鲍配子识别蛋白的研究

配子相互作用的生化机制对于进一步阐明生殖过程具有重要作用,它是深入了解细胞内识别的理想体系。精卵细胞相互作用包括一系列的步骤,开始于精子与卵细胞外被的接触,终止于两性细胞的融合及精子核进入卵细胞质中,而精卵细胞的识别具有建立于各自性细胞表面成分基础上的种的特异性,鲍则是研究精卵识别的好材料。鲍精子在发生顶体反应时释放出两种蛋白质——细胞溶素(1ysin)和18ku糖蛋白(spl8),其中的细胞溶素与其卵黄膜上的受体紧密结合,并利用非酶反应在卵黄膜上穿一个小孔,整个精子则从此孔穿过卵黄膜与卵细胞融合;spl8释放后则覆盖到精子细胞膜表面,起到溶解卵细胞脂质体的作用,即spl8介导精、卵细胞膜的融合。鲍卵细胞膜上存在细胞溶素受体,它是大的不分支的糖蛋白分子,占据了卵黄膜30％的组分,可以专一性地与细胞溶素相结合。这些配子识别蛋白共同进化且速度很快,其中细胞溶素和18ku糖蛋白通过正向选择进化,而细胞溶素受体进行协同进化。     

Gamete production, sex hormone secretion, and mating behavior uncoupled

A common observation for seasonally breeding vertebrates is that the reproductive processes of gamete production, sex steroid hormone secretion, and mating behavior coincide, and further, that sex steroid hormones activate mating behavior. The postulate of hormone-dependence of mating behavior is based primarily on detailed studies of laboratory and domesticated species. However, comparison of a wide array of vertebrates reveals numerous exceptions to this supposed rule. Consideration of these species indicates that there is no fixed or intrinsic causal association among gamete production, sex hormone secretion, and mating behavior within any of the classes of vertebrates.     

Gamete physiology, fertilization and egg activation in teleost fish

The fertilization and activation of fishoocytes are vital, but unfortunatelyoverlooked, processes in fisheries research.This paper sets out to review our presentunderstanding of these important events inteleost fish and, drawing comparisons withmammalian research, to highlight areas in whichresearch effort is urgently required. Presently, the commercial culture of manyimportant freshwater, but especially marine,teleosts is beset by problems associated withfertilization, hatching and early embryonicdevelopment. These problems have beenparticularly acute in certain species leadingto the application of spawning inductiontechnologies in an effort to optimizeproduction. Increased knowledge of theprocesses of egg activation and fertilizationin these groups of fish is likely to makesignificant contribution to commercialaquaculture. Studies of a wide variety ofanimal and plant species has demonstrated thatdevelopment at fertilization is triggered by anincrease in intracellular Ca²⁺concentration within the egg that occurs aseither a single transient or a series ofdistinctive oscillations depending upon thespecies under investigation. This increase inintracellular Ca²⁺ activates the egg andalso appears to play an important role in laterembryonic development. Teleost reproductivestrategies and more importantly, teleostoocytes and spermatozoa, exhibit a remarkablevariety of adaptations. Currently, studies ofegg activation in teleosts are confined tolaboratory species such as medaka Oryziaslatipes and zebrafish Brachydanio rerio.Nevertheless, even between these two species,although an increase in intracellular Ca²⁺appears to be the trigger in both cases, themechanism of Ca²⁺ release may be quitedifferent. Activation in medaka is initiatedonly through direct contact with conspecificsperm, suggesting the involvement of asperm-specific factor, while zebrafish eggsappear to require only contact with theexternal spawning medium. In view of the highlyvariable fertility rates evident in manycommercially cultured teleosts, it could bevery rewarding to investigate the mechanism ofegg activation in representative teleost groupsusing the findings and theories emerging fromother animal groups as a starting point. Inorder to successfully conduct such aninvestigation, it will be necessary to employ acombination of physiological, molecular andrecombinant approaches.     

Gamete recognition during fertilization in a red alga,Antithamnion nipponicum

Summary Fertilization in the marine red algaAntithamnion nipponicum is a highly specific process involving non-motile male gametes, spermatia, and female receptive structures, carpogonia. FITC-lectin and Calcofluor white ST labelling show that the outer cell walls of spermatia differ from vegetative cells in carbohydrate composition. Specific binding of the lectins to spermatial walls was confirmed by lectin-gold labelling on thin sections. Gametic recognition inAntithamnion nipponicum is based on the interaction of a surface carbohydrate on the spermatia with a surface carbohydrate receptor on the trichogynes. Spermatial binding to trichogynes is inhibited by pre-incubation with concanavalin A and trichogyne receptors are blocked by the complementary carbohydrate -D-methyl mannose. The inhibitory effects of concanavalin A to spermatial binding of trichogynes is reversed by preincubation with -D-methyl mannose. The combination of long spermatial appendages and a carbohydrate-carbohydrate receptor-based gamete recognition mechanism make fertilization in this species an efficient process.     

The Genic Nature of Gamete Eliminator in Rice

The two cultivated rice species, Oryza sativa and Oryza glaberrima, are morphologically alike but are reproductively isolated from each other by hybrid sterility. The hybrid is male sterile but partially female fertile. Backcross experiments were conducted to introduce an alien factor controlling hybrid sterility from O. glaberrima (W025) into O. sativa (T65wx) and examine the genetic basis. An extracted sterility factor, closely linked to the wx locus, induced gametic abortion due to allelic interaction and was tentatively designated as S(t). The segregation patterns for infertility was explained by assuming that W025 and T65wx carried S(t) and S(t)a, respectively, and gametes with S(t)a aborted only in the heterozygote (S(t)/S(t)a) although the elimination of female gametes was incomplete. Thus, S(t) seemed to be intermediate between a gamete eliminator and pollen killer. However, S(t) was proven to be likely the same as S1 which was formerly reported as gamete eliminator in a different genetic background of O. sativa. In addition, a chromosomal segment containing S1 (or S(t] caused a marked suppression of crossing over around it, suggesting the presence of an inversion. Further, female transmission of S1a increased as the segment containing S1 became small by recombination. After S1 was further purified by successive backcrosses up to the BC15 generation, it became pollen killer. The present results give evidence that a profound sterility gene such as gamete eliminator can be made from accumulation of pollen killer and its modifier(s) when pollen killer and modifier(s) are linked, they behave as a gene complex in the hybrid.     

Gamete segregation in female carriers of Robertsonian translocations

Eleven female carriers of either 45,XX,der(13;14) (q10;q10) or 45,XX, der(14;21)(q10;q10) underwent hormonal stimulation with the purpose of producing enough oocytes for in-vitro fertilization and preimplantation genetic diagnosis. Polar body biopsy was performed in those oocytes and FISH with painting probes was applied in their metaphase-like first polar body chromosomes. In this way, unbalanced, normal and balanced oocytes could be distinguished and segregation modes ascertained. der(14;21)(q10;q10) produced 42% unbalanced, 37% normal and 21% balanced oocytes (n = 86) while der(13;14)(q10;q10) generated 33% unbalanced, 51% normal and 16% balanced oocytes (n = 69). In both translocations the number of normal oocytes was significantly higher than the number of balanced oocytes. However, while the frequency of unbalanced events involving chromosome 13 and 14 was similar in der(13;14)(q10;q10), there were significantly more abnormalities involving chromosome 21 than 14 in the der(14;21) (q10;q10) cases. When comparing survival rates to term, trisomies from Robertsonian origin seem to survive more often than those originated by non-disjunction in non-translocation carriers. The meiotic segregation patterns found in female Robertsonian translocations are different from those described in male carriers, with higher rates of unbalanced gametes in females than in males.     

Gamete activity in mating strains of Chlamydomonas eugametos

The gamete activity of compatible mating strains of the isogamous, heterothallic species Chlamydomonas eugametos was investigated. Gamete activity was optimum within 4 h after flooding of agar slants and was maintained over a 24-h period. When male and female mating strains were mixed in proportions of 1:4, 2:3, 1:1, 3:2, and 4:1, the results based on zygote yield, indicated the strains exhibited different degrees of gamete activity. The male strain consistently showed less gamete activity than the female strain in a variety of culture conditions.     

Immunological Characterization of Gamete Autolysins in Chlamydomonas reinhardtii

Polyclonal antibodies were raised in rabbits against the C. reinhardtii cell wall lytic enzyme, autolysin, which dissolves the cell wall of gametes prior to cell fusion. The purified immunoglobulins react with both the native and the deglycosylated forms of this gametic autolysin and specifically inhibit enzyme activity. In addition, the immunoglobulins selectively detect the gametic autolysin in immunoblots of crude extracts and do not cross-react with the autolysin of the vegetative cells. The antibodies have been used to study the time of synthesis of the enzyme during gametogenesis and to compare gametic autolysins of different strains of Chlamydomonas.     

The Gamete Pigments of Hormosira banksii (Phaeophyta)

The gamete pigments of Hormosira banksii have been separatedby thin-layer chromatography and identified by absorption spectrophotometry.Male gametes contain predominantly ß-carotene, andfemale gametes predominantly chlorophyll and fucoxanthin. Severalminor pigments have also been tentatively identified.     

Defined Media for Growth and Gamete Production by the Green Alga, Oedogonium cardiacum

Defined media consisting of inorganic salts and vitamin B₁₂ are described for the male and female filaments of the green alga, Oedogonium cardiacum. These media provide for a maximal growth rate and for the induction of oogonia and antheridia under the prescribed conditions. The maximal amounts of growth, based on dry weight measurements, compare favorably with other green algae.     

The cell biology of fertilization: Gamete attachment and fusion

Fertilization is defined as the union of two gametes. During fertilization, sperm and egg fuse to form a diploid zygote to initiate prenatal development. In mammals, fertilization involves multiple ordered steps, including the acrosome reaction, zona pellucida penetration, sperm–egg attachment, and membrane fusion. Given the success of in vitro fertilization, one would think that the mechanisms of fertilization are understood; however, the precise details for many of the steps in fertilization remain a mystery. Recent studies using genetic knockout mouse models and structural biology are providing valuable insight into the molecular basis of sperm–egg attachment and fusion. Here, we review the cell biology of fertilization, specifically summarizing data from recent structural and functional studies that provide insights into the interactions involved in human gamete attachment and fusion.

IntroductionDuring sexual reproduction, the oocyte and sperm fuse to generate a new and unique embryo. The journey of a sperm to an egg ends in the ampulla of the female oviduct. From there, the sperm must overcome a number of physical and biochemical barriers. After undergoing the acrosome reaction and binding the ova, the sperm penetrates through the cumulus oophorus cells and the zona pellucida (ZP) to reach the perivitelline space (PVS) and oocyte membrane. Upon fusion of the sperm and egg membranes, the sperm nucleus and organelles are incorporated into the egg cytoplasm.An understanding of the mechanisms of mammalian fertilization is crucial to treat infertility and develop new methods of birth control. Infertility affects 15% of couples globally, and in one third of these cases, the underlying cause is unknown (Gelbaya et al., 2014). Developments in assisted reproductive technologies have provided couples with new options to conceive but may have epigenetic side effects (Mani et al., 2020). Furthermore, only 40% of couples manage to have a child despite 2 yr of treatment. Safety, efficacy, and acceptability of contraceptives are also critically important, but many current female contraceptive methods have side effects that limit long-term use (Aitken et al., 2008), while male contraceptives are limited to condoms or vasectomy (Kanakis and Goulis, 2015). A better understanding of the molecular players involved in fertilization is necessary to drive innovation in both assisted reproductive technologies and contraception.In this review, we will first briefly review the events that prepare the gametes for fertilization. We will then discuss how recent studies of genetically altered mice and structural biology efforts have shed light on the molecular mechanisms of sperm–egg attachment and fusion. We will also discuss the gaps in current knowledge and suggest new perspectives and future directions in the search for other protein factors involved at the gamete fusion synapse.Cell biology of gametesFertilization requires proper gametogenesis (oogenesis in the female and spermatogenesis in the male), which produces haploid cells and introduces diversity. Primordial germ cells (PGC) are the embryonic precursors to spermatocytes and ova. The cells produced by the first few divisions of the fertilized egg are totipotent and capable of differentiating into any cell type, including germ cells. PGCs originate within the primary ectoderm of the embryo and then migrate into the yolk sac. Between weeks 4 and 6, the PGCs migrate back into the posterior body wall of the embryo, where they stimulate cells of the adjacent coelomic epithelium and mesonephros to form primitive sex cords and induce the formation of the genital ridges and gonads. The sex (gonadal) cords surround the PGCs and give rise to the tissue that will nourish and regulate the development of the maturing sex cells (ovarian follicles in the female and Sertoli cells in the male).EggOogenesis is a complex differentiation process by which mature functional ova develop from germ cells (Fig. 1 A; Edson et al., 2009). In humans, oogenesis begins in the ovary at 6–8 wk of fetus development, when PGCs differentiate into oogonia. By the 12th week, several million oogonia enter prophase, the first meiotic division and become dormant until shortly before ovulation (Hayashi et al., 2020). Due to their large and watery nuclei, these cells are referred to as germinal vesicles (Pan and Li, 2019). These primary oocytes become enclosed by follicle cells to form primordial follicles. The number of primordial follicles peaks at ∼7 million by the fifth month of fetal life, with ∼700,000 left at birth and 400,000 by puberty (Marcozzi et al., 2018). All of the egg cells that the ovaries will release are already present at birth.Open in a separate windowFigure 1.Gametogenesis and fertilization. (A–C) Illustration of oogenesis and follicle development (A), spermatogenesis (B), and the major steps in fertilization (C): (1) initial contact, (2) acrosome reaction, (3) ZP penetration, (4) sperm–egg fusion, (5) entry of sperm nucleus, (6) cortical reaction, and (7) fusion of the sperm and egg nuclei. The oocyte with its ZP measures 130 μm in diameter. Created with BioRender.During each menstrual cycle, hormones from the hypothalamic–pituitary–gonadal axis restart the division of the primary oocytes in meiosis I and follicular development (Atwood and Vadakkadath Meethal, 2016). Primary follicles develop into secondary follicles, containing each growing oocyte surrounded by two or more layers of proliferating follicle cells. ZP glycoproteins are secreted by the oocyte of the primary follicle and possibly the follicular cells (Törmälä et al., 2008). Although these glycoproteins form a physical barrier between the follicle cells and the oocyte, follicle cells and the oocyte remain connected through transzonal cytoplasmic projections from the follicle cells until fertilization (Makabe et al., 2006). A reciprocal dialog between the oocyte and its surrounding follicular cells coordinates the different phases of follicular development and the maintenance of meiotic arrest (Dalbies-Tran et al., 2020). Oocyte-derived microvilli control female fertility by optimizing ovarian follicle selection in mice (Zhang et al., 2021). The epithelium of 5–12 primary follicles proliferates to form a multilayered capsule around the oocyte. A few of these growing follicles continue to enlarge in response to follicle-stimulating hormone (FSH; Visser and Themmen, 2014). A single follicle becomes dominant, and the others degenerate by atresia (Atwood and Vadakkadath Meethal, 2016). Meiosis of the oocyte in the mature preovulatory follicle is blocked until a surge in levels of FSH and luteinizing hormone that occurs midway through the menstrual cycle. The membrane of the germinal vesicle nucleus breaks down, the chromosomes align in metaphase, and the oocyte expels its first polar body. The secondary oocyte then begins the second meiotic division, which is arrested at the meiotic metaphase II stage until ovulation (Gougeon, 1996). Ovulation depends on the breakdown of the follicle wall and occurs ∼38 h after the increase in levels of FSH and luteinizing hormone (Holesh et al., 2021). The disruption of the follicle wall expulses the oocyte, which is captured by the fimbriated mouth of the oviduct and moved into the ampulla. The oocyte retains its ability to be fertilized for ∼24 h and completes meiosis only if it is fertilized.SpermIn contrast to oogenesis, which is complete before birth, spermatogenesis is a continuous process that begins at puberty (Fig. 1 B). In humans, spermatogenesis takes 74 d to complete; thus, multiple spermatogenesis events occur simultaneously to allow for continual sperm production. Spermatogenesis occurs in the testis in a stepwise manner, beginning with diploid spermatogonia at the basal surface of seminiferous tubules and ending with mature elongated spermatozoa that are released in tubule lumens in a process called spermiation (Clermont, 1972; Yang and Oatley, 2014). During spermatogenesis, mitosis results in gene amplification, meiosis results in genome reduction, and finally maturation occurs (Hess and Renato de Franca, 2008). At this stage, sperm are not motile and are fertilization incompetent. Two additional sperm maturational processes are required outside the testis. First, sperm undergo a maturation process during epididymal transit (Bedford et al., 1973) involving posttranslational modifications of previously synthesized proteins and acquisition of proteins from the epididymal epithelium (James et al., 2020; see text box). After ejaculation into the female reproductive tract, dilution triggers additional changes in sperm, collectively termed capacitation (see text box), that prepare the sperm for the acrosome reaction.Epididymal maturationSperm exchange with the epididymal epithelium occurs by direct interaction with epithelial cells, by interaction with soluble proteins in the epididymal fluid or via extracellular exosome-like vesicles released by epithelial cells called epididymosomes (James et al., 2020). The purposes of this exchange are to redistribute sperm proteins and change the composition and lipid balance of the sperm membrane. These changes take place during the transit from the epididymis initial segment, through the caput and the corpus, to the cauda where sperm are stored (Cornwall, 2009). Epididymal transit lasts 10–12 d in mammals, but storage is dependent on sexual activity. Since fertilization is not immediate, fertilizing capacities of the spermatozoa are preserved by decapacitation factors that are active in the epididymis. An example of a decapacitation factor is SPINK3, which is secreted by seminal vesicles; it impairs sperm membrane hyperpolarization and calcium influx through CatSper (Zalazar et al., 2020). Epididymal plasma and sperm represent only a small fraction (5%) of semen in men (Batruch et al., 2011). Two thirds of the volume of semen comes from the seminal vesicles and the other third from the prostate. These secretions protect the sperm and prevent early maturation.Sperm capacitationMore than 70 yr ago, Austin and Chang described capacitation as the changes required for sperm to fertilize oocytes in vivo (Austin, 1952; Chang, 1951). Once sperm enter the female reproductive tract, they undergo capacitation. Capacitation results in hyperactivation of sperm movement and initiation of the acrosome reaction (Saling et al., 1979; Florman and First, 1988). During capacitation, stabilizing or decapacitation factors that are adsorbed on the sperm plasma membrane are removed (Bedford and Chang, 1962). These agents that initiate removal of decapacitation factors are electrolytes, energy substrates, and proteins such as seminal plasma protein or albumin. Removal of decapacitation factors increases sperm plasma membrane fluidity, allowing an increase in the permeability to calcium, chloride, and bicarbonate ions (Gangwar and Atreja, 2015). Sperm motility depends on the membrane potential, intracellular pH, and balance of intracellular ions (reviewed in Nowicka-Bauer and Szymczak-Cendlak, 2021). The most important ion for this function is Ca²⁺ (Hwang et al., 2019). This secondary messenger is an important signaling pathways activator that regulates sperm motility (Finkelstein et al., 2020). The activation of soluble adenyl cyclases generates cyclic adenosine monophosphate that in turn activates serine/threonine protein kinase A, which induces a cascade of protein phosphorylation initiating the induction of sperm motility (Chen et al., 2000). Protein phosphorylation, sperm hyperactivation, and the acrosome reaction are used in vitro to evaluate capacitation. Capacitation can be induced in vitro by incubation in medium containing calcium, bicarbonate ions, and serum albumin (Touré, 2019).Mammalian sperm capacitation occurs during sperm migration in the female tract. Mammalian males ejaculate millions of sperm cells into the female reproductive tract, but only a few hundred sperm at most reach the oocytes. This massive elimination process likely prevents polyspermy (reviewed in Kölle, 2015). Selection of human sperm during the journey begins in the acidic environment of the vagina. In the cervix, only morphologically normal sperm can migrate. Some sperm immediately pass into the cervical mucus, whereas the remaining sperm becomes a part of the coagulum. The next selection occurs at the uterus–tubal junction, the connection between the uterus and the oviduct that represents a major obstacle for sperm migration (Kölle, 2015). Experiments in mice indicate that sperm motility alone is insufficient for sperm migration through the uterus–tubal junction (Fujihara et al., 2018). Uterine contractions facilitate sperm transport as do molecular interactions. Several proteins, such as ADAM3 and other ADAM family members, are known to be involved in this step in mice (Yamaguchi et al., 2009; Xiong et al., 2019); most ADAM proteins have human orthologues.Spermatogenesis takes place in a species-specific cycle called the seminiferous epithelial cycle and is regulated in particular through the hypothalamic–pituitary–testicular axis. Indeed, at puberty, the testes (interstitial steroidogenic Leydig cells) secrete an increased amount of testosterone, which triggers growth of the testes, maturation of the seminiferous tubules, and the commencement of spermatogenesis. The Sertoli cells are the major somatic cells present in the seminiferous tubules and are considered to be the main regulators of spermatogenesis. They orchestrate spermatogenesis by supporting spermatogonial stem cells, determining the testis size, organizing meiotic and postmeiotic development and sperm output, supporting androgen production by maintaining the development and function of Leydig cells, and regulating other aspects of testis function like peritubular myoid cells, immune cells, and the vasculature, which participate in the maintenance of the spermatogonial stem cell niche.Acrosome reactionThe acrosome is a secretory vesicle located on the anterior region of sperm that originates from the spermatid Golgi apparatus. An acrosomal granule is formed by the fusion of proacrosomal vesicles in the vicinity of the nucleus. The region increases in size and spreads over the anterior part of the nucleus. The acrosome reaction is driven by SNARE complexes and results in the exocytosis of the contents of the acrosome upon fusion of the plasma membrane with the outer acrosomal membrane (Fig. 1 C; reviewed in Okabe, 2016; De Blas et al., 2005). The timing of the acrosome reaction is critical. Only sperm that have undergone this reaction are fertilization competent, but when a high proportion of sperm undergo the acrosome reaction prematurely, success of in vitro fertilization is low (Wiser et al., 2014). Several studies indicate that only a fraction of sperm is capable of undergoing spontaneous acrosome reaction. In human and mice sperm samples, 15–20% of cells undergo spontaneous acrosome reaction (Nakanishi et al., 2001), whereas only 20–30% undergo progesterone-induced acrosome reaction (Stival et al., 2016), suggesting physiological heterogeneity of sperm population. In addition, Inoue et al. demonstrated that acrosome-reacted mouse spermatozoa recovered from the PVS can fertilize other eggs (Inoue et al., 2011).Based on in vitro data, it was thought that the acrosome reaction occurs when the sperm contacts the ZP, particularly the ZP3 protein (Litscher and Wassarman, 1996). Using transgenic mice that express fluorescent markers in the acrosome (Nakanishi et al., 1999) and the midpiece mitochondria (Hasuwa et al., 2010), real-time observation of acrosomal exocytosis was possible. These experiments showed that most mouse spermatozoa capable of fertilization had undergone the acrosome reaction before contact with the oocyte ZP (Jin et al., 2011). Most spermatozoa begin to react in the isthmus of the oviduct before reaching the ampulla (Hino et al., 2016; La Spina et al., 2016). Contact with the ZP in vitro probably makes it possible to complete a partial acrosome reaction. The most important function of the acrosome reaction is to induce changes in the sperm membrane (Okabe, 2016). The relocations of IZUMO1 and SPACA6, proteins essential for sperm–egg fusion, that occur after the acrosome reaction are illustrative examples of these changes (Sosnik et al., 2009; Barbaux et al., 2020; Satouh et al., 2012). The presence of these proteins on the sperm membrane, in addition to the classic markers Pisum sativum agglutinin, Peanut agglutinin lectins, or CD46, can be used as markers for the acrosome reaction (Ito and Toshimori, 2016). The acrosome and its disruption are both crucial for effective fertilization, as low fertilization rates are observed upon intracytoplasmic sperm injection of acrosome-intact sperm (Morozumi and Yanagimachi, 2005) or round spermatozoa lacking acrosomes (Dávila Garza and Patrizio, 2013).ZP penetrationThe ZP is a physical barrier between the oocyte and the follicular cells that forms from glycoproteins secreted from the primary follicles (Fig. 1 C). The human ZP consists of four glycoproteins (hZP1–hZP4; Harris et al., 1994). Mice, which have been used for most of the ZP studies in mammals, express only three ZP glycoproteins (mZP1–mZP3; Litscher and Wassarman, 2007). Analysis of mouse lines expressing human ZP proteins demonstrated that only hZP2 is important in human sperm–egg binding (Gupta, 2021). Experiments using purified native or recombinant human ZP proteins have shown that hZP1, hZP3, and hZP4 bind to the capacitated human spermatozoa and induce the acrosome reaction (Gupta, 2021). ZP1 is required for the structural integrity of the ZP (Chakravarty et al., 2008). To better understand the roles of ZP glycoproteins, further studies, particularly on ZP protein glycosylation, are needed. The species-specific binding of the ZP to sperm is presumably related to these carbohydrate moieties (Clark, 2014). The sialyl-Lewis(x) sequence is the major carbohydrate ligand for human sperm–egg binding (Pang et al., 2011). The current hypothesis that hZP1, hZP3, and hZP4 bind to capacitated sperm and hZP2 binds to sperm with intact acrosomes will need to be revisited due to the recent demonstration that the acrosome reaction takes place before ZP contact. Regardless, the role of the ZP in preventing polyspermy is clear. Indeed, ZP hardening is due to ZP2 cleavage by ovastacin, a protease released into the PVS by cortical granules after the first sperm–egg fusion (Burkart et al., 2012).Sperm–egg attachment and membrane fusionAfter penetration of the ZP, the sperm enters the PVS and can attach and fuse with the egg plasma membrane. The development of genetic knockout animal models has proven critical in determining the importance of various sperm and egg proteins in sperm–egg attachment and fusion. Surprisingly, genetic knockout studies revealed that many factors originally thought to be important for fertilization were in fact not necessary (reviewed in Okabe, 2018, 2015). The proteins from sperm and egg that are essential for sperm–egg membrane interaction and fusion are listed in

Determination of n+1 Gamete Transmission Rate of Trisomics and Location of Gene Controlling 2n Gamete Formation in Chinese Cabbage (Brassica rapa)

A set of trisomics of Chinese cabbage was used for determining the n+1 gamete transmission rate and locating the gene controlling 2n gamete formation on the corresponding chromosome. The results showed that the transmission rates of extra chromosomes in different trisomica varied from 0% to 15.38% by male gametes and from 0% to 17.39% by female gametes. Of the nine F2 populations derived from the hybridizations between each triaomic and Bp058 (2n gamete material), only Tri-4×Bp058 showed that the segregation ratio of plants without 2n gamete formation to plants with 2n gamete formation was 10.38:1, which fitted the expected segregation ratio of the trisomics (AAa) based on the 7.37% of n+1 gamete transmission through female and 5.88% through male. In other populations the segregation ratios varied from 2.48:1 to 3.72:1, which fitted the expected 3:1 segregation ratio of the bisomice (Aa). These results suggested that the gene controlling 2n gamete formation in Chinese cabbage Bp058 was located on chromosome 4. Further trisomic analysis based on the chromosome segregation and the incomplete stochastic chromatid segregation indicated that the gene locus was tightly linked to the centromere.