Spontaneous and sperm-induced activation of oocytes in LT/Sv strain mice

The oocytes of LT/Sv strain mice are unique in that a high proportion of them (∼40% in this study) are ovulated before reaching metaphase of the second meiotic division (metaphase II). The remaining oocytes of LT/Sv mice are ovulated at metaphase II, as in other strains of mice. When recently ovulated oocytes were cultured in vitro for 11–12 h, those ovulated at metaphase II remained at this stage, whereas those ovulated at metaphase of the first meiotic division (metaphase I) commonly resumed meiosis during in vitro aging. These oocytes extrude the polar body and form a diploid pronucleus. This oocyte activation is not coupled with cortical granule exocytosis. The oocytes ovulated at metaphase II are fully capable of normal fertilization, whereas those ovulated at metaphase I are not. Approximately 50% of metaphase I oocytes penetrated by spermatozoa remain at this stage, and sperm nuclei frequently undergo premature chromosome condensation. Only 13% of spermpenetrated metaphase I oocytes formed a diploid female pronucleus and a haploid male pronucleus by 4 h after insemination. These results demonstrate that the two types of ovulated LT/Sv oocytes have different potentials to undergo either spontaneous or sperm-induced activation.     

Defective calcium release during in vitro fertilization of maturing oocytes of LT/Sv mice

Oocytes of LT/Sv mice have anomalous cytoplasmic and nuclear maturation. Here, we show that in contrast to the oocytes of wild-type mice, a significant fraction of LT/Sv oocytes remains arrested at the metaphase of the first meiotic division and is unable to undergo sperm-induced activation when fertilized 15 hours after the resumption of meiosis. We also show that LT/Sv oocytes experimentally induced to resume meiosis and to reach metaphase II are unable to undergo activation in response to sperm penetration. However, the ability for sperm-induced activation developed during prolonged in vitro culture. Both types of LT/Sv oocytes, i.e. metaphase I and those that were experimentally induced to reach metaphase II, underwent activation when they were fertilized 21 hours after germinal vesicle breakdown (GVBD). Thus, the ability of LT/Sv oocytes to become activated by sperm depends on cytoplasmic maturation rather than on nuclear maturation i.e. on the progression of meiotic division. We also show that sperm penetration induces fewer Ca(2+) transients in LT/Sv oocytes than in control wild-type oocytes. In addition, we found that the levels of mRNA encoding different isoforms of protein kinase C (alpha, delta and zeta), that are involved in meiotic maturation and signal transduction during fertilization, differed between metaphase I LT/Sv oocytes which cannot be activated by sperm, and those which are able to undergo activation after fertilization. However, no significant differences between these oocytes were found at the level of mRNA encoding IP(3) receptors which participate in calcium release during oocyte fertilization.     

The ovulation and activation of primary and secondary oocytes in LT/Sv strain mice

Eggs were isolated from the oviducts or ovaries of LT/Sv strain mice in order to investigate the pathways taken by them following spontaneous or induced parthenogenetic activation. The chromosome preparations from the ovarian oocytes that matured in vitro to metaphase I were all morphologically normal. Of 42 recently ovulated eggs that failed to activate parthenogenetically in culture, 57% on nuclear densitometric analysis were found to have the normal 2C amount of DNA, and 1N (haploid) number of chromosomes present, and were arrested at metaphase II. Somewhat unexpectedly, 43% had a 4C amount of DNA, and 2N (diploid) number of chromosomes present, had been arrested at metaphase I, and were evidently ovulated as primary oocytes. Following parthenogenetic activation, the majority of oocytes extruded a polar body and developed a single pronucleus. The activated eggs could be divided into two sub-populations according to the diameter (and therefore volume) of the pronucleus—in one group this was about one-third greater than in the other. The chromosome constitution of the two groups was determined separately at the first cleavage mitosis. Those with a normal-sized pronucleus were invariably haploid, while those with an enlarged pronuclear volume were invariably found to be diploid. The chromosomes in the diploid spreads often appeared to be associated in homologous pairs. We conclude that almost uniquely in LT/Sv strain females eggs may be activated parthenogenetically at either stage of meiotic maturation giving rise to diploid or haploid embryos, respectively.     

Mouse oocytes gradually develop the capacity for activation during the metaphase II arrest

Metaphase II (M II) mouse oocytes were subjected to a parthenogenetic stimulus (8% ethanol) or fertilized in vitro at various times following the extrusion of the first polar body. The oocytes progressively develop the ability for full activation. Their responsiveness to activation stimuli not only increases, but also changes qualitatively with time. Newly arrested oocytes do not respond at all; then, when the ability to undergo meiotic anaphase II first develops, the response is defective: following extrusion of the second polar body (II PB), the oocyte does not enter interphase but arrests again at metaphase (M III-arrest). Finally, oocytes gain the ability for full activation including the entry to interphase. Depending on the type of activating stimulus, oocytes exhibit the capacity for full activation at different ages. The oocyte arrest in M III is similar to M II and can be released by subsequent activation. Such oocytes undergo anaphase III, extrude a third polar body (III PB), and form an aneuploid female pronuclei.     

Ovulation and fertilization of primary and secondary oocytes in LT/Sv strain mice

In this study, the chromosome constitution of both unfertilized oocytes and fertilized eggs isolated from the oviducts of LT/Sv strain mice were analyzed. Air-dried chromosome preparations from unfertilized oocytes revealed that about one-third of those examined were ovulated as primary oocytes. These were arrested at metaphase of the first meiotic division and exhibited the characteristic “tetrad” chromosome configuration. The remaining two-thirds of the unfertilized oocytes were ovulated at metaphase of the second meiotic division. The fertilized eggs were isolated from the oviducts of LT/Sv females previously mated to (C57BL × CBA) F1 hybrid males. Analysis of the fertilized eggs at metaphase of their first cleavage mitosis revealed that about one-third of the eggs examined were digynic triploids, whereas the remaining two-thirds had the normal diploid chromsome constitution. In the triploids, the 40 female chromosomes present (mouse, n = 20) were derived from a single diploid pronucleus formed after the extrusion of a first polar body, and following the monospermic fertilization of primary oocytes. The female pronuclear-derived chromosomes invariably exhibited “homologous pairing,” and these were associated at their centromeres. The ovulation, penetration, and subsequent fertilization of primary oocytes is an extremely unusual phenomenon in mammals and only appears to occur on a regular basis in LT/Sv mice. The premature “cytoplasmic maturation” of these oocytes is of interest, as they clearly have the same developmental capacity as secondary oocytes. The significance of these observations in relation to folliculogenesis and litter size in LT/Sv mice is discussed.     

Evidence that protein kinase C (PKC) participates in the meiosis I to meiosis II transition in mouse oocytes.

Oocytes from LTXBO mice exhibit a delayed entry into anaphase I and frequently enter interphase after the first meiotic division. This unique oocyte model was used to test the hypothesis that protein kinase C (PKC) may regulate the meiosis I-to-meiosis II transition. PKC activity was detected in LTXBO oocytes at prophase I and increased with meiotic maturation, with the highest (P < 0.05) activity observed at late metaphase I (MI). Treatment of late MI-stage oocytes with the PKC inhibitor, bisindolylmaleimide I (BIM), transiently reduced (P < 0.05) M-phase-promoting factor (MPF) activity and promoted (P < 0.05) progression to metaphase II (MII), while mitogen-activated protein kinase (MAPK) activity remained elevated during the MI-to-MII transition. Confocal microscopy analysis of LTXBO oocytes during this transition showed PKC-delta associated with the meiotic spindle and then with the chromosomes at MII. Inhibition of PKC activity also prevented untimely entry into interphase, but only when PKC activity was reduced in oocytes before the progression to MII and thus indicates that the transition into interphase is directly associated with the delayed triggering of anaphase I. Moreover, the defect(s) that initiate activation occur upstream of MAPK, as suppression of PKC activity failed to prevent activation by Mos(tm1Ev)/ Mos(tm1Ev) LTXBO oocytes expressing no detectable MAPK activity. In summary, PKC participates in the regulatory mechanisms that delay entry into anaphase I in LTXBO oocytes, and the disruption promotes untimely entry into interphase. Thus, loss of regulatory control over PKC activity during oocyte maturation disrupts the critical MI-to-MII transition, leading to a precocious exit from meiosis.     

Unusual cytoskeletal and chromatin configurations in mouse oocytes that are atypical in meiotic progression

Meiotic maturation progresses atypically in oocytes of strain LT/Sv and l/LnJ mice. LT/Sv occytes show a high frequency of metaphase l-arrest and parthenogenetic activation. l/LnJ oocytes display retarded kinetics of meiotic maturation and a high frequency of metaphase l-arrest. Some l/LnJ oocytes fail to resume meiosis. Changes in the configuration of chromatin, microtubules, and centrosomes are associated with specific stages of meiotic progression. In this study, the configuration of these subcellular components was examined in LT/Sv, l/LnJ, and C57BL/6J (control) oocytes either freshly isolated from large antral follicles or after culture for 15 hr to allow progression of spontaneous meiotic maturation. Differences were found in the organization of chromatin, microtubules, and centrosomes in LT/Sv and l/LnJ oocytes compared to control oocytes. For example, rather than exhibiting multiple cytoplasmic and nuclear centrosomes as in the normal germinal vesicle-stage oocytes, LT/Sv oocytes typically contain a single large centrosome. In contrast, l/LnJ oocytes displayed many small centrosomes. The microtubules of normal germinal vesicle-stage oocytes were organized as arrays or asters, but microtubules were shorter in LT/Sv oocytes and absent from l/LnJ oocytes. After a 15-hr culture, centrosomal material of normal metaphase II oocytes was organized at both spindle poles. In contrast, metaphase l-arrested LT/Sv oocytes exhibited an elongated spindle with centrosomal material appearing more organized at one pole of the spindle. Both control and LT/Sv oocytes displayed cytoplasmic centrosomes. Metaphase l-arrested l/LnJ oocytes rarely had cytoplasmic centrosomes but exhibited centrosomal foci at the spindle periphery. Thus, oocytes that are atypical in the progression of meiotic maturation displayed aberrant configurations of microtubules and centrosomes, which are thought to participate in the regulation of meiotic maturation.     

Microinjected centromere [corrected] kinetochore antibodies interfere with chromosome movement in meiotic and mitotic mouse oocytes [published erratum appears in J Cell Biol 1990 Dec;111(6 Pt 1):following 2800]

Kinetochores may perform several functions at mitosis and meiosis including: (a) directing anaphase chromosome separation, (b) regulating prometaphase alignment of the chromosomes at the spindle equator (congression), and/or (c) capturing and stabilizing microtubules. To explore these functions in vivo, autoimmune sera against the centromere/kinetochore complex are microinjected into mouse oocytes during specific phases of first or second meiosis, or first mitosis. Serum E.K. crossreacts with an 80-kD protein in mouse cells and detects the centromere/kinetochore complex in permeabilized cells or when microinjected into living oocytes. Chromosome separation at anaphase is not blocked when these antibodies are microinjected into unfertilized oocytes naturally arrested at second meiotic metaphase, into eggs at first mitotic metaphase, or into immature oocytes at first meiotic metaphase. Microtubule capture and spindle reformation occur normally in microinjected unfertilized oocytes recovering from cold or microtubule disrupting drugs; the chromosomes segregate correctly after parthenogenetic activation. Prometaphase congression is dramatically influenced when antikinetochore/centromere antibodies are introduced during interphase or in prometaphase-stage meiotic or mitotic eggs. At metaphase, these oocytes have unaligned chromosomes scattered throughout the spindle with several remaining at the poles; anaphase is aberrant and, after division, karyomeres are found in the polar body and oocyte or daughter blastomeres. Neither nonimmune sera, diffuse scleroderma sera, nor sham microinjections affect either meiosis or mitosis. These results suggest that antikinetochore/centromere antibodies produced by CREST patients interfere with chromosome congression at prometaphase in vivo.     

Analysis of the mechanism(s) of metaphase I-arrest in strain LT mouse oocytes: delay in the acquisition of competence to undergo the metaphase I/anaphase transition.

Fully grown oocytes of most laboratory mice progress without interruption from the germinal vesicle (GV) stage to metaphase II, where meiosis is arrested until fertilization. In contrast, many oocytes of strain LT mice arrest precociously at metaphase I and often undergo subsequent spontaneous parthenogenetic activation. Cytostatic factor (CSF), which prevents the degradation of cyclin B and maintains high maturation-promoting factor (MPF) activity, is required for maintenance of metaphase I-arrest in LT oocytes, similar to its requirement for maintaining metaphase II-arrest in normal oocytes. However, CSF does not instigate metaphase I-arrest since a temporary metaphase I-arrest occurs in MOS-null LT oocytes. This paper addresses the mechanism(s) that may instigate metaphase I-arrest and tests the hypothesis that there may be one or more defects in LT oocytes that delay their acquisition of competence to trigger the cascade of processes that normally drive entry into and progression through anaphase I. To test this hypothesis, MPF activity was artificially abrogated by treating oocytes with a general protein kinase inhibitor, 6-DMAP, at various times during the progression of meiosis I. This allowed a comparison of the time at which LT and normal oocytes become competent to undergo the metaphase I/anaphase transition even if oocytes were arrested at metaphase I when 6-DMAP-treatment was begun. There were no differences between LT and control oocytes in the kinetics of MPF suppression by 6-DMAP. However, it was found that LT oocytes do not acquire competence to undergo the metaphase I/anaphase transition in response to 6-DMAP until 50-60 min after normal oocytes. A similar delay was observed in strain CX8-4 oocytes, which also have a high incidence of metaphase I-arrest, but not in strain CX8-11 oocytes, which exhibit a low incidence of metaphase I-arrest. MOS-null LT oocytes also exhibit a delay in acquisition of competence to undergo the metaphase I/anaphase transition. Thus, a delay in competence to undergo the metaphase I/anaphase transition in response to 6-DMAP-treatment correlates with metaphase I-arrest. It is therefore hypothesized that the observed delay in acquisition of competence to enter anaphase I may instigate the sustained metaphase I-arrest in LT oocytes by allowing CSF activity to rise to a level that prevents cyclin B degradation and maintains high MPF activity before anaphase can be initiated by normal triggering mechanisms.     

Spontaneous and induced activation of rat oocytes

Ovulated rat oocytes undergo spontaneous activation during in vitro culture. After extrusion of the second polar body, they do not enter interphase but are arrested again in next metaphase-like stage (M III arrest). The present study demonstrates that puromycin and chloral hydrate can trigger transition to interphase of metaphase II and spontaneously (incompletely) activated rat oocytes. The response of oocytes to these activators depends on their stage at the time of application of a stimulus. Metaphase II oocytes enter interphase at 86.8% when treated with puromycin and in 28.7% after chloral hydrate activation. Oocytes activated with chloral hydrate at the time of spontaneously induced anaphase II enter interphase at 64.8%, but after reaching the stage of telophase II their capability to shift to interphase is again low (28.8%). Finally, M III oocytes cannot be forced to enter interphase by either chloral hydrate or puromycin treatment. This study shows that resumption of the second meiotic division and transition to interphase--the two processes that normally occur in succession as a response to oocyte activatin--can be experimentally separated.     

Evidence that multifunctional calcium/calmodulin-dependent protein kinase II (CaM KII) participates in the meiotic maturation of mouse oocytes

Calcium-dependent signaling pathways are thought to be involved in the regulation of mammalian oocyte meiotic maturation. However, the molecular linkages between the calcium signal and the processes driving meiotic maturation are not clearly defined. The present study was conducted to test the hypothesis that the multi-functional calcium/calmodulin-dependent protein kinase II (CaM KII) functions as one of these key linkers. Mouse oocytes were treated with a pharmacological CaM KII inhibitor, KN-93, or a peptide CaM KII inhibitor, myristoylated AIP, and assessed for the progression of meiosis. Two systems for in vitro oocyte maturation were used: (1) spontaneous gonadotropin-independent maturation and (2) follicle-stimulating hormone (FSH)-induced reversal of hypoxanthine-mediated meiotic arrest. FSH-induced, but not spontaneous germinal vesicle breakdown (GVB) was dose-dependently inhibited by both myristoylated AIP and KN-93, but not its inactive analog, KN-92. However, emission of the first polar body (PB1) was inhibited by myristoylated AIP and KN-93 in both oocyte maturation systems. Oocytes that failed to produce PB1 exhibited normal-appearing metaphase I chromosome congression and spindles indicating that CaM KII inhibitors blocked the metaphase I to anaphase I transition. Similar results were obtained when the oocytes were treated with a calmodulin antagonist, W-7, and matured spontaneously. These results suggest that CaM KII, and hence the calcium signaling pathway, is potentially involved in regulating the meiotic maturation of mouse oocytes. This kinase both participates in gonadotropin-induced resumption of meiosis, as well as promoting the metaphase I to anaphase I transition. Further evidence is therefore, provided of the critical role of calcium-dependent pathways in mammalian oocyte maturation.     

Ultrastructural analysis of abnormalities in the morphology of the second meiotic spindle in ethanol-induced parthenogenones

A high frequency of parthenogenetic activation occurs when ovulated mouse oocytes are briefly exposed to a dilute solution of ethanol in vitro. Cytogenetic analyses of parthenogenones at metaphase of the first cleavage division have confirmed that parthenogenetic activation, per se, does not increase the incidence of chromosome segregation errors during the completion of the second meiotic division. Ethanol-induced activation, however, significantly increases the incidence of aneuploidy. The ultrastructural changes that occur in the morphology and organization of the second meiotic spindle apparatus in ethanol- and hyaluronidase-activated oocytes is reported here. Abnormalities in the arrangement of microtubule arrays and chromosome position were principally observed in ethanol-activated oocytes at anaphase and telophase of the second meiotic division, but were only rarely observed in hyaluronidase-activated oocytes. It is proposed that the abnormalities in spindle morphology and chromosome displacement observed in ethanol-activated oocytes represent the initial events that lead to chromosome segregation errors following exposure to this agent.     

Granulosa Cell Deficient Follicles

The diameters of oocytes in follicles having a single layer of granulosa cells were measured hi four week old mice of various strains. There is a unique population of these follicles hi strains LT/Sv and C58/J in which the oocytes are significantly larger than the oocytes in single granulosa cell layered follicles of other common strains (C57BL/6J, BALB/cJ, and DBA/2J). These unique follicles are referred to as granulosa cell deficient (GCD) follicles since oocytes of these sizes are usually found in follicles with more than a single layer of granulosa cells. The parthenogenetic embryos that give rise to ovarian teratomas in strain LT/Sv are usually found in GCD-follicles. Some of the ova of strains LT/Sv and LTXBP, but not the ova of the other strains, are capable of spontaneous parthenogenetic activation after meiotic maturation. Although the ovulated ova of strain LTXBP are capable of spontaneous parthenogenetic development, the frequency of GCD-follicles and teratocarcinogenesis is low. Therefore, the frequency of ovarian teratocarcinogenesis is correlated with the simultaneous occurrence of two atypical conditions: first, the capability of the matured ova to undergo spontaneous parthenogenetic activation and, second, the high frequency of GCD-follicles.
GCD-follicles containing oocytes with a diameter greater than 65 μm were studied by electron microscopy. The follicles are usually enclosed within a layer of flattened theca-like cells. A basal lamina separates these cells from a single layer of cuboidal granulosa cells. Granulosa cell processes traverse the zona pellucida to contact the oocyte which shows ultrastructural characteristics typical of oocytes in the final growth stages. It is proposed that the GCD-follicles are competent to participate in the normal functions of follicular cells relating to oocyte growth and meiotic maturation.     

Preimplantation embryonic development of spontaneous mouse parthenotes after oocyte meiotic maturation in vitro

Of eggs ovulated in LT/Sv mice, 10–20% undergo spontaneous parthenogenetic activation, and 40–50% of the parthenotes develop to blastocysts when cultured in simple defined medium from the one-cell stage. Similar percentages of oocytes isolated from Graafian follicles undergo parthenogenetic activation after spontaneous maturation in simple defined medium, but embryonic development proceeds no further than the two-cell stage. The simple defined medium that supported preimplantation development of ovulated eggs and spontaneous maturation of extrafollicular oocytes contained no serum, free amino acids, or vitamins. The present experiments were conducted to determine what conditions during spontaneous maturation of extrafollicular oocytes could promote the ability of oocytes to develop to blastocysts after parthenogenetic activation and mimic the environment of preovulatory follicles. Cumulus-enclosed oocytes that were matured in simple medium supplemented with fetal bovine serum (FBS) developed to blastocysts after spontaneous parthenogenetic activation. Furthermore, minimum essential medium (MEM), a complex medium containing free amino acids and vitamins, could substitute completely for FBS for maturing oocytes from (C57BL/6J × LT/Sv)F₁ mice, and to a lesser extent for maturing LT/Sv oocytes. Therefore, even though germinal vesicle breakdown in mouse oocytes and preimplantation development of mouse eggs can occur in the absence of an exogenous supply of free amino acids and vitamins, a complete, or normal, mouse oocyte maturation cannot. These results also demonstrated that gonadotropins are not necessary during oocyte meiotic maturation for parthenogenetically activated eggs to develop through the preimplantation stages. Luteinizing hormone or 17β-estradiol in MEM during oocyte maturation had no effect on the subsequent development of parthenotes. In contrast, follicle stimulating hormone (FSH) and progesterone in the maturation medium decreased the number of ova that subsequently cleaved, and FSH decreased the number of cleaved eggs that developed to blastocysts.     

A metaphase pause: Hormone-induced maturation progresses through a long pause at the first meiotic metaphase in oocytes of the starfish, Pisaster ochraceus

In several species of starfish, it has been reported that the meiotic divisions in fertilized oocytes occur precociously compared to those in unfertilized oocytes. The nature of the 'acceleration' of meiosis was studied using Pisaster ochraceus oocytes. The extent of the acceleration of first polar body formation was found to be completely dependent on the time of fertilization (or artificial activation); fertilization at about 100 min after 1–methyladenine application accelerated meiosis I the most, while earlier or later fertilization resulted in a smaller extent of accelerations of meiosis I. Observation of isolated meiotic spindles and fluorescent visualization of meiotic spindles in whole oocytes showed that progression of meiosis I in Pisaster oocytes pauses transiently at metaphase I for more than 40min unless they are activated. The activation shortened the duration of metaphase I, which resulted in the acceleration of first polar body formation. A new term 'metaphase pause' is proposed to define this long duration of metaphase I in starfish oocytes.     

The metaphase II arrest in mouse oocytes is controlled through microtubule-dependent destruction of cyclin B in the presence of CSF.

In unfertilized eggs from vertebrates, the cell cycle is arrested in metaphase of the second meiotic division (metaphase II) until fertilization or activation. Maintenance of the long-term meiotic metaphase arrest requires mechanisms preventing the destruction of the maturation promoting factor (MPF) and the migration of the chromosomes. In frog oocytes, arrest in metaphase II (M II) is achieved by cytostatic factor (CSF) that stabilizes MPF, a heterodimer formed of cdc2 kinase and cyclin. At the metaphase/anaphase transition, a rapid proteolysis of cyclin is associated with MPF inactivation. In Drosophila, oocytes are arrested in metaphase I (M I); however, only mechanical forces generated by the chiasmata seem to prevent chromosome separation. Thus, entirely different mechanisms may be involved in the meiotic arrests in various species. We report here that in mouse oocytes a CSF-like activity is involved in the M II arrest (as observed in hybrids composed of fragments of metaphase II-arrested oocytes and activated mitotic mouse oocytes) and that the high activity of MPF is maintained through a continuous equilibrium between cyclin B synthesis and degradation. In addition, the presence of an intact metaphase spindle is required for cyclin B degradation. Finally, MPF activity is preferentially associated with the spindle after bisection of the oocyte. Taken together, these observations suggest that the mechanism maintaining the metaphase arrest in mouse oocytes involves an equilibrium between cyclin synthesis and degradation, probably controlled by CSF, and which is also dependent upon the three-dimensional organization of the spindle.     

Participation of the ubiquitin-proteasome pathway in rat oocyte activation

The role of the ubiquitin-proteasome pathway (UPP) in mitosis is well known. However, its role in meiotic division is still poorly documented, especially in the activation of mammalian oocytes. In this study, the role of proteasome in the spontaneous and parthenogenetic activation of rat oocytes was investigated. We found that ALLN, an inhibitor of proteasome, when applied to metaphase II oocytes, inhibited spontaneous activation, blocked extrusion of the second polar body (PB) and caused the withdrawal of the partially extruded second PB. ALLN also inhibited the parthenogenetic activation induced by cycloheximide, but had no effect on the formation of pronuclei in activated eggs. In metaphase and anaphase, ubiquitin and proteasome localized to the meiotic spindle, concentrating on both sides of the oocyte-second PB boundary during PB extrusion. This pattern of cellular distribution suggests that UPP may have a role in regulating nuclear division and cytokinesis. Ubiquitin was seen to form a ring around the pronucleus, whereas proteasome was evenly distributed in the pronuclear region. Taken together, our results indicate that (1) UPP is required for the transitions of oocytes from metaphase II to anaphase II and from anaphase II to the end of meiosis; and (2) the UPP plays a role in cytokinesis of the second meiotic division.     

Cytoplasmic modification of the nuclear lamina during pronuclear-like transformation of mouse blastomere nuclei.

During the successive interphases of cleaving mouse embryos the nuclear periphery diminishes its reactivity to anti-lamin A and C antibodies. This developmentally regulated characteristic can be modified by exposure of the blastomere nuclei to metaphase II (M II) oocyte cytoplasm followed by activation. In the current study we define the cytoplasmic conditions necessary for this modification of 8-cell and 16-cell stage nuclei in hybrids obtained by fusion with metaphase II arrested oocytes, oocytes at various time points after parthenogenetic activation, naturally fertilized eggs (zygotes) and interphase 2-cell embryo blastomeres. The intensity of fluorescence obtained with anti-lamins A/C in the blastomere nuclei increases as a result of fusion with freshly activated oocytes or early zygotes (first 3.0-5.5 h in the case of parthenogenetic activation), and not when eggs or 2-cell blastomeres advanced in interphase are used as partners for fusion. This transformation of the A/C lamin pattern is correlated with the ability to promote pronucleus-like growth of blastomere nuclei in hybrids. Blastomere nuclei introduced into M II-arrested oocytes undergo premature chromatin condensation and dissolution of the nuclear lamina. The results are discussed with regard to certain particularities of the first embryonic interphase of the mouse and the potential involvement of nuclear lamins in pronuclear growth.     

Differential regulation of cyclin B1 degradation between the first and second meiotic divisions of bovine oocytes

During mammalian oocyte maturation, two consecutive meiotic divisions are required to form a haploid gamete. For each meiotic division, oocytes must transfer from metaphase to anaphase, but maturation promoting factor (cyclin-dependent kinase 1/cyclin B1) activity would keep the oocytes at metaphase. Therefore, inactivation of maturation promoting factor is needed to finish the transition and complete both these divisions; this is provided through anaphase-promoting complex/cyclosome-dependent degradation of cyclin B1. The objective of this study was to examine meiotic divisions in bovine oocytes after expression of a full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion, coupled with the fluorochrome Venus, by microinjecting their complementary RNA (cRNA). Overexpression of full-length cyclin B1-Venus inhibited homologue disjunction and first polar body formation in maturing oocytes (control 70% vs. overexpression 16%; P < 0.05). However at the same levels of expression, it did not block second meiotic metaphase and cleavage of eggs after parthenogenetic activation (control: 82% pronuclei and 79% cleaved; overexpression: 91% pronuclei and 89% cleaved). The full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion caused metaphase arrest in both meiotic divisions, whereas degradation of securin was unaffected. Roscovitine, a potent cyclin-dependent kinase 1 (CDK1) inhibitor, overcame this metaphase arrest in maturing oocytes at 140 μM, but higher doses (200 μM) were needed to overcome arrest in eggs. In conclusion, because metaphase I (MI) blocked by nondegradable cyclin B1 was distinct from metaphase II (MII) in their different sensitivities to trigger CDK1 inactivation, we concluded that mechanisms of MI arrest differed from MII arrest.