Regulation of human and mouse oocyte maturation in vitro with 6-dimethylaminopurine

It has been postulated that premature shortening of the oocyte growth phase due to the recovery of oocytes from small diameter follicles may be responsible for the developmental anomalies associated with in-vitro maturation. 6-Dimethylaminopurine (DMAP) was used to artificially lengthen the pre-maturation period of oocyte growth, in vitro, by inhibiting germinal vesicle breakdown in mouse and human oocytes. DMAP inhibited the meiotic maturation of mouse and human oocytes and the inhibition was fully reversible. The timing of polar body extrusion was accelerated in mouse oocytes following the withdrawal of DMAP; however, the kinetics of nuclear maturation in human oocytes was unaffected by exposure to DMAP. All mouse and human DMAP-treated oocytes that matured to metaphase II expressed histone H1 kinase activity. Fertilization rates in both DMAP-treated and control mouse and human oocytes were comparable, and human embryonic development was similar in control and DMAP-treated oocytes. However, blastocyst development was significantly reduced in DMAP-treated mouse oocytes (P < 0.05). It is concluded that lengthening the prematuration growth phase, by temporarily inhibiting kinase activity with DMAP, does not directly improve oocyte developmental competence but provides a useful tool for further investigating meiotic and developmentally related events in vitro by manipulating meiotic resumption.     

The immunomodulatory effects induced by dietary Zearalenone in pregnant rats

Context: Zearalenone (ZEN) is a common contaminant that is present in feedstuff of high humidity and high temperatures.

Objective: The aim of this study was to investigate the effects of diets contaminated with different concentrations of ZEN on immunomodulation in early pregnant rats.

Materials and methods: Forty-eight pregnant Sprague Dawley (SD) rats were randomly divided into four treatment groups fed on a diet supplemented with one of four concentrations of ZEN: 0?mg/kg (ZEN 0), 50?mg/kg (ZEN 50), 100?mg/kg (ZEN 100) and 150?mg/kg (ZEN 150). The pregnant rats were fed ZEN-treated diets from gestation days 0 to 7 and a basal diet from gestation days 8 to 20.

Results: ZEN exposure (ZEN 100 and 150) caused significant decreases in splenic coefficients, viability of splenocyte and T-cell proliferation and induced histopathological damage in the spleen of early pregnant rats compared with other groups. Levels of IgG and IgA were decreased, while IgM was increased, in high doses of ZEN (ZEN 100 and ZEN 150) compared with other groups. ZEN 150 caused increases in white blood cells and hemoglobin and induced a significant decrease in platelets in blood of the pregnant rats compared with other groups. ZEN 150 increased the mRNA expression levels of interleukin (IL)-6, IL-18 and IL-1β and decreased the mRNA expression levels of interferon-γ, tumor necrosis factor-α and IL-10 in the spleen of pregnant rats compared with ZEN 0.

Conclusion: High doses of ZEN-induced immunomodulatory effects on early pregnant rats by altering immunological parameters.     

Lactobacillus paracasei BEJ01 prevents immunotoxic effects during chronic zearalenone exposure in Balb/c mice

Abstract

Background and aim: Zearalenone (ZEN) is an estrogenic mycotoxin produced by numerous Fusarium species in pre- or post-harvest cereals. ZEN displays a potent estrogenicity in livestock and also causes severe immunological problems. The aims of this study were to isolate a new ZEN-degrading micro-organism for biological detoxification, to examine its ability to degrade ZEN in liquid medium, and to evaluate its potential for in vivo preventitive effects against ZEN (as would occur with contaminated feed)-induced immunomodulation in mice.

Materials and methods: Lactobacillus paracasei BEJ01 (LP) isolated from Tunisian artisanal butter was found to display significant binding ability to ZEN in phosphate-buffered saline (i.e. 96.6%) within 24?h of incubation. The in vivo study was conducted using Balb/c mice that received either vehicle (control), LP only (at 2?×?10^9?cfu/l, ～2?mg/kg BW), ZEN alone (at 40?mg/kg BW), or ZEN?+?LP daily for 15?d.

Results: Compared to control mice, ZEN treatment led to significantly decreased body weight gains and decrements in all immune parameters assessed. The addition of LP to ZEN strongly reduced the adverse effects of ZEN on each parameter. In fact, mice receiving ZEN?+?LP co-treatment displayed no significant differences in the assayed parameters as compared to the control mice. The exposures to the bacteria alone had no adverse effects in the mice.

Conclusion: From these data, we conclude that LP bacteria could be beneficial in human and animals for protection against immunotoxicity from ZEN at high levels and during chronic exposures.     

Reconstruction of mouse oocytes by germinal vesicle transfer: maturity of host oocyte cytoplasm determines meiosis.

We evaluated the maturational competence of mouse oocytes reconstructed by the transfer and electrofusion of germinal vesicles (GV) into anuclear cytoplasts of GV stage oocytes (both auto- and hetero-transfers), metaphase II stage oocytes or zygotes. Following in-vitro culture, the maturation rates of the reconstructed oocytes to metaphase II did not significantly differ between auto- and hetero-transfers (40/70 versus 95/144 respectively); these rates also did not differ from those of control oocytes (57/97) which were matured in vitro without micromanipulation and electrofusion. In contrast, when a GV was transferred into an enucleated metaphase II oocyte or a zygote, only a few reconstructed oocytes underwent germinal vesicle breakdown (5/30 and 2/21 respectively); moreover, none reached metaphase II stage. Cytogenetic and immunofluorescence analyses were conducted on hetero-GV oocytes that extruded a first polar body. Each oocyte showed two groups of chromosomes, one in the cytoplast and one in the polar body, as well as a bipolar spindle with twenty univalent chromosomes. Our findings suggest that oocytes reconstructed by GV transfer into a cytoplast of the same developmental stage mature normally in vitro through metaphase II. Such oocytes may be a useful research model to elucidate the cytoplasmic and nuclear mechanisms regulating meiosis and the relationships between meiotic errors and age-related changes in the oocyte.     

The Fusarium toxin deoxynivalenol disrupts phenotype and function of monocyte-derived dendritic cells in vivo and in vitro

The trichothecene mycotoxin deoxynivalenol (DON) causes systemic immuno-suppression in pigs and possibly also in humans after chronic dietary exposure. Since the outcome of every immune response is largely controlled by dendritic cells (DC), we hypothesised that a direct influence of DON on DC function might play a role in mediating DON immunotoxicity. To test this hypothesis, a 2x2 factorial design study was performed. Pigs were fed a control diet or a diet containing DON (DON-diet); monocyte-derived DC (MoDC) from these pigs were then treated with DON in vitro or left untreated. Phenotype and function of the MoDC were analysed. In vitro DON-treatment of MoDC from pigs fed the control diet resulted in a down-regulation of CD80/86 and CD40. This was associated with an activation of the mitogen-associated protein kinases ERK1/2 and JNK. The endocytic activity of MoDC was decreased after in vitro DON-exposure while their T cell stimulatory capacity was not altered. MoDC derived from pigs that had been fed the DON-diet failed to up-regulate MHC-II in response to LPS/TNFalpha. Dietary exposure of pigs to DON inhibited endocytosis of FITC-dextran by MoDC, but did not influence T cell stimulatory capacity. ERK1/2 and JNK were constitutively activated in MoDC from pigs fed the DON-diet. If MoDC derived from pigs fed the DON-diet were exposed to DON in vitro, this resulted in an up-regulation of MHC-II and CD80/86, but not CD40. In comparison to untreated MoDC from pigs fed DON-diet, endocytic capacity was further down-regulated, whereas mitogen-activated protein kinase activation was increased. In summary, DON disrupts porcine DC function in vitro and in vivo, which might contribute to the immunosuppressive effects of this mycotoxin.     

A synthetic analogue of meiosis-activating sterol (FF-MAS) is a potent agonist promoting meiotic maturation and preimplantation development of mouse oocytes maturing in vitro

BACKGROUND: Follicular fluid-meiosis-activating sterol (FF-MAS) is a factor present in the pre-ovulatory follicle during the time of oocyte maturation. In mouse oocytes maturing in vitro, FF-MAS promotes the completion of meiotic maturation to metaphase II (MII) and improves competence to complete the 2-cell stage to blastocyst transition. We produced analogues of FF-MAS and selected three on the basis of potency to promote the resumption of meiosis by mouse oocytes maintained in meiotic arrest by hypoxanthine. The objective of this study was to determine whether these FF-MAS analogues also affect the quality of oocytes maturing in vitro with respect to the completion of meiotic maturation and augmenting the frequency of development to the blastocyst stage after fertilization in vitro. METHODS: Cumulus cell-enclosed oocytes were isolated from the small antral follicles of 18 or 20 day post-natal mice. These oocytes normally have a reduced competence to complete meiotic maturation and preimplantation embryo development. Oocytes were isolated at the germinal vesicle stage and matured in vitro using media supplemented with 0.1% ethanol, 1 micromol/l FF-MAS, or 0.1-10 micromol/l FF-MAS analogues ZK255884 (884), ZK255933 (933) and ZK255991 (991). Oocytes that progressed to MII were fertilized in vitro and the percentage developing to the 2-cell and blastocyst stages was determined. RESULTS: At 1 micromol/l, 991 and 933 increased the portion of oocytes progressing to MII, whereas the lowest dose of 991 and 884 was ineffective. Treatment of maturing oocytes with either 0.1 or 1 micromol/l 933 dramatically increased oocyte competence to complete preimplantation development. CONCLUSIONS: The synthetic analogue of FF-MAS, ZK255933, is a potent agonist that improves the quality of mouse oocytes matured in vitro. This compound may therefore have therapeutic value for treatment of oocytes from women undergoing therapy for infertility owing to poor oocyte quality.     

Cortical granules behave differently in mouse oocytes matured under different conditions

BACKGROUND: To better understand the differences between in vivo (IVO) and in vitro (IVM) matured oocytes, we studied the chronological changes in cortical granule (CG) distribution and nuclear progression during maturation, and the competence of CG release and embryo development of mouse oocytes matured under different conditions. METHODS: Oocytes matured in vivo or in different culture media were used and CG distribution and release were assessed by fluorescein isothiocyanate-labelled Lens culinaris agglutinin and laser confocal microscopy. RESULTS: Tempos of nuclear maturation and CG redistribution were slower, and competence for CG exocytosis, cleavage and blastulation were lower in the IVM oocytes than in the IVO oocytes. These parameters also differed among oocytes matured in different culture media. Hypoxanthine (HX, 4 mM) blocked germinal vesicle breakdown (GVBD), postponed CG migration and prevented CG-free domain (CGFD) formation. Cycloheximide (CHX) facilitated both GVBD and CG migration, but inhibited CGFD formation. The presence of serum in maturation media enhanced CG release after aging or activation of oocytes. Maintenance of germinal vesicle intact for some time by a trace amount (0.18 mM) of HX was beneficial to oocyte cytoplasmic maturation. CONCLUSION: CGs behaved differently in mouse oocytes matured under different conditions, and cytoplasmic maturity was not fully achieved in the IVM oocytes.     

TNF-α对小鼠卵母细胞体外成熟的影响

目的探讨TNF-α对小鼠卵母细胞体外成熟的影响。方法收集未成熟卵母细胞,在含不同浓度TNF-α的培养液中进行培养,浓度梯度为0(作为对照),5,10,100,200,400和600ng/ml培养3、9、12h,所有卵母细胞体外培养12h后统计MⅡ期成熟卵母细胞数量。结果当TNF-α浓度≥5ng/ml作用3h,与对照组相比,卵母细胞生发泡破裂(GVBD)比例及成熟率明显降低(分别为89.54%与73.12%及78.43%与50.54%,P0.05)。将已经发生生发泡破裂的小鼠卵母细胞移到含有不同浓度TNF-α的培养液中培养9h,当TNF-α浓度≥10ng/ml时,小鼠卵母细胞自GVBD发育到MⅡ的比例为74.55%,明显低于对照87.59%(P0.05)。TNF-α作用12h组,当TNF-α浓度≥5ng/ml时,MⅡ期卵母细胞比例显著低于对照组(P0.01)。结论TNF-α明显抑制卵母细胞的体外成熟,包括抑制生发泡的破裂及第一极体的排放,并具有一定的剂量依赖性,低剂量组还具有时间依赖性。     

Evidence that obesity alters the quality of oocytes and embryos

Infertility is more common in overweight and obese women, with reproductive impairments occurring at many levels of the hypothalamic-ovarian-uterine axis. These impairments lead primarily to longer times to conception and decreased pregnancy rates and have resulted in increasing numbers of overweight and obese women seeking assisted reproduction technologies, such as in vitro fertilization or IVF. Even after undertaking IVF procedures obese women have decreased pregnancy rates compared to moderate weight women, suggesting there may be intrinsic differences in the oocytes of these patients. Definitive data is lacking however, and thus the effect of obesity on oocyte quality remains one of the biggest controversies in reproductive medicine. This review summarizes the studies to date which have yielded information about the effects of obesity on human oocyte quality and pre-implantation embryo development. In addition recent results from our laboratory which clearly demonstrate that diet-induced obesity in mice impairs oocyte developmental competence are discussed.     

线粒体能量代谢和DNA复制对小鼠卵子体外成熟、受精及发育的影响

目的 探讨胞质线粒体的氧化磷酸化（OXPHOS）活性或线粒体DNA复制在卵子成熟、受精和胚胎发育过程中的作用。方法 通过在小鼠体外成熟培养液中引入不同浓度的羰基氰4-(三氟甲氧基)苯腙 (FCCP, 10 nmol/L和100nmol/L)或2′,3′-双脱氧胞苷(ddC,10μmol/L和100μmol/L),抑制线粒体OXPHOS活性或线粒体DNA复制,统计分析各组卵子的体外生发泡破裂（GVBD）率、核成熟率、受精率及囊胚形成率,以分析线粒体功能抑制对卵子成熟、受精和胚胎发育的影响。 结果 线粒体OXPHOS活性和DNA复制功能在卵子和胚胎中所发挥的作用并不完全相同。FCCP抑制线粒体OXPHOS活性可显著降低卵子的核成熟率和囊胚形成率;但对卵子的GVBD的发生率和受精率无显著影响。而ddC抑制线粒体DNA复制不影响卵子的体外成熟和受精,但可显著抑制囊胚的形成。 结论 OXPHOS活性主要影响卵子成熟及胚胎发育;线粒体DNA复制则主要影响胚胎发育;而线粒体功能抑制不影响卵子的成熟启动和体外受精。     

Effects of Acute Fluorene-9-Bisphenol Exposure on Mouse Oocyte in vitro Maturation and Its Possible Mechanisms

Fluorene-9-bisphenol (BHPF), a substitute of bisphenol A (BPA) used in the production of the so-called “BPA-free” plastics, has now been shown to be released from commercial plastic bottles into drinking water and has strong anti-estrogenic activity in mice, which suggests that BHPF is also an environmental toxin. However, whether BHPF exposure has effects on mouse oocyte development is unknown. In this study, the influence of acute exposure to BHPF (50–150 μM, 12 hr) on mouse oocyte maturation and its possible mechanisms were investigated. Of note, 50-μM BHPF had no effects on the maturation of mouse oocytes, whereas 100- and 150-μM BHPF significantly blocked germinal vesicle breakdown and led to the failure of first polar body extrusion. Particularly, 100-μM BHPF exposure severely decreased the cellular adenosine triphosphate in a time-dependent manner, which finally brought out the loss of spindles. In addition, the actin cytoskeleton was also impaired. The defective mitochondrial dynamics and decreased mitochondrial DNA implied the damage of mitochondria in BHPF-treated oocytes. Increased PINK1, Beclin1, and LC3B protein level and decreased TOMM20 and TOMM17A protein level illustrated that mitophagy was induced, which also confirmed that BHPF exposure impaired the cellular mitochondria. Moreover, BHPF induced reactive oxygen species accumulation and early apoptosis. Oocyte quality was also impaired by BHPF exposure through altering histone modifications evidenced by increased H3K9me3 and H3K27me3 levels. Collectively, our results indicated that BHPF exposure disrupted mouse oocyte maturation and reduced oocyte quality through affecting cytoskeleton architecture, mitochondrial function, oxidative stress, apoptosis, and histone modifications. Environ. Mol. Mutagen. 60:243–253, 2019. © 2018 Wiley Periodicals, Inc.     

Osmotic responses and tolerance limits to changes in external osmolalities, and oolemma permeability characteristics, of human in vitro matured MII oocytes

BACKGROUND: Oocyte cryopreservation remains a realistic objective, provided that more systematic approaches are applied, such as thorough analysis of the oocyte oolemma permeability to water and diverse cryoprotectants. METHODS: We prospectively investigated volume changes over time at different temperatures (30 degrees C, 22 degrees C and 8 degrees C) of human metaphase II (MII) oocytes (obtained in stimulated ICSI cycles and matured in vitro from the germinal vesicle stage) when exposed to changes in external osmolality. We also investigated human in vitro matured (IVM) oocytes membrane permeability characteristics at 22 degrees C to 1,2-propanediol (PG) and dimethylsulphoxide (DMSO) and at 30 degrees C, 22 degrees C and 8 degrees C to ethylene glycol (EG), and calculated corresponding oocyte oolemma permeability coefficients (Lp and Pcpa). Furthermore, we investigated the osmotic tolerance limits of IVM oocytes exposed to changes in external osmolality as assessed by their developmental competence during the course of 72 h after ICSI. RESULTS: The results of our studies describe human oocyte membrane permeability coefficients for EG at 30 degrees C (2.85+/-0.15x10(-3) cm/min), 22 degrees C (1.17+/-0.60x10(-3) cm/min) and 8 degrees C (0.37+/-0.15x10(-3) cm/min). Furthermore, at 22 degrees C the EG oolemma permeability coefficient was lower than that of PG and DMSO (1.17+/-0.60x10(-3) cm/min versus 2.15+/-0.70x10(-3) and 1.56+/-0.38x10(-3) cm/min, respectively). Our results also indicate, that human IVM MII oocytes tolerated exposure to solutions in the range of 39-2264 mOsmol/kg H2O as assessed by the oocytes' developmental competence after exposure. CONCLUSIONS: The results of the present study may contribute to a better understanding of the biology and cryobiology of human oocytes, and to the design of better and more robust cryopreservation (freezing or vitrification) protocols.     

Regulation of mitochondrial polarity in mouse and human oocytes: the influence of cumulus derived nitric oxide

Whether exogenous factors influenced the level of mitochondrial polarity (DeltaPsim) in the subplasmalemmal cytoplasm of the oocyte was investigated with denuded and cumulus-enclosed human and mouse oocytes between the germinal vesicle and metaphase II stage. Co-culture of denuded oocytes with cumulus masses or primary cumulus cell cultures demonstrated a 'proximity' effect with respect to the detectable level of DeltaPsim in the oocyte. The specificity and reversibility of this effect on subplasmalemmal mitochondria were shown by repeated repositioning between cellular and acellular regions, which sequentially down- or up-regulated DeltaPsim. Experimental studies with a nitric oxide (NO) donor and inhibitor of NO synthase indicate that NO produced by cumulus cells has a regulatory influence on DeltaPsim in the subplasmalemmal cytoplasm of the corresponding oocyte. Culture of denuded and cumulus-enclosed (intact) oocytes in low and high oxygen atmospheres suggests that competition between oxygen and NO at the mitochondrial level may regulate the level of DeltaPsim and maintain mitochondria homeostasis in the pre-ovulatory oocyte, with a shift to higher polarity occurring after ovulation. The role of exogenous influences on oocyte DeltaPsim is discussed with respect to the regulation of developmental processes in the oocyte and early embryo.     

Centrosome and microtubule dynamics during early stages of meiosis in mouse oocytes

Centrosomes, major regulatory sites for the microtubule (MT) nucleation, are regulated in a dynamic manner throughout the process of meiotic maturation. Recently, centrosome orientation in mouse oocytes has been demonstrated in metaphase I through metaphase II. However, centrosomal protein expression in concordance with MT polymerization in earlier stages of oocyte maturation from germinal vesicle stage (GV) to prometaphase I still remains unclear. The present study aims to assess the centrosome-microtubule remodelling during the onset of meiosis based on strict criteria of nuclear maturation. Six consecutive stages were determined for scoring the oocytes as unrimmed nucleolus (UR), partially rimmed nucleolus (PR), fully rimmed nucleolus (FR), nuclear lamina dissolution (NLD), disappearance of nucleolus (DON), and chromatin condensation (CC). A centrosomal protein, pericentrin, was found tightly localized adjacent to nuclear lamina in UR, lacking any MT nucleation activity. In concordance with the competency to resume meiosis, an increase in the amount and nucleation capacity of pericentrin is noted. In FR, cytoplasmic MT almost disappeared while de-novo microtubule polymerization was found in small aggregates of pericentrin localized around the nucleus. Towards the end of DON and CC, a sudden burst of pericentrin was noted with an extreme MT nucleation activity in an organized fashion that is essential for the rapid formation of first meiotic spindle. The results show that centrosomes display precisely controlled spatio-temporal changes during the onset of meiotic maturation. Accumulation of centrosomal proteins to a single locus followed by a sequestration to several spots might be evidence of a mechanism by which the proper distribution of centrosomal material during nuclear breakdown and subsequently formation of spindle are regulated in concordance with the nuclear maturation.     

Developmental competence of oocytes after ICSI in the rhesus monkey

Oocyte quantity and quality are critical to assisted reproductive technology (ART), yet few assessments beyond counting metaphase II (MII) oocytes exist. In this study, 30 +/- 2 oocytes per cycle were recovered from rhesus monkeys subjected to follicular stimulation with human gonadotrophins, of which 15 +/- 1 were MII. Oocyte quality was investigated by monitoring the developmental potential of oocytes subjected to intracytoplasmic sperm injection (ICSI). Despite uniform fertilization rates (71 +/- 4%), progression of embryos to blastocysts varied when expressed as a monthly average, from 20 to 85%, with lows from February to April and again in October, which could be attributed to developmental failure of a significant number of oocyte cohorts (14 of 55). Blastocyst rates, after elimination of failed cohorts, were uniform over time (59 +/- 4%). Neither culture conditions, the number of follicular stimulations, nor the individual sperm or oocyte donor were associated specifically with developmental failure, suggesting that intrinsic differences between stimulation cycles account for the observed variation in developmental potential. The in-vivo developmental competence of ICSI-produced embryos grown to blastocysts in vitro was also assessed. Two ongoing pregnancies and the birth of a normal female, 'Blastulina', represent landmarks in efforts to expand the use of ART in the rhesus monkey.     

Distribution of alpha3, alpha5 and alpha(v) integrin subunits in mature and immature human oocytes

The distribution of three integrin subunits, alpha3, alpha5 and alpha(v), in immature and mature human oocytes has been examined using immunofluorescence and confocal microscopy. The results demonstrate that both alpha5 and alpha(v) are present at the germinal vesicle stage, while alpha3 was only detected in oocytes after germinal vesicle breakdown, in metaphase I and II stage oocytes. The cortical concentration of integrin subunits alpha3 and alpha5 is consistent with their localization in the oolemma. In contrast, the homogeneous distribution of alpha(v) throughout the oocyte suggests the existence of cytoplasmic reservoirs of this protein in the oocyte.      

Assessment of nuclear and cytoplasmic maturation in in-vitro matured human oocytes

BACKGROUND: With improved prospects for the use of human oocyte in-vitro maturation in assisted reproductive technologies, the need to define more clearly the coordination of nuclear and cytoplasmic maturation has arisen. METHODS: Immunofluorescence and confocal microscopy were used to evaluate cell cycle-dependent modifications in chromatin and microtubules in human germinal vesicle oocytes (n = 455) undergoing in-vitro maturation. RESULTS: Four distinct classes of germinal vesicle stage oocytes were identified based on the expression of G2/interphase characteristics, but, of these, only one class of oocytes was competent to complete meiotic progression to metaphase-II in vitro. The majority of germinal vesicle stage oocytes resumed meiosis within 6 h (88.9%) of culture and exhibited an accelerated pace of progression to metaphase-II (66.7%) over 24 h, but in general were unable to maintain meiotic arrest and defaulted into interphase within 24 h of polar body emission. Characterization of microtubule dynamics and chromatin phosphorylation demonstrates specific cell cycle deficiencies in in-vitro matured human oocytes. CONCLUSION: This work forms a basis for future studies aimed at optimizing nuclear and cytoplasmic maturation during in-vitro maturation.     

Cinemicrography of mouse oocyte maturation utilizing nomarski differential-interference microscopy

The analysis of in vitro mouse oocyte maturation using Nomarski differential-interference optics and time-lapse cinemicrography provides information on the morphological events of oocyte maturation and their duration with greater precision than previously available. Furthermore, some previously unreported morphological events are described. The breakdown of the germinal vesicle was preceded for 41 minutes by extreme activity of the germinal vesicle membrane. The perivitelline space formed precociously by expansion of the zona pellucida rather than by shrinkage of the oocyte. Condensation of the bivalents, centromeric repulsion, formation of the metaphase I plate, and growth of the spindle apparatus were observed. Metaphase I, defined as the period from formation of the metaphase plate to the beginning of anaphase, lasted four hours and twenty-seven minutes. Anaphase chromosome separation began 11 hours and 15 minutes after disintegration of the nucleolus, and was complete within ten minutes. Polar body formation began before the completion of anaphase. The mid-body formed between the separating anaphase dyads, and moved with the telophase spindle to the newly formed oocyte-polar body boundary.