Oocyte Meiotic Progression Research Articles

Calcium Release at Fertilization: Artificially Mimicking the Oocyte's Response to Sperm.

The mechanism of sperm-induced calcium release has been the subject of many studies since the development in the late 1950s of in vitro culture systems that support mammalian fertilization. Despite efforts to elucidate the nature of the signal from the sperm that triggers both the early and late events of oocyte activation, the precise mechanism remains unresolved. Now, with the advent of somatic nuclear transfer technologies, the need to better understand this unique process has been recognised. Nuclear transfer embryos must be induced to commence development artificially because the activating signal from the sperm is absent. The primary activating stimulus is a large increase in the concentration of intracellular-free calcium and numerous physical and chemical treatments have been found to induce calcium changes that initiate the events of oocyte activation. Although live cloned offspring have been produced in a number of species, the overall efficiencies of the nuclear transfer procedures described thus far are unacceptably low and phenotypic anomalies are common. With the aim of improving these efficiencies, researchers are developing artificial activation treatments which induce oocyte responses that mimic those induced by fertilizing sperm. One strategy is to replicate the pattern of calcium change more closely. Another strategy is to couple an activating stimulus with treatments that inhibit maturation (or M-phase) promoting factor (MPF) activity, which regulates meiotic progression in oocytes. This paper reviews what is understood of calcium release at fertilization and describes the treatments that have been used to induce oocyte activation artificially in parthenogenetic and nuclear transfer studies. The relative effectiveness of the strategies employed to mimic the oocyte's response to sperm are discussed.

Mouse‐rabbit germinal vesicle transfer reveals that factors regulating oocyte meiotic progression are not species‐specific in mammals

A series of experiments were designed to evaluate the meiotic competence of mouse oocyte germinal vesicle (GV) in rabbit ooplasm. In experiment 1, an isolated mouse GV was transferred into rabbit GV-stage cytoplast by electrofusion. It was shown that 71.8% and 63.3% of the reconstructed oocytes completed the first meiosis as indicated by the first polar body (PB1) emission when cultured in M199 and M199 + PMSG, respectively. Chromosomal analysis showed that 75% of matured oocytes contained the normal 20 mouse chromosomes. When mouse spermatozoa were microinjected into the cytoplasm of oocytes matured in M199 + PMSG and M199, as many as 59.4% and 48% finished the second meiosis as revealed by the second polar body (PB2) emission and a few fertilized eggs developed to the eight-cell stage. In experiment 2, a mouse GV was transferred into rabbit MII-stage cytoplast. Only 13.0–14.3% of the reconstructed oocytes underwent germinal vesicle breakdown (GVBD) and none proceeded past the MI stage. When two mouse GVs were transferred into an enucleated rabbit oocyte, only 8.7% went through GVBD. In experiment 3, a whole zona-free mouse GV oocyte was fused with a rabbit MII cytoplast. The GVBD rates were increased to 51.2% and 49.4% when cultured in M199 + PMSG and M199, respectively, but none reached the MII stage. In experiment 4, a mouse GV was transferred into a partial cytoplasm-removed rabbit MII oocyte in which the second meiotic apparatus was still present. GVBD occurred in nearly all the reconstructed oocytes when one or two GVs were transferred and two or three metaphase plates were observed in ooplasm after culturing in M199 + PMSG for 8 hr. These data suggest that cytoplasmic factors regulating the progression of the first and the second meioses are not species-specific in mammalian oocytes and that these factors are located in the meiotic apparatus and/or its surrounding cytoplasm at MII stage. J. Exp. Zool. 289:322–329, 2001. © 2001 Wiley-Liss, Inc.

Mouse-rabbit germinal vesicle transfer reveals that factors regulating oocyte meiotic progression are not species-specific in mammals

A series of experiments were designed to evaluate the meiotic competence of mouse oocyte germinal vesicle (GV) in rabbit ooplasm. In experiment 1, an isolated mouse GV was transferred into rabbit GV-stage cytoplast by electrofusion. It was shown that 71.8% and 63.3% of the reconstructed oocytes completed the first meiosis as indicated by the first polar body (PB1) emission when cultured in M199 and M199 + PMSG, respectively. Chromosomal analysis showed that 75% of matured oocytes contained the normal 20 mouse chromosomes. When mouse spermatozoa were microinjected into the cytoplasm of oocytes matured in M199 + PMSG and M199, as many as 59.4% and 48% finished the second meiosis as revealed by the second polar body (PB2) emission and a few fertilized eggs developed to the eight-cell stage. In experiment 2, a mouse GV was transferred into rabbit MII-stage cytoplast. Only 13.0-14.3% of the reconstructed oocytes underwent germinal vesicle breakdown (GVBD) and none proceeded past the MI stage. When two mouse GVs were transferred into an enucleated rabbit oocyte, only 8.7% went through GVBD. In experiment 3, a whole zona-free mouse GV oocyte was fused with a rabbit MII cytoplast. The GVBD rates were increased to 51.2% and 49.4% when cultured in M199 + PMSG and M199, respectively, but none reached the MII stage. In experiment 4, a mouse GV was transferred into a partial cytoplasm-removed rabbit MII oocyte in which the second meiotic apparatus was still present. GVBD occurred in nearly all the reconstructed oocytes when one or two GVs were transferred and two or three metaphase plates were observed in ooplasm after culturing in M199 + PMSG for 8 hr. These data suggest that cytoplasmic factors regulating the progression of the first and the second meioses are not species-specific in mammalian oocytes and that these factors are located in the meiotic apparatus and/or its surrounding cytoplasm at MII stage.

Immunocytogenetic detection of normal and abnormal oocytes in human fetal ovarian tissue in culture.

This study aimed to: (i) determine whether oocytes are present in cultures of human fetal ovary; (ii) identify whether meiotic anomalies are evident; and (iii) assess whether preparation or culture conditions influence oocyte survival and meiotic progression. Ovaries were collected from fetuses after termination at 13-16 weeks. Oocyte assessment utilized antibodies specific for synaptonemal complex proteins (associated with chromosomes only during meiosis), and antibodies to centromeric proteins. Fragments of tissue were cultured in minimal essential medium + 10% serum +/- follicle stimulating hormone (100 mIU/ml). The sera were fetal calf serum (FCS), FCS for embryonic stem cells (ES-FCS) and human female serum. The numbers and stages of oocytes were assessed after 7-40 days, and particular arrangements of chromosome synapsis identified. Results in fresh tissue included oocytes at leptotene, zygotene, pachytene and diplotene in three of five samples. Four specimens remained viable in vitro, and three had detectable oocytes after culture. The numbers of oocytes and the proportions of zygotene and pachytene cells increased with time in culture. The proportion of degenerate cells in culture was initially higher than in fresh samples, but declined subsequently. More oocytes were detected in ES-FCS and human serum than in FCS. We conclude that human oocytes survive in culture and that progression through prophase I continues.

Effects of Phosphatidylinositol 3-Kinase Inhibitors, Wortmannin and LY294002, on Germinal Vesicle Breakdown (GVBD) in Porcine Oocytes.

To determine whether phosphatidylinositol 3-kinase (PI 3-kinase) cascade plays any role in mammalian oocyte meiotic progression, porcine oocytes were matured in vitro with PI 3-kinase specific inhibitors, wortmannin and LY294002. In cumulus-oocyte complexes (COCs), the addition of 10-8, 10-7 or 10-6 M wortmannin or 0.5, 5.0 or 7.5 ×10-5 M LY294002 to the medium significantly reduced the proportion of oocytes exhibiting germinal vesicle breakdown (GVBD) compared with the inhibitor-free control. In denuded oocytes (DOs), no significant differences in the incidence of GVBD were found between 10-8 M wortmannin treatment and controls, and between 10-8 and 10-7 M treatments, respectively. LY294002 treatment at all concentrations tested did not significantly affect GVBD in DOs. When COCs were cultured in the presence of wortmannin or LY294002, the proportions of oocytes arrested at GVII were significantly higher than the control. The degree of cumulus cell expansion was significantly decreased with wortmannin or LY294002 treatment compared to the control. We concluded that activation of PI 3-kinase in cumulus cells is closely associated with meiotic resumption of the porcine oocytes and the cumulus cell expansion.

Maturation, fertilization, and development of marmoset monkey oocytes in vitro.

This study was conducted to investigate 1) the capacity of in vitro-matured (IVM) marmoset oocytes to be fertilized and to support embryonic development in vitro and 2) oocyte meiotic maturation in relation to in vivo FSH administration, follicle size, and oocyte-cumulus cell status. Pairs of ovaries were collected on Day 4 of the follicular phase from adult females receiving either 1) human FSH (3 IU; n = 5) or 2) control (saline; n = 5) daily for 4 days. Antral follicles were excised from ovaries and separated into classes according to size: class 1 (660-840 microm), class 2 (> 840-1000 microm), class 3 (> 1000-1400 microm), and class 4 (> 1400 microm). A total of 823 partially naked and cumulus-enclosed oocytes (CEOs) were released from follicles and cultured in vitro. Cumulus cells remaining after 22 h were removed, metaphase II (MII) oocytes were inseminated with epididymal sperm, and resulting embryos were cultured until developmental arrest. Fluorescence microscopy was used to assess oocyte meiotic and embryo developmental progression. Oocyte germinal vesicle breakdown (GVB)- and MII-competencies increased significantly with follicular size (p < 0.01 and p < 0.0001, respectively), although they were independent of oocyte-cumulus cell associations. After 24 and 32 h in vitro, 69% and 93%, respectively, of CEOs with MII competence had completed meiotic maturation, and the rate of nuclear maturation increased progressively with follicle size (p < 0.01) and with the association of cumulus cells (p < 0.01). In vivo FSH priming slightly improved oocyte GVB- and MII-competencies (p < 0.01 and p < 0.05, respectively) and decreased the time required to achieve MII (p < 0.01). IVM oocytes from all follicle sizes fertilized (78-92%) in vitro, with 27% developing to morula- and 4% to blastocyst-stage embryos. This study demonstrates for the first time that IVM New World primate oocytes are able to support advanced preimplantation embryonic development in vitro. Oocyte meiotic competence and the time course of nuclear maturation are profoundly influenced by their follicular origin, and marginally by FSH treatment.

Meiotic progression of mouse oocytes throughout follicle growth and ovulation in vitro.

Intact ovarian follicles can be grown in culture but the normality of their oocyte development has not yet been established. This work examined meiotic progression in oocytes from mouse ovarian follicles grown in vitro and in vivo. Follicles of initial diameter 112-279 microns were isolated from ovaries of B6 CBA F1 mice and cultured individually in 25 microliters minimal essential medium-alpha containing 5% mouse serum, 100 mIU/ml follicle stimulating hormone and various supplements. Oocytes were isolated from follicles of various sizes after culture for < or = 5 days and oocyte chromatin was examined by fluorescence microscopy with Hoechst 33258. The stage of meiosis was classed as prophase I [germinal vesicle (GV) stages I-IV], metaphase I, anaphase I or metaphase II and the results were compared with oocytes from fresh follicles of unprimed or gonadotrophin-primed immature mice, and mature mice in oestrus. Small pre-antral follicles grew in vitro to intact pre-ovulatory follicles and some ovulated spontaneously despite negligible luteinizing hormone (LH). Progression through GV stages I-IV in vitro was related to initial and final follicle diameter, follicle growth rate and antrum formation. All the normal stages of GV development were observed in vitro; however, the cultured follicles were significantly larger than the freshly dissected follicles for stages II and III of GV development. GV breakdown had occurred in 7/11 analysable ovulated oocytes and 3/69 intra-follicular oocytes. We conclude that, under the conditions employed, the majority of oocytes cultured in intact immature follicles retain the GV.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of gossypol on in vitro bovine oocyte maturation and steroidogenesis in bovine granulosa cells

Abstract Recently, we demonstrated an adverse effect of gossypol on steroidogenesis of bovine luteal, porcine granulosa and rat adrenocortical cells as well as on bovine, rat and hamster early embryonic development. The purpose of this study was to evaluate the effects of varying dosages of gossypol on bovine oocytes and granulosa cells from small and large follicles. Oocytes and bovine granulosa cells from small follicles were harvested from 3- to 8-mm follicles, and bovine granulosa cells from larger follicles (>15 mm) were used. Bovine oocytes were cultured in the presence of 10, 50 and 150 μg/ml gossypol for 26 to 28 h. Both bovine granulosa cells and bovine granulosa cells from larger follicles were treated with 0.1, 0.5, 2.5 and 12.5 μg/ml gossypol in the presence or absence of human chorionic gonadotropin (hCG) for 6 h. Cumulus expansion, meiotic progression of oocytes, progesterone production and intracellular cAMP formation in granulosa cells were measured. Gossypol significantly decreased cumulus expansion and inhibited nuclear maturation of oocytes. In bovine granulosa cells, gossypol inhibited cAMP formation only at 12.5 μg/ml. In bovine granulosa cells from larger follicles, hCG significantly increased both progesterone and cAMP levels. Treatment with gossypol significantly decreased hCG-induced cAMP formation in a dose-dependent manner. Basal cAMP level and progesterone secretion were significantly inhibited by only 12.5 μg/ml gossypol. However, inhibition of hCG-induced progesterone secretion was more dramatic. These results indicate that gossypol is capable of inhibiting cumulus expansion and nuclear maturation of bovine oocytes, and progesterone production and cAMP formation in bovine granulosa cells from larger follicles.