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The relationship between G proteins and luteinizing hormone activated meiotic resumption, during oocyte maturation, is analyzed.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.06.034 Byline: Noriyo Takeda (a)(b), Keiichiro Kyozuka (b), Ryusaku Deguchi (a) Keywords: Jellyfish; Cyclic AMP; Meiosis; GVBD; cAMP-dependent protein kinase; Umbrella-free medusa; Spawning; 8-Br-cAMP; Caged cAMP; H-89 Abstract: In medusae of the hydrozoan Cytaeis uchidae, oocyte meiotic maturation and spawning occur as a consequence of dark-light transition. In this study, we investigated the mechanism underlying the initiation of meiotic maturation using in vitro (isolated oocytes from ovaries) and in vivo (ovarian oocytes in medusae) systems. Injection of cAMP derivatives into isolated oocytes induced meiotic maturation in a dose-dependent manner. Meiotic maturation was also achieved in isolated oocytes preloaded with caged cAMP and exposed to UV irradiation. The caged cAMP/UV irradiation-induced meiotic maturation was completely inhibited by blockers of protein kinase A (PKA), H-89, KT5720, and Rp-cAMPS. The medusae from which most parts of the umbrella were removed (umbrella-free medusae) survived for at least 2 weeks, during which time oocyte meiotic maturation and spawning occurred. When H-89 and Rp-cAMPS were injected into ovarian oocytes of umbrella-free medusae within 3 min of dark-light stimulation, meiotic maturation was inhibited or delayed. An increase in intracellular cAMP was confirmed by FlCRhR, a fluorescent cAMP indicator, in ovarian oocytes exposed to dark-light transition as well as in isolated oocytes stimulated by caged cAMP/UV irradiation. These results indicate that the cAMP/PKA signaling pathway positively contributes to light-triggered physiological oocyte meiotic maturation in Cytaeis uchidae. Author Affiliation: (a) Department of Biology, Miyagi University of Education, Aoba-ku, Sendai, Miyagi 980-0845, Japan (b) Research Center for Marine Biology, Asamushi, Graduate School of Life Science, Tohoku University, Asamushi, Aomori 039-3501, Japan Article History: Received 15 April 2006; Revised 16 June 2006; Accepted 21 June 2006

A study examines the role of yolk protein hydrolysis in the entry of water into the oocyte of fish. Meiosis process is considered in the study.

Effects of Aurora kinase on meiotic chromosome segregation during spermatogenesis in male mouse are presented.

Oocytes from most animals arrest twice during the meiotic cell cycle. The universally conserved prophase I arrest is released by a maturation hormone that allows progression to a second arrest point, typically metaphase I or II. This second arrest allows for short-term storage of fertilization-competent eggs and is released by signaling that occurs during fertilization. Nematodes are unique in that the maturation hormone is secreted by sperm rather than by the mother's somatic tissues. We have investigated the nature of the second arrest in matured but unfertilized Caenorhabditis elegans embryos using time-lapse imaging of GFP-tubulin or GFP-histone. Unfertilized embryos completed anaphase I but did not form polar bodies or assemble meiosis II spindles. Nevertheless, unfertilized embryos assembled female pronuclei at the same time as fertilized embryos. Analysis of embryos fertilized by sperm lacking the SPE-11 protein indicated that fertilization promotes meiotic cytokinesis through the SPE-11 protein but assembly of the meiosis II spindle is initiated through an SPE-11-independent pathway. Keywords: Meiosis; Fertilization; Cyclin B; Meiotic exit

We analyzed progression through the meiotic maturation in oocytes manipulated to replace the prophase oocyte nucleus with the nucleus from a cumulus cell, a pachytene spermatocyte or the pronucleus from a fertilized egg. Removal of the oocyte nucleus led to a significant reduction in histone H1 kinase activity. Replacement of the oocyte nucleus by a pronucleus followed by culture resulted in premature pseudomeiotic division and occasional abnormal cytokinesis; however, histone H1 kinase activity was rescued, microtubules formed a bipolar spindle, and chromosomes were condensed. In addition to the anomalies observed after pronuclear transfer, those after transfer of the nucleus from a cumulus cell or spermatocyte included a dramatically impaired ability to form the bipolar spindle or to condense chromosomes, and histone H1 kinase activity was not rescued. Expression of a cyclin B-YFP in enucleated oocytes receiving the cumulus cell nucleus rescued histone H1 kinase activity, but spindle formation and chromosome condensation remained impaired, indicating a pleiotropic effect of oocyte nucleus removal. However, when the cumulus cell nucleus was first transformed into pronuclei (transfer into a metaphase II oocyte followed by activation), such pronuclei supported maturation after transfer into the oocyte in a manner similar to that of normal pronuclei. These results show that the oocyte nucleus contains specific components required for the control of progression through the meiotic maturation and that some of these components are also present in pronuclei. Keywords: Prophase oocyte; Oocyte nucleus; Pronucleus; Meiotic maturation; Nuclear transfer; MPF

The evolutionarily conserved partitioning defective (PAR) protein PAR-3 is pivotal for establishing and maintaining cell polarity. During mammalian oocyte maturation, the radially symmetric oocyte is transformed into a highly polarized metaphase II (MII)-arrested egg. We therefore examined several aspects of PAR-3 expression during oocyte maturation. We cloned two novel PAR-3 transcripts from an oocyte library that likely encode proteins of [M.sub.r] = 73 K and 133 K that are phosphorylated during maturation. PAR-3, which is found throughout the GV-intact oocyte, becomes asymmetrically localized during meiosis. Following germinal vesicle breakdown, PAR-3 surrounds the condensing chromosomes and associates with the meiotic spindles. Prior to emission of the first and second polar bodies, PAR-3 is located within a central subdomain of the polarized actin cap, which overlies the spindle. This cortical PAR-3 localization depends on intact microfilaments. These results suggest a role for PAR-3 in establishing asymmetry in the egg and in defining the future site of polar body emission. Keywords: Mouse egg; PAR-3; Cortical actin; Meiosis

In vertebrates, unfertilized eggs are arrested at meiotic metaphase II (meta-II) by cytostatic factor (CSF), with Cdc2 activity maintained at a constant, high level. CSF is thought to suppress cyclin B degradation through the inhibition of the anaphase-promoting complex/cyclosome (APC/C)-Cdc20 while cyclin B synthesis continues in unfertilized eggs. Thus, it is a mystery how Cdc2 activity is kept constant during CSF arrest. Here, we show that the APC/C-Cdc20 can mediate cyclin B degradation in CSF-arrested Xenopus eggs and extracts, in such a way that when Cdc2 activity is elevated beyond a critical level, APC/C-Cdc20-dependent cyclin B degradation is activated and Cdc2 activity consequently declines to the critical level. This feedback control of Cdc2 activity is shown to be required for keeping Cdc2 activity constant during meta-II arrest. We have also shown that Mos/MAPK pathway is essential for preventing the cyclin B degradation from inactivating Cdc2 below the critical level required to sustain meta-II arrest. Our results indicate that under CSF arrest, Mos/MAPK activity suppresses cyclin B degradation, preventing Cdc2 activity from falling below normal meta-II levels, whereas activation of APC/C-Cdc20-mediated cyclin B degradation at elevated levels of Cdc2 activity prevents Cdc2 activity from reaching excessively high levels. Keywords: Cytostatic factor; Metaphase II arrest; Xenopus egg; Cyclin B degradation; The anaphase-promoting complex/cyclosome; Cdc20; Mos; MAP kinase

Unfertilized eggs usually lack maternal centrosomes and cannot develop without sperm contribution. However, several insect species lay eggs that develop to adulthood as unfertilized in the absence of a preexisting centrosome. We report that the oocyte of the parthenogenetic viviparous pea aphid Acyrthosiphon pisum is able to self-organize microtubule-based asters, which in turn interact with the female chromatin to form the first mitotic spindle. This mode of reproduction provides a good system to investigate how the oocyte can assemble new centrosomes and how their number can be exactly monitored. We propose that the cooperative interaction of motor proteins and randomly nucleated surface microtubules could lead to the formation of aster-like structures in the absence of pre-existing centrosomes. Recruitment of material along the microtubules might contribute to the accumulation of pericentriolar material and centriole precursors at the focus of the asters, thus leading to the formation of true centrosomes. The appearance of microtubule asters at the surface of activated oocytes could represent a possible common mechanism for centrosome formation during insect parthenogenesis. Keywords: Parthenogenesis; Asters; Centrosome inheritance; Spindle formation; Aphids

A-kinase-anchoring proteins (AKAP) help regulate the intracellular organization of cyclic AMP-dependent kinase (PKA) and actin within somatic cells. Elevated levels of cAMP also help maintain meiotic arrest in immature oocytes, with AKAPs implicated as critical mediators but poorly understood during this process. Here we test the hypothesis that the AKAP WAVE1 is required during mammalian fertilization, and identify a nuclear localization of WAVE1 that is independent of actin and actin-related proteins (Arp). Immunofluorescence and immunoprecipitation experiments show a redistribution of WAVE1 from the cortex in germinal vesicle (GV) oocytes to cytoplasmic foci in oocytes arrested in second meiosis (Met II). Following sperm entry, WAVE1 relocalizes to the developing male and female pronuclei. Association of WAVE1 with a regulatory subunit of PKA is detected in both Met II oocytes and pronucleate zygotes, but interaction with Arp 2/3 is observed only in Met II oocytes. WAVE1 redistributes to the cytoplasm upon nuclear envelope breakdown at mitosis, and concentrates at the cleavage furrow during embryonic cell division. Blocking nuclear pore formation with microinjected wheat germ agglutinin does not inhibit the nuclear localization of WAVE1, suggesting that this event precedes nuclear envelope formation. Neither depolymerization nor stabilization of actin affects WAVE1 distribution. Microtubule stabilization with Taxol, however, redistributes WAVE1 to the centrosome, and anti-WAVE1 antibodies prevent both the nuclear distribution of WAVE1 and the migration and apposition of pronuclei. These findings show that WAVE1 sequestration to the nucleus is required during fertilization, and is an actin-independent event that relies on dynamic microtubules but not nuclear pores. Keywords: AKAP; WAVE1; Meiosis; Fertilization; Embryo

An important event in the development of the germline is the initiation of meiotic development. In Caenorhabditis elegans, the conserved GLP-1/Notch signaling pathway regulates the proliferative versus meiotic entry decision, at least in part, by spatially inhibiting genes in the gld-1 and gld-2 parallel pathways, which are proposed to either inhibit proliferation and/or promote meiotic development. Mutations that cause constitutive activation of the GLP-1 pathway, of inactivation of both the gld-1 and gld-2 parallel pathways, result in a tumorous germline in which all cells are thought to be proliferative. Here, to analyze proliferation and meiotic entry in wild-type and mutant tumorous germlines, we use anti-REC-8 and anti-HIM-3 specific antibodies as markers, which under our fixation conditions, stain proliferative and meiotic cells, respectively. Using these makers in wild-type animals, we find that the border of the switch from proliferation to meiotic entry is staggered in late-larval and adult germlines. In wild-type adults, the switch occurs between 19 and 26 cell diameters from the distal end, on average. Our analysis of mutants reveals that tumorous germlines that form when GLP-1 is constitutively active are completely proliferative, while tumors due to inactivation of the gld-1 and gld-2 pathways show evidence of meiotic entry. Genetic and time course studies suggest that a third pathway may exist, parallel to the GLD-1 and GLD-2 pathways, that promotes meiotic development. Keywords: Germline development; Proliferation; Meiotic entry; Germline rumor; GLP-1/Notch; gld-1; gld-2; nos-3

Maintenance of meiotic prophase arrest in fully grown vertebrate oocytes depends on an elevated level of cAMP in the oocyte. To investigate how the cAMP level is regulated, we examined whether the activity of an oocyte G protein of the family that stimulates adenylyl cyclase, [G.sub.s], is required to maintain meiotic arrest. Microinjection of a dominant negative form of [G.sub.s] into Xenopus and mouse oocytes, or microinjection of an antibody that inhibits the [G.sub.s] G protein into zebrafish oocytes, caused meiosis to resume. Together with previous studies, these results support the conclusion that [G.sub.s]-regulated generation of cAMP by the oocyte is a common mechanism for maintaining meiotic prophase arrest in vertebrate oocytes. Keywords: Meiotic prophase arrest; Oocyte maturation; Heterotrimeric (G proteins; Zebrafish; Xenopus; Mouse

One current theory of the Golgi apparatus views its organization as containing both a matrix fraction of structural proteins and a reservoir of cycling enzymes. During mitosis, the putative matrix protein GM130 is phosphorylated and relocalized to spindle poles. When the secretory pathway is inhibited during interphase, GM130 redistributes to regions adjacent to vesicle export sites on the endoplasmic reticulum (ER). Strikingly, meiotic maturation and fertilization in nonrodent mammalian eggs presents a unique experimental environment for the Golgi apparatus, because secretion is inhibited until after fertilization, and because the centrosome is absent until introduced by the sperm. Here, we test the hypothesis that phosphorylated GM130 associates not with meiotic spindle poles, but with ER clusters in the mature bovine oocyte. At the germinal vesicle stage, phosphorylated GM130 is observed as fragments dispersed throughout the cytoplasm. During meiotic maturation, GM130 reorganizes into punctate foci that associate near the ER-resident protein calreticulin and is notably absent from the meiotic spindle. GM130 colocalizes with Sec23, a marker for ER vesicle export sites, but not with Lens culinaris agglutinin, a marker for cortical granules. Because disruption of vesicle transport has been shown to block meiotic maturation and embryonic cleavage in some species, we also test the hypothesis that fertilization and cytokinesis are inhibited with membrane trafficking disruptor brefeldin A (BFA). Despite Golgi fragmentation after BFA treatment, pronuclei form and unite, and embryos cleave and develop through the eight-cell stage. We conclude that, while the meiotic phosphorylation cycle of GM 130 mirrors that of mitosis, absence of a maternal centrosome precludes Golgi association with the meiotic spindle. Fertilization introduces the sperm centrosome that can reorganize Golgi proteins, but neither fertilization nor cytokinesis prior to compaction requires a functional Golgi apparatus. Keywords: Golgi apparatus; Oocyte; Meiosis; Fertilization; Phosphorylation

The molecular events regulating hormone-induced oocyte activation and meiotic maturation are probably best understood in Xenopus laevis. In X. laevis, progesterone activates the G2-arrested oocyte, induces entry into M phase of meiosis I (MI) and resumption of the meiotic cell cycles, and leads to the formation of a mature, fertilizable egg. Oocytes of Xenopus tropicalis offer several practical advantages over those of X. laevis, including faster and more synchronous meiotic cell cycle progression, less seasonal variability, and the availability of transgenic approaches. Previous work found several similarities in the pathways regulating oocyte maturation in the two species. Here, we report several additional ones that are conserved in X. tropicalis. (1) Injection of Mos mRNA into G2-arrested oocytes activates the MAP kinase cascade and induces the G2/MI transition. (2) Injection of the [beta] subunit of the kinase CK2 (a negative regulator of Mos and oocyte activation) delays the G2/MI transition. (3) Elevating PKA activity blocks progesterone-induced maturation; repressing PKA activity induces entry into MI in the absence of progesterone. (4) LF (anthrax lethal factor), which cleaves certain MAP kinase kinases, strongly reduces both the rate and extent of entry into MI. In contrast to the one previously reported major difference between oocytes of the two species, we find that injection of egg cytoplasm ('MPF activity') into G2-arrested X. tropicalis oocytes induces entry into meiosis I even when protein synthesis is blocked, just as it does in oocytes of X. laevis. These results indicate that much of what we have learned from studies of X. laevis oocytes holds for those of X. tropicalis, and suggest that X. tropicalis oocytes offer a good experimental system for investigating certain questions that require a rapid, synchronous progression through the G2/meiosis I transition.

In most animals, successful segregation of female meiotic chromosomes involves sequential associations of the meiosis I and meiosis II spindles with the cell cortex so that extra chromosomes can be deposited in polar bodies. The resulting reduction in chromosome number is essential to prevent the generation of polyploid embryos after fertilization. Using time-lapse imaging of living Caenorhabditis elegans oocytes containing fluorescently labeled chromosomes or microtubules, we have characterized the movements of meiotic spindles relative to the cell cortex. Spindle assembly initiated several microns from the cortex. After formation of a bipolar structure, the meiosis I spindle translocated to the cortex. When microtubules were partially depleted, translocation of the bivalent chromosomes to the cortex was blocked without affecting cell cycle timing. In oocytes depleted of the microtubule-severing enzyme, MEI-1, spindles moved to the cortex, but association with the cortex was unstable. Unlike translocation of wild-type spindles, movement of MEI-1-depleted spindles was dependent on FZY-1/CDC20, a regulator of the metaphase/anaphase transition. We observed a microtubule and FZY-1/CDC20-dependent circular cytoplasmic streaming in wild-type and mei-1 mutant embryos during meiosis. We propose that, in mei-1 mutant oocytes, this cytoplasmic streaming is sufficient to drive the spindle into the cortex. Cytoplasmic streaming is not the normal spindle translocation mechanism because translocation occurred in the absence of cytoplasmic streaming in embryos depleted of either the orbit/CLASP homolog, CLS-2, or FZY-1. These results indicate a direct role of microtubule severing in translocation of the meiotic spindle to the cortex.

Fertilization-induced intracellular calcium ([Ca.sup.2+]) oscillations stimulate the onset of mammalian development, and little is known about the biochemical mechanism by which these [Ca.sup.2+] signals are transduced into the events of egg activation. This study addresses the hypothesis that transient increases in [Ca.sup.2+] similar to those at fertilization stimulate oscillatory [Ca.sup.2+]/calmodulin-dependent kinase II (CaMKII) enzyme activity, incrementally driving the events of egg activation. Since groups of fertilized eggs normally oscillate asynchronously, synchronous oscillatory [Ca.sup.2+] signaling with a frequency similar to fertilization was experimentally induced in unfertilized mouse eggs by using ionomycin and manipulating extracellular calcium. Coanalysis of intracellular [Ca.sup.2+] levels and CaMKII activity in the same population of eggs demonstrated a rapid and transient enzyme response to each increase in [Ca.sup.2+]. Enzyme activity increased 370% during the first [Ca.sup.2+] rise, representing about 60% of maximal activity, and had decreased to basal levels within 5 min from the time [Ca.sup.2+] reached its peak value. Single fertilized eggs monitored for [Ca.sup.2+] had a mean increase in CaMKII activity of 185%. One and two ionomycin-induced [Ca.sup.2+] transients resulted in 39 and 49% mean cortical granule (CG) loss, respectively, while CG exocytosis and resumption of meiosis were inhibited by a CaMKII antagonist. These studies demonstrate that changes in the level of [Ca.sup.2+] and in CaMKII activity can be studied in the same cell and that CaMKII activity is exquisitely sensitive to experimentally induced oscillations of [Ca.sup.2+] in vivo. The data support the hypothesis that CaMKII activity oscillates for a period of time after normal fertilization and temporally regulates many events of egg activation. Keywords: Egg; Intracellular calcium; CaMKII; Cell cycle; Secretion; Meiosis; Fertilization

The intracellular levels of cAMP play a critical role in the meiotic arrest of mammalian oocytes. However, it is debated whether this second messenger is produced endogenously by the oocytes or is maintained at levels inhibitory to meiotic resumption via diffusion from somatic cells. Here, we demonstrate that adenylyl cyclase genes and corresponding proteins are expressed in rodent oocytes. The mRNA coding for the AC3 isoform of adenylyl cyclase was detected in rat and mouse oocytes by RT-PCR and by in situ hybridization. The expression of AC3 protein was confirmed by immunocytochemistry and immunofluorescence analysis in oocytes in situ. Cyclic AMP accumulation in denuded oocytes was increased by incubation with forskolin, and this stimulation was abolished by increasing intraoocyte [Ca.sub.2+] with the ionophore A23187. The [Ca.sub.2+] effects were reversed by an inhibitor of [Ca.sub.2+], calmodulin-dependent kinase II. These regulations of cAMP levels indicate that the major cyclase that produces cAMP in the rat oocyte has properties identical to those of recombinant or endogenous AC3 expressed in somatic cells. Furthermore, mouse oocytes deficient in AC3 show signs of a defect in meiotic arrest in vivo and accelerated spontaneous maturation in vitro. Collectively, these data provide evidence that an adenylyl cyclase is functional in rodent oocytes and that its activity is involved in the control of oocyte meiotic arrest. Keywords: cAMP; Adenylyl cyclase; Oocyte; Meiosis; Cell cycle

The number of germ cells reaches the maximum just prior to entry into meiosis, yet decreases dramatically by a few days after birth in the female mouse, rat, and human. Previous studies have reported a major loss at the pachytene stage of meiotic prophase during fetal development, leading to the hypothesis that chromosomal pairing abnormalities may be a signal for oocyte death. However, the identification as well as the quantification of germ cells in these studies have been questioned. A recent study using Mouse Vasa Homologue (MVH) as a germ cell marker reached a contradictory conclusion claiming that oocyte loss occurs in the mouse only after birth. In the present study, we established a new method to quantify murine germ cells by using Germ Cell Nuclear Antigen-1 (GCNA-I) as a germ cell marker. Comparison of GCNA-1 and MVH immunolabeling revealed that the two markers identify the same population of germ cells. However, nuclear labeling of GCNA-1 was better suited for counting germ cells in histological sections as well as for double labeling with the antibody against synaptonemal complex (SC) proteins in chromosome spreading preparations. The latter experiment demonstrated that the majority of GCNA-1-labeled cells entered and progressed through meiotic prophase during fetal development. The number of GCNA-1-positive cells in the ovary was estimated by counting the labeled cells retained in chromosome spreading preparations and also in histological sections by using the ratio estimation method. Both methods demonstrated a continuous decline in the number of GCNA-I-labeled cells during fetal development when the oocytes progress through meiotic prophase. These observations suggest that multiple causes are responsible for oocyte elimination. Keywords: Mouse fetal ovary; Germ cell; Oocyte; GCNA-1; MVH; Meiotic prophase

Sea urchins are members of a limited group of animals in which meiotic maturation of oocytes is completed prior to fertilization. This is different from oocytes of most animals such as mammals and amphibians in which fertilization reactivates an arrested meiotic cycle. Using a recently developed technique for in vitro maturation of sea urchin oocytes, we analyzed the role of cyclin B, the regulatory component of maturation-promoting factor, in the control of sea urchin oocyte meiotic induction and progression. Oocytes of the sea urchin Lytechinus variegatus accumulate significant amounts of cyclin B mRNA and protein during oogenesis. We analyzed cyclin B synthetic requirements in oocytes and early embryos by inhibiting cyclin B synthesis with DNA and morpholino antisense oligonucleotides. Cyclin B synthesis is not necessary for the entry of G2-arrested oocytes into meiosis; however, it is required for the proper progression through meiotic divisions. Surprisingly, mature sea urchin eggs contain significant cyclin B protein following meiosis that serves as a maternal store for early cleavage divisions. We also find that cyclin A can functionally substitute for cyclin B in early embryos but not in oocytes. These studies provide a foundation for understanding the mechanism of meiotic maturation independent of the zygotic cell cycle. Keywords: Meiosis; Egg; Cyclin B; MPF, Embryo

Resumption of meiosis in Oocytes of Xenopus tropicalis required translation but not transcription, and was marked by the appearance of a white spot and a dark ring, coincident with entry into metaphase I and the onset of anaphase I, respectively. Cyclin [B.sub.2]/[p34.sup.cdc2] activity increased prior to the first meiotic division, declined at the onset of anaphase I, and subsequently increased again. The capacity of egg cytoplasm to induce germinal vesicle breakdown (GVBD) was inhibited by cycloheximide, despite the fact that these oocytes contained cyclin [B.sub.2]/[p34.sup.cdc2] complexes. However, cycloheximide-treated oocytes underwent GVBD following injection of constitutively active mitogen-activated protein kinase (MAPK) kinase 2 (MEK2), [p33.sup.Ringo], or [DELTA]90 cyclin B. MAPK activity increased just prior to the first meiotic division and remained stable thereafter. Although injection of constitutively active MEK2 induced GVBD, treatment with the MEK inhibitors U0126 or anthrax lethal factor delayed GVBD and prevented spindle formation. Interestingly, the ability of egg cytoplasm to induce GVBD was unaffected by the inhibition of MEK activity. Our results indicate that the synthesis of a novel or short-lived protein(s) which acts in a MEK-independent fashion is required in order for egg cytoplasm to induce GVBD in X. tropicalis oocytes. Key Words: Xenopus tropicalis; oocyte; meiosis; MPF; MAPK.

To reach fertilization competence, sperm undergo an incompletely understood series of morphological and molecular maturational processes, termed capacitation, involving, among other processes, protein tyrosine phosphorylation and increased intracellular calcium. Hyperactivated motility and an ability to undergo the acrosome reaction serve as physiological end points to assess successful capacitation. We report here that acidic (pI 4.0) 86-kDa isoforms of a novel, polymorphic, testis-specific protein, designated calcium-binding tyrosine phosphorylation-regulated protein (CABYR), were tyrosine phosphorylated during in vitro capacitation and bound [sup.45]Ca on 2D gels. Acidic 86-kDa calcium-binding forms of CABYR increased during in vitro capacitation, and calcium binding to these acidic forms was abolished by dephosphorylation with alkaline phosphatase. Six variants of CABYR containing two coding regions (CR-A and CR-B) were cloned from human testis cDNA libraries, including five variants with alternative splice deletions. A motif homologous to the RII dimerization domain of PK-A was present in the N-terminus of CR-A in four CABYR variants. A single putative EF handlike motif was noted in CR-A at aas 197-209, while seven potential tyrosine phosphorylation-like sites were noted in CR-A and four in CR-B. Pro-X-X-Pro (PXXP) modules were identified in the N- and C-termini of CR-A and CR-B. CABYR localizes to the principal piece of the human sperm flagellum in association with the fibrous sheath and is the first demonstration of a sperm protein that gains calcium-binding capacity when phosphorylated during capacitation. Key Words: CABYR; spermatozoa; capacitation; calcium-binding protein; fibrous sheath; tyrosine phosphorylation; postmeiotic expression.

Following fertilization, the oocyte remodels the sperm chromatin into the male pronucleus. As a component of this process, during meiotic maturation, oocytes develop an activity that transfers histones onto sperm DNA. To further characterize this activity, we tested whether oocytes at different stages of growth could, upon entry into metaphase of maturation, transfer histones onto sperm DNA, as judged by chromatin morphology and immunocytochemistry. Meiotically competent growing oocytes, which spontaneously enter metaphase upon culture, tranferred histones onto sperm chromatin, whereas incompetent oocytes did not, even when treated with okadaic acid to induce germinal vesicle breakdown (GVBD) and chromosome condensation. When incompetent oocytes were cultured until they acquired the ability to undergo GVBD, only a small proportion also developed histone-transfer activity during maturation. However, this proportion significantly increased when the oocytes were cultured as granulosa-oocyte complexes. The failure of histone-transfer activity to develop in incompetent oocytes treated with okadaic acid was not linked to low H1 kinase activity nor rescued by injected histones. Because competent, but not incompetent, oocytes produce natural calcium oscillations, incompetent oocytes were exposed to Sr[Cl.sub.2]. One-third of treated oocytes produced at least one [Ca.sup.2+] oscillation and, following insemination, the same proportion transferred histones onto sperm DNA. Histone transfer did not occur in oocytes pretreated with the [Ca.sup.2+]. chelator, BAPTA-AM. These results indicate that the ability to develop histone-transfer activity is acquired by growing oocytes near the time of meiotic competence, that it is separable from this event, and that it may be regulated through a [Ca.sup.2+]-dependent process. Key Words: sperm; chromatin; histones; oocyte growth; meiotic competence; calcium.

The topography of microtubule assembly events during meiotic maturation of animal oocytes demands tight spatial control and temporal precision. To better understand what regulates the timing and location of microtubule assembly, synchronously maturing mouse oocytes were evaluated with respect to [gamma]-tubulin, pericentrin, and total tubulin polymer fractions at specific stages of meiotic progression. [gamma]-Tubulin remained associated with cytoplasmic centrosomes through diakinesis of meiosis-1. Following chromatin condensation and perinuclear centrosome aggregation, [gamma]-tubulin relocated to a nuclear lamina-bounded compartment in which meiosis-1 spindle assembly occurred. [gamma]-Tubulin was stably associated with the meiotic spindle from prometaphase-1 through to anaphase-2, but also exhibited cell cycle-specific relocalization to cytoplasmic centrosomes. Specifically, anaphase onset of both meiosis-1 and -2 was characterized by the concomitant appearance of [gamma]-tubulin and microtubule nucleation in subcortical centrosomes. Brief pulses of taxol applied at specific cell cycle stages enhanced detection of [gamma]-tubulin compartmentalization, consistent with a [gamma]-tubulin localization-dependent spatial restriction of microtubule assembly during meiotic progression. In addition, a taxol pulse during meiotic resumption impaired subsequent [gamma]-tubulin sorting, resulting in monopolar spindle formation and cell cycle arrest in meiosis-1; despite cell cycle arrest, polar body extrusion occurred roughly on schedule. Therefore, sorting of [gamma]-tubulin is involved in both the timing of location of meiotic spindle assembly as well as the coordination of karyokinesis and cytokinesis in mouse oocytes. Key Words: [gamma]-tubulin; centrosome; spindle; nuclear lamina; oocyte; meiosis; nuclear; cytoplasmic maturation.

The germinal vesicle (GV) of starfish oocytes stays just beneath the oocyte cortex at the presumptive animal pole during the long period of oogenesis. We subjected oocytes to a centrifugal force field to detach the GV from the cortex. The association between the cortex and the GV persisted and withstood a small amount of centrifugal acceleration at 200g. The GV was eventually separated from the cortex at 700g. The amount of acceleration sufficient for the GV separation was lowered when the oocytes were pretreated with Nocodazole and was increased by Taxol pretreatment. Observation of microtubular structures with an anti-(alpha)-tubulin antibody revealed the presence of a complex of spots and radiating arrays as was described by J. J. Otto and T. E. Schroeder (1984, Dev. Biol. 101, 274-281) and called the premeiotic aster. Nocodazole shortened the astral arrays, and Taxol enhanced them. These observations indicate that the premeiotic aster works as a device to hold the GV in an eccentric position just beneath the oocyte cortex.

Meiosis, the fundamental evolutionarily conserved differentiative process by which haploid gametes are produced, is a complex and tightly regulated nuclear process. The murine Meig1 gene was previously shown to have a germ cell-specific transcript which is abundantly expressed during meiosis, in both males and females, suggesting that it is involved in meiotic processes. Protein analysis revealed that MEIG1 appears in multiple phosphorylated forms, including two dimeric forms of M(sub r) 31000 and 32000, which exhibit a developmentally regulated switch in their relative abundance. The tyrosine-phosphorylated M(sub r) 31000 form becomes the dominant form once the cells enter meiosis. In this study we show that the M(sub r) 31000 dimeric form appears in the nuclear fraction of testicular protein extract, whereas the M(sub r) 32000 dimeric form and the monomeric forms of MEIG1 remain cytoplasmic. The appearance in the nuclear fraction is developmentally regulated, coinciding with progression of the first spermatogenic wave through meiotic prophase I. Utilizing immunocytochemistry we show that nuclear localization is apparent in primary spermatocytes through their maturation into elongated spermatozoa, but not in either somatic cells or germ cells from early postnatal pups. We also show that MEIG1 associates specifically with meiotic chromosomes in vivo. These results indicate that in germ cells, the M(sub r) 31000 dimeric form enters the nucleus during the first meiotic prophase and binds to the meiotic chromatin. Possible nuclear functions, as well as possible modes of nuclear localization, are discussed.

Although maturation or M-phase-promoting factor (MPF) was originally identified as a cytoplasmic activity responsible for induction of maturation or meiosis reinitiation in oocytes, MPF is now thought to be the universal trigger of G2/M-phase transition in all eukaryotic cells, and its activity is ascribed to cyclin B*Cdc2 kinase. Here, the activation process of cyclin B*Cdc2 at meiosis reinitiation in starfish oocytes is compared with that at G2/M-phase transition in mitotic somatic cells. Based on this comparison, the role of cyclin B*Cdc2 in the original cytoplasmic MPF activity is reexamined.

Incubation of isolated germinal vesicles in the homogenate from maturing starfish oocytes resulted in synchronous germinal vesicle breakdown (GVBD), and chromosome condensation and gathering within 30 min. GVBD in this cell-free system required aerobic conditions. The endogenous ATP-generation system was preserved in the homogenate and effective under aerobic conditions, and thus exogenous ATP was not added to the homogenate. Injection of the homogenate into immature starfish oocytes induced meiotic maturation without 1-methyladenine, indicating high activity of maturation-promoting factor (MPF) in the homogenate. MPF activity in the homogenate was stable for 2 h at room temperature, while it disappeared within 1 h in the supernatant prepared by centrifugation of the homogenate. This disappearance of MPF activity is regulated by cyclin B destruction, similar to that seen in vivo. Key Words: starfish; oocyte; GVBD; cell free.

Oocytes of wild-type mice are ovulated as the secondary oocytes arrested at metaphase of the second meiotic division. Their fertilization or parthenogenetic activation triggers the completion of the second meiotic division followed by the first embryonic interphase. Oocytes of the LT/Sv strain of mice are ovulated either at the first meiotic metaphase (M I) as primary oocytes or in the second meiotic metaphase (M II) as secondary oocytes. We show here that during in vitro maturation a high proportion of LT/Sv oocytes progresses normally only until metaphase I. In these oocytes MAP kinase activates shortly after histone H1 kinase (MPF) activation and germinal vesicle breakdown. However, MAP kinase activation is slightly earlier than in oocytes from wild-type F1 (CBA/H x C57Bl/10) mice. The first meiotic spindle of these oocytes forms similarly to wild-type oocytes. During aging, however, it increases in size and finally degenerates. In those oocytes which do not remain in metaphase I the extrusion of first polar bodies is highly delayed and starts about 15 h after germinal vesicle breakdown. Most of the oocytes enter interphase directly after first polar body extrusion. Fusion between metaphase I LT/Sv oocytes and wild-type mitotic one-cell embryos results in prolonged M-phase arrest of hybrids in a proportion similar to control LT/Sv oocytes and control hybrids made by fusion of two M I LT/Sv oocytes. This indicates that LT/Sv oocytes develop cytostatic factor during metaphase I. Eventually, anaphase occurs spontaneously and the hybrids extrude the polar body and form pronuclei in a proportion similar as in controls. In hybrids between LT/Sv metaphase I oocytes and wild-type metaphase I! oocytes (which contain cytostatic factor) anaphase I proceeds at the time observed in control LT/Sv oocytes and hybrids between two M I LT/Sv oocytes, and is followed by the parthenogenetic activation and formation of interphase nuclei. Also the great majority of hybrids between M I and M II wild-type oocytes undergoes the anaphase but further arrests in a subsequent M-phase. These observations suggest that an internally triggered anaphase I occurs despite the presence of the cytostatic activity both in LT/Sv and wild-type M I oocytes. Anaphase I triggering mechanism must therefore either inactivate or override the CSF activity. The comparison between spontaneous and induced activation of metaphase I LT/Sv oocytes shows that mechanisms involved in anaphase I triggering are altered in these oocytes. Thus, the prolongation of metaphase I in LT/Sv oocytes seems to be determined by delayed anaphase I triggering and not provoked directly by the cytostatic activity. Key Words: CSF; LT/Sv mice; MAP kinase; microtubules; meiotic anaphase; meiotic maturation; MPF.

We report functional analysis of [Gamma]Tub37CD, a maternally synthesized [Gamma]-tubulin that is highly expressed during oogenesis and utilized at centrosomes in precellular embryos. Two [Gamma]Tub37CD mutants contained missense mutations that altered residues conserved in all [Gamma]-tubulins and [Alpha]- and/or [Beta]-tubulins. A third [Gamma]Tub37CD missense mutant identified a conserved motif unique to [Gamma]-tubulins. A fourth [Gamma]Tub37CD mutant contained a nonsense mutation and the corresponding premature stop codon generated a protein null allele. Immunofluorescence analysis of laid eggs and activated oocytes derived from the mutants revealed microtubules and meiotic spindles that were close to normal even in the absence of [Gamma]Tub37CD. Eggs lacking the maternal [Gamma]-tubulin were arrested in meiosis, indicative of a deficiency in activation. Analysis of meiosis with in vitro activation techniques showed that the cortical microtubule cytoskeleton of mature wild-type eggs was reorganized upon activation and expressed as transient assembly of cortical asters, and this cortical reorganization was altered in [Gamma]Tub37CD mutants. In precellular embryos of partial loss of function mutants, spindles were frequently abnormal and cell cycle progression was inhibited. Thus, [Gamma]Tub37CD functions differentially in female meiosis and in the early embryo; while involved in oocyte activation, it is apparently not required or plays a subtle role in formation of the female meiotic spindle which is acentriolar, but is essential for assembly of a discrete bipolar mitotic spindle which is directed by centrosomes organized about centrioles. Key Words: tubulin; mitosis; meiosis; centrosome; spindle.

An increasing body of evidence indicates that the oocyte plays an active role in the control of ovarian follicle development in mammals. In the present study, we have examined the role of oocytes in regulating granulosa cell proliferation. Rat and bovine oocytes cocultured with rat granulosa cells stimulated granulosa cell DNA synthesis and DNA content in the cultures. FSH or cAMP further amplified this effect. Poor-quality oocytes showed a marked decrease in their stimulatory effect. Stimulation of DNA synthesis by bovine oocytes seems to be cell-type specific, since Swiss 3T3 fibroblasts and CCL-64 mink lung epithelial cells were not responsive, while primary cultures of rat and bovine granulosa cells and the bovine granulosa cell line BGC-1 showed significant responses. Oocyte-conditioned medium produced only a slight stimulation of rat granulosa cell DNA synthesis. However, the effect of oocyte coculture was dependent on the total incubation volume, suggesting that the growth promoting activity was mediated by a soluble factor. The stimulation elicited by bovine oocytes was evident even in the presence of maximally effective doses of transforming growth factor-[Beta] or tumor necrosis factor-[Alpha], indicating that neither of these growth factors was responsible for this effect. In vitro maturation of bovine oocytes was associated with a marked decrease in the stimulatory activity. This decrease was partially prevented when maturation was blocked by addition of cycloheximide. Comparison of the developmental pattern of the secretion of the growth promoting activity with that of the cumulus expansion-enabling factor indicated that both activities can be dissociated. Our data suggest the existence of a very labile factor produced by the oocyte before completion of the first meiotic division that promotes granulosa cell proliferation.

We have isolated a cDNA clone encoding a germ cell specific protein from an expression cDNA library prepared from the mouse testis, using testis-specific polyclonal antibodies. Sequence analysis of the cDNA revealed that the deduced amino acid sequence consisted of 284 residues, including a nominal repeat structure in the N-terminal region. Northern blot analysis revealed the presence of a transcript of 1.3 kb exclusively expressed in the testis and ovary, but at relatively low levels in the ovary. In contrast, no other tissues and organs expressed significant levels of the transcript. Expression of the mRNA in the testis was first detected on day 14 in postnatal development. Western blot analysis showed the presence of the protein with a molecular weight of approximately 40 kDa and an isoelectric point of 4.9. The protein was exclusively found in the testis and ovary, but in a far lesser amount in the ovary as was the case with the transcript. Immunohistochemical examination revealed that the protein was predominantly present in the cytoplasm in pachytene spermatocytes through to round spermatids. However, during the disappearance of the nuclear envelope at both the first and second meiotic divisions, the protein was localized around the metaphase chromosomes and spindles. Because of this, the name meichroacidin which stands for male meiotic metaphase chromosome-associated acidic protein is proposed for this antigen. The highly regulated stage-specific expression of meichroacidin and its specific association with the metaphase chromosomes and spindles suggest that the protein plays important roles in male meiosis.

In starfish, fertilization occurs naturally at late meiosis I. In the absence of fertilization, however, oocytes complete meiosis I and II, resulting in mature eggs arrested at the pronucleus stage, which are still fertilizable. In this study, we isolated cDNAs of starfish cyclin A and Cdc2, and monitored extensively the cell cycle dynamics of cyclin A and cyclin B levels and their associated Cdc2 kinase activity, Tyr phosphorylation of Cdc2, and Cdc25 phosphorylation states throughout meiotic and early embryonic cleavage cycles in vivo. In meiosis I, cyclin A was undetectable and cyclin B/Cdc2 alone exhibited histone H1 kinase activity, while thereafter both cyclin A/Cdc2 and cyclin B/Cdc2 kinase activity oscillated along with the cell cycle. Cyclin B-, but not cyclin A-, associated Cdc2 was subjected to regulation via Tyr phosphorylation, and phosphorylation states of Cdc25 correlated with cyclin B/Cdc2 kinase activity with some exceptions. Between meiosis I and II and at the pronucleus stage, cyclin A and B levels remained low, Cdc2 Tyr phosphorylation was undetectable, and Cdc25 remained phosphorylated depending on MAP kinase activity, showing a good correlation between these two stages. Upon fertilization of mature eggs, Cdc2 Tyr phosphorylation reappeared and Cdc25 was dephosphorylated. In the first cleavage cycle, under conditions which prevented Cdc25 activity, cyclin A/Cdc2 was activated with a normal time course and then cyclin B/Cdc2 was activated with a significant delay, resulting in the delayed completion of M-phase. Thus, in contrast to meiosis I, both cyclin A and cyclin B appear to be involved in the embryonic cleavage cycles. We propose that regulation of cyclin A/Cdc2 and cyclin B/Cdc2 is characteristic of meiotic and early cleavage cycles.

Recent findings on cell division suggest that differences exist in spindle organization not only between mitotic and meiotic systems, but also between female and male meiosis. In mammals, this has been difficult to demonstrate due to lack of appropriate methods. By taking advantage of the strict organization and ordered kinetics of mammalian spermatogenesis, we harvested highly enriched populations of dividing mouse and rat spermatocytes using transillumination-assisted microdissection of seminiferous tubules. In the spermatocytes, we examined the localization and distribution of microtubules, centrosomes, and kinetochores at different phases of the first meiotic division using immunohistochemistry with antibodies against [Alpha]-tubulin, [Gamma]-tubulin, and CENP-E, respectively. Fluorescence and confocal microscope analysis of dividing spermatocytes provides evidence that the formation of the male mammalian meiosis I spindle differs from that of female meiosis and mitosis. A short (1-2 [[micro]meter]) bipolar aggregate of microtubules is nucleated by two adjacent centrosomes located next to the nucleus. After nuclear envelope breakdown, adjacent centrosomes and the short spindle become surrounded by the mass of paired meiotic chromosomes. At prometaphase the distance between the centrosomes increases resulting in elongation of the microtubule arrays and eventually formation of a full-length metaphase spindle (12-14 [[micro]meter]). based on these results we suggest a model for spindle morphogenesis in mammalian spermatocytes.

FGF signaling is critical for establishing the Xenopus laevis embryonic body plan and requires the expression of functional FGF receptor during early embryogenesis. FGF receptor-1 (XFGFR) maternal mRNA is present in immature oocytes, but the protein is not expressed until oocyte maturation. In this report we demonstrate that endogenous XFGFR translation begins just prior to germinal vesicle breakdown and that translation depends on completion of earlier meiotic events. We show that the previously identified XFGFR 3[prime]UTR translation inhibitory element (TIE), which is necessary and sufficient for repressing translation in the immature oocyte, also regulates the onset of translation during oocyte maturation. In addition we demonstrate that cytoplasmic polyadenylation of XFGFR RNA is regulated independently of TIE-mediated translation and is not sufficient to activate the translation of XFGFR. These experiments reveal that polyadenylation and translational activation are separable events in this mRNA, each of which is timed and regulated independently. Key Words: translational control; 3[prime] untranslated region; polyadenylation; maternal mRNA; meiosis.

The resumption of meiosis in the developing starfish oocyte is the result of intracellular signaling events initiated by 1-methyladenine stimulation. One of the earliest detectable kinase activities during meiotic maturation of starfish oocytes is a protein kinase C or PKC-like activity. In this study, several isoforms of protein kinase C were cloned from the oocyte; however, the most abundant PKC-like maternal transcript corresponds to protein kinase C-related kinase 2 (PRK2). PRK2 is expressed in the immature oocyte and at least until germinal vesicle breakdown. Subcellular localization of PRK2 revealed a cytoplasmic distribution in the immature oocyte, which, during meiotic maturation, remained in the cytoplasm but also localized to the disintegrating germinal vesicle. Significantly, PRK2 is phosphorylated in vivo in response to 1-methyladenine which precedes MPF activation, making PRK2 a candidate regulator of early signaling events of meiotic maturation.

During male meiosis in wild-type Arabidopsis the pollen mother cell (PMC) undergoes two meiotic nuclear divisions in the absence of cell division. Only after telophase II is a wall formed which partitions the PMC into four microspores. Each microspore undergoes two subsequent mitotic divisions to produce one vegetative cell and two sperm cells in the mature pollen grain. In this paper we describe the isolation and the phenotypic characterization of mutations in the STUD (STD) gene, which is specifically required for male-specific cytokinesis after telophase II of meiosis. Although the male meiotic nuclear divisions are normal in std mutant plants, no walls are formed resulting in a tetranucleate microspore. Despite the absence of cell division in the PMC, postmeiotic development in the coenocytic microspore proceeds relatively normally, resulting in the formation of large pollen grains which contain four vegetative nuclei and up to eight sperm cells. Interestingly, these enlarged pollen grains which contain multiple vegetative nuclei and extra sperm cells behave as single male gametophytes, producing only single pollen tubes and resulting in partial male fertility in std mutant plants. Characterization of the process of pollen development and pollen function in std mutants thus reveals two different types of developmental regulation. Each of the four nuclei found in a std microspore following meiosis is capable of independently undergoing the complete mitotic cell division (including cytokinesis) which the single nucleus of a wild-type microspore would normally undertake. The ability of the four meiotic products to independently continue through mitosis does not depend on their division into separate cells, but is controlled by some subcellular component found within the coenocytic micropsore. By contrast, the mature std pollen grain functions as a unit and produces only a single pollen tube despite the presence of multiple nuclei within the vegetative cell, suggesting that this process is controlled at the cellular level independently of the extra subcellular components.

Germ cells harvested from mouse embryonic genital ridges were mixed with disaggregated embryonic lung cells, and the reaggregates were cultured for 4-7 days. Germ cells derived from female embryos 10.5-13.5 days postcoitum (dpc) entered and progressed through meiotic prophase in vitro as in vivo, although with a 12- to 24-hr delay. If the cultures were maintained for 2-3 weeks, the germ cells developed into growing oocytes. When germ cells were taken from male embryos 10.5 and 11.5 dpc, they too entered and progressed through meiotic prophase, but germ cells from later embryos (12.5 and 13.5 dpc) developed as prospermatogonia, as in male genital ridges in vivo. When 11.5 dpc male genital ridges were disaggregated, reaggregated, and cultured in the same way as the lung reaggregates, the germ cells again entered meiotic prophase. We conclude that the male genital ridge at about 12 dpc produces a factor that inhibits entry of germ cells into meiosis, and that production of this factor is disrupted by prior disaggregation of the genital ridge. If a meiotic inducing substance is required for entry of germ cells into meiosis, it must be present in the male genital ridge as well as in the female genital ridge, and probably also in the lung.

Full-grown mouse oocytes spontaneously resume meiosis in vitro when released from their follicular environment. By contrast, growing oocytes are not competent to resume meiosis; the molecular basis of meiotic competence is not known. Entry into M phase of the eukaryotic cell cycle is controlled by MPF, a catalytically active complex comprising [p34.sup.cdc2] kinase and cyclin B. Incompetent oocytes contain levels of cyclin B comparable to those in competent oocytes, while their level of [p34.sup.cdc2] is markedly lower; [p34.sup.cdc2] accumulates abruptly at the end of oocyte growth, at the time of meiotic competence acquisition. We show here that this change in [p34.sup.cdc2] concentration is not secondary to a corresponding change in the concentration of the cognate mRNA, indicating that translational control may be involved. Microinjection of translatable [p34.sup.cdc2] mRNA into incompetent oocytes yielded high levels of the protein, but it did not lead to resumption of meiosis. Similarly, microinjection of cyclin B1 mRNA resulted in accumulation of the protein, but not in the acquisition of meiotic competence. By contrast, the microinjection of both [p34.sup.cdc2] and cyclin B1 mRNAs in incompetent oocytes induced histone H1 and MAP kinase activation, germinal vesicle breakdown, and entry into M-phase including the translational activation of a dormant mRNA. Thus, endogenous cyclin B1 in incompetent oocytes is not available for interaction with [p34.sup.cdc2], suggesting that a posttranslational event must occur to achieve meiotic competence. Microinjection of either [p34.sup.cdc2] or cyclin B1 mRNAs accelerated meiotic reinitiation of okadaic acid-treated incompetent oocytes. Taken together, these results suggest that acquisition of meiotic competence by mouse oocytes is regulated at both translational and posttranslational levels.

How sperm trigger activating calcium transients in eggs remains a central, unresolved question in fertilization biology. To determine if a soluble sperm factor can generate a fertilization-like calcium response in the absence of sperm-egg binding, aqueous extracts of sperm from the nemertean worm Cerebratulus lacteus were mixed with [Ca.sup.2+]-sensitive fluorescent dyes and injected into unfertilized, metaphase-I-arrested oocytes. Based on confocal imaging analyses, unfertilized oocytes that had been injected with sperm extract routinely produced oscillating [Ca.sup.2+] waves and resumed meiotic maturation in a manner that closely resembled normal fertilization. Calcium oscillations and maturation were typically lacking in control oocytes that had been (i) injected with buffer alone or with buffer containing added calcium, (ii) given external treatments of the sperm factor, or (iii) injected with extracts made from cells other than sperm. Boiling or protease treatment essentially abolished the potency of the sperm extract, and nonboiled extracts retained full activity in > 10-kDa fractions, but not in

Mutations in the genes encoding the Kit tyrosine kinase receptor or kit ligand (KL) cause numerous phenotypic defects, including sterility. In the postnatal ovary, Kit is expressed on the oocyte surface and KL is produced by the surrounding granulosa cells, but its function in these cells is still unknown. The purpose of this study was to determine the role KL/Kit interactions play in the regulation of oocyte meiosis. Here, we demonstrate that meiotically arrested rat oocytes that are microinjected with Kit antisense oligonucleotides have decreased Kit expression. This decreased expression is associated with an increased ability of these oocytes to resume meiosis compared with those microinjected with missense oligonucleotides or buffer alone. In addition, oocytes cultured in the presence of KL were delayed in their resumption of meiosis, but KL could not enhance the meiosis inhibitory effects of dibutyryl cAMP, suggesting that KL operates through a mechanism that is independent of cAMP. Human chorionic gonadotropin-induced meiotic resumption in oocytes was accompanied by a shift in follicular granulosa cell KL expression from membrane-bound to soluble forms and a loss of expression of both forms of KL in cumulus cells. Thus, KL-activated Kit inhibits meiotic progression, and the in vivo luteinizing hormone-stimulated resumption of meiosis may negate Kit activity by a localized decrease in KL expression and by altering the form of KL produced within the follicle.

Analogs of cyclic adenosine 5'-monophosphate (cAMP) were tested in paired combinations with type I or type II protein kinase isozymes A (PKA) found within the mouse oocyte-cumulus cell complex to determine whether cAMP-dependent protein kinase isozymes influence the response of the complex to cAMP. The potency of the isozymes were characterized according to their ability to prevent germinal vesicle breakdown and to cumulus expansion in response to PKA stimulation.

A study of calcium-independent transition from metaphase to interphase in mouse eggs treated with phorbol esters revealed that in the absence of an increase in Ca(super 2 plus)i, activation of protein kinase C can induce the transition from metaphase to interphase in mouse eggs. An elevation of calcium ion concentration is not essential for the phorbol myristate acetate (PMA)-initiated release from metaphase.

The large release of calcium and subsequent cortical granule exocytosis at fertilization may depend on structural reorganization of the endoplasmic reticulum (ER). A comparison was made between the ER of live metaphase II mouse eggs and prophase I-arrested oocytes. In the mature eggs, many dense accumulations of membrane were present in the cortex but absent deeper in the cytoplasm, with a fine reticular network of ER throughout the cell. Immature oocytes had few ER accumulations in the cortex, but larger clusters were found in the deeper cytoplasm.

Brefeldin A is a fungal metabolite which acts on cell cycle regulatory elements. It indirectly activates cdc2 protein kinase in Xenopus oocytes and meiosis progresses till metaphase II. The amount of Cdc2 protein kinase, M-phase factor, cyclic B, MAP kinase and changes in the protein synthesis in oocytes treated with brefeldin A are similar to that in progesterone-induced oocytes. The maturation effect of brefeldin A is affected by drugs which affect cAMP metabolism. Brefeldin A disrupts the Golgi apparatus' protein transport.

The protein kinase p34cdc2 is produced in the G2 meiotic phase in pig oocytes but is not associated with its cyclin B subunit until the first metaphase (MI). The p34cdc2-cyclin B complex continues increasing throughout the second metaphase (MII). In oocytes from non-mammalian species, the p34cdc2-cyclin B complex is present during the GV stage.

A study of the in vitro-matured bovine oocytes to parthenogenic activation using protein phosphorylation inhibiting- or intracellular calcium-enhancing compounds reveals that protein kinase blockade with DMAP subsequent to an ionomycin-stimulated calcium transient leads to the entry of secondary oocytes into embryonic cell cycles, circumventing the second reduction division. Restoration of meiosis and second polar body extrusion require only an increase in Ca2+(i) while pronuclear formation requires the prolongation of metaphase II arrest. Further treatment with DMAP eliminates karyokinesis by inhibiting the second reduction division. The oocytes enter interphase, get back into the cell cycles and form blastocytes. The nature of parthenogenic stimuli and the age of the oocyte influence the possibility of global activation.