Angiotensin II reverses the inhibitory action produced by theca cells on bovine oocyte nuclear maturation

The presence of prorenin, renin, angiotensinogen, angiotensin-converting enzyme, angiotensin II (Ang II) and Ang II receptors in the ovary is suggestive of a functional ovarian renin-angiotensin system (RAS). In cattle, the expression of Ang II is greatest in large follicles, suggesting that it is important during follicular growth and maturation. The present study was designed to investigate the role of Ang II in bovine oocyte nuclear maturation. Bovine cumulus-oocyte complexes (COCs) were cultured with or without follicular cells and Ang II or saralasin (Ang II antagonist). In the absence of follicular cells, Ang II at 0, 10(-11), 10(-9) and 10(-7) M did not affect the percentage of oocytes reaching the germinal vesicle breakdown (GVBD), metaphase I (MI) and metaphase II (MII) stage after 7-h (41.3 +/- 4.3, 35.3 +/- 4.0, 31.3 +/- 9.7, 38.7 +/- 8.6), 12-h (31.6 +/- 7.0, 34.7 +/- 6.1, 31.7 +/- 5.3, 28.9 +/- 9.1; mean +/- S.E.M.) and 18-h (44.9 +/- 7.3, 58.4 +/- 8.4, 53.1 +/- 7.4, 44.9 +/- 7.3) of culture, respectively. Similarly, saralasin at 0, 10(-11), 10(-9) and 10(-7) M did not affect the percentage of oocytes reaching MII stage after 18-h of culture (37.6 +/- 7.4, 34.4 +/- 7.7, 30.0 +/- 10.8 and 31.2 +/- 5.1, respectively). The theca cells (MII = 22.9%) or medium conditioned with follicular cells (GV = 65.5%, MI = 23.6%) inhibited oocyte maturation; however, theca cells (MII = 35.5 +/- 4.9; P < 0.05) or medium conditioned with follicular cells (GV = 34.6%, MI = 52.7%; P < 0.01) were not able to inhibit nuclear maturation when Ang II (10(-11) M) was present in the culture system. Theca cells remained viable during the culture period when Ang II was present. Therefore, results supported the idea of a role of Ang II in blocking the inhibitory effect of theca cells on nuclear maturation of bovine oocytes.     

Nuclear maturation of domestic cat ovarian oocytes in vitro.

Using the domestic cat as a model for salvaging genetic material from rare Felidae, we collected oocytes from ovarian tissue and placed them in 1 of 3 treatments to observe time-related, meiotic changes of in vitro oocyte maturation. Oocytes obtained from ovaries collected at ovario-hysterectomy were assigned to 1 of 3 treatment groups: 1) modified Krebs-Ringer bicarbonate buffer (mKRB) + 4% BSA and 5 micrograms/ml FSH (+FSH, n = 499); 2) mKRB + 4% BSA (-FSH, n = 502); or 3) mKRB + 5% natural estrus cat serum (NE, n = 873). They were placed in the respective media in a 5% CO2 humidified environment at 38 degrees C. Beginning at 16 h, oocytes were removed at 4-h intervals through 48 h, and the meiotic status was evaluated by means of cytogenetic analysis. On the basis of chromosomal analysis, each cell was placed into one of the following categories: metaphase II (MII); metaphase I (MI); pre-MI (germinal vesicle [GV], GV breakdown, or diakinesis); degenerate or unidentifiable. The percentage of oocytes with degenerate chromatin increased over time in all culture treatments, but was always greatest (p less than 0.05) in the NE group. In the +FSH and -FSH treatments, the proportion of oocytes with nuclear material reaching MII increased with time in culture to 32 h and was equal to or greater than the proportion of oocytes with pre-MI + MI chromatin at this time interval (-FSH, 55%; +FSH, 38%).(ABSTRACT TRUNCATED AT 250 WORDS)     

The new system of shorter porcine oocyte in vitro maturation (18 hours) using ≥8 mm follicles derived from cumulus-oocyte complexes

Despite recent efforts to improve in vitro maturation (IVM) systems for porcine oocytes, developmental competence of in vitro-matured oocytes is still suboptimal compared with those matured in vivo. In this study, we compared oocytes obtained from large (≥8 mm; LF) and medium (3–7 mm; MF) sized follicles in terms of nuclear maturation, intracellular glutathione and reactive oxygen species levels, gene expression, and embryo developmental competence after IVM. In the control group, cumulus-oocyte complexes (COCs) were aspirated from MF and matured for 22 hours with hormones and subsequently matured for 18 to 20 hours without hormones at 39 °C, 5% CO₂in vitro. In the LF group, COCs were obtained from follicles larger than 8 mm and were subjected to IVM for only 18 hours. The ovaries have LF were averagely obtained with 1.7% per day during 2012 and it was significantly higher in the winter season. The results of the nuclear stage assessment of the COCs from the LFs are as follows: before IVM (0 hours); germinal vesicle stage (15.2%), metaphase I (MI) stage (55.4%), anaphase and telophase I stages (15.8%), and metaphase II (MII) stage (13.6%). After 6 hours IVM; germinal vesicle (4.2%), MI (43.6%), anaphase and telophase I (9.4%), and MII (42.8%). After 18-hour IVM; MI (9.7%) and MII (90.3%). Oocytes from LF showed a significant (P < 0.001) increase in intracellular glutathione (1.41 vs. 1.00) and decrease in reactive oxygen species (0.8 vs. 1.0) levels compared with the control. The cumulus cells derived from LFs showed lower (P < 0.1) mRNA expression of COX-2 and TNFAIP6, and higher (P < 0.1) mRNA expression of PCNA and Nrf2 compared with the control group-derived cumulus cells. After parthenogenetic activation, in vitro fertilization and somatic cell nuclear transfer (SCNT) using matured oocytes from LFs, the embryo development was significantly improved (greater blastocyst formation rates and total cell numbers in blastocysts) compared with the control group. In conclusion, oocytes from LFs require only 18 hours to complete oocyte maturation in vitro and their developmental competence is significantly greater than those obtained from MFs. Although their numbers are limited, oocytes from LFs might offer an alternative source for the efficient production of transgenic pigs using SCNT.     

In vitro maturation,fertilization and development of domestic cat oocytes recovered from ovaries collected at three stages of the reproductive cycle

This study was conducted to examine the effect of the donor cat's reproductive cycle stage on in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro development of oocytes recovered from ovaries that were collected and stored at 35 degrees C for a short period (1-6 h). Based on the presence or absence of follicles and corpora lutea, the ovarian pairs collected were classified into inactive, follicular, or luteal stages. Nuclear status of 161 cumulus-oocyte complexes (COCs) were examined immediately after recovery; 91.3% of the oocytes were found to be at the immature germinal vesicle (GV) stage, and 3.7% of the oocytes were at metaphase II (MII) stage. The percentage of the oocytes at the GV stage was significantly lower in the follicular stage than in the inactive stage (P < 0.01). Of the oocytes from the follicular stage, 9.1% were at MII stage. After culture for 24 h, however, the proportions of oocytes that reached metaphase I and MII were not different among the reproductive cycle stages of the ovaries collected (P > 0.05). After co-incubation with sperm, 63.1% of oocytes were fertilized, but there were no significant differences among the reproductive cycle stages of the ovaries with respect to the proportions of normal and polyspermic fertilization. However, the number of oocytes reaching cleavage stage and development to the morula and blastocyst stages from follicular stage ovaries were significantly lower (P < 0.05) than those obtained from inactive and luteal stage ovaries. These results indicate that the reproductive cycle stage of donor cat ovaries, stored at 35 degrees C, has no apparent effects on the frequencies of maturation and fertilization of oocytes, but influences developmental competence of the oocytes following IVM or IVF.     

Development of in vitro embryo production systems for red deer (Cervus elaphus). Part 2. The timing of in vitro nuclear oocyte maturation

The time course of in vitro red deer nuclear oocyte maturation was determined. Ovaries were obtained at slaughter and oocytes were aspirated from follicles greater than 2mm in diameter. Oocytes with compact cumulus cells were matured in 50 microl microdrops (10 per drop) under mineral oil containing TCM 199 supplemented with 0.33 mM pyruvate, 10 microg LH and FSH, 1 microg oestradiol and 10% foetal bovine serum. Oocytes were matured at 39 degrees C and 5% CO(2) in air. At 3h intervals (0-27 h) oocytes were removed from incubation, cumulus expansion scored and removed, and fixed oocytes in ethanol:acetic acid (3:1) for 48 h. Oocytes were stained with lacmoid (1%) and nuclear maturation assessed. Oocytes were arrested in the germinal vesicle (GV) stage at aspiration and up to 6h of incubation. The nuclear membrane began to disperse after 6h and by 10.6+/-0.6h of incubation 75% of the oocytes exhibited germinal vesicle breakdown (GVBD). The mean time for 50% of the oocytes to reach metaphase one (MI) and metaphase two (MII) was 11.7+/-0.4 and 24.8+/-0.9h, respectively. Cumulus oophorus were tightly compacted at aspiration and did not begin expansion until 12h of culture. Full expansion was complete by 18 h of culture. Corona radiata cells did not begin expansion until 15 h and were fully expanded by 24h. Results indicate that in vitro red deer oocyte maturation follows a similar time course of nuclear maturation as reported for bovine and ovine oocytes.     

Effect of osmotic stress on the developmental competence of germinal vesicle and metaphase II stage bovine cumulus oocyte complexes and its relevance to cryopreservation

The effects of osmotic stress on germinal vesicle (GV) and metaphase II (MII) stage bovine cumulus oocyte complexes (COCs) were evaluated by first exposing them to various anisotonic NaCl solutions (75, 150, 600, 1200, 2400, and 4800 +/- 5 mOsm/kg) for 10 min and then returning them to isotonic TL-Hepes solution (270 +/- 5 mOsm/kg) at 20 +/- 2 degrees C. Percentages of oocyte maturation, fertilization, polyspermy, cleavage, and blastocyst formation were measured as endpoints. Exposure to anisotonic conditions had a significant (P < 0.05) effect on the developmental competence of both GV and bovine MII COCs. Oocytes at the GV stage were more sensitive to anisotonic stress than MII oocytes (P < 0.05). None of the GV oocytes developed to the blastocyst stage after exposure to hypertonic conditions (2400 or 4800 mOsm solutions), while exposure to hypotonic conditions (75 or 150 mOsm solutions) resulted in significantly lower (P < 0.05) blastocyst formation (9% and 13%, respectively) compared to the isotonic control (25%). A dramatic decrease to 4% development to blastocyst was observed for MII oocytes following exposure to a 4800 mOsm solution. Blastocyst formation of MII oocytes which were exposed to 75, 150, 600, 1200, or 2400 mOsm solutions were similar (15%, 20%, 18%, 14%, and 13%, respectively; P > 0.05), but lower (P < 0.05) than those in the control group (29%). Exposing GV oocytes to anisotonic conditions increased polyspermic fertilization (P < 0.05), although MII oocytes were not similarly affected (P > 0.05). These data support the hypothesis that osmotic stress is detrimental to bovine oocytes and must be considered when developing optimized cryopreservation procedures for these cells. Mol. Reprod. Dev. 55:212-219, 2000.     

Maturation of porcine oocytes after cooling at the germinal vesicle stage

Maturation of porcine oocytes was examined after oocytes were cooled at the germinal vesicle stage. Cumulus-oocyte complexes (COCs) collected from medium-sized follicles were cooled at 24 degrees C or 4 degrees C for 5, 30 or 120 min in a solution with or without 1.5 M dimethylsulfoxide (DMSO). After rewarming, COCs were cultured in maturation medium at 39 degrees C, 5% CO2 in air for 44 h. Meiotic spindle organisation (by immunostaining and confocal microscopy), nuclear maturation (by orcein staining) and cytoplasmic maturation (by intracellular glutathione assay) of oocytes were examined after maturation. When COCs were cooled at 24 degrees C for various times in the medium without DMSO, a tendency to decreased spindle formation, nuclear maturation and cytoplasmic maturation was observed, but there was no statistical difference compared with controls. Addition of DMSO during cooling inhibited subsequent nuclear maturation and spindle formation. When COCs were cooled at 4 degrees C, both nuclear and cytoplasmic maturation as well as spindle formation were inhibited in most oocytes in a time-dependent manner. DMSO during cooling did not have any beneficial effect on subsequent oocyte maturation and spindle formation. These results suggest that porcine oocytes are very sensitive to a drop in the temperature before exposure to culture. Cooling oocytes before maturation inhibits their subsequent spindle organisation, nuclear and cytoplasmic maturation. Addition of DMSO to the cooling solution did not protect porcine oocytes from cooling-induced damage.     

Roles of cAMP in regulation of both MAP kinase and p34(cdc2) kinase activity during meiotic progression, especially beyond the MI stage

Time-dependent changes in the level of adenosine cyclic AMP (cAMP) in porcine oocytes during meiotic progression from the germinal vesicle stage (GV stage) to the metaphase II stage (MII stage) were examined using reversed-phase HPLC with UV detection. The concentration of cAMP in oocytes reached a peak at 8 hr of cultivation of cumulus-oocyte complexes (COCs), but it was dramatically decreased after 12-hr cultivation. After a 28-hr cultivation period, the level of cAMP in the oocytes had significantly reduced further, and the basal level of cAMP was observed in oocytes cultured at 32 hr and for up to 48 hr. When phosphatidylinositol 3-kinase (PI 3-kinase) or protein kinase C (PKC) in cumulus cells [which were required for meiotic progression to the MII stage in porcine oocytes (Shimada and Terada, 2001: Biol Reprod 64:1106-1114)] was suppressed by each specific inhibitor following initial 24-hr cultivation of COCs, cAMP level in the oocytes was significantly increased. After 24-hr cultivation in the maturation medium, COCs, which were cultured for an additional 24 hr in the presence of either forskolin or 3-isobutyl-1-methylxanthine (IBMX), exhibited a significant increase in the oocyte cAMP level to the similar level of that in oocytes cultured with PI 3-kinase inhibitor or PKC inhibitor, and the addition of each agent significantly suppressed meiotic progression from the MI to the MII stage and the activity of mitogen-activated protein kinase (MAPK) and p34(cdc2) kinase. These results demonstrated that when transported into oocytes from the cumulus cells via gap junctions, cAMP plays an important role not only in meiotic resumption, but also in the regulation of meiotic progression beyond the MI stage in porcine 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.     

Phosphatidylinositol 3-kinase and Akt participate in the FSH-induced meiotic maturation of mouse oocytes

Phosphatidylinositol 3-kinase (PI3K) is known to play critical roles in signal transduction processes related to a variety of cellular activities. In the present study, we investigated the role of PI3K during meiotic maturation in mouse oocytes using a specific inhibitor, LY294002. In follicle-stimulating hormone (FSH)-induced reversal of hypoxanthine-mediated meiotic arrest of cumulus oocyte complexes (COCs), LY294002 suppressed germinal vesicle breakdown (GVBD), first polar body (PB1) emission, and cumulus expansion. To examine the effect of LY294002, denuded oocytes (DOs) were cultured in medium containing follicular fluid meiosis-activating sterol (FF-MAS) since absence of gonadotropin receptors in oocytes has been reported and FSH did not stimulate meiotic maturation of DOs in the presence of hypoxanthine. In FF-MAS-induced maturation of DOs, LY294002 suppressed PB1emission, but not GVBD. In spontaneous gonadotropin-independent oocyte maturation, LY294002 had no effect on COCs and DOs. Akt/protein kinase B, a serine-threonine kinase, is a key downstream effector of the PI3K pathway. Therefore, we also examined the distribution of Akt during FSH-induced meiotic maturation. The distribution of Ser(473) phosphorylated Akt was similar to the localization of microtubules, while Thr(308) phosphorylated Akt was present in the pericentriolar materials (PCM) in metaphase I (MI) and II (MII) oocytes. LY294002 decreased the amount of Thr(308) phosphorylated Akt to very low to undetectable levels in MI and MII oocytes. Ser(473) phosphorylated Akt showed aberrant distribution and very low to undetectable levels of expression in LY294002-treated MI and MII oocytes, respectively. These results suggest that PI3K and Akt participate in mouse meiotic maturation.     

Antibody microarray analyses of signal transduction protein expression and phosphorylation during porcine oocyte maturation

Kinex antibody microarray analyses was used to investigate the regulation of 188 protein kinases, 24 protein phosphatases, and 170 other regulatory proteins during meiotic maturation of immature germinal vesicle (GV+) pig oocytes to maturing oocytes that had completed meiosis I (MI), and fully mature oocytes arrested at metaphase of meiosis II (MII). Increases in apparent protein levels of protein kinases accounted for most of the detected changes during the GV to MI transition, whereas reduced protein kinase levels and increased protein phosphorylation characterized the MI to MII transition. During the MI to MII period, many of the MI-associated increased levels of the proteins and phosphosites were completely or partially reversed. The regulation of these proteins were also examined in parallel during the meiotic maturation of bovine, frog, and sea star oocytes with the Kinex antibody microarray. Western blotting analyses confirmed altered expression levels of Bub1A, IRAK4, MST2, PP4C, and Rsk2, and the phosphorylation site changes in the kinases Erk5 (T218 + Y220), FAK (S722), GSK3-beta (Y216), MEK1 (S217 + S221) and PKR1 (T451), and nucleophosmin/B23 (S4) during pig oocyte maturation.     

The induction of reversible and irreversible chromosome decondensation by protein synthesis inhibition during meiotic maturation of mouse oocytes

We investigated the effects of puromycin on mouse oocyte chromosomes during meiotic maturation in vitro. Puromycin treatment for 6 hr at 100 μg/ml almost completely, but reversibly, suppressed [³⁵S]methionine incorporation into oocyte protein at all stages of maturation tested. Nevertheless, oocytes treated at the germinal vesicle stage underwent germinal vesicle breakdown (GVBD) and chromosome condensation. These oocytes completed nuclear maturation to metaphase II (MII) if the inhibitor was withdrawn. Prolonged (24-hr) treatment, however, caused the chromsomes to degenerate. The chromosomes of oocytes treated shortly after GVBD for 6 hr remained condensed, but the oocytes failed to form a polar body. However, 24-hr treatment caused the chromosomes to decondense to form an interphase nucleus. Oocytes treated near MI for 6 hr gave off a polar body during the treatment, and their chromosomes decondensed to form a nucleus, which remained as long as the treatment was continued. However, if the puromycin was withdrawn, the chromosomes recondensed to a state morphologically similar to that at MII. Thus, the chromosome decondensation induced by protein synthesis inhibition at MI was reversible. Oocytes treated at MII, several hours after first polar body formation, also underwent chromosome decondensation to form a nucleus. In the continuous presence of puromycin, the chromosomes remained decondensed, but neither DNA synthesis nor mitosis occurred. However, following puromycin withdrawal, these occytes synthesised DNA and underwent mitosis. Thus, protein synthesis inhibition at MII, by parthenogenetically activating the oocytes, caused irreversible chromosome decondensation. Based on these observations, we discussed the roles of protein synthesis in the regulation of oocyte chromosome behaviour during meiotic maturation.     

Biochemical studies on goat oocytes: Timing of nuclear progression, effect of protein inhibitor and pattern of polypeptide synthesis during in vitro maturation

Temporal progression of nuclear events of goat oocytes matured in vitro was studied by adding a specific inhibitor to the culture medium at different time points, to investigate protein synthesis requirements and its pattern during in vitro maturation. Goat cumulus-oocyte complexes (COCs) were matured in vitro in TCM 199, fixed at different time intervals and stained with orcein to assess nuclear changes. The germinal vesicle (GV) stage was found to be present at 0 h, chromosomal condensation stage was observed at 8 h, metaphase I at 12 to 14 h, and metaphase II was begun after 16 h of maturation and was nearly completed at 24 h. Protein synthesis inhibitor, cycloheximide, blocked oocyte maturation at germinal vesicle breakdown(GVBD), if added to the maturation medium between 0 to 4 h, suggesting that protein synthesis is required for GVBD. The transition from metaphase I to metaphase II was also protein synthesis-dependent, as observed when cycloheximide was used between 8 to 10 h of culture. When cycloheximide was added from 12 h of culture onwards, nuclear progression to metaphase II was progressively restored, but many chromosomal abnormalities were noted. Changes in the protein synthesis pattern were studied by radiolabeling of oocytes with [(35)S]-methionine at 0, 7, 12 and 24 h of culture, corresponding with GV, GVBD, metaphase I and metaphase II stages. A polypeptide of 28.1 KDa appeared as a major band at the GV stage, and its size decreased greatly and disappeared after the GVBD stage. Three new polypeptides (35, 36.5 and 39 KDa) appeared at GVBD and were detectable at metaphase II. In conclusion, the synthesis of proteins is required for the maintenance and transition of goat oocytes from GV to metaphase II during in vitro maturation.     

Susceptibility of bovine germinal vesicle-stage oocytes from antral follicles to direct effects of heat stress in vitro

Delineation of maternal versus direct effects of heat stress in reducing development at the germinal vesicle (GV) stage is challenging, because oocytes spontaneously resume meiosis after removal from antral follicles. The use of S-roscovitine (inhibitor of p34(cdc2)/cyclin B kinase) to hold bovine oocytes at the GV stage without compromising early embryo development was previously validated in our laboratory. The objective of the present study was to assess the direct effects of an elevated temperature commonly seen in heat-stressed dairy cows on cumulus-oocyte complexes (COCs) held at the GV stage using 50 microM S-roscovitine. During roscovitine culture, GV-stage COCs (antral follicle diameter, 3-8 mm) were cultured at 38.5 or 41 degrees C. Thereafter, oocytes were removed from roscovitine medium and allowed to undergo in vitro maturation, fertilization, and culture. Zona pellucida hardening (solubility to 0.5% pronase), nuclear stage (Hoechst 33342), cortical granule type (lens culinaris agglutinin-fluorescein isothiocyanate [FITC]), and early embryo development were evaluated. Culture of GV-stage COCs at 41 degrees C increased the proportion that had type III cortical granules and reduced the proportion that progressed to metaphase II after in vitro maturation. Effects of 41 degrees C on zona pellucida hardening, fertilization (penetration, sperm per oocyte, pronuclear formation, and monospermic and putative embryos), and cleavage of putative zygotes were not noted. However, culture of GV-stage COCs at 41 degrees C for 6 h decreased the proportion of 8- to 16-cell embryos, whereas 41 degrees C for 12 h reduced blastocyst development. In summary, antral follicle COCs are susceptible to direct effects of elevated body temperature, which may account in part for reduced fertility in heat-stressed cows.     

Mitochondrial distribution and meiotic progression in canine oocytes during in vivo and in vitro maturation

The objective was to evaluate mitochondrial distribution, and its relationship to meiotic development, in canine oocytes during in vitro maturation (IVM) at 48, 72, and 96 h, compared to those that were non-matured or in vivo matured (ovulated). The distribution of active mitochondria during canine oocyte maturation (both in vitro and in vivo) was assessed with fluorescence and confocal microscopy using MitoTracker Red (MT-Red), whereas chromatin configuration was concurrently evaluated with fluorescence microscopy and DAPI staining. During IVM, oocytes exhibited changes in mitochondrial organization, ranging from a fine uniform distribution (pattern A), to increasing clustering spread throughout the cytoplasm (pattern B), and to a more perinuclear and cortical distribution (pattern C). Pattern A was mainly observed in germinal vesicle (GV) oocytes (96.4%), primarily in the non-matured group (P < 0.05). Pattern B was seen in all ovulated oocytes which were fully in second metaphase (MII), whereas in IVM oocytes, ∼64% were pattern B, irrespective of duration of culture or stage of nuclear development (P > 0.05). Pattern C was detected in a minor percentage (P < 0.05) of oocytes (mainly those in first metaphase, MI) cultured for 72 or 96 h. In vitro matured oocytes had a minor percentage of pattern B (P < 0.05) and smaller mitochondrial clusters in IVM oocytes than ovulated oocytes, reaching only 4, 11, and 17% of MII at 48, 72, and 96 h, respectively. Thus, although IVM canine oocytes rearranged mitochondria, which could be related to nuclear maturation, they did not consistently proceed to MII, perhaps due to incomplete IVM, confirming that oocytes matured in vitro were less likely to be competent than those matured in vivo.     

Ovine cumulus cells estradiol-17Beta production in the presence or absence of oocyte

The objective of the present study was to compare the in vitro production of estradiol-17Beta (E(2)) by cumulus cells in the presence or absence of ovine oocyte. Moreover, the relationship between the concentration of produced estradiol-17Beta and oocyte nuclear maturation was assessed. Ovaries collected from the local abattoir were transported to the laboratory in saline at 30-35 degrees C within 1-3 h after collection. The oocytes of follicles, 2-6 mm in diameter, were recovered by aspiration. The oocytes with evenly granulated cytoplasm and which were surrounded with at least three layers of cumulus cells were selected and subjected to culture in pre-incubated oocyte culture medium (OCM). Before culturing, the selected oocytes were randomly divided into five treatment groups: Group 1, cumulus enclosed oocytes cultured in OCM (Group COCs); Group 2, denuded oocytes cultured in OCM (Group D); Group 3, denuded oocytes co-cultured with a cumulus cell-monolayer in OCM (Group D+M); Group 4, denuded oocytes co-cultured with previously cultured (for 26 h) cumulus cell-monolayer (10(5) cells/ml) in refreshed OCM (Group D+M(26)); Group 5, cumulus cell-monolayer (10(5) cells/ml) cultured in OCM (Group M). After an incubation period (26 h at 38.6 degrees C, 5% CO(2) and 100% humidity), the media were collected and kept at -20 degrees C until hormonal assay. The concentration of E(2) was determined by RIA method. For assessment of nuclear status, the completely denuded oocytes were subjected to DAPI staining. The highest percentage of metaphase II (MII) stage oocytes was observed in Group N (91%) and the lowest percentage was observed in Group D (6%) and Group D+M(26) (6%). The mean production of E(2) was highest and lowest in Group D+M (378.69+/-54.34 pg/ml) and Group D+M(26) (109.15+/-8.24 pg/ml), respectively. The production of E(2) was significantly (P<0.01) higher in Group D+/-M when compared with Groups M and D+/-M(26). Regarding the nuclear maturation, the percentage of MII stage oocytes was significantly (P<0.001) higher in Group COCs compared to the other groups. The results suggest that steroidogenic activity of cumulus cells in in vitro condition can be influenced by the pattern of connection between cumulus cells and the oocyte. Moreover, the nuclear maturation of oocytes is not influenced by the different production levels of E(2).     

Maturity at collection and the developmental potential of rhesus monkey oocytes

The purpose of this study was to evaluate the in vitro fertilizability of rhesus monkey oocytes and the developmental capacity of the resulting embryos as they relate to oocyte maturation at the time of follicular aspiration. Animals were hyperstimulated with human follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH), with follicular aspiration performed 27 h after administration of an ovulatory stimulus (1000 IU human chorionic gonadotropin [hCG] or 3 x 100 micrograms gonadotropin-releasing hormone [GnRH]). In 7 animals exhibiting a continuously rising pattern of serum estradiol through Day 10 of hyperstimulation, 45 germinal vesicle-intact (GV), 106 metaphase I (MI), and 24 metaphase II (MII) oocytes were collected and cultured in vitro. Upon reaching MII, oocytes were inseminated with 5 x 10(4) motile sperm/ml. Twenty-four percent of GV oocytes cultured in vitro matured to MII with 11 inseminated and none fertilized. Seventy-three percent of MI oocytes matured to MII in vitro with 50% inseminated and 32% fertilized. Oocytes collected at MII stage and inseminated underwent fertilization at a high rate of efficiency (93%). Pronuclear to 8-cell stage embryos were frozen and, upon thawing, 67% (10/15) survived with all blastomeres intact. Frozen-thawed embryos (2- to 6-cell) were transferred to the oviducts of 4 recipients (2 embryos/recipient) during the early luteal phase (1-3 days post LH surge) of natural menstrual cycles. Three twin pregnancies resulted. Thus, a positive correlation exists between the degree of nuclear maturation of rhesus monkey oocytes at collection and their potential for fertilization in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nuclear and microtubule dynamics of G2/M somatic nuclei during haploidization in germinal vesicle-stage mouse oocytes

During the haploidization process, it is expected that diploid chromosomes of somatic cells will be reduced to haploid for the generation of artificial gametes. In the present study, we aimed to use enucleated mouse oocytes at the germinal vesicle-stage (G2/M) as recipients for somatic cells that are also synchronized to the G2/M stage for haploidization. The reconstructed oocytes were then induced to undergo meiosis in vitro and observed for their nuclear morphology and microtubule network formation at various expected stages of the meiotic division. Following in vitro maturation, more than half (62/119, 52.1%) of the reconstructed oocytes completed the first round of meiosis-like division, as evidenced by the extrusion of pseudopolar bodies (PBs). However, accelerated PB extrusion, approximately 3-4 h earlier than that by control oocytes occurred. Furthermore, abnormally large pseudo-PBs, as large as four times the normal PB sizes, were observed. During the process of in vitro maturation at both the expected stages of metaphase I (MI) and metaphase II (MII), condensed chromosomes were observed in 38.7% and 55.2% of oocytes, respectively. However, two other types of nuclear configurations were also observed: 1) uneven distribution of chromatin and 2) an interphase-like nucleus, indicating deficiencies in chromosome condensation. Following oocyte activation, more than half (21/33, 63.6%) of the reconstructed oocytes with pseudo-PBs formed separated pseudopronuclei (PN), suggesting formation of functional spindles. The formation of bipolar spindle-like microtubule network at both the expected MI and MII stages during in vitro maturation was confirmed by immunohistochemistry. In summary, this study demonstrated that a high proportion of G2/M somatic nuclei appear to undergo meiosis-like division, in two successive steps, forming a pseudo-PB and two separate pseudo-PN upon in vitro maturation and activation treatment. Moreover, the enucleated geminal vesicle cytoplast retained its capacity for meiotic division following the introduction of a somatic G2/M nucleus.     

Somatic nucleus remodelling in immature and mature Rassir oocyte cytoplasm

Successful production of cloned animals derived from somatic cells has been achieved in sheep, cattle, goats, mice, pigs, rabbits, etc. But the efficiency of nuclear transfer is very low in all species. The present study was conducted to examine somatic nucleus remodelling and developmental ability in vitro of rabbit embryos by transferring somatic cells into enucleated germinal vesicle (GV), metaphase I (MI) or metaphase II (MII) oocytes. Microtubules were organized around condensed chromosomes after the nucleus had been transferred into any of the three types of cytoplasm. A bipolar spindle was formed in enucleated MII cytoplasm. Most of the nuclei failed to form a normal spindle within GV and MI cytoplasm. Some chromosomes scattered throughout the cytoplasm and some formed a monopolar spindle. Pseudopronucleus formation was observed in all three types of cytoplasm. Reconstructed embryos with MI and MII cytoplasm could develop to blastcysts. Nuclei in GV cytoplasm could develop only to the 4-cell stage. These results suggest that (1) GV material is important for nucleus remodelling after nuclear transfer, and (2) oocyte cytoplasm has the capacity to dedifferentiate somatic cells during oocyte maturation.