The Ultracytochemical Localzation of Acid Phosphatase Activity in Wheat During Fertilization

No acid phosphatase activity was observed in the mature embryo sac of wheat (Triticum aestivum) except the chalazal cytoplasm Of the central cell before fertilization. During fertilization, acid phosphataseactivity was observed in the following loci: part of chromatin of the egg nucleus and most of the mitochondria in the egg cytoplasm; the perinuclear spaces of the egg and sperm nuclei at the fusion of the egg and sperm nuclei; the chalazal cytoplasm and some vacuoles of the degenerated synergid; two sperm nuclei within the cytoplasm of female cells; the cell wall of each cell of the embryo sac and that of the nucellar cells surrounding the embryo sac. No acid phosphatase was observed in the two-celled proembryo. Dense enzyme reaction product was localized in the chromatin of the free nuclei at early stage of the endosperm. The characteristic of acid phosphatase distribution during fertilization may be associated with the physiological change of the egg Cell, the reorganization of mitochondria in the egg cell cytoplasm, the degeneration of one of the two synergids, the physiological state of the sperm nuclei and the nuclear membrane fusion of the egg and sperm nuclei.     

Human sperm centrosomal function during fertilization, a novel assessment for male sterility

In human fertilization, the sperm introduces the centrosome-the microtubule organizing center-and microtubules are organized within the inseminated egg from the sperm centrosome. These microtubules form a radial array, the sperm aster, the functioning of which is essential for pronuclear movement for the union of the male and female genomes. We established functional assay for human sperm centrosomal function, by using heterologus ICSI system with bovine and rabbit eggs. After human sperm incorporation into mammalian egg, we observed that the sperm aster was organized from sperm centrosome, and the sperm aster enlarged as the sperm nuclei underwent pronuclear formation. The normal human sperm aster formation rate at 6 h post-ICSI were 60.0% in bovine egg and 36.1% in rabbit egg, respectively. However, sperm aster formation rate following heterologus ICSI into bovine eggs with teratozoospermia (globozoospermia, dysplasia of fibrous sheath) were low. These data indicate that human sperm centrosomal function is low in abnormal shaped sperm. Wherus, elucidation of human sperm centrosomal function can lead us to find a new type of failure in "post ICSI events in fertilization".     

小麦受精过程中酸性磷酸酶的超微细胞化学定位

小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ ）受精前成熟胚囊,除胚囊中央细胞的合点端细胞质中有酸性磷酸酶外,其余部位均未发现酸性磷酸酶。受精时期,以下部位存在酸性磷酸酶活性：卵细胞的细胞核内一部分染色质和细胞质中大部分线粒体;精、卵核融合时两核的核周腔内;退化助细胞合点端细胞质和一些液泡内;进入雌性细胞中的两个精核;胚囊各成员细胞的细胞壁及胚囊周围珠心细胞的细胞壁。二细胞原胚中未见有酸性磷酸酶。早期胚乳游离核染色质上有酸性磷酸酶。小麦受精过程酸性磷酸酶的分布特点可能与卵细胞生理状态的变化和细胞质中线粒体的改组、助细胞的退化、精核的生理状态以及精核与卵核的核膜融合等有关。     

The developments between gametogenesis and fertilization: ovulation and female sperm storage in Drosophila melanogaster

In animals with internal fertilization, ovulation and female sperm storage are essential steps in reproduction. While these events are often required for successful fertilization, they remain poorly understood at the developmental and molecular levels in many species. Ovulation involves the regulated release of oocytes from the ovary. Female sperm storage consists of the movement of sperm into, maintenance within, and release from specific regions of the female reproductive tract. Both ovulation and sperm storage elicit important changes in gametes: in oocytes, ovulation can trigger changes in the egg envelopes and the resumption of meiosis; for sperm, storage is a step in their transition from being "movers" to "fertilizers." Ovulation and sperm storage both consist of timed and directed cell movements within a morphologically and chemically complex environment (the female reproductive tract), culminating with gamete fusion. We review the processes of ovulation and sperm storage for Drosophila melanogaster, whose requirements for gamete maturation and sperm storage as well as powerful molecular genetics make it an excellent model organism for study of these processes. Within the female D. melanogaster, both processes are triggered by male factors during and after mating, including sperm and seminal fluid proteins. Therefore, an interplay of male and female factors coordinates the gametes for fertilization.     

Clinical utility of freeze-all approach in ART treatment: A mini-review

A significant proportion of couples at reproductive age rely on assisted reproductive technology to overcome infertility. In vitro fertilisation (IVF) involves typically the use of exogenous gonadotropins to stimulate the ovary to produce oocytes, which are collected surgically. After fertilization by conventional IVF or intracytoplasmic sperm injection (ICSI), embryos are cultured in the embryology laboratory for a few days before being replaced into the uterus (fresh embryo transfer). Spare embryos can be vitrified and stored in liquid nitrogen to be transferred in a subsequent cycle. Over the years, concerns have arisen about possible adverse outcomes of transferring embryos back to the uterus immediately after controlled ovarian stimulation (COS) as regards to obstetrical and perinatal outcomes. It has been suggested that high hormonal levels during COS could create a relatively hostile environment for embryo implantation whilst increasing the risk of ovarian hyperstimulation syndrome (OHSS). With the remarkable improvement of vitrification as an alternative to the slow-freezing technique for human embryos, a new strategy the so-called “freeze-all” (FA) or “elective frozen embryo transfer” (eFET) was introduced. This approach involves COS, followed by the elective cryopreservation of the entire cohort of viable embryos to be transferred to the uterus in subsequent cycles in a possibly more physiological environment, thus avoiding the supra-physiologic hormonal levels observed during COS. The initial reports suggested that this policy could lead to improved pregnancy rates and reduced perinatal complications, which resulted in a steady increase and widespread use of FA globally. However, as data accumulated, it became clear that the use of FA to unselected couples undergoing ART offered no additional benefits over the conventional approach. Nonetheless, current evidence based on randomized controlled trials and observational studies indicates that FA might be justified in selected clinical scenarios, such as those involving the risk of OHSS. By contrast, there is a lack of evidence to support the FA policy for other indications, such as implantation failure or high progesterone levels on the trigger day. This review summarizes the clinical effectiveness of FA with the main focus on the health of offspring.     

Double fertilization inEphedra trifurca,a non-flowering seed plant: The relationship between fertilization events and the cell cycle

Summary Fertilization inEphedra trifurca was examined with a combination of light and fluorescence microscopy. Developmental analysis clearly indicates that double fertilization events, similar to those described inE. nevadensis, regularly occur during the process of sexual reproduction inE. trifurca. In addition to the typical fusion of a sperm nucleus and egg nucleus, a second fertilization event occurs between the second sperm nucleus from an individual pollen tube and the ventral canal nucleus. Both of the fertilization events take place within the confines of an individual egg cell of the female gametophyte. Microspectrofluorometric data demonstrate that each nucleus involved in a sexual fusion event proceeds through the synthesis phase of the cell cycle and increases its DNA content from 1C to 2C before the process of nuclear fusion is completed. Photometric data also confirm that the product of the second fertilization event is equal in DNA content (4C) to the zygotic nucleus derived from the first fertilization event, and is prepared to enter into mitosis as a fully functional diploid nucleus.Abbreviations DAPI 4;,6-diamidino-2-phenylindole - RFU relative fluorescence units     

Aquaporin3 is a sperm water channel essential for postcopulatory sperm osmoadaptation and migration

In the journey from the male to female reproductive tract, mammalian sperm experience a natural osmotic decrease (e.g., in mouse, from ~415 mOsm in the cauda epididymis to ~310 mOsm in the uterine cavity). Sperm have evolved to utilize this hypotonic exposure for motility activation, meanwhile efficiently silence the negative impact of hypotonic cell swelling. Previous physiological and pharmacological studies have shown that ion channel-controlled water influx/efflux is actively involved in the process of sperm volume regulation; however, no specific sperm proteins have been found responsible for this rapid osmoadaptation. Here, we report that aquaporin3 (AQP3) is a sperm water channel in mice and humans. Aqp3-deficient sperm show normal motility activation in response to hypotonicity but display increased vulnerability to hypotonic cell swelling, characterized by increased tail bending after entering uterus. The sperm defect is a result of impaired sperm volume regulation and progressive cell swelling in response to physiological hypotonic stress during male-female reproductive tract transition. Time-lapse imaging revealed that the cell volume expansion begins at cytoplasmic droplet, forcing the tail to angulate and form a hairpin-like structure due to mechanical membrane stretch. The tail deformation hampered sperm migration into oviduct, resulting in impaired fertilization and reduced male fertility. These data suggest AQP3 as an essential membrane pathway for sperm regulatory volume decrease (RVD) that balances the "trade-off" between sperm motility and cell swelling upon physiological hypotonicity, thereby optimizing postcopulatory sperm behavior.     

An update of the regulatory factors of sperm migration from the uterus into the oviduct by genetically manipulated mice

Mammalian reproductive processes involve spermatogenesis, which occurs in the testis, and fertilization, which takes place in the female genital tract. Fertilization is a successive, multistep, and extremely complicated event that usually includes sperm survival in the uterus, sperm migration through the uterotubal junction (UTJ) and the oviduct, sperm penetration through the cumulus cell layer and the zona pellucida, and sperm–egg fusion. There may be a complex molecular mechanism to ensure that the above processes run smoothly. Previous studies have discovered essential factors for these fertilization steps through in vitro fertilization experiments. However, recent gene disruption approaches in mice have revealed that many of the factors previously described as important for fertilization are largely dispensable in gene‐knockout animals, and some previously unknown factors are emerging. As a result, the molecular mechanisms of fertilization, especially sperm migration from the uterus into the oviduct, have recently been revised by the emergence of genetically modified animals. In this review, we only focus on and update the essential genes for sperm migration through the UTJ and describe recent advances in our knowledge of the basis of mammalian sperm migration.     

Neural and hormonal control of muscular activity of the spermatheca in the locust, Locusta migratoria

The spermatheca in insects is a tubular structure within the female that acts as a repository for spermatozoa deposited by the male during copulation. The spermatozoa remain viable within the spermatheca for extended periods of time, and are then delivered to the site of fertilization during oviposition (egg-laying). Thus, the production of viable offspring is dependent upon the coordination of events associated with fertilization, including the passage of the egg through the lateral and common oviducts and the passage of spermatozoa along the spermathecal duct. The egg and the spermatozoa are propelled along their respective tracts by contractions of the visceral muscles intrinsic to the oviduct and spermatheca. The neural and hormonal control of muscular activity of the locust oviducts has been well reviewed, with more recent studies examining the control over the spermatheca. This review highlights more recent literature, including new data, for neural and hormonal control of muscular activity of the spermatheca of the locust, Locusta migratoria, making reference to examples in other insects where relevant. A variety of neuronal types project to the spermatheca in L. migratoria, and a variety of neuroactive chemicals, including neuropeptides and amines, influence contraction. A comparison is made between the control of oviducts and spermatheca in L. migratoria with regard to their neural substrate and the composition of neuroactive chemicals.     

Long-term sperm storage in the Siberian crane (<Emphasis Type="Italic">Grus leucogeranus</Emphasis> pallas): Analysis of paternity and relatedness under artificial insemination

Using ten microsatellite loci, paternity analysis has been conducted for 71 individuals of the Siberian crane (Grus leucogeranus Pallas) obtained under artificial insemination in Oka Crane Breeding Center in 2001–2014. The fathers of 39 chicks were the sires whose sperm was used for insemination directly before fertilized egg laying. Paternity of 23 fertilizations belonged to the sires whose sperm was used in the beginning or middle of insemination cycle. Nine cases of fertilization resulted from natural copulation of artificially inseminated females with their social partners. The terms of sperm storage in the female’s reproductive ducts before fertilization were 0–6 days in the case of paternity of the last sperm donor and 2–15 days in the case of competing sperm by previous donors. Genetic relatedness by microsatellite loci between breeders of the captive Siberian crane population does not prevent fertilization and does not always lead to inbreeding depression.     

Ocean acidification during prefertilization chemical communication affects sperm success

Ocean acidification (OA) poses a major threat to marine organisms, particularly during reproduction when externally shed gametes are vulnerable to changes in seawater pH. Accordingly, several studies on OA have focused on how changes in seawater pH influence sperm behavior and/or rates of in vitro fertilization. By contrast, few studies have examined how pH influences prefertilization gamete interactions, which are crucial during natural spawning events in most externally fertilizing taxa. One mechanism of gamete interaction that forms an important component of fertilization in most taxa is communication between sperm and egg‐derived chemicals. These chemical signals, along with the physiological responses in sperm they elicit, are likely to be highly sensitive to changes in seawater chemistry. In this study, we experimentally tested this possibility using the blue mussel, Mytilus galloprovincialis, a species in which females have been shown to use egg‐derived chemicals to promote the success of sperm from genetically compatible males. We conducted trials in which sperm were allowed to swim in gradients of egg‐derived chemicals under different seawater CO₂ (and therefore pH) treatments. We found that sperm had elevated fertilization rates after swimming in the presence of egg‐derived chemicals in low pH (pH 7.6) compared with ambient (pH 8.0) seawater. This observed effect could have important implications for the reproductive fitness of external fertilizers, where gamete compatibility plays a critical role in modulating reproduction in many species. For example, elevated sperm fertilization rates might disrupt the eggs' capacity to avoid fertilizations by genetically incompatible sperm. Our findings highlight the need to understand how OA affects the multiple stages of sperm‐egg interactions and to develop approaches that disentangle the implications of OA for female, male, and population fitness.     

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.     

Downregulation of egg cell-secreted EC1 is accompanied with delayed gamete fusion and polytubey

One major player known to be essential for successful gamete interactions during double fertilization in Arabidopsis thaliana is the recently identified family of egg cell-secreted EC1 proteins. Both gamete fusion events are affected in EC1-deficient female gametophytes. Here, we show that the number of ovules with unfused sperm cells is considerably higher than the number of undeveloped seeds in the same ec1-RNAi knockdown lines. We found that some sperm cells are able to fuse with the female gametes even 2 to 3 days after pollination, as reflected by delayed embryo and endosperm development, and by polytubey. We propose that the egg cell secretes EC1 proteins upon sperm arrival to promote rapid sperm activation, thereby accelerating gamete fusion and preventing polytubey.     

被子植物助细胞的发育和功能

助细胞是被子植物雌配子体的组成细胞之一,是大多数被子植物完成受精作用的关键环节之一。在受精过程中,助细胞吸引花粉管向雌配子体生长,并接受花粉管长入细胞程序死亡助细胞中。接下来的花粉管停止生长和花粉管顶端破裂释放出2个精细胞的过程可能也依赖于助细胞。另外,雄性生殖单位的解体、提供精细胞的运动轨道和启动精细胞合成DNA的生物学事件也可能与助细胞有关。助细胞的形态结构已研究得比较清楚,但有关助细胞的发育机理和它在受精过程中的作用则仍不明确。近年来对助细胞的研究又取得了一些新结果,尤其是一些分子生物学的研究结果为揭示助细胞的功能提供了重要线索。该文总结了这方面的研究成果,并对助细胞的生殖功能进行了分析。     

Male mice deficient for germ-cell cyritestin are infertile

Cyritestin is a membrane-anchored sperm protein belonging to the ADAM (f1.gif" BORDER="0"> f2.gif" BORDER="0">isintegrin and f1.gif" BORDER="0"> f3.gif" BORDER="0">etalloprotease) family of proteins, which are proposed to be involved in cell-cell adhesion through binding to integrin receptors. Several lines of evidence support a role of cyritestin and other members of this protein family in the fusion of sperm and the egg plasma membrane. In an effort to elucidate the physiological function of cyritestin, we have disrupted its locus by homologous recombination. Male homozygous null mutants are infertile, even though spermatogenesis, mating, and migration of sperm from the uterus into the oviduct are normal. In vitro experiments showed that infertility is due to the inability of the cyritestin-deficient sperm to bind to the zona pellucida. However, after removal of the zona pellucida, sperm-egg membrane fusion monitored by the presence of pronuclei and generation of 2- and 4-cell embryos did not reveal any differences from the wild-type situation. These results demonstrate that cyritestin is crucial in the fertilization process at the level of the sperm-zona pellucida interaction.     

Helical nature of sperm swimming affects the fit of fertilization-kinetics models to empirical data

Models of fertilization kinetics rely upon estimates of the swimming velocity of sperm to predict collision rates between egg and sperm. Most investigators measure sperm swimming velocity without accounting for the helical motion of sperm, thereby obtaining an inflated estimate of the velocity with which sperm approach eggs. In turn, models of fertilization predict inflated rates of sperm/egg collision. I observed sea urchin sperm colliding with eggs, quantified the rate of sperm/egg collision, and measured sperm velocity as a component of the helix through which they swim. I also adjusted the "target size" of eggs to reflect the diameter of the helix. My estimate of sperm swimming velocity is an order of magnitude lower than other estimates for the same species. By using helical parameters in fertilization kinetics models and accounting for dead sperm in laboratory trials, I was able to accurately predict lower rates of sperm/egg collision. Moreover, making these adjustments in the model increased the estimated proportion of sperm that initiate fertilization by 6- to 7-fold, suggesting that a better understanding of sperm swimming might lead to a more complete understanding of fertilization biology and natural selection on gamete traits.     

被子植物受精机制的研究进展

被子植物的受精是一个复杂而精巧的过程。花粉管到达子房,通过退化助细胞进入胚囊,释放出两个精细胞。原来在花粉管中相互联结的两个精细胞在退化助细胞中分开,一个与卵细胞融合,另一个与中央细胞融合,完成双受精。目前对双受精过程中有关雌、雄配子识别的机制还知之甚少。本文介绍了目前被子植物精、卵细胞融合前后的细胞周期变化、退化助细胞的功能、精细胞在退化助细胞中迁移的研究动态、精细胞的倾向受精和卵细胞的激活等被子植物受精生物学领域中的一些新的研究成果和发展趋势。     

In vitro fertilization in ctenophores: sperm entry, mitosis, and the establishment of bilateral symmetry in Beroe ovata.

We have found ways to control in vitro fertilization in a ctenophore (Beroe ovata) for the first time. This is based on the existence of a partial block to self-fertilization at the time of gamete release which can be overcome by removal of the egg envelope. It has allowed us to exploit the excellent optical properties of Beroe eggs to make detailed observations on all events from sperm penetration or penetrations in these physiologically polyspermic eggs to first cleavage, and to extend our initial observations (Carré and Sardet, 1984). Sperm entry is characterized by local modifications of the egg cortex in a 70-microns zone around the penetration site or sites. Upon sperm entry, the egg surface contracts and relaxes locally, then a fertilization cone forms and disappears. These events are accompanied by localized exocytosis, growth of a ring of microvilli, thickening of the egg cortex, and gathering of mitochondria around the sperm pronuclei. The female pronucleus then migrates beneath the egg surface toward one or successive sperm pronuclei. The fusion of pronuclei, sperm and egg chromatin intermixing, and mitosis were also observed with exceptional clarity. Furthermore, we have noticed that the direction of the last trajectory of the female pronucleus tends to define the orientation of the mitotic spindle, and as a consequence the position of first unipolar cleavage furrow. This in turn determines the future sagittal plane of the embryo and of the adult B. ovata.     

Gamete interaction in the white sturgeon Acipenser transmontanus: a morphological and physiological review

Synopsis Sturgeon gametes differ from those of most fish in that the sperm possess acrosomes that undergo exocytosis and filament formation while the eggs possess numerous micropyles. Acipenser transmontanus eggs are encased by multilayered envelopes that consist of outer adhesive jelly coats and three structured layers interior to the jelly. The glycoprotein jelly layer only becomes adhesive upon exposure to freshwater. The layer interior to the jelly, layer 3, is the other carbohydrate-containing component of the egg envelope. This layer consists of a water-insoluble glycoprotein that, upon freshwater exposure, is hydrolyzed by a trypsinlike protease to yield a water-soluble, lower molecular weight carbohydrate-containing component. This component can be identified in the surrounding medium when unfertilized eggs are incubated in freshwater. This egg water component elicits acrosome reactions only in homologous sperm. The A. transmontanus sperm acrosome reaction is a Ca⁺⁺ and/or Mg⁺⁺ dependent event that includes the formation of a 10 μ long fertilization filament. A. transmontanus fertilization can occur at low sperm per egg ratios; however, crossfertilization of A. transmontanus eggs with lake sturgeon, A. fluvescens, sperm results in a very low number of fertilized eggs, even at high sperm per egg ratios. The morphological, physiological, and biochemical phenomenon reviewed in this paper are related to the environment in which they occur. Also, the possible role of the acrosome and the presence of numerous micropyles are discussed.     

A consistently successful procedure for in vitro fertilization of golden hamster eggs

Complete details are described for the first time of the procedures used in the author's laboratory for obtaining in vitro fertilization (IVF) of golden hamster eggs leading to the first cleavage division. These IVF procedures have been developed during the past 20 years and are very reproducible: IVF of at least 75% of eggs is routinely achieved, and on average 65% of inseminated eggs undergo the first cleavage division in vitro. These results can easily be obtained by inexperienced investigators. The ease and reproducibility of the hamster IVF procedures make them very suitable for studies of sperm:egg interaction and associated events. Studies in the author's laboratory have included analysis of sperm fertilizing ability under chemically defined conditions, the presence of sperm acrosome reaction stimulating factors in the egg investments, maturation of oocytes in vitro, the block to polyspermy, and the contribution of egg aging to fertilization anomalies. In addition, the motility of hamster sperm under chemically defined conditions is used in a routine screening protocol for detecting contaminants in the culture milieu. Golden hamster gametes of Ter several distinct advantages for IVF studies, including the large size of the sperm acrosome, the persistence of the very large sperm tail in the ooplasm for many hours following fertilization, and the translucence of the ooplasm, which facilitates observation of the sperm tail and pronuclei. The female golden hamster exhibits a regular 4 day estrous cycle, with distinctive indications of estrus and postestrus phases. Because of the advantages of using the golden hamster, the procedures described in this report may be useful to other investigators wishing to conduct research using IVF. Essentially the same IVF procedures can be used with monkey and bovine gametes.