Competence Of Mouse Oocytes Research Articles

Reducing CYP51 inhibits follicle-stimulating hormone induced resumption of mouse oocyte meiosis in vitro

Meiosis activating sterol, produced directly by lanosterol 14-alpha-demethylase (CYP51) during cholesterol biosynthesis, has been shown to promote the initiation of oocyte meiosis. However, the physiological significance of CYP51 action on oocyte meiosis in response to gonadotrophins' induction remained to be further explored. Herein, we analyzed the role of CYP51 in gonadotrophin-induced in vitro oocyte maturation via RNA interference (RNAi). We showed that although both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) significantly induced meiotic resumption in follicle-enclosed oocytes (FEOs), the effect of LH on oocyte meiosis resumption in FEOs was weaker than FSH. Moreover, both FSH and LH were able to upregulate CYP51 expression in cultured follicular granulosa cells when examined at 8 h or 12 h posttreatments, respectively. Interestingly, whereas knockdown of CYP51 expression via small interference RNA (siRNA) moderately blocked (23% reduction at 24 h) FSH-induced oocyte maturation [43% germinal vesicle breakdown (GVBD) rate in RNAi vs. 66% in control, P < 0.05] in FEOs, similar treatments showed no apparent effects on LH-induced FEO meiotic maturation (58% GVBD rate in RNAi vs. 63% in control, P > 0.05). Moreover, the results in a cumulus-enclosed oocytes (CEOs) model showed that approximately 30% of FSH-induced CEOs' meiotic resumption was blocked upon CYP51 knockdown by siRNAs. These findings suggest that FSH, partially at least, employs CYP51, and therefore the MAS pathway, to initiate oocyte meiosis.

Oct-4 regulates the expression of Stella and Foxj2 at the Nanog locus: implications for the developmental competence of mouse oocytes

Our knowledge of what determines the mammalian oocyte developmental competence is meagre. By comparing the transcriptional profiles of developmentally competent surrounded nucleolus (SN) and incompetent not surrounded nucleolus (NSN) mouse MII oocytes, we recently demonstrated that Oct-4 and Stella are key factors in the establishment of the oocytes' developmental competence. Using RT-PCR, microarray and immunocytochemistry assays, we analysed expression of genes and proteins in oocytes isolated throughout folliculogenesis and classified based on their SN- or NSN-type of chromatin organization. We show that: (1) Oct-4 and Stella are expressed concurrently at the beginning of oocytes' growth and only in SN oocytes; (2) Germ Cell Nuclear Factor is a putative regulator of Oct-4 expression in MII oocytes; (3) the function of Oct-4 is directed at the Nanog locus, regulating the expression of Stella and Foxj2. (1) A number of factors that act upstream and downstream of Oct-4 emerge as candidate players in the acquisition of the oocyte's developmental competence; (2) we define molecular markers that identify a specific group of ovarian oocytes (SN) that have a potential to acquire developmental competence; (3) the presence of SN and NSN oocytes in human ovaries extends the interest of these results to the field of human reproduction.

Influence of gonadotropin surge on gene expression and developmental competence of mouse oocytes

Influence of gonadotropin surge on gene expression and developmental competence of mouse oocytes

The Contribution of Germinal Vesicle Materials to the Determination of Developmental Competence in Mouse Oocytes.

The Contribution of Germinal Vesicle Materials to the Determination of Developmental Competence in Mouse Oocytes.

Maternal Oct-4 is a potential key regulator of the developmental competence of mouse oocytes

BackgroundThe maternal contribution of transcripts and proteins supplied to the zygote is crucial for the progression from a gametic to an embryonic control of preimplantation development. Here we compared the transcriptional profiles of two types of mouse MII oocytes, one which is developmentally competent (MIISN oocyte), the other that ceases development at the 2-cell stage (MIINSN oocyte), with the aim of identifying genes and gene expression networks whose misregulated expression would contribute to a reduced developmental competence.ResultsWe report that: 1) the transcription factor Oct-4 is absent in MIINSN oocytes, accounting for 2) the down-regulation of Stella, a maternal-effect factor required for the oocyte-to-embryo transition and of which Oct-4 is a positive regulator; 3) eighteen Oct-4-regulated genes are up-regulated in MIINSN oocytes and are part of gene expression networks implicated in the activation of adverse biochemical pathways such as oxidative phosphorylation, mitochondrial dysfunction and apoptosis.ConclusionThe down-regulation of Oct-4 plays a crucial function in a sequence of molecular processes that leads to the developmental arrest of MIINSN oocytes. The use of a model study in which the MII oocyte ceases development consistently at the 2-cell stage has allowed to attribute a role to the maternal Oct-4 that has never been described before. Oct-4 emerges as a key regulator of the molecular events that govern the establishment of the developmental competence of mouse oocytes.

249 FOLLICLE-STIMULATING HORMONE AND DIBUTYRYL cAMP TREATMENTS DURING IN VITRO MATURATION IMPROVE THE DEVELOPMENTAL COMPETENCE OF MOUSE OOCYTES

The high level of cyclic adenosine monophosphate (cAMP), which is provided to the oocytes from cumulus cells via gap junctional complexes in cumulus-enclosed oocytes (CEOs), is known to contribute to meiotic arrest at the germinal vesicle (GV) stage of CEOs. However, whether intraoocyte cAMP during the period of in vitro maturation (IVM) affects postfertilization developmental competence of mouse oocytes still remains unclear. The aim of this study was to examine the effects of FSH or dibutyryl cAMP (dbcAMP) treatment during IVM on in vitro development of mouse oocytes after in vitro fertilization (IVF). Whether a junctional association between cumulus cells and the oocyte would be essential for a cytoplasmic maturation-promoting effect was also examined. CEOs were isolated from and eCG-primed 3-week-old ICR mouse by rupturing preovulatory follicles with needles in M16 medium with 5% FCS and essential and nonessential amino acids (basal medium). IVM media used were basal medium without (control) or with 100 µm dbcAMP or 1 IU mL–1 FSH. Carbenoxolone (100 µm, CBX), an inhibitor of gap junction, was used to inhibit a junctional association between cumulus cells and the oocyte. Denuded oocytes (DOs) were prepared by repeatedly pipetting in basal medium with 0.2% hyaluronidase. CEOs and DOs were cultured in IVM media at 37�C under 5% CO2 in air for 16.5 h, and then transferred to TYH medium (a modified Krebs-Ringer bicarbonate medium) containing 0.4% BSA, followed by insemination with capacitated sperm. After 6 h of IVF, inseminated oocytes were cultured in KSOM medium with 0.3% BSA. Development to the 2-cell and blastocyst stages was estimated at 24 h and 120 h after IVF, respectively. All experiments were done in 3 replicates, and the statistical analysis was carried out by ANOVA and Fisher's protected least-squares difference (PLSD) test. When CEOs were matured in IVM media, the rates of postfertilization development to the 2-cell and blastocyst stages of oocytes matured in the control medium were very low(29% and 13%, respectively), whereas those of oocytes matured with FSH or dbcAMP significantly increased (FSH: 61% and 52%, dbcAMP: 63 and 57%, respectively, v. control; P &lt; 0.05). Next, when CEOs were matured in basal medium with 1 IU mL–1 FSH and 100 µm CBX, the developmental rate to the 2-cell stage (56%) was similar to that in medium with FSH alone (61%) but the rate to the blastocyst stage (40%) was little lower compared with that in medium with FSH alone (52%), although not significantly different (P &gt; 0.05). Furthermore, when DOs were matured in IVM media, the developmental rates to the blastocyst stage after IVF of the oocytes matured with FSH or dbcAMP significantly increased (FSH: 25%, dbcAMP: 15%; P &lt; 0.05) compared with those in control medium (7%). Taken together, it is suggested that increasing the concentration of intraoocyte cAMP during the IVM period is important to improve the developmental competence after IVF of mouse oocytes, and that the competence is acquired in part in a cumulus-oocyte junctional communication-independent manner.

Reduced oxygen concentration improves the developmental competence of mouse oocytes following in vitro maturation

Reduced atmospheric oxygen concentration is beneficial to embryo development; however, optimal oxygen concentration for oocyte maturation remains undetermined. Likewise, there is no consensus of appropriate medium supplementation during maturation. The objective of this study was to determine whether oxygen tension (20% or 5% O2) and epidermal growth factor (EGF) affect oocyte metabolism and subsequent embryo development. Cumulus-oocyte complexes (COCs) were collected from 28-day-old equine chorionic gonadotropin (eCG) primed or unprimed F1 (C57BL/6xCBA) mice. COCs were matured in defined medium in one of four groups: 20% O2, 20% O2 + EGF, 5% O2, 5% O2 + EGF. In vivo matured COCs were also collected for analysis. COCs from unprimed mice, matured in 5% O2 +/- EGF or 20% O2 + EGF had higher metabolic rates than COCs matured in 20% O2 (P < 0.05). COCs from primed mice had higher metabolic rates when matured in the presence of EGF, regardless of oxygen tension (P < 0.01). Oxygen uptake and mitochondrial membrane potential were higher for in vivo matured oocytes and oocytes matured under 5% O2 compared to oocytes matured under 20% O2 (P < 0.05). Blastocyst formation was not different between maturation groups (primed or unprimed); however, embryo cell numbers were 20-45% significantly higher when COCs were matured at 5% O2 (P < 0.05). Results suggest that oocytes matured in physiological concentrations of oxygen have improved development and metabolic activity, more closely resembling in vivo maturation. These findings have implications for oocyte maturation in both clinical and research laboratories.

Evaluation of Pre-maturity of Mouse Oocytes Ovulated from Prepubertal Females using an In Vitro Fertilization Technique

ABSTRACT The cytogenetic normality and developmental competence of mouse oocytes derived from prepubertal females were investigated to determine ooplasmic maturity after in vitro fertilization (IVF) and to examine the ability of the resultant blastocysts to develop to term. To estimate the effect of body weight and age on ovulation, prepubertal female (BALB/c × C57BL/6J) F1 mice, 20–30 days of age, were classified into two groups according to body weight as follows: a light group (L) of 8.5–12 g and a heavy (H) group of 13–16 g. The IVF blastocysts were fixed as chromosome samples, and some of the blastocysts were transferred to pseudopregnant recipients. The implantation rates and number of fetuses were subsequently evaluated at 20 days postcoitus. The average number of ovulated oocytes differed significantly, with 46.2, 18.1, and 37.1 in the H, L, and control groups, respectively. All fertilization rates were high, and there were no significant differences. However, 16.8% of zygotes from the L group wer...

O-198: Leukemia inhibitory factor (LIF) induce cumulus expansion in immature human and mouse oocytes and improves mouse fertilization and delivery rates when it is present during mouse in vitro oocyte maturation (IVM)

Studies have demonstrated that LIF concentrations are higher in preovulatory follicles than in developing follicles and a positive correlation exists between follicular fluid LIF concentration and embryo quality. Therefore we decided to examine the role of LIF during human and mouse IVM on cumulus expansion and oocyte competence. Mouse and human oocyte cumulus expansion was evaluated during IVM. Mouse oocyte competence was determined by IVF, IVC and embryo transfer (ET). Adult B6CBAF1/J females were primed with 5 IU of PMSG. Oocytes were collected 48 hs later. Immature human oocytes (4 donors, 25 oocytes) were collected after COH with rhFSH at the time of anticipated hCG administration. IVM medium was Alfa-MEM (Gibco 32571-036) supplemented with 10 % HSS (human oocytes) or FCS (mouse oocytes) and 1.5 IU rhCG. Oocyte treatments during IVM were ± rFSH and ± LIF (mouse 18 h and human 48 h) and cumulus expansion was evaluated. Mouse IVM oocytes, but not human oocytes continued to IVF and IVC for 4 days. Experimental groups: IVM oocytes ± 0.2 IU rFSH; IVM oocytes ± rFSH + LIF (0.1, 1, 1000 ng/ml); and in vivo matured (ovulated) oocytes. Mouse embryo development in vitro was recorded to Day 4, and blastocysts from all groups (395 blastocysts), were transferred to the uterine horn of 2.5-Day pseudopregnant females. Delivery rates were recorded. Logarithmic transformed data were analyzed by ANOVA and Tukey’s test. Results are expressed as Mean ± SE. LIF alone in the IVM medium induced cumulus expansion in human and mouse oocytes. There were no significantly differences between the standard IVM medium supplemented with rFSH vs. supplemented with LIF (P>0.05). But, when 1000 ng/ml LIF was added in combination with rFSH, a significant increase in cleavage rate, embryo development rate (Blastocyst/Total oocytes) and birth rate was observed when compared to oocytes matured with FSH alone (P<0.05). The birth rate of the IVM + rFSH + 1000 ng/ml LIF group was comparable to the in vivo matured control (endogenously ovulated oocytes) (P>0.05). In summary, LIF induced cumulus expansion similarly in human and mouse cumulus-oocyte complexes, and IVM with rFSH + LIF during mouse IVM significantly improved oocyte competence as measured by cleavage rate, blastocyst development and birth rate Our results demonstrate that LIF induces cumulus expansion in human and mouse oocytes and has a profound effect on mouse IVM oocyte competence improving live birth rates. These results strongly suggest that addition of LIF and FSH to IVM media may improves the efficiency of human IVM in ART.

Choline Replacement for Sodium in Vitrification and Thaw Solutions Improves the Viability and Embryonic Competence of Mouse Oocytes

Although Metaphase II (MII) oocytes survive freeze-thaw in sodium-based vitrification solutions and complete pre-implantation development, the efficiency at which they complete full term development is still low. Replacing Na+ with choline in slow freezing protocols for MII oocytes has been reported to significant improve survival rates and developmental potential. This study examines whether choline replacement also improves these parameters for vitrified MII mouse oocytes.

257. Addition of glycine to vitrification solutions protects oocyte and embryo physiology and health

Cryopreservation procedures for oocytes result in a significant reduction in viability. Although cryopreservation procedures cause dehydration and therefore osmotic stress, the role of osmolytes in solutions has not been considered and they have therefore not been included for routine use. The aim of this study was to assess the effects of the addition of the osmolyte glycine to vitrification solutions on the health and developmental competence of mouse oocytes. Oocytes were collected from F1 female mice and cryopreserved using cryoloop vitrification with or without glycine, with fresh oocytes examined as controls (n = 2086). Mitochondrial distribution and membrane potential as well as the morphology of the spindles and chromosomes were assessed. Oocytes were fertilised to assess their ability to develop into blastocysts, which were then assessed for their expression of Glut1, Glut3 and IGF2 by real-time RT-PCR. Statistical analysis was performed using a generalised linear model followed by multiple comparisons using an LSD test. Vitrification without glycine perturbed mitochondrial distribution (mean pixel intensity of outer region:inner region, 1.58±0.20, P&lt;0.01) and mitochondrial membrane potential (mean pixel intensity 0.56±0.01, P&lt;0.01) compared to control oocytes (2.34±0.24 and 0.52±0.01, respectively). The addition of glycine prevented these changes (1.97±0.16 and 0.53±0.01, respectively). Vitrification without glycine resulted in 52% of spindles and chromosomes appearing normal while this was increased to 69% with the addition of glycine, however in both treatments these abnormalities appeared to recover after culture for 2 h. Vitrification did not affect fertilisation and blastocyst development however expression of Glut3 was decreased 2.9 fold in blastocysts resulting from oocytes vitrified in the absence of glycine (P&lt;0.01). The data presented suggests that the addition of glycine results in fewer perturbations in oocyte physiology and gene expression of the subsequent blastocysts and should therefore be considered for routine inclusion in solutions for the cryopreservation of oocytes.

Meiotic competence of mouse oocytes reconstructed by replacing the germinal vesicle with a male pronucleus and somatic nucleus

ABSTRACTThe present study examines the contribution of the nucleus to meiotic competence in mouse oocytes that were reconstructed using nuclear transfer. Three types of reconstructed oocytes were produced: MP‐GV, by transplanting the male pronucleus (MP) into germinal vesicle (GV) stage oocytes; 3T3‐GV, by transplanting the nucleus of a National Institute of Health (NIH) 3T3 cell into a GV stage oocyte; and 3T3‐MII, by transplanting the nucleus of an NIH 3T3 cell into a metaphase II (MII) stage oocyte. The fusion rates differed, but not significantly, in the MP‐GV, 3T3‐GV, and 3T3‐MII groups (77, 63, 56%, respectively). Then, meiotic competence was compared in MP‐GV, 3T3‐GV and non‐manipulated GV stage oocytes as a control. Nuclear envelope breakdown occurred in all the reconstructed oocytes, as well as the control ones. The percentage of first polar body extrusion differed between the MP‐GV (100%), 3T3‐GV (72%), and control (67%) groups. DNA staining with Hoechst 33342 revealed that in the MP‐GV‐group oocytes that had reached MII stage, the chromosomes were condensed and aligned in a regular array similar to the normal metaphase plate. By contrast, in 3T3‐GV group oocytes, the condensed chromosomes were irregularly scattered in the cytoplasm. These results suggest that the donor nucleus affects meiotic competence in reconstructed oocytes.

Meiosis-activating sterol promotes the metaphase I to metaphase II transition and preimplantation developmental competence of mouse oocytes maturing in vitro.

The objective of this study was to determine the effects of a sterol found in ovarian follicular fluid, known as meiosis-activating sterol (FF-MAS), on the maturation of mouse oocytes in vitro. Possible effects of FF-MAS in promoting the metaphase I (MI) to metaphase II (MII) transition (nuclear maturation) and the competence of oocytes to complete preimplantation embryo development to the blastocyst stage (cytoplasmic maturation) were assessed. Cumulus cell-enclosed oocytes that were compromised in their ability to undergo nuclear maturation and subsequent development because of the age or genotype of the female were isolated at the germinal vesicle stage and matured in vitro using media supplemented with 0 to 20 microM FF-MAS. Oocytes that progressed to MII were inseminated in vitro, and the percentages developing to the 2-cell and blastocyst stages were determined. The sterol was omitted from the media used for oocyte insemination or preimplantation development. FF-MAS promoted a significantly higher percentage of oocytes in all groups to progress to MII in vitro. Moreover, FF-MAS treatment of oocytes maturing in vitro dramatically increased the competence of all but one of the groups of oocytes to complete preimplantation development. Therefore, FF-MAS improved mouse oocyte quality by promoting both nuclear and cytoplasmic maturation in vitro.

251SEARCH FOR GENES INVOLVED IN DEVELOPMENTAL COMPETENCE IN MOUSE OOCYTES USING SUPPRESSION SUBTRACTIVE HYBRIDIZATION

During the first month after birth, synchronous follicular growth occurs in the ovary of immature mice (first wave). Previously, we showed that mouse oocytes during the first wave were more competent developmentally in older females (Suzuki O et al., 2002 Theriogenology 57, 628 abst), although the numbers of mature oocytes did not differ with female age (17, 18, and 24 days old). In this study, we examined the genetic factors that affect the developmental competence of mouse oocytes during the first wave using suppression subtractive hybridization (SSH). Oocytes collected from 17- and 24-day-old B6D2F1 females (D17 and D24, respectively) without hormonal treatment were matured in Waymouth medium supplemented with pyruvate (0.23mM), antibiotics, bovine fetuin (1mgmL−1), and polyvinylpyrrolidone (3mgmL−1). After 17-h culture at 37°C in an atmosphere of 5% CO2, 5% O2, and 90% N2, total RNA was isolated from oocytes whose germinal vesicles had broken down (mature oocytes), separately, in three independent culture groups per age (each group contained oocytes from four animals) using Cell-to-cDNA Cell Lysis Buffer (Ambion, Austin, TX, USA). Some of the total RNA from each independent group was pooled by age (total of RNA from approximately 100 oocytes per age) and used for SSH. A SMART cDNA Synthesis Kit (Clontech, Palo Alto, CA, USA) was used to reverse-transcribe total RNA to cDNA. SSH was performed with a PCR-Select cDNA Subtraction Kit (Clontech). The subtracted PCR products were cloned into pGEM-T vector (Promega, Madison, WI, USA). Clones from the subtracted library (D24−D17) were sequenced and their identities were examined using the NCBI BLAST search. The differential expression of candidate genes preferentially expressed in mature D24 oocytes suggested by SSH was confirmed with cDNA transcribed separately in the three independent culture groups per age using real-time quantitative PCR with an ABI Prism 7900HT with TaqMan technology (Applied Biosystems, Foster City, CA, USA). Of 513 clones sequenced, the top six preferentially-expressed candidate genes in more developmentally-competent D24 oocytes were spindlin (20 clones), bmi-1 (4 clones), cyclin B1 (4 clones), E330034G19Rik (4 clones), Jagged1 (4 clones), and Ndfip2 (4 clones). The expression of spindlin in mature D24 oocytes (relative threshold cycle: −3.8±0.7, mean±SD) was confirmed to be approximately 11-fold higher than in D17 oocytes (−0.3±1.5) when GAPDH was used as an internal control (P&amp;lt;0.05, t-test). Quantitative analyses of mRNA expression of the remaining genes are now under way. Our results suggest that spindlin is one of the key factors leading to the acquisition of developmental competence in mouse oocytes during folliculogenesis. Supported by JSPS KAKENHI (No.145716000) and MHLW.

Effect of phosphodiesterase type 3 inhibitor on developmental competence of immature mouse oocytes in vitro.

In vitro use of arresters of meiosis could improve cytoplasmic maturation of immature oocytes by controlling the period of prophase I. Phosphodiesterases (PDE) are responsible for the breakdown and concomitant inactivation of the cyclic nucleotides cAMP and cGMP and are implicated in the regulation of oocyte meiotic maturation. Selective inhibitors of phosphodiesterase type 3 (PDE3) prevent meiotic resumption of mammalian oocytes. This study evaluated the impact of meiosis arrest by PDE3 inhibitor, Org 9935, on developmental competence of geminal vesicle (GV)-stage oocytes from small antral follicles. Cumulus-oocyte complexes (COC), retrieved from antral follicles 24 h after eCG exposure and cultured in the presence of PDE3 inhibitor (10 microM) for an additional 24 h, remained arrested in the meiotic prophase. The GV configuration of oocytes before and after the arrest by PDE3 inhibitor was examined. After the period of meiosis arrest, a significantly increased proportion of oocytes had acquired a nucleolus surrounded by a condensed chromatin rim at the GV, which is a morphological correlate of transcriptional repression. Removal of inhibitor resulted in 90.6% +/- 8.3% of oocytes with the first polar body extruded. Fertilization was significantly improved in oocytes that had been arrested compared with oocytes collected 24 h after eCG and undergoing in vitro maturation immediately. Embryonic preimplantation and live offspring rates of arrested oocytes were higher, although not significantly, than those of nonarrested oocytes. These results suggest that a temporal block of meiosis by PDE3 inhibitor promotes developmental competence of mice oocytes retrieved from small antral follicles.

Acquisition of meiotic competence in mouse oocytes: absolute amounts of p34(cdc2), cyclin B1, cdc25C, and wee1 in meiotically incompetent and competent oocytes.

M-Phase promoting factor (MPF) is a complex of p34(cdc2) and cyclin B. Results of previous studies in which relative mass amounts of these cell cycle regulators were determined suggested that the accumulation of p34(cdc2), rather than cyclin B, could be a limiting factor in the acquisition of meiotic competence in mouse oocytes. Nevertheless, in the absence of measurements of the absolute amount of these components of MPF, it is possible that the molar amount of p34(cdc2) is in excess to that of cyclin B, i.e., the accumulation of p34(cdc2) is not a limiting factor. We report measurements of the absolute mass of p34(cdc2) and cyclin B1, as well as the two proximal regulators of MPF, namely cdc25C and wee1, in meiotically incompetent and competent mouse oocytes. We find that the numbers of molecules of p34(cdc2), cyclin B1, cdc25C, and wee1 in meiotically incompetent oocytes are 1.4 x 10(6), 11.3 x 10(6), 24.6 x 10(6), 15. 6 x 10(6), respectively, and in meiotically competent oocytes the numbers are 14.3 x 10(6), 95.5 x 10(6), 80.0 x 10(6), 40.1 x 10(6), respectively. Thus, the concentration of cyclin B1 is always in excess to that of p34(cdc2), and this is consistent with the hypothesis that the accumulation of p34(cdc2) plays a role in the acquisition of meiotic competence. Last, the concentration of cdc25C is greater than that of wee1 and the concentration of each is greater than that of p34(cdc2) in both meiotically incompetent and competent oocytes.

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.

Factors affecting the developmental competence of mouse oocytes grown in vitro: follicle-stimulating hormone and insulin.

This study was undertaken to test the hypothesis that FSH treatment of cultured oocyte-granulosa cell complexes promotes acquisition of competence to complete preimplantation embryo development. Oocyte-granulosa cell complexes were isolated from the preantral follicles of 12-day-old mice and cultured for 10 days in serum-free medium, supplemented with insulin (5 microgram/ml), transferrin (5 microgram/ml), and selenium (5 ng/ml) and containing a highly potent preparation of FSH (0-5 ng/ml). Oocytes were matured and fertilized in vitro and embryos cultured to determine the frequency of development to the blastocyst stage. There was no effect of FSH on oocyte size, general morphology, or competence to resume meiosis. However, addition of FSH to medium containing insulin had a deleterious effect on the percentage of mature oocytes competent to develop to the blastocyst stage. Deletion of insulin from the medium for culture of oocyte-granulosa cell complexes prevented the deleterious effect of FSH, but FSH still did not promote acquisition of competence to complete preimplantation development. Culture of oocyte-granulosa cell complexes with FSH resulted in elevated expression of LH receptor (LHR) mRNA by granulosa cells and stimulated the production of functional LHRs, whether or not insulin was present. However, FSH-induced expression of LHR mRNA reached a maximum steady-state level by 4 days of culture in the presence of insulin, but this level was not reached until 10 days of culture without insulin. Granulosa cells encompassing growing mouse oocytes in vivo do not express LHR mRNA. Thus, expression of LHR mRNA by granulosa cells closely associated with growing oocytes in vitro indicates inappropriate or ambiguous development. In conclusion, conditions occurring during oocyte growth can have profound detrimental effects on oocyte developmental competence to complete preimplantation development, even when oocyte growth, general morphology, and competence to resume meiosis appear unaffected.

Factors affecting the developmental competence of mouse oocytes grown in vitro: oxygen concentration.

The purpose of this study was to assess the effects of oxygen concentration on the developmental competence of mouse oocytes grown in vitro because oxygen has been shown to affect the nuclear maturation of oocytes (Haidri et al., 1971: J Reprod Fertil 26:409-411) and preimplantation embryo development (Whitten, 1971: Adv Biosci 6:129-139). Oocyte--granulosa cell complexes were isolated from the preantral follicles of 12-day-old mice and cultured for 10 days in serum-free medium equilibrated with 5, 10, 15, or 20% O2. Five percent CO2 was used for all groups. Oocytes from all groups were then matured and fertilized, and preimplantation embryos cultured using 5% O2. With increased oxygen tension, there were dramatic decreases in the percentage of (1) oocytes that survived in vitro culture, (2) surviving oocytes that could resume meiosis and undergo germinal vesicle breakdown (GVB), and then cleave to the two-cell stage, and (3) two-cell-stage embryos that completed the blastocyst transition. For example, 42% of the oocytes grown in a 5% O2 atmosphere cleaved to the two-cell stage compared with only 3% when the oocytes grew in an atmosphere of 20% O2. These dramatic effects of elevated oxygen were mitigated by either increased numbers of the oocyte-granulosa cell complexes in the culture or increased concentration of oxygen late in the culture period, after the oocytes became surrounded by greater numbers of granulosa cells. When the culture period was extended for 4 days beyond the standard 10 day cultures used in the experiments described above, there was an increased occurrence of precocious GVB or failure of the somatic cells to maintain meiotic arrest. This was prevented by increased oxygen concentration. Nevertheless, extending the time of culture even in the presence of elevated oxygen failed to increase oocyte growth to the equivalent of in vivo-grown oocytes or to improve the developmental competence of the in vitro-grown oocytes. It was concluded that concentrations of O2 above 5% have a deleterious effect on oocyte development during the early stages of culture. However, increasing the concentration on O2 during the later stages, in conjunction with other treatments to promote normal granulosa cell development and function and their interaction with the oocyte, may be critical to promote normal oocyte development in vitro.

Centrosome phosphorylation and the developmental expression of meiotic competence in mouse oocytes

Previous studies suggested that the transition from an incompetent to a competent meiotic state during the course of oogenesis in the mouse involved a G 2 M - like cell cycle transition (Wickramasinghe et al., 1991. Dev. Biol. 143, 162). The present studies tested the hypothesis that centrosome phosphorylation, an event normally induced by MPF, is required for this developmental transition and the expression of meiotic competence in cultured growing mouse oocytes. Multiple fluorescence labeling techniques were used to evaluate centrosome number, phosphorylation status, and microtubule nucleating capacity in competent and incompetent oocytes. Experimental conditions were established for reversibly altering the phosphorylation status of the centrosomes and the effects of these treatments on meiotic resumption were examined. Phosphorylated centrosomes nucleating short microtubules were observed in competent oocytes, whereas nonphosphorylated centrosomes and interphase microtubule arrays were found in incompetent oocytes. Upon recovery from nocodazole-induced microtubule depolymerization, short microtubules formed from centrosomes in competent oocytes, whereas long microtubules reappear in the cytoplasm of incompetent oocytes. Perturbation of the phosphorylation state of oocytes with activators of protein kinase A or protein kinase C resulted in the formation of long interphase microtubules in competent oocytes while centrosome phosphorylation was maintained. Treatment of competent oocytes with the phosphorylation inhibitor 6-dimethylaminopurine also led to formation of long microtubules, although under these conditions centrosomes were dephosphorylated. When competent oocytes were treated simultaneously with puromycin and the phosphodiesterase inhibitor isobutyl methylxanthine (IBMX) for 6 hr, centrosomes became dephosphorylated; centrosomes were rephosphorylated when competent oocytes were further cultured in IBMX without puromycin. Conditions that induced centrosome dephosphorylation in competent oocytes resulted in the loss of the ability to express meiotic competence in culture, whereas maintenance of centrosome phosophorylation in these oocytes was correlated with the ability to resume meiosis. These results suggest that the G 2 M transition that occurs when mouse oocytes progress from an incompetent to a competent state in vivo involves the phosphorylation of centrosomes and that the maintenance of centrosome phosphorylation is required for the in vitro expression of meiotic competence.