The Periodic Changes in Microvilli Density in Activated Xenopus Eggs That Correspond to the Cleavage Cycle

In order to obtain the cytological basis for the periodic flattening and rounding-up of activated amphibian eggs, the surface ultrastructure and the cortical microfilament organization were studied in Xenopus laevis . Scanning electron microscopy (SEM) of the egg surface revealed that the density of microvilli at the animal pole region decreased significantly when the periodic flattening started, but increased again concomitantly with the commencement of the rounding-up. Isolated pieces of the cortices stained with rhodamine-phalloidin exhibited the periodic disorganization and reorganization of a meshwork with bright dots probably corresponding to microvilli, in good synchrony with the decrease and increase of the microvilli density. Study of appropriate batches of eggs in which the moving front of surface contraction waves (SCWs; 1) can be localized revealed that the decrease and increase of the microvilli density correspond to SCW-1 and -2, respectively. SEM and the cytochemical examination of the eggs from which the germinal vesicle (GV) had been removed revealed that none of these changes occurred in the enucleated eggs. These observations suggest that the GV-dependent regulation of the microfilament organization in an egg cortex constitutes the cytological basis for the SCWs and for the periodic flattening and rounding-up of denuded eggs.     

Egg exudate-induced reduction of sperm lysin sensitivity in the vitelline coat after fertilization of Bufo japonicus and its participation in polyspermy block

The jellyless eggs of Bufo japonicus or those from which the vitelline coats (VCs) had been removed (denuded eggs) were electrically activated. The exudate that accompanied egg activation (AEX) was collected to study its role in preventing polyspermy. When dejellied (but VC intact) eggs were treated with AEX, the eggs lost not only fertilizability but also the sensitivity of their VCs to the sperm lysin. By contrast, denuded eggs treated with AEX were fertilizable; even activated eggs were highly fertilizable, provided they were deprived of their VCs and inseminated 30 min after activation. The loss of sensitivity to sperm lysin occurred in VCs 3-5 min after activation either in De Boer's or 1/20 De Boer's solution. The activity of AEX to reduce the sensitivity of VCs to sperm lysin was heat-sensitive and dependent on Ca2+, but it was not affected at all by the variety of protease inhibitors used. The activity was lost by the preincubation of AEX with fragmented VCs in the presence of Ca2+, suggesting Ca(2+)-dependent binding of AEX molecules to the VC at fertilization. Immunocytochemical studies employing anti-AEX rabbit serum showed that the pertinent antigens were localized in the cortical granules of unfertilized eggs and in both the inner surface of VCs and the perivitelline space of fertilized eggs. We conclude that the AEX-induced loss of lysin sensitivity in VCs and the deposition of cortical granule materials on the inner wall of VCs constitute a slow and permanent block to polyspermy.     

Activity of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) after parthenogenetic activation of ovine oocytes

The maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) are the key regulators of both meiotic and mitotic cell cycles. Knowledge of the dynamics of these two kinases during the transition from meiosis to mitosis would be of great importance for cloning by nuclear transfer. In this study, experiments were designed to assay the changes of MPF and MAP kinase activity of in vitro matured ovine oocytes after chemical activation and culture in 0 mM or 2 mM 6-dimethylaminopurine (6-DMAP) for 12 h. Moreover, to determine the biological significance of the fluctuations of MPF, activated oocytes were fused with GV-staged partners. The biochemical results showed that the high MPF activity of MII oocytes fell to basal level precipitously within the first hour after activation, started to increase at 6-8 h, rising to 80 +/- 4% of MII after 12 h. MAPK activity decreased to a low level 4 h after activation, increased between 6-12 h, but remained below 30 +/- 3.6% of MII values. The incubation with 6-DMAP had no effect on the kinetics of MPF and MAP kinase activity. Fusion of MII oocytes to GV partners induced rapid breakdown of the GV, whereas no breakdown occurred when GV were fused with eggs in the first hours post activation. Interestingly, the high biochemical levels of MPF activity at 8-12 h after activation were not able to induce GVBD in fusion partners.     

Plasma membrane block to sperm entry occurs in mouse eggs upon parthenogenetic activation

The ability of parthenogenetically activated mouse eggs to establish a plasma membrane (PM) block to sperm penetration was studied. Zona-free eggs preloaded with Hoechst 33342 were activated by exposure to ethanol or OAG (1-oleoyl-2-acetyl-sn-glycerol) and inseminated after different periods. Eggs challenged with sperm at 30- or 60-min postactivation displayed a fertilization frequency significantly lower than that of control eggs. Conversely, when insemination was carried out at 120-min postactivation, the proportion of fertilized eggs was equivalent to that observed in the control group. Moreover, we report that when the eggs were induced to resume meiosis without any notable loss of CGs (egg exposure to OAG at 100 μM external Ca²⁺ or to heat shock), a normal ability to be penetrated was recorded at 30-min postactivation. Similar behaviour was exhibited by eggs that underwent a CG exocytosis close to that triggered by sperm in absence of nuclear activation (microinjection of inositol 1,4,5-trisphosphate into the egg at 1 μM cytosolic concentration). Present data support the conclusion that parthenogenetically activated mouse eggs are capable of a transitory PM block response that requires both CG exocytosis and meiosis resumption to occur. © 1994 Wiley-Liss, Inc.     

EFFECT OF DNP ON ACCELERATION OF THE CLEAVAGE FOLLOWING INSUFFICIENT PARTHENOGENETIC ACTIVATION IN THE SEA URCHIN EGG*

Unfertilized eggs of sea urchins, Hemicentrotus pulcherrimus and Pseudocentrotus depressus, were treated with 4–5% butyric acid-sea water for 40–60 sec so that they were activated partheno-genetically without visible cortical changes. When these insufficiently activated eggs were inseminated 90–120 min after butyric acid-treatment, they divided much earlier than the control eggs in the first cleavage cycle. In the present paper, it becomes clear that if eggs are put into m /2,000-m /16,000 DNP-sea water at 60 min after insufficient activation and 30 min later, returned to normal sea water and then inseminated, they still show acceleration of the first cleavage in the same degree as the eggs which are not treated with DNP, while if eggs are exposed to DNP for 30 min prior to the insufficient activation or within 60 min after the activation, they do not show any acceleration of the cleavage. From these results, it may be concluded that some preparations for cleavage acceleration which are arrested by DNP become ready in the eggs at an early period in the first cleavage cycle and these preparations cannot be cancelled by DNP-treatment once they have been completed.     

Characterization of ribosomal S6 protein kinase p90rsk during meiotic maturation and fertilization in pig oocytes: mitogen-activated protein kinase-associated activation and localization

Mitogen-activated protein kinase (MAPK) becomes activated during the meiotic maturation of pig oocytes, but its physiological substrate is unknown. The 90-kDa ribosome S6 protein kinase (p90rsk) is the best known MAPK substrate in Xenopus and mouse oocytes. The present study was designed to investigate the expression, phosphorylation, subcellular localization, and possible roles of p90rsk in porcine oocytes during meiotic maturation, fertilization, and parthenogenetic activation. This kinase was partially phosphorylated in oocytes at germinal vesicle (GV) stage through a MAPK-independent mechanism, but its full phosphorylation is dependent on MAPK activity. After fertilization or electrical activation, p90rsk was dephosphorylated shortly before pronucleus formation, which coincided with the inactivation of MAPK. A protein phosphatase inhibitor, okadaic acid, accelerated the phosphorylation of p90rsk during meiotic maturation and induced its rephosphorylation in activated eggs. MAPK kinase (MAPKK or MEK) inhibitor U0126 inhibited the activation of MAPK and p90rsk in both cumulus-enclosed and denuded pig oocytes, but prevented GV breakdown (GVBD) only in cumulus-enclosed oocytes. Active MAPK and p90rsk were detected in pig cumulus cells, and U0126 induced their dephosphorylation. In meiosis II arrested eggs, U0126 led to the inactivation of MAPK and p90rsk, as well as the interphase transition of the eggs. P90rsk was distributed evenly in GV oocytes, but it accumulated in the nucleus before GVBD. It was localized to the meiotic spindle after GVBD and concentrated in the spindle mid zone during emission of the polar bodies. All these results suggest that p90rsk is downstream of MAPK and plays functional roles in the regulation of nuclear status and microtubule organization. Although MAPK and p90rsk activity are not essential for the spontaneous meiotic resumption in denuded oocytes, activation of this cascade in cumulus cells is indispensable for the gonadotropin-induced meiotic resumption of pig oocytes.     

CYCLIC SURFACE CHANGES IN THE NON-NUCLEATE EGG FRAGMENT OF XENOPUS LAEVIS

Fertilized uncleaved eggs of Xenopus laevis were divided into nucleate and non-nucleate egg fragments. Both fragments, together with the whole egg of the same batch, were observed by time-lapse cinematography.
Two kinds of cyclic surface changes, (1) rounding-up and relaxing movements and (2) surface contraction waves, accompanying each cleavage in the whole eggs and the nucleate fragments, were also observed even in the non-nucleate fragments although they do not cleave.
Cleavage intervals of the whole egg and the nucleate fragment were nearly equal, but the rounding-up intervals of the non-nucleate fragment were slightly but definitely longer than the cleavage intervals of the nucleate fragment and the whole egg.     

Changes in calpain during meiosis in the rat egg

Resumption of meiosis at fertilization is mediated by increased levels of calcium which activate several calcium-dependent enzymes. Calpain, a neutral calcium-activated thiol protease, is present in the cytoplasm of many cells. Its activation is associated with limited autolysis and relocalization in the cell. Calpain is thought to participate in the regulation of mitosis and resumption of meiosis in Xenopus oocytes. In this study we followed the activation and localization of calpain during maturation and fertilization in rat eggs using a polyclonal antibody raised against chicken muscle calpain. A band of 80 kDa was detected in GV oocytes and its level increased in unfertilized MII eggs. At the early stages of fertilization, we observed a transient decrease in the level of calpain which was regained at the pronuclear stage. Adding Ca²⁺ to lysate of MII eggs resulted in an additional band, representing the degraded fragment of the activated protein. In eggs activated by ionomycin, calpain level decreased, followed by an increase in a dynamic similar to that observed in fertilized eggs. Egg activation also led to changes in calpain localization. A homogenous distribution was observed in GV and in MII eggs, while in activated eggs it was localized predominantly overlying the metaphase plate. In the current study we demonstrate the presence of calpain in the rat egg. During maturation, calpain level increases; however, during egg activation, in response to [Ca²⁺]_i changes, calpain undergoes autolysis, translocation, and fluctuation in its level. We therefore suggest a correlation between calpain activation and fertilization. Mol. Reprod. Dev. 48:119–126, 1997. © 1997 Wiley-Liss, Inc.     

The changes in sperm nuclei after penetrating fish oocytes matured without germinal vesicle material in their cytoplasm

The processes occurring from sperm penetration to chromosome formation in the cytoplasm of Oocytes matured in vitro, after removal of the germinal vesicle (GV) and before hormonal stimulation, were observed with electron microscope. The dechorionated oocytes, matured without the participation of the GV material, responded to sperm penetration by initiating a cortical reaction within 20 seconds after insemination. The pentrating sperm nuclei transformed to male pronuclei with vesiculation of the nuclear membrane, chromatin decondensation, and formation of a pronuclear membrane. Before cleavage, however, no chromosome formation was observed in these oocytes. Instead, the fully grown pronuclei change to a picnotic chromatin mass without or with an only fragmented nuclear membrane, then disappeared. On the contrary, sperm nuclei that penetrated into the cytoplasm of naked eggs containing GV material during maturation underwent pronuclear and chromosomal formation. Judging from these observation in Oryzias oocytes, the GV material seems to be unnecessary for the formation of pronucleus from the compact sperm nucleus, but is essential for the process of chromosomal formation.     

Behaviour of the vitelline envelope in Bufo arenarum oocytes matured in vitro in blockade to polyspermy

During activation of amphibian eggs, cortical granule exocytosis causes elaborate ultrastructural changes in the vitelline envelope. These changes involve modifications in the structure of the vitelline envelope and formation of a fertilization envelope (FE) that can no longer be penetrated by sperm. In Bufo arenarum, as the egg traverses the oviduct, the vitelline envelope is altered by a trypsin-like protease secreted by the oviduct, which induces an increased susceptibility of the vitelline envelope to sperm lysins. Full-grown oocytes of B. arenarum, matured in vitro by progesterone, are polyspermic, although cortical granule exocytosis seems to occur within a normal chronological sequence. These oocytes can be fertilized with or without trypsin treatment, suggesting that the vitelline envelope is totally sperm-permeable. Vitelline envelopes without trypsin treatment cannot retain either gp90 or gp96. This suggests that these glycoproteins are involved in the block to polyspermy and that trypsin treatment of matured in vitro oocytes before insemination is necessary to enable vitelline envelopes to block polyspermy. The loss of the binding capacity in vitelline envelopes isolated from B. arenarum oocytes matured in vitro with trypsin treatment and activated by electric shock suggests that previous trypsin treatment is a necessary step for sperm block to occur. When in vitro matured oocytes were incubated with the product of cortical granules obtained from in vitro matured oocytes (vCGP), vitelline envelopes with trypsin treatment were able to block sperm entry. These oocytes exhibited the characteristic signs of activation. These results support the idea that B. arenarum oocytes can be activated by external stimuli and suggest the presence of unknown oocyte surface receptors linked to the activation machinery in response to fertilization. Electrophoretic profiles obtained by SDS-PAGE of solubilized vitelline envelopes from oocytes matured in vitro revealed the conversion of gp40 (in vitro matured oocytes, without trypsin treatment) to gp38 (ascribable to trypsin activity or cortical granule product activity, CGP) and the conversion of gp70 to gp68 (ascribable to trypsin activity plus CGP activity). Taking into account that only the vitelline envelopes of in vitro matured oocytes with trypsin treatment and activated can block sperm entry, we may suggest that the conversion of gp70 to gp68 is related to the changes associated with sperm binding.     

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.     

Spermatozoan response to the toad egg matured after removal of germinal vesicle.

The germinal vesicle (GV) was removed from toad oocytes at various times after treatment with progesterone, and enucleated eggs were inseminated under conditions that ensured fertilization of nucleated control eggs. The eggs enucleated before the initiation of GV break-down did not show genuine cleavage. Cytological examinations revealed, however, that spermatozoa enter the eggs enucleated even before the hormone treatment, without subsequent formation of pronuclei or DNA synthesis. The same lack of response was observed when several detergent-pretreated sperm were injected into enucleated eggs. When GV material was injected back into enucleated oocytes, the injected spermatozoa underwent transformation and DNA synthesis, although in variable degrees, in the egg cytoplasm. It is concluded that the egg becomes fertilizable independently of the GV during the hormone-induced maturation process. After entering the egg, however, spermatozoa require GV material for their participation in the developmental process.     

Development of pronuclei from human spermatozoa injected microsurgically into frog (Xenopus) eggs

Human spermatozoa were demembranated with Triton X-100 (TX) and injected into the mature eggs of Xenopus laevis. The nuclei of these spermatozoa decondensed and developed into pronuclei. Chromosomes did not appear in the eggs until the end of a 5-hr incubation period. When the demembranated human spermatozoa were further treated with dithiothreitol (DTT) before they were injected into the eggs, the sperm nuclear decondensation and pronuclear development took place considerably faster than in spermatozoa treated with the detergent alone. By the end of the 5-hr incubation period, decondensed chromatin threads or chromosome-like structures appeared, but none of the eggs cleaved. When human spermatozoa were injected into full-grown ovarian oocytes with intact germinal vesicle (GV) or oocytes which had matured without GV, the nuclei of a proportion of TX-treated and all TX-DTT-treated sperm decondensed but showed no sign of developing into pronuclei. Sperm nuclei injected into maturing oocytes formed condensed chromatin fragments as long as the oocytes were not activated, but they transformed into pronuclei when the oocytes were stimulated with electric shock. These results indicate that the cytoplasmic factors responsible for the decondensation of human sperm nuclei are present in egg cytoplasm independent of GV-materials. We also suggest that the factors controlling development of decondensed sperm nuclei into pronuclei are dependent on GV materials.     

A Factor That Interrupts the Cell Cycle at G2 or Prophase is Present in Full-Grown Frog Oocytes

Full-grown amphibian oocytes that had been arrested at meiotic prophase I contained an activity that prevented the cell cycle from progressing beyond a G₂-like stage. Injection of the contents of germinal vesicles (GV-content) or cytoplasm obtained from oocytes of the frog Rana rugosa prevented fertilized eggs of Cynops pyrrhogaster or Bufo japonicus from cleaving. The nuclei in the arrested eggs consisted of thin chromosomes and nucleolus-like particles enclosed within clear nuclear membrane and their volume increased as a function of time after injection. Cycling of maturation-promoting factor (MPF) did not occur in the injected eggs, but DNA synthesis was not disturbed. The injection of exogenous MPF into the eggs induced the reinitiation of the cell cycle with progression to the M phase and subsequent cleavage. Furthermore, the injection into the full-grown oocytes of Bufo inhibited induction of the maturation of oocytes by progesterone. These results demonstrate that a factor that arrests the cell cycle either at a G₂-like stage of mitosis or at prophase in meiosis is present both in the GV and cytoplasm of frog oocytes. We refer to this factor as a G₂-specific cytostatic factor (G₂-CSF). G₂-CSF may play an important role not only in the physiological arrest at prophase I in meiosis, but also in regulation of the G₂/M transition in the cell cycle of early embryonic cells.     

Ooplasmic influence on nuclear function during the metaphase II-interphase transition in mouse oocytes.

Nuclear and pronuclear transfer procedures were used to assess the functional competence of the nucleus and cytoplasm of mouse germinal vesicle-stage oocytes denuded of granulosa cells and matured in vitro or in vivo before artificial activation using a sequential treatment of A23187 + cycloheximide. Following activation, in vitro-matured oocytes were "fertilized" by inserting a male pronucleus (PN), cultured to the 2-cell stage, and then transferred to the oviducts of foster mothers. No live births were noted, whereas a 17% live birth rate was observed when in vivo-matured oocytes were used. The developmental competency of other zygotes was similarly assessed following the exchange of haploid PN of matured and activated eggs with the female PN of fertilized zygotes. When PN of oocytes subjected to maturation and activation in vitro were transferred, only 1 of 79 reconstructed zygotes developed to term. In contrast, the live birth rate was 21% (11 of 53) for zygotes reconstructed with PN from in vivo-matured oocytes. Moreover, a live birth rate of 23% (8 of 35) was observed for reconstructed zygotes with female PN from "hybrid" oocytes created by transferring the metaphase II nuclei of in vitro-matured oocytes into enucleated, in vivo-matured oocytes before activation. Such results suggest that the nucleus of an in vitro-matured oocyte can support embryonic development, but only when it is activated in the proper ooplasmic milieu. The cellular factors creating this ooplasmic milieu appear to develop normally in vivo during follicle maturation to metaphase II, but they fail to do so when the oocytes are denuded of granulosa cells and cultured in vitro before the final stages of maturation. In parallel studies, male and female PN of in vivo-fertilized zygotes were inserted into oocytes that were activated and enucleated following either in vitro or in vivo maturation. Live birth rates were comparable at 19% (5 of 27) and 18% (9 of 49), respectively, suggesting that, regardless of the environment of the final stages of oocyte maturation, the resultant ooplasm is competent to support all aspects of embryonic development once activation and PN formation has been completed. Such findings only point further toward the importance of the condition of the ooplasmic milieu at the time of chemical activation. Whether a similar situation exists when eggs are activated following sperm penetration remains to be determined.     

Activation of Xenopus laevis eggs in the absence of intracellular Ca activity by the protein phosphorylation inhibitor, 6-dimethylaminopurine (6-DMAP).

Xenopus laevis eggs pricked or microinjected with water or saline in medium containing a limited quantity of free Ca (1.0 to 2.0 microM) remain unactivated for at least 6 hr, even after transfer to oocyte medium containing Ca at higher concentrations (0.5-1.0 mM). These injected eggs, when later pricked in oocyte medium or exposed to A23187 or urethane are fully capable of activation. This confirms the observations of Wangh ('89). However, eggs injected in this Ca-limited medium (CaLM) with 6-DMAP as well as those simply exposed to this drug undergo changes characteristic of activation, including cortical contraction, cortical granule breakdown, a loss of MPF and CSF activities, and pronuclear formation. The time required for 6-DMAP to induce egg activation is inversely correlated to its concentration. Interestingly, eggs that have been injected with EGTA, and thus are unable to respond to activation stimuli such as pricking and A23187 or urethane treatment, can also be activated by exposure to 6-DMAP. In contrast, eggs exposed to or injected with a 6-DMAP analogue (6-aminopurine or puromycin) or a protein synthesis inhibitor (cycloheximide or emetine or puromycin) are not activated. As well, eggs injected in CaLM with 6-DMAP simultaneously with a phosphatase inhibitor (NaF or ammonium molybdate) fail to become activated. Although 6-DMAP-activated eggs remain at the pronucleus stage so long as 6-DMAP is present, they resume cell cycle activities after the drug is withdrawn. They form cleavage furrows, disassemble pronuclear envelopes, and recondense chromosomes. Also, MPF activity reappears and cycles at least twice, peaking each time shortly before cleavage furrow formation. These results suggest that activation of Xenopus eggs arrested at metaphase II by inhibition of protein phosphorylation does not require intracellular Ca release and that maintenance of the egg at metaphase II depends upon continuous protein phosphorylation.     

The cell cycle can occur in starfish oocytes and embryos without the production of transferable MPF (maturation-promoting factor)

All cells undergoing the transition from interphase to metaphase have been postulated to contain a "maturation-promoting factor" (MPF) capable of causing meiotic maturation when injected into immature oocytes. We have shown in an accompanying paper (A. Picard, M. C. Harricane, J. C. Labbe, and M. Doreé, 1988, Dev. Biol. 128, 121-128) that the basic oscillator driving the cell cycle still operates in maturing starfish oocytes and fertilized eggs in the absence of germinal vesicle (GV) material. Under such conditions of enucleation, we now show, however, that MPF activity cannot be detected after hormonal stimulation of prophase-arrested oocytes in Astropecten or after the normal time of second meiotic cleavage in Marthasterias. In contrast, cell cycles occur with the production of transferable MPF activity in embryos from which both pronuclei have been removed after fertilization. Reinjection of the entire contents of a GV after the normal time of second meiotic cleavage restores the ability of cytoplasm to induce meiotic maturation in immature recipient oocytes after transfer. Transduction of the hormonal stimulus at the level of the plasma membrane, stimulation of the phosphorylation of cytoplasmic proteins, and activation of a cycling Ca2+- and cyclic nucleotide-independent histone kinase still occur in the absence of GV material. Since previous studies have demonstrated that the presence of GV material in the recipient oocytes is absolutely required in starfish for the amplification of microinjected MPF (Kishimoto et al., 1981; Picard and Doree, 1984), we propose that some unidentified component of the GV is required, at least after the normal time of second meiotic cleavage in donor oocytes and at any time in recipient oocytes, for the successful transfer of MPF activity in starfish.     

Developmental potential of selectively enucleated immature mouse oocytes upon nuclear transfer

Selective enucleation (SE) was applied to germinal vesicle (GV) oocytes by removing the chromatin attached to nuclear envelope, and leaving the liquid contents of GV in the cytoplast. However, after reconstruction with 1/8 blastomeres or fetal fibroblasts (FFs) neither the maturation efficiency nor the frequency of normal (asymmetric) division was improved as compared with completely enucleated (CE) oocytes. Chromosomal aberrations introduced with somatic nuclei were not rescued in SE oocytes either. On the other hand, timing of maturation division in SE GV oocytes, but not in CE GV oocytes, reconstructed with GV-karyoplasts was like in the control. After maturation and fertilization in vitro, SE oocytes reconstructed with 1/8 blastomeres developed nucleolated donor pronuclei, contrary to CE oocytes. The latter could be rescued with nucleoli-containing nucleus, but not anucleolate nucleus, from a 1/2 blastomere. SE oocytes reconstructed with FFs contained nucleolated pronuclei upon activation, unlike CE GV oocytes. These experiments show that the ooplast nucleolar material and/or embryonic nucleolus are indispensable for pronuclei formation. SE oocytes reconstructed with 1/8 blastomeres or FFs failed to cleave after activation or in vitro fertilization. Control GV oocytes enucleolated before fertilization seized cleavage at the 6-cell stage, as oppose to intact GV oocytes, which in 50.9% yielded morulae/blastocysts. These results suggest that ooplast nucleolar material is essential for the cleavage divisions. Activation of cumulus-enclosed SE GV oocytes matured in hormone-supplemented medium and fused to 1/2 blastomere-karyoplasts, yielded morulae, and blastocysts in 45.5% and 23.4% of reconstructed oocytes, respectively.     

EFFECTS OF CHEMICAL REAGENTS ON THE CYCLIC CHANGES OF CORTEX AND CYTOPLASM IN THE ACTIVATED ENUCLEATED EGG-FRAGMENTS OF THE SEA URCHIN*

Unfertilized eggs of sea urchins. Anthocidaris crassispina and Hemicentrotus pulcherrimus, were separated by centrifugation into two fractions (nucleated light and enucleated heavy fragments). The enucleated egg-fragments were activated by treatment with 1 M urea and then put into sea water solutions of the following three reagents; colcemid, cytochalasin B and Monogen at a concentration by which cleavage was suppressed. It was then examined whether the egg-fragments can exhibit cyclic changes of cytoplasm and cortex in correlation with the cleavage cycle in normally fertilized eggs without any influence of nuclear activity. The results obtained clearly showed that colcemid can suppress the cyclic appearance of cytoplasmic changes, but not that of cortical changes; on the contrary, in cytochalasin B- and Monogen-treated fragments, the periodicity in cortical activities is suppressed, while the periodic changes in the cytoplasm appear according to a timeschedule of the cleavage cycle. Therefore, it may be said that: 1) cyclic changes can occur in both the cytoplasm and the cortex independently, without the direct influence of nuclear activity; 2) if either of them is arrested, the cleavage does not take place; 3) the normal cleavage requires the simultaneous occurrence of periodic activities both in the cortex and in the cytoplasm after fertilization.     

Effect of cumulus cell removal of in vitro matured bovine oocytes prior to in vitro fertilization on subsequent cleavage rate

The aim of this study is to identify the effect of cumulus cells removal prior to the in vitro fertilization of matured bovine oocytes on cleavage rate. Denuded, matured oocytes were fertilized in presence or absence of loose cumulus cells, cumulus cell conditioned IVF medium (CCCM), charcoal-treated CCCM and charcoal-treated CCCM supplemented with progesterone at a final concentration of 150 ng/ml. After 18 h of incubation with sperm, the presumptive embryos were cultured on a BRL monolayer and the percentage of cleaved embryos was evaluated on Day 4. Removal of cumulus cells prior to IVF significantly reduced the cleavage rate (25% for denuded oocytes versus 56% for cumulus-oocyte complexes (COCs)). The addition of loose cumulus cells partially restored the effect of denudation (cleavage rate: 37% for denuded oocytes supplemented with loose cumulus cells versus 27% for denuded oocytes and 58% for COCs). CCCM also had a positive effect on the cleavage rate of oocytes denuded prior to IVF (36% for denuded oocytes fertilized in CCCM versus 14% for denuded oocytes). Treating the CCCM with charcoal resulted in complete loss of its effect on cleavage rate (18% for denuded oocytes fertilized in charcoal-treated CCCM versus 34% for denuded oocytes fertilized in CCCM). The addition of progesterone to charcoal-treated CCCM partially restored the reduction of the cleavage rate caused by charcoal treatment (27% for denuded oocytes fertilized in charcoal-treated CCCM supplemented with progesterone versus 14% for denuded oocytes fertilized in charcoal-treated CCCM and 36% for denuded oocytes fertilized in CCCM). In conclusion, removal of cumulus cells prior to IVF adversely affects the cleavage rate through loss of a factor secreted by these cells. This factor probably is progesterone.