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PubMed2003

Inhibin B predicts oocyte number and the ratio IGF-I/IGFBP-1 may indicate oocyte quality during ovarian hyperstimulation for in vitro fertilization.

PURPOSE: To perform a retrospective analysis of 62 age-matched IVF-treated women in order to investigate whether levels of inhibin B, IGF-I, and IGFBP-1 in serum 2 days before oocyte retrieval and in follicular fluid at the day of oocyte retrieval might be useful as indicators of the ovarian ability to produce oocytes (ovarian reserve). METHODS: Patients were allocated into three groups on the basis of the number of oocytes retrieved. Group 1 ("low responders") had 0-3 oocytes, group 2 ("normal responders") had 6-11 oocytes, and group 3 ("high responders") had 12 oocytes or more. Levels of inhibin B, IGF-I, and IGFBP-1 in follicular fluid and in serum obtained 2 days before oocyte retrieval were analyzed and correlated to clinical parameters including estradiol levels, progesterone levels, follicle size, follicle number, and oocyte number. RESULTS: We found significant differences in inhibin B levels in the three groups. Inhibin B levels in follicular fluid and serum was strongly correlated to the number of oocytes retrieved (p < 0.01). The number of oocytes retrieved were also correlated to total FSH dose (p < 0.05), to estradiol 2 days before and at ovum pick-up (p < 0.05), to progesterone at ovum pick-up (p < 0.0001), to progesterone at embryo transfer (p < 0.05), and to the number of follicles (size 12-15 mm, p < 0.001, size > 15 mm, p < 0.01). Serum inhibin B also correlated to follicular fluid inhibin B (p < 0.01). Inhibin B was not correlated to pregnancy. In contrast, the ratio IGF-I/IGFBP-1 in serum as well as in follicular fluid was significantly higher in women who became pregnant (p < 0.05). CONCLUSIONS: The results show that inhibin B in serum 2 days before oocyte retrieval predicts number of oocytes retrieved. Since inhibin B in serum before oocyte retrieval in ovarian hyperstimulation was strongly predictive of the number of oocytes retrieved, it appears useful as a marker for ovarian response. Inhibin B did not predict treatment outcome, whereas the ratio IGF-I/IGFBP-1 in serum and follicular fluid was significantly higher in women who became pregnant. The ratio IGF-I/IGFBP-1 may thus reflect oocyte quality.

Adult↗
PubMed1999

In-vitro matured metaphase-I oocytes have a lower fertilization rate but similar embryo quality as mature metaphase-II oocytes after intracytoplasmic sperm injection.

About 4% of all the oocytes denuded prior to intracytoplasmic sperm injection (ICSI) are in metaphase-I (MI). Frequently, these oocytes achieve meiosis after a few hours of in-vitro culture and are available for ICSI on the day of oocyte retrieval. In this retrospective study, the aim was to evaluate the fertilization rate and the developmental capacity of these in-vitro matured MI oocytes. After controlled ovarian stimulation using human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (HCG) in 896 ICSI cycles, 1210 MI-to-MII-matured oocytes were injected approximately 4 h after in-vitro culture and 8803 MII oocytes were injected immediately, or later, after denudation. The fertilization rate of in-vitro matured oocytes was significantly lower than that of mature MII oocytes (52.7 and 70.8% respectively, P < 0.00l). Embryo quality was only slightly different as regards the numbers of good quality embryos: 47.4% good quality embryos were obtained in the in-vitro matured oocyte group, whereas 53.2% good quality embryos were obtained in the MII oocyte group (P < 0.05). The same proportions of excellent (5.7 and 7.0%, NS) and fair quality (17.6 and 15.3%, NS) embryos were obtained for in-vitro matured and mature oocytes respectively. Embryos derived from in-vitro matured oocytes were transferred only if they were of better quality or if there were not enough mature oocyte derived embryos available. Fifteen transfers involved only embryos derived from in-vitro matured oocytes: 11 single embryo transfers and four transfers of two embryos, resulting in one singleton pregnancy and the birth of a healthy baby. It may be concluded that in cycles with few MII oocytes it might be worthwhile to inject in-vitro matured MI oocytes in order to increase the number of embryos available for transfer.

Cell Differentiation↗
PubMed1988

Chromosome analysis of preovulatory human oocytes and oocytes failing to cleave following insemination in vitro.

These investigations were carried out within a clinical in vitro fertilization and egg replacement program. A gradual fixation method for chromosome preparations of animal oocytes, when modified and applied to human oocytes, gave numerical analyzability in about 80%. Of preovulatory oocytes recovered in stimulated cycles, 53% of oocytes with numbers of chromosomes in the haploid range had an apparently normal set of 23 chromosomes. Oocytes with a very low number of chromosomes or in first meiosis were also found. In a separate investigation the chromosome constitution of oocytes not showing clinical signs of fertilization 48 hours after insemination in vitro was also studied. Of the oocytes with numbers of chromosomes in the haploid range, 50% had a normal 23,X karyotype. A different distribution of abnormal chromosome preparations was found, compared with the preovulatory oocytes. Twelve oocytes (16%) carrying between 10 and 18 chromosomes were found among the inseminated oocytes, but none in the other study. Moreover, 15 preparations displayed no chromosomes in the inseminated oocytes, whereas in the uninseminated group, none lacked chromosomes completely. These results may indicate how unfertilized human oocytes in vitro undergo chromosome loss and degeneration and could represent different stages of degeneration. Among both preovulatory and unfertilized oocytes the distribution between hyper- and hypohaploid oocytes was of particular interest in that hypohaploid chromosome complements were in excess. This finding supports the hypothesis that aberration in the number of chromosomes in an oocyte is caused not only by nondisjunction but also by anaphase lag, since nondisjunction alone would result in a 1: 1 ratio between hyper- and hypohaploid complements. In conclusion, human oocytes recovered for in vitro fertilization seem to have an incidence of numerical chromosome abnormalities in the region of 40-50%. This is close to the figures estimated on the basis of observations made in spontaneous abortions and could be a major explanation for the low developmental potential of human cleavage stage ova.

Cell Division↗
PubMed2003

Oocyte transport: Developmental competence of bovine oocytes arrested at germinal vesicle stage by cycloheximide under air.

The effects of the medium (TCM 199 or SOFaa) and temperature (20 or 39 C) during meiotic arrest by cycloheximide (CHX) under air on the developmental competence of bovine oocytes after in vitro maturation (IVM) and fertilization (IVF) were investigated. Oocytes were maintained in meiotic arrest by 10 microg/ml CHX in a 50-microl droplet of 25-mM HEPES-buffered TCM 199 (H199) at 39 C or synthetic oviduct fluid (HSOFaa) at 20 or 39 C in air for 24 h. After release from the arrest, the oocytes was matured and fertilized in vitro and their developmental competence was examined. The developmental rate of oocytes arrested in HSOFaa at 20 C to the blastocyst stage was similar to that of non-arrested oocytes but was significantly higher (P<0.05) than that of oocytes arrested at 39 C in H199 or in HSOFaa. In consideration of oocyte transport conditions, we also investigated the meiotic arrest of oocytes maintained in a 0.25-ml straw by CHX individually with 10 microl HSOFaa or as a group (40-50 oocytes) with 170-200 microl HSOFaa at 20 C in air for 24 h. After release from meiotic arrest, the developmental competence of these oocytes was assessed similarly. The developmental rate of oocytes treated with CHX individually was similar to that of those treated with CHX in 50-microl droplet of HSOFaa at 20 C. However, the developmental rate of oocytes treated with CHX as a group was lower than that of oocytes treated with CHX in a 50-microl droplet. Five blastocysts developed from oocytes maintained in meiotic arrest in a plastic straw were transferred to five recipient heifers. Consequently, three recipients became pregnant and 2 calves were delivered. The results of the present study indicate that bovine oocytes treated with CHX in HSOFaa at 20 C under air retain the same developmental competence as non-arrested oocytes.

Air↗
PubMed2005

Cryopreserved oocytes of infertile couples undergoing assisted reproductive technology could be an important source of oocyte donation: a clinical report of successful pregnancies.

BACKGROUND: Surplus oocytes in assisted reproduction treatment cycles could be saved and donated to other couples. ICSI is usually performed for oocytes that have been stored frozen, considering possible exocytosis of cortical granules (CG). The unavoidability of ICSI merits further study. METHODS: We used a slow method to freeze excess oocytes from infertile couples. After thawing, oocytes were fertilized by either IVF or ICSI according to semen parameters. Some oocytes were examined for CG. RESULTS: Twenty-eight infertile couples cryopreserved a proportion of their oocytes and 12 thawed their oocytes. Three couples used their own oocytes, whereas nine donated their oocytes to nine other couples for 12 cycles. The survival rate from thawing was 90% (73/81). The fertilization rate using IVF (83%) was similar to ICSI (82%). Seven pregnancies (47% per cycle) were achieved; one used her own oocytes and six received donated oocytes. Five women delivered six babies including one set of twins. Two pregnancies aborted. The frozen-thawed oocytes (15/15) revealed no exocytosis of CG. CONCLUSIONS: To freeze oocytes of infertile couples undergoing assisted reproduction treatment may help other couples. Our successful experience may facilitate oocyte banks to become a reality. Both IVF and ICSI are valuable for frozen oocytes.

Adult↗
PubMed2005

Developmental capability of denuded bovine oocyte in a co-culture system with intact cumulus-oocyte complexes: role of cumulus cells, cyclic adenosine 3',5'-monophosphate, and glutathione.

Cumulus oophorus cells have been implicated in the regulation of female gamete development, meiotic maturation, and oocyte-sperm interaction. Nevertheless, the specific role of cumulus cells (CCs) during the final stages of oocyte maturation and fertilization processes still remains unclear. Several studies have been conducted in order to clarify the role of follicular cells using culture systems where denuded oocytes (DOs) were co-cultured with isolated CCs, or in the presence of conditioned medium. However, those attempts were ineffective and the initial oocyte competence to become a blastocyst after fertilization was only partially restored. Aim of the present study was to analyze the effect of the interactions between somatic cells and the female gamete on denuded oocyte developmental capability using a system of culture where CCs were present as dispersed CCs or as intact cumulus-oocyte complexes (COCs) in co-culture with oocytes freed of CC investment immediately after isolation from the ovary. Moreover, we analyzed the specific role of cyclic adenosine 3'-5' monophosphate (cAMP) and glutathione (GSH) during FSH-stimulated maturation of denuded oocyte co-cultured with intact COCs. Our data confirm that denuded oocyte has a scarce developmental capability, and the presence of dispersed CCs during in vitro maturation (IVM) does not improve their developmental competence. On the contrary, the co-presence of intact COCs during denuded oocyte IVM partially restores their developmental capability. The absence of CCs investment causes a drop of cAMP content in DOs at the beginning of IVM and the addition of a cAMP analog in the culture medium does not restore the initial oocyte developmental competence. The relative GSH content of denuded oocyte matured in presence of intact COCs is consistent with the partial recovery of their developmental capability. However, the complete restoration of a full embryonic developmental potential is achieved only when DOs are co-cultured with intact COCs during both IVM and in vitro fertilization (IVF). Our results suggest that the direct interaction between oocyte and CCs is not essential during IVM and IVF of denuded oocyte. We hypothesize that putative diffusible factor(s), produced by CCs and/or by the crosstalk between oocyte and CCs in the intact complex, could play a key role in the acquisition of developmental competence of the denuded female gamete.

Animals↗
PubMed2005

Vitrification of calf oocytes: effects of maturation stage and prematuration treatment on the nuclear and cytoskeletal components of oocytes and their subsequent development.

This study was designed to establish the effects of the meiotic stage of bovine oocytes and of a prematuration treatment with roscovitine (ROS) on their resistance to cryopreservation. Oocytes from prepubertal calves at the stages of germinal vesicle breakdown (GVBD) or at metaphase II (MII) were vitrified by the open pulled straw (OPS) method. In another experiment, oocytes were kept under meiotic arrest with 50 microM ROS for 24 hr and vitrified at the GVBD stage. After warming, some oocyte samples were fixed, stained using specific fluorescent probes and examined under a confocal microscope. The remaining oocytes were fertilized, and cleavage and blastocyst rates recorded. Significantly lower cleavage rates were obtained for the vitrified GVBD and MII oocytes (9.9% and 12.6%, respectively) compared to control oocytes (73.9%). Significantly worse results in terms of cleavage rates were obtained when GVBD calf oocytes were exposed to cryoprotectants (CPAs: ethylene glycol plus dimethyl sulfoxide, DMSO) (13.1%) or vitrified (1.6%) after a prematuration treatment with ROS, when compared to untreated control oocytes (68.7%) or ROS-control oocytes (56.6%). None of the vitrification procedures yielded blastocysts, irrespective of the initial meiotic stage or previous prematuration treatment. Compared to the control oocytes, significantly fewer oocytes exhibited normal spindle configuration after being exposed to CPAs or after vitrification of either GVBD or MII calf oocytes. These results indicate that the vitrification protocol has a deleterious effect on the meiotic spindle organization of calf oocytes cryopreserved at both the GVBD and MII stage, which impairs the capacity for further development of the embryos derived from these vitrified oocytes. Prematuration treatment with ROS has no beneficial effect on the outcome of vitrification by the OPS method.

Actins↗
PubMed2006

Denuding and centrifugation of maturing bovine oocytes alters oocyte spindle integrity and the ability of cytoplasm to support parthenogenetic and nuclear transfer embryo development.

The effects of cumulus cell removal and centrifugation of maturing bovine oocytes on nuclear maturation and subsequent embryo development after parthenogenetic activation and nuclear transfer were examined. Removal of cumulus cells at 4, 8, and 15 hr after in vitro maturation (IVM) or the centrifugation of denuded oocytes had no effect on maturation rates. Oocytes treated at 0 hr of IVM had a lower expulsion rate (50%) of the first polar body (PB1). The removal of cumulus cells and centrifugation affected the pattern of spindle microtubule distribution and division of chromosomes. There were almost no spindle microtubules allocated to PB1 and the spindles were swollen in anaphase I and telophase I oocytes. Approximately 20% of PB1 oocytes contained tripolar or multipolar spindles. After activation, oocytes denuded with or without centrifugation at 8 hr of IVM resulted in the lowest rate of development (3.0%). Denuded oocytes at 4, 15, and 24 hr of IVM with centrifugation or not resulted in similar blastocyst development rates (9.6%-13.2%). However, centrifugation of oocytes denuded at the beginning of IVM resulted in lower blastocyst development rate (8.1%, P < 0.05) than the noncentrifuged oocytes (17.3%). After nuclear transfer, the blastocyst development rates of oocytes denuded and centrifuged at 0, 4, and 8 hr of IVM were not different when compared to the same patch of noncentrifuged oocytes. However, oocytes denuded and centrifuged at 15 hr of IVM resulted in lower (P < 0.05) blastocyst development rates than the noncentrifuged oocytes. The results of this study suggest that removal of cumulus cells and centrifugation of denuded oocytes affect the spindle pattern. Embryo development of denuded and centrifuged oocytes may differ depending on the time of removal of cumulus cells.

Animals↗
PubMed1993

Secretion of cumulus expansion enabling factor by mouse oocytes: relationship to oocyte growth and competence to resume meiosis.

Fully grown mouse oocytes secrete a factor that enables the surrounding cumulus cells to undergo cumulus expansion upon stimulation of the cumulus cells with follicle-stimulating hormone in vitro. The secretion of the enabling factor by the oocytes is developmentally regulated; it is normally secreted in significant amounts only by oocytes that are competent of undergoing spontaneous germinal vesicle breakdown (GVB) and resuming the first meiotic division. The objective of this study was to determine the relationship, if any, among secretion of this factor, oocyte growth, and the competence to undergo GVB. Three experimental systems were used to assess these relationships. First, 20 to 25% of the oocytes isolated from the small antral follicles of mutant hypogonadal mice were GVB-incompetent even though they had achieved a size typical of GVB-competent oocytes. These large GVB-incompetent oocytes secreted almost as much cumulus expansion enabling factor as the GVB-competent oocytes. Second, GVB-incompetent oocytes cultured under conditions that promote the acquisition of GVB competence but not oocyte growth acquired the ability to secrete the cumulus expansion enabling factor. Third, the precocious activation of maturation promoting factor (MPF) in GVB-incompetent oocytes by okadaic acid did not stimulate the secretion of cumulus expansion enabling factor. It is concluded that the secretion of cumulus expansion enabling factor by oocytes is (1) independent of oocyte growth, (2) independent of competence to undergo GVB, and (3) not induced by the precocious activation of MPF. Thus, although the secretion of the enabling factor normally coincides with the time of acquisition of competence to undergo GVB, the ability to secrete the enabling factor is independent of oocyte maturation. Nevertheless, factors that promote the oocyte's program that leads to competence to undergo GVB probably also promote the ability to secrete the enabling factor.

Animals↗
PubMed1994

Cumulus and oocyte maturation and in vitro and in vivo fertilization of oocytes in relation to follicular steroids, prolactin, and glycosaminoglycans throughout the estrous period in superovulated heifers with a normal LH surge, no detectable LH surge, and progestin inhibition of LH surge.

Crossbred heifers (n = 103) were synchronized to estrus with prostaglandin (PGF2 alpha) and superovulated with follicle stimulating hormone (FSH-P). Animals were ovariectomized every 12 hr after the PGF2 alpha injection (n = 7 to 9/time) up to 108 hr to monitor the follicular, hormonal, and oocyte changes associated with follicular development and ovulation. Twenty-eight animals were implanted with Norgestomet implants 12 hr before PGF2 alpha and ovariectomized at 72, 84, 96, and 108 hr post PGF2 alpha injection to monitor effects of progesterone and suppression of the luteinizing hormone (LH) surge on oocyte maturation and quality. Follicular fluid was collected and analyzed for progesterone, estradiol, prolactin, and glycosaminoglycan content in conjunction with cumulus maturation and nuclear stage of oocyte maturation. Analysis of in vivo matured oocytes by in vitro fertilization was carried out at 60, 72, 84, and 96 hr post PGF2 alpha and in vitro matured oocytes at 12 to 108 hr post PGF2 alpha. No developmental changes in cumulus cells surrounding the oocyte of small follicles was noted (< or = 4 mm dia) indicating a static population. Medium (> 4 < or = 8 mm) and large size (> 8 mm) follicles developed to the corona radiata and loose cumulus stages in animals in which an LH surge was detected but cumulus status remained primarily in the tight cumulus stage for animals without an LH surge. The estradiol-to-progesterone ratio for tight cumulus (TC), corona radiata (CR), and loose cumulus (LC) stages was 1.8 +/- .1, 1.0 +/- .1, and .4 +/- .2, respectively (P < .01). Nuclear maturation of oocytes in small follicles from animals without a detectable LH surge seem to indicate early maturation (48 to 72 hr post PGF2 alpha) in conjunction with a high percent of degenerate oocytes not seen in animals exhibiting an LH surge. Oocytes from medium size follicles matured to germinal vesicle breakdown (GVBD) and early meiosis (metaphase I; MI) stages of development in all treatments. Most oocytes were degenerate in Norgestomet-implanted animals. Oocytes from large follicles (> 8 mm dia) from animals exhibiting an LH surge were in MI and metaphase II (MII) stages (48 to 84 hr post PGF2 alpha) in preparation of ovulation whereas oocytes from animals not exhibiting an LH surge had oocytes that early matured to MII (48 to 72 hr post PGF2 alpha), later regressing to degenerate oocytes (84 to 108 hr). Follicular progesterone, estradiol, and prolactin increased with oocyte maturation, particularly in medium and large follicles.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2006

Developmental competence of bovine oocytes is not related to apoptosis incidence in oocytes, cumulus cells and blastocysts.

The number of follicles undergoing atresia in an ovary is very high, and isolation of cumulus-oocyte complexes (COCs) from such atretic follicles may impair subsequent embryo development in vitro. Our aim was to study if stringent selection by morphological assessment of COCs can improve embryo development, and to evaluate whether oocyte diameter is related with apoptotic ratio in oocytes and blastocysts. COCs from slaughtered cattle were recovered by follicle aspiration and classified depending on oocyte diameter: (A) <110 microm; (B) 110-120 microm; (C) >120 microm. COCs were matured, fertilized and cultured in vitro. Early and late stages of apoptosis were detected by Annexin-V and TUNEL staining, respectively, in denuded oocytes, COCs and blastocysts. Immature oocytes from Group A showed higher apoptotic ratio assessed by TUNEL assay, and the COCs corresponding to this group also showed a higher proportion of apoptotic cumulus cells. After maturation, no differences were present in the incidence of apoptosis among oocytes from different groups, but COCs corresponding to the largest diameter showed less apoptotic cumulus cells. In addition, the percentage of apoptotic oocytes decreased during in vitro maturation in all groups. Apoptotic cell ratio (ACR) in blastocysts was not related to oocyte diameter. In conclusion, oocyte selection and oocyte morphological evaluation prior to maturation was not sufficient to select non-atretic oocytes. When oocyte diameter was used as an additional selection the embryonic developmental potential increased together with oocyte diameter, but this improvement was not related to a lower incidence of apoptosis in the largest oocytes.

Animals↗
PubMed2004

Influence of sperm:oocyte ratio during in vitro fertilization of in vitro matured cumulus-intact pig oocytes on fertilization parameters and embryo development.

The present study was conducted to evaluate the influence of sperm:oocyte ratio during in vitro fertilization (IVF) of in vitro matured cumulus-intact oocytes on fertilization parameters and embryo development in pigs. In vitro matured oocytes surrounded by intact cumulus cells (COC) were inseminated with frozen-thawed spermatozoa at different sperm:oocyte ratios (2000:1, 3000:1, 4000:1, 6000:1, and 8000:1). Denuded oocytes inseminated with 2000 frozen-thawed spermatozoa:oocyte were the control group. A total of 2546 oocytes in five replicates were exposed to spermatozoa for 6 h and then cultured in embryo culture (EC) medium for 6 h (pronuclear formation) or 7 days (blastocyst formation: BF). The penetration rate increased in the COC groups with the sperm:oocyte ratio, reaching the highest rates with 8000:1 spermatozoa:oocyte (72.1 +/- 6.5%), similar to the control (73.5 +/- 3.5%). However, the monospermy was highest with the lower spermatozoa:oocyte rates (82.6-94.8%) and decreased drastically (P<0.05) in the COC group fertilized with 8000 sperm:oocyte (36%). The efficiency of fertilization (number of monospermic oocytes/total number of inseminated oocytes) showed no difference among the COC groups (20-30%) but they were significantly lower (P<0.007) than those obtained by the control group (43.7 +/- 2%). Embryo development was highest in the control group (58% for cleavage and 23% for BF) but not significantly different with the 6000 and 8000 sperm:oocyte COC groups (47 and 50% for cleavage and 19 and 17% for BF, respectively). These results indicate that the use of COC for IVF involves a drop in the efficiency of the fertilization and the necessity to increase the frozen-thawed sperm:oocyte ratio three to four times more to obtain similar embryo development to denuded oocytes.

Animals↗
PubMed1988

Regulation of oocyte maturation: communication in the rat cumulus-oocyte complex.

This study was undertaken to analyse the time relationships between breakdown of communication in the cumulus-oocyte complex and reinitiation of meiosis in the oocyte. In addition the possibility was examined that under conditions of established communication, cAMP is transferred from the cumulus cells to the oocyte. Coupling in the cumulus-oocyte complex and maturation of the oocyte were examined, both in vitro in follicles exposed to LH and in vivo following injection of the hormone to immature, pregnant mare's serum gonadotrophin-primed rats. Transfer analysis was performed by determination of cAMP content in cumulus-enclosed as compared to cumulus-free oocytes incubated with forskolin. It was discovered that after 1 h of culture in the presence of LH, coupling in the cumulus-oocyte complexes had decreased to 45% of its initial level, while only 10% of the oocytes had resumed meiosis by this time. The decrease in coupling in cumulus-oocyte complexes in vivo was 20% by 2 h after administration of the hormone, with all the oocytes being immature at this time. Cyclic AMP determinations revealed that cumulus-enclosed oocytes contained concentrations of cAMP three-fold higher than cumulus-free oocytes incubated under similar conditions. This study demonstrates that, in the rat, (i) a decrease in coupling precedes reinitiation of meiosis and (ii) in the presence of established communication, cAMP is transferred from the cumulus cells to the oocyte. These findings suggest that a decrease in communication can serve as the signal for oocyte maturation, possibly by preventing transfer of cAMP from the cumulus cells to the oocyte.

Animals↗
PubMed1993

Glutathione concentration during maturation and after fertilization in pig oocytes: relevance to the ability of oocytes to form male pronucleus.

The present study examined the kinetics of glutathione (GSH) concentration during maturation and after fertilization in pig oocytes and its relevance to the ability of pig oocytes to form a male pronucleus after in vitro fertilization. The GSH concentration was significantly higher in pig oocytes matured in Waymouth medium than in pig oocytes matured in either modified (m) TCM-199 or mTLP media. The addition of 0.04-0.57 mM cysteine (CySH) to mTLP significantly increased both the GSH concentrations in oocytes matured in vitro and the rate of male pronucleus formation as compared to those in oocytes cultured in mTLP alone. When pig oocytes were cultured 12, 24, or 36 h in mTLP plus 0.14 mM CySH, their GSH concentrations were significantly higher than in uncultured oocytes. After fertilization, the GSH concentration in pig oocytes declined significantly. GSH concentrations in oocytes matured in vivo did not differ from those in oocytes matured in mTLP plus 0.14 or 0.57 mM CySH. The results indicate that 1) the composition of maturation medium affects the GSH concentration in pig oocytes; 2) the addition of CySH to maturation medium permits GSH synthesis by the pig oocytes; 3) GSH levels in pig oocytes change during maturation and after fertilization; and 4) GSH synthesis during oocyte maturation is an important factor for promoting their ability to form a male pronucleus after fertilization.

Animals↗
PubMed1995

Selection of follicles, preculture oocyte evaluation, and duration of culture for in vitro maturation of equine oocytes.

Equine oocytes (n = 537) were collected from slaughterhouse ovaries (n = 118 mares) by scraping the internal follicular wall. Preculture record was made of the appearance of oocyte investments (no cumulus, corona radiata only, compact cumulus, expanded cumulus), appearance of cytoplasm (homogeneous, condensed heterogeneous/fragmented), and nuclear maturation stages (germinal vesicle, germinal-vesicle breakdown, metaphase I, metaphase II, degenerated). There was no difference between follicles > 30 mm and follicles < or = 30 mm in the preculture frequency distribution among the 5 nuclear stages; 96% were at either the germinal vesicle or germinal-vesicle breakdown stages. Oocytes from follicles 5 to 30 mm were cultured in modified TCM-199 for 18, 24, 36 and 48 h. Postculture nuclear maturation classifications were immature (germinal vesicle, germinal-vesicle breakdown, and metaphase I), mature (metaphase II or secondary oocyte), and degenerated. The frequency distribution of oocytes among the 3 postculture maturation classifications changed (P < 0.05) at 18 h (15% mature oocytes), changed (P < 0.05) further at 24 h (55% mature oocytes), with no additional change for 36 or 48 h. The only preculture cytoplasm group that affected the postculture results was the heterogeneous/fragmentation group which had a high proportion of postculture degenerated oocytes (67%); however, only 4% of oocytes were in this group. Luteal status of the mare had an effect (P < 0.05) on the frequencies of the maturation classifications, but not enough to be useful in selecting oocytes. Consistency of the follicle and the type of oocyte investment did not alter significantly the maturation frequencies. The frequency of degenerated oocytes after culture was high under the following conditions: 1) diameter of the follicle from which the oocyte was selected was 5 to 10 mm (44% degenerated oocytes), 2) the largest follicle per pair of ovaries was < or = 10 mm (63%), and 3) the mare was pregnant (66%). These results were probably related to the reported high frequency of atretic follicles in the 5- to 10-mm population. In summary, oocytes from individual follicles < or = 10 mm or from follicles in which the largest follicle per mare was < or = 10 mm were the poorest candidates for in vitro maturation.

Journal Article↗
PubMed1983

Regulation of mouse oocyte meiotic maturation: implication of a decrease in oocyte cAMP and protein dephosphorylation in commitment to resume meiosis.

Mouse oocytes are reversibly inhibited from resuming meiotic maturation in vitro by cAMP phosphodiesterase inhibitors such as 3-isobutyl-1-methyl xanthine (IBMX) and cAMP analogs such as dibutyryl cAMP (dbcAMP). Oocytes cultured in IBMX-containing medium were transferred to and cultured in IBMX-free medium for various periods of time prior to their return to either IBMX- or dbcAMP-containing medium. Results from these experiments defined a period of time in which oocytes became committed to resuming meiosis. Forskolin, which elevated the intracellular oocyte cAMP concentration, transiently inhibited oocytes from resuming meiosis. Levels of cAMP were determined in oocytes incubated in medium that allows resumption of meiosis. The level of oocyte cAMP decreased significantly during the time in which oocytes become committed to resuming meiosis. This decrease in oocyte cAMP was not observed in oocytes inhibited from resuming meiosis by IBMX. In addition, cAMP levels were determined in preovulatory antral follicles, cumulus cell-oocyte complexes, and oocytes during gonadotropin-induced resumption of meiosis in vivo. A decrease in oocyte cAMP preceded resumption of meiosis as manifested by germinal vesicle breakdown (GVBD). This decrease apparently occurred before or during a period of time in which follicle and cumulus cell cAMP were increasing. Associated with commitment to resume meiosis was a characteristic set of changes in oocyte phosphoprotein metabolism that preceded GVBD. These changes are, to date, some of the first reported biochemical changes that precede GVBD. Results from these experiments are discussed in terms of a possible role cAMP may play in regulation of resumption of meiosis in mammals.

1-Methyl-3-isobutylxanthine↗
PubMed2004

The effect of harvesting technique on efficiency of oocyte collection and different maturation media on the nuclear maturation of oocytes in camels (Camelus dromedarius).

The purpose of this investigation was to develop an efficient method for harvesting oocytes from dromedary camel ovaries and to examine the effect of different maturation media on their subsequent maturation in vitro. Oocytes were collected by aspirating the follicular contents using a needle attached to a syringe (Method I, n=163 ovaries) or to a constant aspirating pressure, applied by a vacuum pump (Method II, n=117 ovaries). Individual follicles were excised from ovaries and follicles were punctured with two needles (Method III, n=117). Oocytes were matured in vitro for 40-42 h. At the end of maturation period, oocytes were denuded of cumulus cells and the proportion of oocytes in metaphase-II (MII) stage was determined. In the second experiment, oocytes collected by the dissection method were matured in Tissue Culture Medium199 (TCM), CR1 or modified Connaught Medical Research Laboratories medium-1066 (CMRL) and their nuclear maturation was evaluated after 40-42 h. The recovery rate of oocytes was higher (P<0.01) with Method III compared with Method I or II (94, 31 and 33%, respectively). A higher proportions of oocytes collected with Method I or II were either completely or partially denuded compared with Method III (31, 14% versus 1%). The proportions of viable oocytes (78, 60 and 70%, respectively) and those showing metaphase II was not different (39, 50 and 46%, respectively, P>0.05) among the three treatment groups. Oocyte maturation rate was higher (P<0.05) when TCM was used compared with CMRL or CR1 medium. There was, however, no difference in the maturation rate for oocytes cultured in CMRL or CR1 medium. It may be concluded that a higher proportion of cumulus enclosed oocytes may be recovered by follicle dissection method compared to aspiration using syringe or pump. The higher recovery rate with a comparable proportion of viable and matured oocytes resulted in the overall increase in the number of matured (MII) oocytes/ovary with follicle dissection procedure compared with aspiration procedures. For in vitro maturation of oocytes, TCM is superior to CR1 and CMRL as basic maturation medium for this species.

Animals↗
PubMed2004

Synergistic roles of BMP15 and GDF9 in the development and function of the oocyte-cumulus cell complex in mice: genetic evidence for an oocyte-granulosa cell regulatory loop.

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-specific growth factors that appear to play key roles in granulosa cell development and fertility in most mammalian species. We have evaluated the role(s) of these paracrine factors in the development and function of both the cumulus cells and oocytes by assessing cumulus expansion, oocyte maturation, fertilization, and preimplantation embryogenesis in Gdf9+/-Bmp15-/- [hereafter, double mutant (DM)] mice. We found that cumulus expansion, as well as the expression of hyaluronon synthase 2 (Has2) mRNA was impaired in DM oocyte-cumulus cell complexes. This aberrant cumulus expansion was not remedied by coculture with normal wild-type (WT) oocytes, indicating that the development and/or differentiation of cumulus cells in the DM, up to the stage of the preovulatory luteinizing hormone (LH) surge, is impaired. In addition, DM oocytes failed to enable FSH to induce cumulus expansion in WT oocytectomized (OOX) cumulus. Moreover, LH-induced oocyte meiotic resumption was significantly delayed in vivo, and this delayed resumption of meiosis was correlated with the reduced activation of mitogen-activated protein kinase (MAPK) in the cumulus cells, thus suggesting that GDF9 and BMP15 also regulate the function of cumulus cells after the preovulatory LH surge. Although spontaneous in vitro oocyte maturation occurred normally, oocyte fertilization and preimplantation embryogenesis were significantly altered in the DM, suggesting that the full complement of both GDF9 and BMP15 are essential for the development and function of oocytes. Because receptors for GDF9 and BMP15 have not yet been identified in mouse oocytes, the effects of the mutations in the Bmp15 and Gdf9 genes on oocyte development and functions must be produced indirectly by first affecting the granulosa cells and then the oocyte. Therefore, this study provides further evidence for the existence and functioning of an oocyte-granulosa cell regulatory loop.

Animals↗