate that UCP1, anti-Ub, and ALLN have little effects on sperm capacitation and acrosome reaction,but they do inhibit fusion of mouse sperm with eggs, which suggests that UPP play an important role in mouse in vitro fertilization.``     

喀什哈尔葡萄花器及受精过程观察

本文对喀什哈尔葡萄花器的构造和开花特点作了详细介绍、剖析,这是对传粉受精的高度适应。受精过程如下:1、授粉后40—50分钟花粉粒萌发。2、授粉后6小时花粉管到达胚囊。3、授粉后8—10小时二个精子分别达卵细胞和次生核。4、授粉后24小时精、卵完成融合,受精作用属有丝分裂前型。5、授粉后20小时精子和次生核完成融合。了解此程为开花受精期的管理提供依据。     

小鼠精子微注射受精卵的体外发育与移植的研究

用显微注射方法,将精子注入小鼠卵母细胞卵周隙中,使精卵体外受精获得受精卵。并对精子注射后卵母细胞的成活率、受精率和受精卵的发育率以及移植后受胎率等方面进行了系统的研究,结果表明:卵子存活率为75.36％(1309/1737),其中264枚卵裂,受精率为20·17％(264/1309),卵裂卵经体外培养后有192枚发育到桑椹胚及胚泡期,发育率为72.73％(192/264)。将其中73枚(桑椹胚10枚,肛泡63枚)胚胎移植到10只假孕受体子宫中,一只受体妊娠产仔9只,仔鼠发育正常。     

Sperm antigen MSH27 participates in sperm-egg membrane fusion

A monoclonal antibody (mAb), MSH27 has been selected from an mAb library which has been prepared with separated mouse sperm heads as antigens. Three aspects of evidence indicated that the MSH27 antigen was involved in the process of sperm-egg membrane fusion. After the acrosome reaction, the antigen was located at sperm equatorial segment and in postacrosomal area, where, it was widely accepted, the sperm-egg membrane fusion initially occurred. In in vitro fertilization, the MSH27 antibody could decrease the index of sperm-egg membrane fusion, but made no effects on sperm approaching and binding to the plasma membrane of eggs. The inhibition showed an antibody concentration-dependent manner, with a rate decrease of 90% at 600 μg/mL immuno-globulin lgM. Furthermore, the antigen was able to affect the sperm-egg membrane fusion directly. The fusion index was obviously reduced after the zona-free eggs were exposed to the antigen purified by immuno-affinity chromatography. These, all together, demonstrated that the MSH27 antigen played an important role in sperm-egg membrane fusion.     

Role for sperm in spatial patterning of the early mouse embryo

Despite an apparent lack of determinants that specify cell fate, spatial patterning of the mouse embryo is evident early in development. The axis of the post-implantation egg cylinder can be traced back to organization of the pre-implantation blastocyst. This in turn reflects the organization of the cleavage-stage embryo and the animal-vegetal axis of the zygote. These findings suggest that the cleavage pattern of normal development may be involved in specifying the future embryonic axis; however, how and when this pattern becomes established is unclear. In many animal eggs, the sperm entry position provides a cue for embryonic patterning, but until now no such role has been found in mammals. Here we show that the sperm entry position predicts the plane of initial cleavage of the mouse egg and can define embryonic and abembryonic halves of the future blastocyst. In addition, the cell inheriting the sperm entry position acquires a division advantage and tends to cleave ahead of its sister. As cell identity reflects the timing of the early cleavages, these events together shape the blastocyst whose organization will become translated into axial patterning after implantation. We present a model for axial development that accommodates these findings with the regulative nature of mouse embryos.