Nascent protein requirement for completion of meiotic maturation and pronuclear development: examination of fertilized and A-23187-activated surf clam (Spisula solidissima) eggs

The involvement of newly synthesized proteins and calcium in meiotic processes, sperm nuclear transformations, and pronuclear development was examined in emetine-treated, fertilized, and A-23187-activated Spisula eggs by observing changes in the morphogenesis of the maternal and paternal chromatin. Emetine treatment (50 micrograms/ml) initiated 30 min before fertilization or A-23187 activation inhibited incorporation of [3H]leucine into TCA-precipitable material and blocked second polar body formation. Sperm incorporation and the initial enlargement of the sperm nucleus were unaffected; however, the dramatic enlargement and transformation of the sperm nucleus into a male pronucleus, which normally follow polar body formation, were delayed 10 to 20 min. Unlike the situation in untreated, control eggs, male pronuclear development took place while the maternally derived chromosomes remained condensed. It was not until approximately 20 min after the normal period of pronuclear development that the maternal chromosomes dispersed and formed a female pronucleus in emetine-treated, fertilized eggs. Formation of pronuclei, however, was unaffected in both emetine-treated, A-23187-activated eggs and fertilized eggs incubated with A-23187. These observations indicate that germinal vesicle breakdown, first polar body formation, and initial transformations of the sperm nucleus are independent of newly synthesized proteins. Inhibition of second polar body formation and the delay in pronuclear development brought about by emetine, as well as the appearance of silver grains over pronuclei in autoradiographs of control eggs incubated with [3H]leucine demonstrate that nascent proteins are involved with the completion of meiotic maturation and the development of male and female pronuclei. The ability of A-23187 to override the inhibitory effects of emetine on pronuclear development suggests that both nascent protein and calcium signals are involved in regulating the status of the maternal and paternal chromatin during pronuclear development.     

Experimental hybridization among Oryzias species. II. Karyogamy and abnormality of chromosome separation in the cleavage of interspecific hybrids between Oryzias latipes and O. javanicus

In interspecific hybridization between Oryzias latipes and O. javanicus, all hybrid embryos failed to develop and died before hatching. Cytological examination of fertilization and early development was performed to discover the cause of lethal development. When O. latipes eggs were inseminated by sperm of O. javanicus, the cortical reaction was induced normally. Chromosomal material in the fertilized eggs was visualized using the DNA-specific fluorochrome Hoechst. The spermatozoon was capable of penetrating into the egg cytoplasm through the micropyle, and the sperm nucleus transformed to the male pronucleus. The female pronucleus that formed after extrusion of the second polar body migrated towards the male pronucleus. The female and the male pronuclei underwent DNA synthesis and encountered each other in the center of the blastodisc, fused with one another and formed a zygote nucleus before breakdown of the nuclear envelope. Metaphase chromosomes with electron dense chromatin regions were abnormally divided into each blastomere in cleavage. The abnormally separating chromatin masses were also labeled by BrdU. The abnormal separation resulting in partial loss of fragmented chromatin might be a cause of abortive development in the interspecific hybrids between O. latipes and O. javanicus.     

Cytoplasmic and sperm nuclear transformations in fertilized ammonia-activated sea urchin (Arbacia punctulata) eggs

Studies examining cytoplasmic and sperm nuclear transformations in sea urchin (Arbacia punctulata) eggs inseminated at different periods after ammonia activation have been caried out at the light- and electron-microscopic levels of observation. Arbaca eggs treated with ammonia-seawater demonstrated chromosome condensation after DNA synthesis and underwent a chromosome cycle similar to that described for Lytechinus [Mazia, 1947]. Cortical granule reaction, fertilization cone formation, and sperm aster development in eggs fertilized at 20 (interphase), 50 (prometaphase), and 180 (interphase) min after ammonia activation were structurally simialr to processes in untreated zygotes. Cyclical changes in the formation of fertilization cones and sperm asters, as reported for eggs fertilized after activation by agents that induce a cortical granule reaction, were not observed. Although sperm nuclear transformations were prolonged (14 vs 18 min), male pronuclei that developed in eggs fertilized 20 min after ammonia activation were morphologically similar to those observed in fertilized, untreated ova and incorporated ³H-thymidine. Sperm incorporated into eggs at 50 min after ammonia activation underwent nuclear envelope breakdown and chromatin despersion; however, ³H-thymidine incorporation was not observed, and male pronuclei rarely developed (less than 5% of all specimens examined). Subsequent to dispersion, the paternal chromatin condensed into chromosomes which were associated with an aster. These results demonstrate that although ammonia-activated eggs inseminated at interphase or prometaphase undergo similar cytoplasmic alterations, sperm nuclear transformations vary with the chromosome cycle of the egg.     

Surface alterations of fertilized surf clam (Spisula solidissima) eggs induced by concanavalin A

Fertilized Spisula eggs, incubated in ConA, were examined at periodic intervals to determine the effects of lectin binding on events of fertilization and cleavage. ConA was localized to specific regions of the vitelline layer and plasma membrane by reacting lectin-treated eggs with horseradish peroxidase and diaminobenzidine. In contrast to eggs, little reaction product was associated with the plasma membrane of spermatozoa. Sperm that fused with ConA-treated eggs failed to move into the cortex of the ovum and were observed as bulbous appendages at the surface of the zygote. Reorganization of sperm nuclei was inhibited, and male pronuclei failed to develop. ConA also inhibited polar body formation and cleavage. The maternally derived chromatin underwent meiosis, and the chromosomes normally taken into the first and second polar bodies were retained within the zygote. All of the maternally derived chromatin was organized within four or more female pronuclei which subsequently entered mitosis. The effects of ConA binding on events at the surface of fertilized Spisula eggs were abrogated by α-methyl-d-mannoside; succinyl-ConA only partially inhibited fertilization-related processes. The effects of ConA are discussed in terms of possible cross-linking of surface components of fertilized Spisula eggs which may inhibit deformation of the zygote cortex.     

Role of Mos/MEK/ERK cascade and Cdk1 in Ca2+ oscillations in fertilized ascidian eggs

Intracellular calcium ion concentration ([Ca(2+)](i)) transients are observed in the fertilized eggs of all species investigated so far, and are critical for initiating several events related to egg activation and cell cycle control. Here, we investigated the role of the Mos/MEK/ERK cascade and Cdk1 on Ca(2+) oscillations in fertilized ascidian eggs. The egg of the ascidian Phallusia nigra shows [Ca(2+)](i) oscillations after fertilization: Ca(2+) waves immediately following fertilization (phase I), and [Ca(2+)](i) oscillations between the first and second polar body extrusions (phase II). Our results show that in P. nigra eggs, ERK activity peaked just before the extrusion of the first polar body, and decreased gradually, eventually disappearing at the extrusion of the second polar body. Cyclin-dependent protein kinase 1(Cdk1) activity decreased to undetectable levels immediately after fertilization, and then periodically increased according to the meiotic and mitotic cell cycle. When the unfertilized eggs were incubated with U0126, an inhibitor of MEK, before insemination, ERK was immediately inactivated, and the phase II [Ca(2+)](i) oscillations disappeared. Alternatively, when the constitutively active Mos protein (GST-Mos) was injected into the unfertilized eggs, ERK activity was preserved for at least 120 min after fertilization, and the phase II [Ca(2+)](i) oscillations lasted for more than 120 min after the second polar body extrusion. These results suggest that ERK activity is necessary for maintaining [Ca(2+)](i) oscillations. GST-ΔN85-cyclin, which maintains Cdk1 activity, caused ERK activity in the eggs to persist for over 120 min after fertilization, and prolonged [Ca(2+)](i) oscillations. Moreover, the effects of GST-ΔN85-cyclin on the egg were abrogated by the application of U0126. Thus, Cdk1-mediated [Ca(2+)](i) oscillations seem to require ERK activity. However, GST-Mos triggered [Ca(2+)](i) oscillations after the second polar body extrusion, whereas GST-ΔN85-cyclin did not, although it prolongs the duration of [Ca(2+)](i) oscillations. Interestingly, GST-ΔN85-cyclin increased the frequency of [Ca(2+)](i) transients in the Mos-induced [Ca(2+)](i) oscillations after the extrusion of the second polar body. Thus, Cdk1 could maintain, but not activate, ERK and [Ca(2+)](i) oscillations. ERK activity and [Ca(2+)](i) oscillations seem to form a negative feedback loop which may be responsible for maintaining the meiotic period.     

DNA replication cycle in parthenogenetically developing eggs of the starfish Asterina pectinifera

Starfish oocytes artificially activated by a calcium ionophore will develop normally if the formation of polar bodies is suppressed. In the present paper, schedules of the DNA replication period (S phase) of these parthenogenotes were explicitly timed using 5-bromo-2'-deoxyuridine (BrdU) and anti-BrdU monoclonal antibody. Their schedule of S phase was identical to that of fertilized eggs. Consequently, an S phase regulation system is triggered even in parthenogenotes raised by dual treatment of egg activation and polar body suppression. The S phase schedule of parthenogenotes confirms the temporal pattern of chromosome duplication, observed by other researchers, leading to tetraploid parthenogenotes. The S phase determination also provides a basis for argument concerning the number of centrioles participating in parthenogenetic development. If polar body formation of activated eggs was not suppressed, the first S phase was normal, but the second S phase did not recur on time. A rigidly regulated system of DNA replication cycle, which should be an essential prerequisite for parthenogenesis, thus requires the content of polar bodies.     

Morphological changes in the oocyte and its surrounding vestments during in vivo fertilization in the dasyurid marsupial Sminthopsis crassicaudata

This light and transmission electron microscopical study shows that the first polar body is given off before ovulation and that part of its cell membrane and that of the surrounding oocyte have long microvilli at the time of its ejection. Several layers of cumulus cells initially surround the secondary oocyte and first polar body, but the ovulated oocytes in the oviducts in the process of being fertilized do not have cumulus cells around them. Partly expelled second polar bodies occur in the oviduct; they are elongated structures that lack organelles and have electron-dense nuclei. A small fertilization cone appears to form around the sperm tail at the time of sperm entry into the egg and an incorporation cone develops around the sperm head in the egg cytoplasm. In three fertilized eggs a small hole was seen in the zona, which was presumably formed by the spermatozoon during penetration. Cortical granules, present in ovarian oocytes, are not seen in fertilized tubal or uterine eggs; release of their contents probably reduces the chances of polyspermy, although at least one polyspermic fertilized egg was seen and several other fertilized eggs had spermatozoa within the zona pellucida. In the zygote the pronuclei come to lie close together, but there was no evidence of fusion. A "yolk mass," which becomes eccentric before ovulation, is extruded by the time the two-cell embryos are formed, but many vacuoles remain in the non-yolky pole of the egg. A shell membrane of variable thickness is present around all uterine eggs but its origin remains undetermined.     

The role of oocyte maturation in the ontogeny of the fertilization site in the hydrozoan Hydractinia echinata

Summary In hydrozoans the sperm will fuse with the egg only at the site of polar body formation. The primary oocyte and maturing oocytes which have produced the first polar body cannot be fertilized even though maturing oocytes which have produced the first polar body attract sperm. These eggs do not acquire the ability to be fertilized until after second polar body formation. If either first or second polar body formation is inhibited or if first and second polar body formation do not take place in close proximity to each other, the fertilization site is not set up. Under normal circumstances the site of polar body formation takes place at the region on the maturing oocyte surface nearest the site where the germinal vesicle resided in the primary oocyte. When maturing oocytes are centrifuged prior to polar body formation, the site of polar body formation is frequently shifted so that it does not correspond to the site where it would be given off under normal circumstances. Under these conditions the shifted site of polar body formation is the only site where the egg can be fertilized, indicating that the fertilization site is selected during oocyte maturation.Oocyte maturation in these hydrozoans is mediated by a hormone released by the somatic cells of gonophores as a consequence of bringing dark adapted gonophores into the light. The hormone acts directly on the oocyte to induce maturation. The oocyte only has to be exposed to the hormone for the first few minutes of the maturation process in order to complete the process of maturation.Dedicated to Professor N.H. Verdonk of the Rijksuniversiteit Utrecht on his 65th birthday     

Association of newly synthesized 3H-arginine-labeled proteins with chromatin structures in fertilization

Zona-free hamster eggs have been fertilized in vitro with boar spermatozoa in a medium enriched by arginine-³H and the sites of localization of newly synthesized arginine-³H–labeled proteins have been investigated using fine-structure autoradiography. It was confirmed that such proteins are synthesized during fertilization and that they accumulate to a notable degree in decondensing sperm chromatin as well as in the chromatin of the female pronucleus and also of the second polar body. A similar process did evidently take place also in defective pronuclei, characterized by a core of a still condensed chromatin and by remaining nuclear membrane. In such male pronuclei the highest concentration of the label was seen just on the border of the condensed chromatin, on the expected site of nuclear protein exchange. It is supposed that, at least in this experimental system, any morphologically detectable sperm decondensation is accompanied immediately by a shift from sperm basic nuclear proteins to other nuclear proteins.     

Influence of the calcium ionophore A23187 on rat egg behavior and cortical F-actin

Rat eggs treated with the calcium ionophore A23187 and subjected to long-term observation by phase microscopy were found to undergo many developmental changes that are normally associated with fertilization. These included cortical granule exocytosis and the abstriction of the second polar body. In addition, time-lapse video microscopy revealed that, unlike untreated eggs, whose surfaces remained relatively immotile, the ionophore-treated eggs underwent a lengthy period of surface undulatory activity. Since all of these events were remarkably similar in timing and morphology to those seen in fertilized eggs, we conclude that A23187 is capable of activating rat eggs. Using NBD-phallacidin, the distribution of F-actin in ionophore-activated eggs was determined. During most of the postactivation period the eggs possessed an uninterrupted, uniform band of polymerized actin encompassing the entire cortex of the egg. However, during a discrete 1.5-h period after the formation of the second polar body, an area adjacent to the region of polar body abstriction exhibited more intense staining than the rest of the cortex. Cytochalasin B treatment caused a dramatic reduction and/or rearrangement in cortical NBD-phallacidin staining in activated eggs as compared to activated controls not exposed to the drug. We observed that all the developmental changes described above could be produced in the absence of exogenous calcium, suggesting that the rat egg possesses internal stores of calcium sufficient to elicit an activational response. We conclude that the ionophore-induced release of free calcium ions into the cytosol stimulates many of the developmental changes that are normally seen during fertilization. These results indicate that calcium influx and cytoskeletal activity are correlated during the activation of this animal egg.     

中国对虾受精过程中精卵核的细胞学变化

中国对虾精子以其棘部顶端随机附着在卵上,精子在凝胶膜形成后,第一极体排出前入卵,精子入卵后,絮状的精核经过重建形成雄原核,中国对虾卵子排放时处于第一次成熟分裂的中期,卵子入海水时,纺锤体的长轴与质膜平行,卵子激活后,纺锤体的长轴开始旋转,旋转至纺鲑体长轴与质膜垂直时,由纺锤丝牵引着染色体向两极移动,外侧的染色体由质膜包裹形成第一极体,受膜举起后,由次级卵母细胞排放出第二极体,此后,单倍雌核重建形成雌原核,雄原核形成早于雌原核,雌雄原核于卵子中央联会形成联合核,受精后的50分钟纺锤丝牵关染色体称向两极,质膜内缢断裂形成两个细胞的胚胎。     

Sequential transformations of human sperm nucleus in human egg

In-vitro insemination of human zona-free oocytes prepared from oocytes that failed to fertilize in an in-vitro fertilization programme was used as an experimental model to study the time course and morphological events during the development of sperm nuclei into male pronuclei. At 30 min after insemination, 22 eggs were cultured in a CO2 incubator for further 3.5 h and 17 eggs were placed individually between a slide and coverslip for randomly repeated microscopical observations in a controlled environment for at least 3.5 h. Simultaneous arrest of maternal meiosis and sperm nuclear development occurred in 36.4% (8/22) eggs cultured in the CO2 incubator and 47.1% (8/17) of those cultured between a slide and coverslip. Sequential transformation of the human sperm nucleus in human eggs was studied in 6 eggs that showed continuous development of sperm nuclei into male pronuclei during at least 3.5 h after insemination. The early sperm nuclear development in human egg ooplasm can be divided into three phases: the sperm nucleus first decondenses (phase 1) then partly recondenses (phase 2) before expanding again to form an early male pronucleus (phase 3). The prepronuclear stages (phases 1 and 2) took about 60 min each and the pronuclear formation (phase 3) began between 120 and 170 min after insemination. Early pronuclear formation was associated with the occurrence of dense outline material, probably a precursor of the future pronuclear membrane, around the recondensed nucleus in re-expansion (phase 3). Between 30 and 60 min after the beginning of phase 3, numerous (greater than 20) dense grains, considered as nucleolar precursors, were clearly visible inside the growing male pronucleus. Moreover, we have examined sperm nuclear changes in some eggs in which the progression of late meiosis was abnormal. Meiotic arrest of maternal chromatin was always associated with arrest of sperm head development. In 75% (6/8) of the eggs arrested in the metaphase II stages and in 87.5% (7/8) of the eggs arrested in late anaphase II, sperm nuclear development was stopped at the decondensed and recondensed stages, respectively. We have always observed male pronuclei when a maternal pronucleus was present in the egg. These observations suggested that maternal chromatin and sperm nuclear development are probably regulated by common factor(s).     

胞浆内精子注射技术生产小鼠

以piezo操作系统为技术支撑 ,在掌握小鼠卵母细胞胞浆内精子注射技术 (ICSI)的基础上 ,进行了ICSI技术生产试管小鼠的尝试。来自成年昆明 (KM)小鼠附睾尾的新鲜精子 ,剪切去尾后 ,直接将精子头注射到B6D2F1小鼠卵母细胞质中 ,注射后 1h ,83.3%的卵母细胞存活。6h时 ,84.0 %的成活卵子成功受精 ,形成原核 ,排出PB2 体外培养的ICSI胚胎 ,卵裂率 (98%vs 94.7% )和 4-细胞期胚胎比率 (89.5%vs 92.1% )均与培养的体内受精卵没有差异 (P >0.05 ) ;但是 ,桑椹胚(63.8%vs84.2% )和囊胚发育率 (25.7%vs68.4% )极显著地 (P <0.01)低于对照组。120枚原核期胚胎移植给 7只假孕受体后 ,4只受孕小鼠共产出 28只ICSI小鼠 (23.3% )。健康成年的 25只ICSI小鼠都没有明显的生理和行为异常。随机选择其中的 20只小鼠 ,分别进行ICSI小鼠间、ICSI与KM小鼠间共 12组的交配 ,结果所有雌鼠妊娠产仔。在成功建立小鼠ICSI技术的基础上,成功获得了我国的首例ICSI小鼠,并且证明这些ICSI小鼠都具有正常的繁殖后代的能力。     

The formation of the pronuclei and their associated perinuclear cytoplasm; the determination of the phases of the first cleavage cycles in Crepidula fornicata (Mollusca,Gastropoda)

Summary

The behaviour of the male and the female pronuclei in Crepidula fornicata is studied, beginning at the formation of the second polar body. Shortly after the extrusion of the second polar body the female pronucleus is formed, and then the male pronucleus enters the yolk-free cytoplasm near the animal pole. Both pronuclei are enveloped by a typical nuclear membrane, and increase in size until the prophase; a zygote nucleus is not formed (“Ascaris type” of fertilization). In the meantime, the chromatin of both pronuclei is arranged in a meshwork in the centre of the pronuclei.

Shortly after the formation of the second polar body a special cytoplasm, the “perinuclear cytoplasm”, is formed in the vicinity of each of the pronuclei. During the early stages of the first cleavage cycle this cytoplasm is composed of numerous Golgi complexes, small dense Golgi vesicles, smooth endoplasmic reticulum vesicles, mitochondria and rosettes of glycogen-like granules. At later stages, when the pronuclei have met and their plasms coalesced, the number of Golgi elements decreases; at the same time, the small dense Golgi vesicles increase in number and aggregate in clusters.

The phases of the first three cleavage cycles are determined by cytophotometry. The nuclear DNA of the male pronucleus and that in the nuclei of the blastomeres of the 2- and the 4-cell stage is reduplicated between 7 and 33% of the normalized cleavage cycles; the G₂-phase is between 33 and 57%, while the mitotic phase occupies the last part of each cleavage cycle and the first 7% of the next cleavage cycle. There is no G j-phase. Since the female pronucleus lies just beneath the polar bodies, its DNA content could not be measured separately.     

The localization of LAP2beta in bovine oocytes after in vitro activation and fertilization

The present study was designed to clarify the localization of LAP2beta and to compare it with those of lamins A/C and B in bovine oocytes after activation and in vitro fertilization (IVF). After fertilization, LAP2beta was not found until telophase II, and was observed around condensed chromatin after the extrusion of the second polar body, but not in activated oocytes. Although the reaction of LAP2beta was temporally negative or weak on the membrane of the growing small pronuclei, it became strong on the fully grown pronuclei of both activated and fertilized oocytes. Examination of the timing of DNA synthesis using bromodeoxyuridine revealed that the expression of LAP2beta on the pronuclear membrane became strong around the end of the DNA synthesis in both activated and fertilized oocytes. Both male and female pronuclei exhibited the same reactivity to all nuclear proteins examined. It was also shown that LAP2beta first assembled around condensed chromatin, followed by the integration of lamins B and A/C as in somatic cells. LAP2beta staining was maintained on the nuclear membrane of the embryonic cells at interphase until the later stage of preimplantational development. There were no differences between parthenogenetic and fertilized embryos in the expression and localization of LAP2beta from the PN-stage oocyte to the blastocyst. The assembly of LAP2beta was observed around the telophase chromatin of both blastocyst and cumulus cells. Thus, it was shown that the timing of the aggregation of LAP2beta at the second meiosis was different from that in the mitosis of blastocyst and somatic cells. LAP2beta was constantly expressed in the nuclear membrane in in vitro fertilized and parthenogenetic embryos as was lamin B, and lamin A/C was expressed stage-dependently in both types of embryos. Lamin A/C was positive in some inner cell mass cells of parthenogenetic blastocysts, but not those of in vitro fertilized embryos.     

Prematurely condensed human sperm chromosomes after in vitro fertilization (IVF)

Summary During an in vitro fertilization (IVF) program 122 inseminated eggs showing polar body extrusion, but neither formation of pronuclei nor cell cleavage were analysed cytogenetically. Nine of these eggs showed prematurely condensed sperm chromosomes of the G₁-phase (G₁-PCC) besides the haploid set of maternal metaphase II chromosomes. This phenomenon can be explained by the permanent arrest of the oocytes at metaphase II after sperm penetration and hence the continuing presence of cytoplasmic chromosome condensing factors which lead to the induction of PCC in the sperm nucleus. The overall frequency of this aberrant type of fertilization was calculated to be in the order of 3–4% of all in vitro fertilized eggs.     

Development of human male pronucleus: Ultrastructure and timing

The process of human male pronuclear formation was studied using an experimental model based on in vitro inseminated human zona-free eggs prepared from oocytes that failed to fertilize in a clinical in vitro fertilization program. The main ultrastructural changes in penetrated sperm nuclei transforming into pronuclei were used to define four stages of pronuclear development. The first two stages, representing partial (Stage 1) and total (Stage 2) sperm chromatin decondensation, appeared as early as 1 hr after mixing of gametes. This rapid initial phase was followed by a more lengthy array of events leading to transformation of decondensed sperm nuclei into fully developed male pronuclei (Stages 3 and 4). Stage 3 was characterized by reformation of the nuclear envelope, reorganization of chromatin, and the assembly of nuclcolar precursors. It was not completed until 12 hr after in vitro insemination when fully developed male pronuclei (Stage 4) were first observed. In some eggs pronuclei did not reach Stage 4 at all. The results of this study provide a morphological background for further research into molecular aspects of human male pronuclear development and its regulation.     

Involvement of mitogen-activating protein kinase and intracellular pH in the duration of the metaphase I (MI) pause of starfish oocytes after spawning

The metaphase I (MI) arrest of starfish oocytes is released after spawning. In this study using starfish Asterina pectinifera , the duration of MI after spawning was ~20 min and ~30 min in fertilized and unfertilized oocytes, respectively. This prolongation of MI in unfertilized oocytes, referred to as the MI pause, was maintained by mitogen-activating protein kinase (MAPK) as well as low intracellular pH (~7.0). Contrary to previous reports, MI arrest was not maintained by MAPK, since it was inactive in the oocytes arrested at MI in the ovary and activated immediately after spawning. Also, cyclin B was not degraded at pH 6.7 in the cell-free preparation without MAPK activity, whereas it was degraded at pH 7.0, suggesting that MI arrest was solely maintained by lower pH (< 7.0). Normal development occurred when the spawned oocytes were fertilized before the first polar body formation, whereas fertilization after the first polar body formation increased the rate of abnormal development. Thus, due to MI pause and MI arrest, the probability for fertilization before the polar body formation might be increased, leading to normal development.     

体外受精和孤雌活化过程中小鼠胚胎细胞骨架的动态变化

为研究微管在体外受精与孤雌活化过程中的动态变化,本实验比较了体外受精胚胎、SrCl2激活的孤雌胚胎和体内受精的原核期胚胎在体外发育的情况,采用免疫荧光化学与激光共聚焦显微术检测卵母细胞孤雌活化过程中及体外受精后微管及核的动态变化,以分析微管在减数分裂过程中的作用及其对早期发育的影响.结果显示,体内受精胚胎的发育率显著高于体外受精和孤雌激活胚胎体外发育率(P<0.05),而体外受精与孤雌激活胚胎在各阶段发育率差异均不显著.在体外受精中,精子入卵,激活卵母细胞,减数分裂恢复,纺锤丝牵拉赤道板卜致密排列的母源染色体向纺锤体两侧迁移;后期将染色体拉向两极;末期时,微管分布于两组已去凝集的母源染色体之间,卵母细胞排出第二极体(the second polarbody,Pb2),解聚的母源染色体形成雌原核.同时,在受精后5～8 h精子染色质发生去浓缩与再浓缩,形成雄原核.在原核形成的同时,胞质星体在雌、雄原核的周围重组形成长的微管,负责雌、雄原核的迁移靠近.孤雌活化过程中,卵母细胞恢复减数分裂,姐妹染色单体分离,被拉向两极,经细胞松弛素B处理后,活化4～6 h,卵周隙中未见Pb2,而在胞质中出现两个混合的单倍体原核,之间由微管相连接,负责两个单倍体原核的迁移靠近.与体外受精相比较,孤雌活化时卵母细胞更容易被激活,减数分裂期间微管的发育早且更完善.     

Ultrastructure of in vivo fertilization in the goat

In vivo fertilization of goat eggs has been studied by electron microscopy. Eggs were recovered from superovulated or natural cyclic goats, 32 to 52 hours after the onset of oestrus; only eggs recovered between 46 and 52 hours were fertilized. Spermatozoa penetrated the zona pellucida tangentially leaving vesiculated products of the acrosome reaction at the zona surface. As sperm penetrated into the ooplasm, the second meiotic division completed and cortical granule exocytosis occurred. However a few unreacted cortical granules usually remained in the cortex of the two fertilized eggs, adjacent to the plasma membrane. After swelling the two pronuclei presented similar ultrastructural morphology: they contained small, compact, agranular nucleoli and unevenly distributed chromatin. The cytoplasm in close vicinity to the apposed pronuclei contained large stacks of annulate lamellae, smooth endoplasmic reticulum, prominent Golgi complexes, as well as dense areas of unidentified material. The abundance of cytoplasmic organelles near the pronuclei might be the expression of intensive metabolic activity. Conversely, in the cortex of fertilized ova several large organelles-free cytoplasmic areas were randomly distributed.