New lights in early steps of in vitro fertilization in plants

Studies using in vitro fertilization systems in animals and lower plants have led to a better understanding of the initial steps of fertilization and their underlying mechanisms. These mechanisms remain to be elucidated in flowering plants. Recent progress related to the development of in vitro fertilization systems using maize as a plant model is presented in this review. Their potential for leading to a better understanding of the process of gametic recognition and fusion and of the early events triggering egg activation and zygote formation are also discussed.     

离体受精作为技术平台在被子植物有性生殖研究中的应用

被子植物的离体受精10a前在玉米中已获得成功,尽管目前只在玉米获得完全成功和小麦获得部分成功,但离体受精技术的研究成果非常显著。目前离体受精技术已被用于其他的研究,如用分离的精细胞和卵细胞筛选配子细胞的特异基因和蛋白质：研究合子细胞被激活的机理：用不同种植物的精、卵细胞体外融合进行新的远缘杂交尝试;利用合子细胞易分裂和胚胎发生特征探索用其作为转基因研究的受体细胞等。以离体受精技术为基础在高等植物发育生物学和生殖生物学领域的基础研究和应用探索显示了巨大潜力。介绍了离体受精技术在被子植物有性生殖的研究成果和应用前景,为研究和利用被子植物有性生殖过程中的生殖细胞特征提供线索。     

Establishment of an in vitro fertilization system in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

In vitro fertilization (IVF) systems using isolated male and female gametes have been utilized to dissect fertilization-induced events in angiosperms, such as egg activation, zygote development and early embryogenesis, as the female gametophytes of plants are deeply embedded within ovaries. In this study, a rice IVF system was established to take advantage of the abundant resources stemming from rice research for investigations into the mechanisms of fertilization and early embryogenesis. Fusion of gametes was performed using a modified electrofusion method, and the fusion product, a zygote, formed cell wall and an additional nucleolus. The zygote divided into a two-celled embryo 15–24 h after fusion, and developed into a globular-like embryo consisting of an average of 15–16 cells by 48 h after fusion. Comparison of the developmental processes of zygotes produced by IVF with those of zygotes generated in planta suggested that zygotes produced by IVF develop and grow into early globular stage embryos in a highly similar manner to those in planta. Although the IVF-produced globular embryos did not develop into late globular-stage or differentiated embryos, but into irregularly shaped cell masses, fertile plants were regenerated from the cell masses and the seeds harvested from these plants germinated normally. The rice IVF system reported here will be a powerful tool for studying the molecular mechanisms involved in the early embryogenesis of angiosperms and for making new cultivars.     

我国被子植物体外受精研究——十年回顾

简要回顾了十余年来我国被子植物体外受精领域的研究历程和主要成就.我国科学家在性细胞的分离, 尤其是生活胚囊和雌配子的分离方面走在国际前列. 在一定程度上我国科学家的开拓性工作引起了国际同行的广泛兴趣和重视, 推动了该领域的迅速发展, 为实现被子植物真正意义的体外受精做出了独特的贡献.十年来, 我国已建立了自己独特的被子植物离体受精实验技术系统并利用该系统与国际同行合作进行了一系列研究, 尤其在探讨配子相互作用、卵细胞激活、避免多精入卵的机制等方面做出了创新性工作, 为正确认识体外受精系统的优越性与局限性, 也为深入探讨受精机制提供了有价值的新资料.目前正以体外受精操作系统结合细胞生物学和分子生物学等多种手段对受精过程中的一些重要事态与机理做进一步探讨.     

我国被子植物体外受精研究—十年回顾

简要回顾了十余年来我国被子植物体外受精领域的研究历程和主要成就。我国科学家在性细胞的分离 ,尤其是生活胚囊和雌配子的分离方面走在国际前列。在一定程度上我国科学家的开拓性工作引起了国际同行的广泛兴趣和重视 ,推动了该领域的迅速发展 ,为实现被子植物真正意义的体外受精做出了独特的贡献。十年来 ,我国已建立了自己独特的被子植物离体受精实验技术系统并利用该系统与国际同行合作进行了一系列研究 ,尤其在探讨配子相互作用、卵细胞激活、避免多精入卵的机制等方面做出了创新性工作 ,为正确认识体外受精系统的优越性与局限性 ,也为深入探讨受精机制提供了有价值的新资料。目前正以体外受精操作系统结合细胞生物学和分子生物学等多种手段对受精过程中的一些重要事态与机理做进一步探讨     

Calcium in sea urchin egg during fertilization

Calcium plays a strikingly important role in two of the major events in developmental biology: cell activation and differentiation. In this review we begin with the location and quantity of intracellular calcium in sea urchin oocytes, and then discuss the changes that occur during fertilization and egg activation, placing special emphasis on the mobilization and redistribution of intracellular calcium. We also discuss the propagation of the calcium wave and the role of the burst of calcium on the process of reorganizing the egg cortex at fertilization.     

Heterochronic expression of sexual reproductive programs during apomictic development in Tripsacum

Some angiosperms reproduce by apomixis, a natural way of cloning through seeds. Apomictic plants bypass both meiosis and egg cell fertilization, producing progeny that are genetic replicas of the mother plant. In this report, we analyze reproductive development in Tripsacum dactyloides, an apomictic relative of maize, and in experimental apomictic hybrids between maize and Tripsacum. We show that apomictic reproduction is characterized by an alteration of developmental timing of both sporogenesis and early embryo development. The absence of female meiosis in apomictic Tripsacum results from an early termination of female meiosis. Similarly, parthenogenetic development of a maternal embryo in apomicts results from precocious induction of early embryogenesis events. We also show that male meiosis in apomicts is characterized by comparable asynchronous expression of developmental stages. Apomixis thus results in an array of possible phenotypes, including wild-type sexual development. Overall, our observations suggest that apomixis in Tripsacum is a heterochronic phenotype; i.e., it relies on a deregulation of the timing of reproductive events, rather than on the alteration of a specific component of the reproductive pathway.     

Egg activation: upstream of the fertilization calcium signal

Interaction of sperm and egg at fertilization induces well-coordinated molecular events including specific recognition between species, adhesion and fusion, that lead to the formation of a zygote, a totipotent cell that develops into a new individual. A calcium signal, common to a great number of species, from marine invertebrates to mammals, is essential to activate the metabolism of the unfertilized oocyte. However, how fertilization triggers this calcium signal and initiates development of the early embryo is far from understood. The signaling pathways activated in eggs may be similar to those described in somatic cells, since changes in intracellular free calcium and in mitosis activating protein (MAP) kinase activity occur in both systems after activation. Several hypotheses are currently proposed, implying a spermatic ligand binding to a specific receptor expressed at the egg surface, or where the fused sperm either allows the transit of external calcium into the egg or injects one (or several) activating factor(s). It is still not known which of these ideas is true. We concentrate in this review on the possible signaling pathways involving IP3 (inositol trisphosphate), since its production is involved in most species to generate the fertilization calcium wave.     

Role of calcium signals in early development

The mammalian egg appears to transduce the duration, amplitude, and temporal presentation of the increase in the intracellular calcium concentration ([Ca(2+)](i)) upon fertilization. These Ca(2+) parameters have important short-term effects on the initiation and completion of early events of egg activation, as well as much later consequences for the extent of peri-implantation development. Recent studies have begun to shed light on how the egg quantitatively interprets the Ca(2+) signal (e.g., by summation of individual Ca(2+) rises) and the mechanisms by which down-stream Ca(2+) effectors, such as Ca(2+)/Calmodulin (CaM)-dependent protein kinase II (CaMKII), utilize this ionic signal to promote biological events that initiate development.     

Osmolarity-regulated swelling initiates egg activation in Drosophila

Egg activation is a series of highly coordinated processes that prepare the mature oocyte for embryogenesis. Typically associated with fertilization, egg activation results in many downstream outcomes, including the resumption of the meiotic cell cycle, translation of maternal mRNAs and cross-linking of the vitelline membrane. While some aspects of egg activation, such as initiation factors in mammals and environmental cues in sea animals, have been well-documented, the mechanics of egg activation in insects are less well-understood. For many insects, egg activation can be triggered independently of fertilization. In Drosophila melanogaster, egg activation occurs in the oviduct resulting in a single calcium wave propagating from the posterior pole of the oocyte. Here we use physical manipulations, genetics and live imaging to demonstrate the requirement of a volume increase for calcium entry at egg activation in ex vivo mature Drosophila oocytes. The addition of water, modified with sucrose to a specific osmolarity, is sufficient to trigger the calcium wave in the mature oocyte and the downstream events associated with egg activation. We show that the swelling process is regulated by the conserved osmoregulatory channels, aquaporins and DEGenerin/Epithelial Na⁺ channels. Furthermore, through pharmacological and genetic disruption, we reveal a concentration-dependent requirement of transient receptor potential M channels to transport calcium, most probably from the perivitelline space, across the plasma membrane into the mature oocyte. Our data establish osmotic pressure as a mechanism that initiates egg activation in Drosophila and are consistent with previous work from evolutionarily distant insects, including dragonflies and mosquitos, and show remarkable similarities to the mechanism of egg activation in some plants.     

Signal transduction at fertilization: the Ca2+ release pathway in echinoderms and other invertebrate deuterostomes

Gamete interaction and fusion triggers a number of events that lead to egg activation and development of a new organism. A key event at fertilization is the rise in intracellular calcium. In deuterostomes, this calcium is released from the egg's endoplasmic reticulum and is necessary for proper activation. This article reviews recent data regarding how gamete interaction triggers the initial calcium release, focusing on the echinoderms (invertebrate deuterostomes) as model systems. In eggs of these animals, Src-type kinases and phospholipase C-gamma are required components of the initial calcium trigger pathway in eggs.     

Cortical granule exocytosis in sea urchin eggs is inhibited by drugs that alter intracellular calcium stores

In sea urchin eggs fertilization is accompanied by cortical granule exocytosis, a secretory event thought to be initiated by release of intracellularly sequestered calcium. We have examined the effect of two drugs on this process: chlortetracycline (CTC), a known chelator of intracellular calcium, and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), an antagonist of intracellular calcium release in both skeletal and smooth muscle. Preincubation of eggs for 10 min with either CTC or TMB-8 blocked sperm entry, inhibited the burst of 45Ca2+ efflux normally seen postinsemination, and prevented fertilization envelope elevation. Half-maximal inhibition occurred with 200 microM CTC and 60 microM TMB-8. Electron microscopy confirmed that cortical granule exocytosis had been blocked, although inhibition was not due to a direct effect on exocytosis. CTC and TMB-8 had no effect on Ca2+-stimulated granule fusion in isolated egg cortices. Rather, these drugs block the early events in egg activation: sperm incorporation and triggering of exocytosis. These two effects appear to be independent since addition of either drug just before insemination permits sperm entry but inhibits calcium release and cortical granule exocytosis.     

A calcium‐mediated actin redistribution at egg activation in Drosophila

Egg activation is the essential process in which mature oocytes gain the competency to proceed into embryonic development. Many events of egg activation are conserved, including an initial rise of intracellular calcium. In some species, such as echinoderms and mammals, changes in the actin cytoskeleton occur around the time of fertilization and egg activation. However, the interplay between calcium and actin during egg activation remains unclear. Here, we use imaging, genetics, pharmacological treatment, and physical manipulation to elucidate the relationship between calcium and actin in living Drosophila eggs. We show that, before egg activation, actin is smoothly distributed between ridges in the cortex of the dehydrated mature oocytes. At the onset of egg activation, we observe actin spreading out as the egg swells though the intake of fluid. We show that a relaxed actin cytoskeleton is required for the intracellular rise of calcium to initiate and propagate. Once the swelling is complete and the calcium wave is traversing the egg, it leads to a reorganization of actin in a wavelike manner. After the calcium wave, the actin cytoskeleton has an even distribution of foci at the cortex. Together, our data show that calcium resets the actin cytoskeleton at egg activation, a model that we propose to be likely conserved in other species.     

Activation of protein kinase C after fertilization is required for remodeling the mouse egg into the zygote

Fertilization of the mammalian egg initiates numerous biochemical and structural changes which remodel the egg into a single-celled zygote. To date, the most extensively studied phenomenon of fertilization in virtually all species has been the relationship between sperm penetration and the induction of the initial rise in intracellular-free calcium ([Ca²⁺]_i) concentration within the egg. In contrast, relatively few studies have focused on the biochemical events following this rise in calcium, and even fewer studies have directly linked the biochemical events to the structural changes which must ensue for proper development of the embryo. In this study, we exploited recently developed technologies to investigate the action of protein kinase C (PKC), a presumed downstream transducer of the initial rise in [Ca²⁺]_i, during fertilization and artificial activation with calcium ionophore or phorbol 12-myristate 13-acetate (PMA). The newly developed myristoylated PKC pseudosubstrate (myrPKCΨ) was used to specifically inhibit PKC, thereby averting the trauma of injecting the egg with nonmyristoylated PKCΨ. Following fertilization, eggs which were pretreated with myrPKCΨ were not capable of forming a second polar body and pronuclear formation was significantly inhibited. Spatial and temporal localization of PKC using confocal microscopy to visualize the PKC reporter dye, Rim-1, demonstrated localization of PKC to the lateral aspects of the forming second polar body after fertilization, or after artificial activation with calcium ionophore or PMA. In vivo biochemical analysis of eggs which were fertilized or artificially activated demonstrated that PKC activity rose at the same time (40 min) as the second polar body formed and then subsided over the next 5 hr post activation. From these data, we conclude that PKC plays an integral role in directing the transformation from egg to embryo. Mol. Reprod. Dev. 46:587–601, 1997. © 1997 Wiley-Liss, Inc.     

Gamete membrane microdomains and their associated molecules in fertilization signaling

Fertilization is the fundamental system of biological reproduction in many organisms, including animals, plants, and algae. A growing body of knowledge has emerged to explain how fertilization and activation of development are accomplished. Studies on the molecular mechanisms of fertilization are in progress for a wide variety of multicellular organisms. In this review, we summarize recent findings and debates about the long-standing questions concerning fertilization: how egg and sperm become competent for their interaction with each other, how the binding and fusion of these gamete cells are made possible, and how the fertilized eggs initiate development to a newborn. We will focus on the structure and function of the membrane microdomains (MDs) of egg and sperm that may serve as a platform or signaling center for the aforementioned cellular functions. In particular, we provide evidence that MDs of eggs from the African clawed frog, Xenopus laevis, play a pivotal role in receiving extracellular signals from fertilizing sperm and then transmitting them to the egg cytoplasm, where the tyrosine kinase Src is present and responsible for the subsequent signaling events collectively called egg activation. The presence of a new signaling axis involving uroplakin III, an MD-associated transmembrane protein, and Src in this system will be highlighted and discussed.     

Fertilization signalling and protein-tyrosine kinases

Fertilization is initiated by species-specific gamete cell recognition, i.e. sperm-egg interaction, followed by a rapid and sustained activation of multiple cellular and biochemical events, collectively called 'egg activation', which is indispensable for successful formation of zygotic nucleus and later embryogenesis. It is well known that sperm-induced egg activation is mediated by a transient release of calcium ions that originates from the sperm entry point and propagates through the entire egg cytoplasm. It is unclear, however, what kind of upstream events prelude to the calcium transient after sperm-egg interaction. Recently, much attention has been paid to the role of protein-tyrosine phosphorylation in egg activation process by a number of studies on some well-established model organisms. These includes marine invertebrates, frogs, and mammals. In this review, we will summarize the recent findings that begin to uncover a 'missing link' between sperm-egg interaction and egg activation with emphasis on the role of egg protein-tyrosine kinases (PTKs) in Xenopus egg fertilization.     

Reconstitution of Src-dependent phospholipase Cgamma phosphorylation and transient calcium release by using membrane rafts and cell-free extracts from Xenopus eggs

We reported previously that egg membrane rafts serve as a subcellular microdomain for sperm-dependent tyrosine kinase signaling in Xenopus fertilization. Moreover, we demonstrated that raft-associated Src tyrosine kinase was activated by sperm in vitro. Here we show that egg rafts incubated with sperm or hydrogen peroxide (H2O2) can promote Src-dependent phosphorylation of phospholipase Cgamma (PLCgamma) and transient calcium release in the extracts of unfertilized Xenopus eggs. In vivo egg activation by sperm or H2O2 also promotes tyrosine phosphorylation and raft-translocalization of PLCgamma. Immunodepletion of PLCgamma from the egg extracts inhibits the raft-dependent calcium release. Rafts prepared from H2O2-activated eggs also promote Src-dependent dephosphorylation of p42 mitogen-activated protein kinase and cell cycle transition from metaphase II to interphase in egg extracts. PLCgamma phosphorylation and calcium release in egg extracts can be promoted by rafts prepared from COS-7 cells expressing the Xenopus Src gene. These results demonstrate that the signaling events elicited by fertilization in Xenopus eggs can be reconstituted in vitro. The development of such experimental platforms will allow us to dissect the molecular mechanism of sperm-dependent activation of raft-associated Src and subsequent up-regulation of PLCgamma and egg activation machinery in Xenopus eggs.     

A rapid change in phosphorylation on tyrosine accompanies fertilization of sea urchin eggs.

Alterations in protein phosphorylation, particularly phosphorylation on tyrosine, frequently accompany cell change and are important agents in the cascades initiated by extracellular signals. This paper examines whether the activation of the sea urchin egg at fertilization involves an early and rapid phosphotyrosine response. Using an anti-phosphotyrosine antibody and a rapid sampling technique, we find a very early increase in the phosphorylation on tyrosine of two proteins of approximately 91 kDa and 138 kDa. A similar phosphorylation occurs after activation of the eggs by the calcium ionophore, ionomycin, suggesting the stimulation of a Ca(2+)-sensitive pathway. The timing and Ca2+ sensitivity suggest a role in the primary signal transduction events of fertilization.