Effect of Ca2+ on deformation, polar body extrusion and pronucleus formation in the egg of the ascidian, Ciona savignyi

Cortical deformation and polar body extrusion are the principal events that occur at fertilization in the ascidian egg. We demonstrated that the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the fertilized egg of Ciona savignyi increased at egg deformation (main peak) and then several small Ca²⁺ spikes (1st spikes) appeared before the first polar body extrusion. Brief Ca²⁺ spikes (2nd spikes), then appeared in the period between the first and second polar body extrusion. When eggs were fertilized in Ca²⁺-free artificial seawater, the main peak and 1st spikes appeared, but the 2nd spikes did not, suggesting that the Ca²⁺ required for the main peak and 1st spikes is released from the intracellular store in this species and that extracellular Ca²⁺ is required for the 2nd spikes. When [Ca²⁺]_i was clamped at a low level (0.03–0.13 μmol/L) by injecting the egg with low-Ca²⁺ buffers and the egg was then inseminated, deformation, polar body extrusion and pronucleus formation were suppressed. In contrast, egg deformation and first polar body extrusion were induced without insemination when [Ca²⁺]_i was 0.9 μmol/L. A higher Ca²⁺ concentration of 1.2–10.1 μmol/L was required for extrusion of the second polar body and pronucleus formation. These data suggest that sequential Ca²⁺ increases (i.e. main peak and 1st and 2nd spikes) are prerequisite for the deformation and polar body extrusion of the egg. Furthermore, in eggs arrested at the second meiotic metaphase after first polar body extrusion by the injection of Ca²⁺ buffer, subsequent injection of excess Ca²⁺ caused formation of an irregular second polar body-like protrusion, suggesting latent arrest at the second meiotic metaphase in the ascidian egg.     

Hydrogen peroxide modulates meiotic cell cycle and induces morphological features characteristic of apoptosis in rat oocytes cultured <Emphasis Type="Italic">in vitro</Emphasis>

Hydrogen peroxide (H₂O₂) is known to induce cell cycle arrest and apoptosis in various somatic cell types cultured in vitro. We hypothesize that this reactive oxygen species (ROS) could modulate cell cycle and induce morphological features characteristics of apoptosis in oocytes cultured in vitro. To test this hypothesis, immature and mature oocytes were cultured in medium containing various doses of H₂O₂ with or without caspase-3 inhibitor for various times. The treatment of H₂O₂ induced germinal vesicle break down (GVBD) in all immature oocytes followed by initiation of shrinkage. Some of immature oocytes (but not mature oocytes) also showed membrane blebbing. On the other hand, H₂O₂ treatment inhibited first polar body emission in mature oocytes just prior to initiation of shrinkage. The cytoplasmic granulation and fragmentation into apoptotic bodies were observed in mature oocytes during later stages of H₂O₂ treatment. The shrinkage was induced by H₂O₂ in a dose- and time-dependent manner in both immature and mature oocytes. Although, H₂O₂-induced degeneration was observed in both immature and mature oocytes after 2.0 hrs of treatment, immature oocytes were more susceptible to undergo quick shrinkage, membrane blebbing and degeneration. Co-addition of caspase-3 inhibitor prevented shrinkage and degeneration of both immature and mature oocytes except membrane blebbing that was observed at higher doses of H₂O₂ after 1.0 hr of culture. Treatment of H₂O₂ induced bax protein expression (3 times), DNA fragmentation and caspase-3 activity (2.5 times) in oocytes undergoing morphological apoptotic changes. These findings clearly suggest that H₂O₂ induced GVBD in immature oocytes, inhibited first polar body extrusion in mature oocytes prior to initiation of morphological changes characteristic of apoptosis such as shrinkage, membrane blebbing and cytoplasmic fragmentation prior to degeneration.     

Mouse oocyte maturation is affected by lithium via the polyphosphoinositide metabolism and the microtubule network

The incubation of mechanically denuded mouse oocytes in medium containing LiCl delayed both germinal vesicle breakdown (GVBD) and polar body extrusion in a dose-dependent and reversible manner. When myo-inositol alone was added to the culture medium, we observed that it accelerated GVBD and increased the rate of polar body extrusion, whereas, when combined with LiCl, the normal timing of GVBD was recovered. In the same way, when inositol trisphosphate (InsP₃) was microinjected into the ooplasma, we observed an important improvement of the rate of GVBD, as compared to control oocytes, and prevention of lithium inhibition. However, neither myo-inositol nor InsP₃ were able to rescue totally the oocytes from the negative effect of lithium on polar body extrusion. Moreover, lithium induced some important changes in microtubule and chromosome organizations. Before extrusion of the first polar body, the reduction of the spindle size or the appearance of short individualized chromosomes dispersed around a large aster of microtubules were often observed, whereas, after polar body extrusion, the spindle appeared smaller and chromosomes were often trapped in the midbody. Thus lithium affects mouse oocyte maturation at two different levels: GVBD and polar body extrusion. Whereas the former seems to be affected via polyphosphoinositide turnover, the latter is InsP₃-independent and seems to be influenced negatively via underdevelopment of microtubular structures. © 1994 Wiley-Liss, Inc.     

Asymmetric division in mouse oocytes: with or without Mos

In both vertebrates and invertebrates, meiotic divisions in oocytes are typically asymmetric, resulting in the formation of a large oocyte and small polar bodies. The size difference between the daughter cells is usually a consequence of asymmetric positioning of the spindle before cytokinesis. Spindle movements are often related to interactions between the cell cortex and the spindle asters [1,2]. The spindles of mammalian oocytes are, however, typically devoid of astral microtubules, which normally connect the spindle to the cortex, suggesting that another mechanism is responsible for the unequal divisions in these oocytes. We observed the formation of the first polar body in wild-type oocytes and oocytes derived from c-Mos knockout mice [3]. In wild-type oocytes, the meiotic spindle formed in the centre of the cell and migrated to the cortex just before polar-body extrusion. The spindle did not elongate during anaphase. In mos-/- oocytes, the spindle formed centrally but did not migrate, although an asymmetric division still took place. In these oocytes, the spindle elongated during anaphase and the pole closest to the cortex moved while the other remained in place. Thus, a compensation mechanism exists in mouse oocytes and formation of the first polar body can be achieved in two ways: either after migration of the spindle to the cortex in wild-type oocytes, or after elongation, without migration, of the first meiotic spindle in mos-/- oocytes.     

Oogenesis and ovarian morphology in pollinating and non-pollinating fig wasps: evidence from adult and immature stages

Hymenopteran insects have meroistic polytrophic ovaries characterised by trophocytes associated with oocytes inside the follicles. In pro-ovigenic species, all oocytes mature before emergence and no trace of oogenesis is visible in adult females. Pro-ovigeny is a rare condition among Hymenoptera, but common in pollinating fig wasps. In the present investigation, we studied adult and pupa females of three fig wasp species with different trophic strategies. We demonstrated that females of Pegoscapus aerumnosus and Idarnes spp. have an unusual ovarian organisation (i.e. each ovariole has only one mature egg and no oocyte) that has led to misleading interpretation of fig wasp reproductive anatomy. The ovaries of these studied species have several ovarioles, recognisable by the presence of nuclei of tunica propria cells surrounding them. Each adult wasp ovariole had one mature egg. None of the pupae had mature eggs, but all of them had follicles with oocytes in different developmental stages. The studied fig wasps are pro-ovigenic, irrespective of their trophic strategy, since there were no signs of ovigeny in adult females. We discuss ecological and phylogenetic factors that could play a role in fig wasps reproductive strategies.     

The Src Homology 2 Domain-Containing Adapter Protein B (SHB) Regulates Mouse Oocyte Maturation

SHB (Src homology 2 domain-containing adapter protein B) is involved in receptor tyrosine kinase signaling. Mice deficient in the Shb gene have been found to exhibit a transmission ratio distortion with respect to inheritance of the Shb null allele among offspring and this phenomenon was linked to female gamete production. Consequently, we postulated that Shb plays a role for oocyte biology and thus decided to investigate oocyte formation, meiotic maturation, and early embryo development in relation to absence of the Shb gene. Oogenesis was apparently accelerated judging from the stages of oocyte development on fetal day 18.5 and one week postnatally in Shb −/− mice; but in adulthood ovarian follicle maturation was impaired in these mice. Completion of meiosis I (first polar body extrusion) was less synchronized, with a fraction of oocytes showing premature polar body extrusion in the absence of Shb. In vitro fertilization of mature oocytes isolated from Shb +/+, +/− and −/− mice revealed impaired early embryo development in the −/− embryos. Moreover, the absence of Shb enhanced ERK (extracellular-signal regulated kinase) and RSK (ribosomal S6 kinase) signaling in oocytes and these effects were paralleled by an increased ribosomal protein S6 phosphorylation and activation. It is concluded that SHB regulates normal oocyte and follicle development and that perturbation of SHB signaling causes defective meiosis I and early embryo development.     

Brefeldin A disrupts asymmetric spindle positioning in mouse oocytes

Polar body formation in oocytes is an extreme form of asymmetric cell division, but what regulates the asymmetric spindle positioning and cytokinesis is poorly understood. During mouse oocyte maturation, the metaphase I spindle forms at the center but then moves to the cortex prior to anaphase I and first polar body emission. We show here that treating denuded mouse oocytes with brefeldin A, an inhibitor of Golgi-based membrane fusion, abolished the asymmetric positioning of the metaphase I spindle and resulted in the formation of two half-size metaphase II eggs, instead of a full-sized egg and a polar body. The normal metaphase II spindle is similarly asymmetrically positioned in the mature egg, where the spindle lies with its axis parallel to the cortex but becomes perpendicular before anaphase II and emission of the second polar body. When ovulated eggs were activated with strontium in the presence of brefeldin A, the metaphase II spindle failed to assume perpendicular position, and the chromosomes separated without the extrusion of the second polar body. Remarkably, symmetric cytokinesis began following a 3 h delay, forming two half-size eggs each containing a pronucleus. BFA-sensitive intracellular vesicular transport is therefore required for spindle positioning in both MI and MII.     

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.     

Embryonic development and endosymbiont transmission mode in the symbiotic clam Lucinoma aequizonata (Bivalvia: Lucinidae)

Summary

Lucinoma aequizonata is a large lucinid clam which lives in reducing mud around 500 m deep. Adults harbor intracellular chemoautotrophic sulfur-oxidizing bacteria in specialized gill cells called bacteriocytes. The embryonic and early larval development of L. aequizonata is described by using light and scanning electron microscopy. Gametes were obtained by injection of 0.2ml of 4 mM serotonin solution in seawater into the posterior adductor muscle. The oocytes, 200 μm in diameter, are surrounded by a glycoprotein capsule which gives to the egg a total diameter of 500μm. The development which occurs at 10°C is slow. The first polar body is detected 2.5 h after contact between sperm and oocytes (T_o+2.5 h), and the first cleavage begins 10 h later (T_o+12.5 h). The following successive cleavages produce a nonciliated morula, then a ciliated gastrula which begins to rotate within the egg-capsule at T_o+4.5 days. At this stage, the first shell pellicle appears on the dorsal side of the embryo. At T₀+8 days, the trochophore larvae develop discrete ciliary bands which constitute the prototroch. Typical straight-hinge veligers, D-shaped larvae, hatch from the egg-capsule 12 days after fertilization. The newly hatched larvae are 240 μm in length and 200μm in height, and the straight hinge 150μm long. To elucidate the symbiont transmission mode, two symbiont-specific primers were designed and used in amplifications by PCR. This primer set was unsuccessful in amplifying symbiont DNA targets from mature gonads, spawned oocytes, eggs, and veligers whereas successful amplifications were obtained from symbiont-containing gill tissues. These data rule out the vertical transmission mode and strongly suggest that the symbionts are environmentally transmitted to the new host generation in L. aequizonata as for all tropical lucinids examined to date.     

Ca and Mg control of cytostatic factors from Rana pipiens oocytes which cause metaphase and cleavage arrest.

A Ca-sensitive cytoplasmic factor is extractable from mature oocytes of Rana pipiens, but not from immature oocytes or fertilized embryos, and is capable of inducing cleavage arrest at metaphase when microinjected into one blastomere of a cleaving embryo. This factor, designated “primary cytostatic factor” (1°CSF), is active in the presence of Mg or EGTA, but is rapidly inactivated by Ca at concentrations as low as 10 μM or by EDTA. 1°CSF is also inactivated in vivo by injection into the cytoplasm of a zygote during the first 45 min following insemination. Another factor, the “secondary cytostatic factor” (2°CSF), develops in vitro during the aging of extracts prepared from immature oocytes, mature oocytes, and fertilized eggs, and is also capable of arresting cleavage at metaphase. 2°CSF is insensitive to exogenous Ca or EDTA, and also remains active when injected into a zygote at any time following insemination. Since the responses of the fertilized egg to 1°CSF and to mature oocyte cytoplasm are quite similar, it appears that 1°CSF is equivalent to the factor which maintains metaphase II arrest in the mature oocyte. The possibility of 1°CSF inactivation by endogenous free Ca at the time of fertilization is discussed in the context of changes in the free Ca concentration.     

Expression of CD9 and CD81 in bovine germinal vesicle oocytes after vitrification followed by in vitro maturation

The present study aimed to investigate the effect of vitrification on the expression of fertilization related genes (CD9 and CD81) and DNA methyl transferases (DNMT1 and DNMT3b) in bovine germinal vesicle (GV) oocytes and their resulting metaphase Ⅱ (MⅡ) stages after in vitro maturation culture. GV oocytes were vitrified using the open-pulled straw method; after warming, they were cultured in vitro. The vitrified-warmed GV oocytes and more developed MII oocytes were used to calculate the maturation rates (first polar body extrusion under a stereomicroscopy), and to detect mRNA expression (qRT-PCR). Fresh GV oocytes and their in vitro-derived MII oocytes served as controls. The results showed that both the maturation rate (54.23% vs. 42.93%) and the relative abundance of CD9 mRNA decreased significantly (p < 0.05) in bovine GV oocytes after vitrification, but the expression of CD81 and DNMT3b increased significantly. After in vitro maturation of vitrified GV oocytes, the resulting MII oocytes showed lower (p < 0.05) mRNA expression of genes (CD9, CD81, DNMT1 and DNMT3b) when compared to the control group (MII oocytes). Altogether, vitrification decreased the maturation rate of bovine GV oocytes and changed the expression of fertilization related genes and DNA methyl transferases during in vitro maturation.     

Redistribution and Increase in Cortical Inositol 1,4,5-Trisphosphate Receptors after Meiotic Maturation of the Mouse Oocyte

Mouse oocytes develop sensitivity to inositol 1,4,5-trisphosphate (IP₃) during oocyte maturation. We recently reported that a change in the organization of the endoplasmic reticulum (ER) during oocyte maturation may contribute to this enhanced sensitivity (Mehlmannet al.,1995,Dev. Biol.170, 607–615). Here, we investigated whether there is an increase in the number of available IP₃receptors after maturation and whether there is a redistribution of IP₃receptors similar to the redistribution of the ER that occurs during maturation. Western blot analysis of the IP₃receptor in oocytes and eggs demonstrated a 1.8-fold increase in immunoreactive mass of the IP₃receptor following oocyte maturation. Microinjection of the function-blocking monoclonal antibody 18A10 inhibited IP₃-induced Ca²⁺release in a concentration-dependent manner in both eggs and oocytes. More antibody was required to inhibit Ca²⁺release to the same extent in eggs compared to oocytes when both were injected with the same concentration of IP₃, suggesting that eggs contain a greater number of functional IP₃receptors. Immunolocalization of the IP₃receptor revealed that receptors were present in large clusters, 1–2 μm in diameter, in the cortex of the mature egg except in a ring-shaped band of cortex adjacent to the meiotic spindle. In contrast, receptor clusters were located around the entire cortex of the immature oocyte and were much smaller (<1 μm); larger patches were sometimes seen, but they did not display the same spherical organization as those in eggs. These results suggest that the number of cortical IP₃receptors increases during mouse oocyte maturation and that this increase may contribute to enhanced Ca²⁺release at fertilization.     

The reproductive biology of the deep-sea asteroids Benthopecten simplex (Perrier), Pectinaster filholi Perrier,and Pontaster tenuispinus Düben & Koren (phanerozonia : benthopectinidae) from the rockall trough

Samples of three species of benthopectinid starfish were collected in a time-series from stations in the northern Rockall Trough. A detailed study was made of the reproductive biology of Benthopecten simplex (Perrier) from a fixed station at 2200 m. Studies of the gametogenic cycles of Pectinaster fllholi Perrier and Pontaster tenuispinus Düben & Koren from less regular and more scattered samplings are also presented.The oogenic cycles of the three species are similar. Growing oocytes line the ovary wall and come to lie in the lumen only when well developed. Accessory cells surrounding young oocytes become more numerous as the oocytes develop. Vitellogenesis begins when oocyte diameter reaches ≈ 250–300 μm and oocytes attain a maximum diameter of 950 μm in Benthopecten simplex, 850 μm in Pectinaster filholi, and 800 μm in Pontaster tenuispinus. Unspent oocytes are broken down by phagocytes and degenerate ovaries become filled with the breakdown products. Internal degeneration of large oocytes is not common.Size frequency data show a dominance of previtellogenic oocytes and a pattern of continuous growth of vitellogenic oocytes. There is no evidence of synchrony of growth between individuals and no suggestion of seasonal reproduction is evident from summated sample data.Spermatogenesis follows the pattern previously established for deep-sea asteroids. A mature male remains in a state of constant ripeness, ready to shed sperm at any chance encounter with a female releasing ripe eggs. There is no evidence of brooding in any of the species and from the large size and yolk content of the egg, we can infer direct lecithotrophic development demersally.     

Effects of gonadotropins upon the incidence and kinetics of meiotic maturation of macaque oocytes in vitro

The specific aim of this study was to determine the effects of gonadotropins in vitro upon the incidence of and precise time interval to germinal vesicle breakdown (GVB) and extrusion of the first polar body (PB1) in oocytes from nonstimulated rhesus monkeys. Cumulus-enclod germinal vesicle (GV) stage oocytes from 10 normal, cycling rhesus monkeys in the follicular phase of the menstrual cycle were cultured with either: (1) 1.0 μg/ml human follicle-stimulating hormone (hFSH), (2) 10 μg/ml human luteinizing hormone (hLH), (3) 1.0 μg/ml hFSH and 10 μg/ml hLH, or (4) no gonadotropins (controls). Oocytes (n = 234) were examined at 3-hr intervals from 0 to 21 hr and at 4-hr intervals from 24 to 52 hr for GVB and PB1. Neither the incidence of GVB (hFSH: 63.5%; hLH: 56.1%; both gonadotropins: 63.1%; no gonadotropins: 53.6%) nor extrusion of PB1 (hFSH: 41.3%; hLH: 36.4%; both gonadotropins: 36.9%; no gonadotropins; 31.9%) differed (P > 0.05) among treatments. The time to GVB was accelerated (P < 0.05) by gonadotropins (hFSH: 10.8 ± 1.7 hr; hLH: 10.1 ± 1.8 hr; both gonadotropins: 8.8 ± 1.1 hr) when compared to controls (17.4 ± 2.0 hr). However, the time interval to extrusion of PB1 did not differ (P > 0.05) among treatments (hFSH: 32.3 ± 1.2 hr; hLH: 35.1 ± 1.4 hr; both gonadotropins: 35.2 ± 1.3 hr; no gonadotropins: 34.1 ± 1.2 hr). The mean interval to extrusion of PB1 was 34.1 ± 0.6 hr. In conclusion, GVB and PB1 extrusions appear to be, in part, independently regulated events in macaque oocytes matured in vitro since the timing of PB1 extrusion is not tightly coupled with the onset of GVB. Although the developmental potential of oocytes may be enhanced by gonadotropins, alternative approaches must be developed to improve the poor competence of oocytes from nonstimulated monkeys to mature in vitro. © 1994 Wiley-Liss, Inc.     

Involvement of Aurora A kinase during meiosis I-II transition in Xenopus oocytes

The Aurora kinase family has been involved both in vivo and in vitro in the stability of the metaphase plate and chromosome segregation. However, to date only one member of this family, the protein kinase Aurora B, has been implicated in the regulation of meiotic division in Caenorhabditis elegans. In this species, disruption of Aurora B results in the failure of polar body extrusion. To investigate whether Aurora A is also required in meiosis, we microinjected highly specific alpha-Aurora A antibodies in Xenopus oocytes. We demonstrated that microinjected oocytes fail to extrude the first polar body and are arrested with condensed chromosomes on a typical metaphase I plate, which has not performed its normal 90 degrees rotation. We additionally found that, although the failure of first polar body extrusion observed in alpha-Aurora A-microinjected oocytes is likely mediated by Eg5, the impairment of the metaphase plate rotation does not involve this kinesin-like protein. Surprisingly, although chromosomes remain condensed at a metaphase I stage in alpha-Aurora A-microinjected oocytes, the cytoplasmic cell cycle events progress normally through meiosis until metaphase II arrest. Moreover, these oocytes are able to undergo parthenogenetic activation. We conclude that Aurora A and Eg5 are involved in meiosis I to meiosis II transition in Xenopus oocytes.     

Spire-type actin nucleators cooperate with Formin-2 to drive asymmetric oocyte division

Oocytes mature into eggs by extruding half of their chromosomes in a small cell termed the polar body. Asymmetric oocyte division is essential for fertility [1], but despite its importance, little is known about its mechanism. In mammals, the meiotic spindle initially forms close to the center of the oocyte. Thus, two steps are required for asymmetric meiotic division: first, asymmetric spindle positioning and second, polar body extrusion. Here, we identify Spire1 and Spire2 as new key factors in asymmetric division of mouse oocytes. Spire proteins are novel types of actin nucleators that drive nucleation of actin filaments with their four WH2 actin-binding domains [2-6]. We show that Spire1 and Spire2 first mediate asymmetric spindle positioning by assembling an actin network that serves as a substrate for spindle movement. Second, they drive polar body extrusion by promoting assembly of the cleavage furrow. Our data suggest that Spire1 and Spire2 cooperate with Formin-2 (Fmn2) to nucleate actin filaments in mouse oocytes and that both types of nucleators act as a functional unit. This study not only reveals how Spire1 and Spire2 drive two critical steps of asymmetric oocyte division, but it also uncovers the first physiological function of Spire-type actin nucleators in vertebrates.     

Melatonin rescues the aneuploidy in mice vitrified oocytes by regulating mitochondrial heat product

Vitrification of germinal vesicle (GV) stage oocytes has been shown to be closely associated with decreased rates of meiosis maturation and increased rates of aneuploidy. However, little is known about the effects of melatonin on these events in mice vitrified GV oocytes. In this study, the effects of melatonin on meiosis maturation potential and the incidence rate of aneuploidy in mouse vitrified oocytes were analyzed by supplementing in vitro maturation (IVM) solution with melatonin at different concentrations. This study, for the first time, showed that the mitochondrial heat production was markedly increased in vitrified oocytes (P < 0.05), which compromised the first polar body extrusion (PBE) of vitrified oocytes (73.3% vs. 85.1%, P < 0.05). However, 10⁻¹¹ mol/L melatonin could significantly decrease mitochondrial heat production and ROS level (9.1 vs. 12.0 pixels, P < 0.05), meanwhile increase ATP level (1.1 vs. 0.88 pmol, P < 0.05) and mtDNA copies (107438 vs. 67869, P < 0.05), which rescued the abnormal chromosome alignment (32% vs. 69%, P < 0.05) and reduced the incidence of aneuploidy (15.6% vs. 38.5%, P < 0.05) in vitrified oocytes. The meiosis maturation ability of vitrified oocytes with melatonin supplementation was similar to that of fresh ones (83.4% vs. 85.1%, P > 0.05). Collectively, our data revealed that melatonin has a protective action against vitrification-induced injuries of oocytes meiosis maturation.     

Cytoplasmic maturation for activation of pig follicular oocytes cultured and arrested at metaphase I.

A large population (62-90%) of pig follicular oocytes can mature to metaphase II after culture for 48 h. However, a proportion (6-22%) remain in an immature stage at metaphase I (metaphase I-arrested). The main objective of this study was to determine whether the cytoplasm of metaphase I-arrested pig oocytes is capable of being activated by sperm penetration or parthenogenetic stimulation. After culture for 48 h, oocytes without a polar body (73% were shown to be at metaphase I after staining) and those with a polar body (94% were at metaphase II) were fertilized in vitro. A total of 69% and 62% of the oocytes were activated to form a female pronucleus, respectively, and the rate of polar body extrusion induced by fertilization in the activated oocytes was 90% (the first polar body) and 95% (the second polar body), respectively. When oocytes without and with a polar body were stimulated with an electric pulse, 53% and 81% of the oocytes were activated, respectively. The rate of polar body extrusion in the activated oocytes was 73% (the first polar body) and 79% (the second polar body), respectively. In contrast, young metaphase I oocytes cultured for 24 h had low (6%) or zero activation rate after in vitro fertilization or electric pulse stimulation. However, about one-third of the young metaphase I oocytes penetrated by spermatozoa after in vitro fertilization responded to electric pulse 12 h after insemination, and almost all (93%) were activated when they were stimulated 24 h after insemination. Patterns of polypeptide synthesis and histone H1 kinase activity were similar in metaphase I-arrested and metaphase II oocytes, and were characterized by increase in a 25 kDa polypeptide and by decrease in kinase activity. Although the first step of meiotic division is impaired, these results indicate that metaphase I-arrested oocytes are mature cytoplasmically.     

MICROINJECTION OF TOAD SPERM INTO OOCYTES UNDERGOING MATURATION DIVISION*

Spermatozoa of Bufo bufo japonicus were briefly treated with Triton X-100 to remove their plasma membrane, and were injected into oocytes at various stages of maturation division. All the sperm injected into mature coelomic eggs transformed into pronuclei and synthesized DNA, as a normally fertilizing sperm does. The sperm injected into oocytes at the germinal vesicle (GV) stage did not show any change as long as the GV remained intact. In the oocytes which were induced to mature by progesterone, the injected sperm displayed characteristic features in synchrony with those of the resident female nucleus. These included the formation of several sperm-derived chromosomes in association with multipolar spindles in the oocytes from the stage of the germinal vesicle breakdown to the first polar spindle; the appearance of swollen, vesicular nuclei without concomitant DNA synthesis in those at the stage of the first polar body emission; and the reappearance of the condensed chromosomes with giant spindles in those at the stage of the second meiotic metaphase. Pricking of these last oocytes induced the formation of several male pronuclei and DNA synthesis. These results prove that the injection of detergent-treated sperm employed here provides an excellent means of studying the cytoplasmic state that regulates nuclear behavior.     

A Tripolar Spindle Formed at Meiosis I Assures the Retention of the Original Ploidy in the Gynogenetic Triploid Crucian Carp, Ginbuna Carassius auratus langsdorfii

A triploid crucian carp, ginbuna ( Carassius auratus langsdorfii ), reproduces by gynogenesis, in which sperm of diploid ginbuna or of other species triggers the development of the triploid eggs, but a male genome makes no contribution to the zygotic genome. Gynogenesis is maintained by two mechanisms: exclusion of male genome during fertilization and retention of somatic ploidy levels during oogenesis. We examined the mechanisms responsible for producing unreduced eggs. Microfluorometry with a DNA staining dye showed that DNA content in the ginbuna oocytes was not reduced in half during meiosis I. Cytological observations revealed that a tripolar spindle was formed at meiosis I and the first polar body was not extruded, whereas an ordinary bipolar spindle was formed and the second polar body was extruded at meiosis II. Activity of histone H1 kinase (as an indicator of maturation-promoting factor) decreased transiently between meiosis I and II, strongly suggesting a "normal" meiotic cycle progression in the ginbuna oocytes. These results have indicated that in the gynogenetic ginbuna the somatic ploidy levels are maintained by inhibiting the first polar body extrusion via the formation of the tripolar spindle at meiosis I.