Activation of PKA, p38 MAPK and ERK1/2 by gonadotropins in cumulus cells is critical for induction of EGF-like factor and TACE/ADAM17 gene expression during in vitro maturation of porcine COCs

<p>Abstract</p> <p>Objectives</p> <p>During ovulation, it has been shown that LH stimulus induces the expression of numerous genes via PKA, p38 MAPK, PI3K and ERK1/2 in cumulus cells and granulosa cells. Our recent study showed that EGF-like factor and its protease (TACE/ADAM17) are required for the activation of EGF receptor (EGFR), cumulus expansion and oocyte maturation of porcine cumulus-oocyte complexes (COCs). In the present study, we investigated which signaling pathways are involved in the gene expression of EGF-like factor and in <it>Tace/Adam17 </it>expression in cumulus cells of porcine COC during <it>in vitro </it>maturation.</p> <p>Methods</p> <p><it>Areg</it>, <it>Ereg</it>, <it>Tace/Adam17</it>, <it>Has2</it>, <it>Tnfaip6 </it>and <it>Ptgs2 </it>mRNA expressions were detected in cumulus cells of porcine COCs by RT-PCR. Protein level of ERK1/2 phosphorylation in cultured cumulus cells was analyzed by westernblotting. COCs were visualized using a phase-contrast microscope.</p> <p>Results</p> <p>When COCs were cultured with FSH and LH up to 2.5 h, <it>Areg</it>, <it>Ereg </it>and <it>Tace/Adam17 </it>mRNA were expressed in cumulus cells of COCs. <it>Areg</it>, <it>Ereg </it>and <it>Tace/Adam17 </it>gene expressions were not suppressed by PI3K inhibitor (LY294002), whereas PKA inhibitor (H89), p38 MAPK inhibitor (SB203580) and MEK inhibitor (U0126) significantly suppressed these gene expressions. Phosphorylation of ERK1/2, and the gene expression of <it>Has2</it>, <it>Tnfaip6 </it>and <it>Ptgs2 </it>were also suppressed by H89, SB203580 and U0126, however, these negative effects were overcome by the addition of EGF to the medium, but not in the U0126 treatment group.</p> <p>Conclusion</p> <p>The results showed that PKA, p38 MAPK and ERK1/2 positively controlled the expression of EGF-like factor and TACE/ADMA17, the latter of which impacts the cumulus expansion and oocyte maturation of porcine COCs via the EGFR-ERK1/2 pathway in cumulus cells.</p

HISTOCHEMICAL OBSERVATIONS OF LIPID DROPLETS IN MOUSE EMBRYOS

Morphology of lipid droplets in 313 embryos of different developmental stages was examined. Embryos were obtained from spontaneously ovulated and superovulated mice of ddY strain. Sudan III was used to stain embryos so as to count the number of lipid droplets. Lipid droplets were classified into three groups according to the diameter : small (≦ 1.0μm), medium (1.1-3.0μm) and large (3.1-5.0μm). From the 2-cell stage to the expanded blastocyst, lipid droplets were found to be abundant. Numerous small lipid droplets were observed in all developmental stages. The number of medium and large lipid droplets increased with the development of embryos. The numbers of medium lipid droplets in the embryos at the 2-cell stage, 8-cell stage and expanded blastocyst were 0.5±0.4,11.2±3.3 and 60.3±3.2,respectively. Large lipid droplets were not observed in the 2-cell and 4-cell stages, but were found to be abundant in the morula and blastocyst. A sudden and significant increase in the number of medium and large lipid droplets was observed from the 8-cell stage to the morula (p<0.01). Similar increase of lipid droplets was also observed in morulae developed from 8-cell embryos in in vitro culture (p<0.01). The number of lipid droplets in expanded blastocysts was not affected by the ovulation methods

Prediction of maturational competence of feline oocytes using supravital staining of cumulus cells by propidium iodide

We examined the relationship between integrityof Cumulus cells and nuclear maturation rate after in vitro culture to determine a non-invasive prediction of the maturationalcompetence of feline oocytes･ Feline cumulus-oocyte complexes (COCs) were collected from either small (400-800l九m) or large (≧800 l⊥m) follicles･ Immediately after collection′ cumulus cells were evaluated morphologically (thickness of cumulus cell layers) and stained with propidium iodide (PI)′ which penetrates only nonviable cells･ Cumulus cells without PI staining were judged as having good membrane integrity･ After evaluation, COCs were cultured for 30 h and their nuclear maturation rate was determined. The nuclear maturation rate of oocytes derived from large follicles (89･8%) was higher (p < 0･05) than that from small follicles (60･8%)･ There was no difference in the maturation rate of oocytes from follicleswith the same size regardless of cumulus morphology ln contrast′ Oocytes that had cumulus cells with good membrane integrity showed a higher mahration rate (93･8%) than oocytes with poor cumulus integrity (76･9%) in large follicles (p < 0･05)･ We conclude that evaluation of membrane integrity of cumulus cells by propidium iodide stainlng Can be used to predict the maturational competence of oocytes

EFFECTS OF SUPPLEMENTATION OF THE MATURATION MEDIA WITH INSULIN ON IN VITRO MATURATION AND IN VITRO FERTILIZATION OF BOVINE OOCYTES

This study was carried out to determine the effects of supplementation of the maturation media with insulin on in vitro maturation and fertilization of bovine oocytes. In Experiment 1,cumulus-intact bovine oocytes were cultured in a maturation medium (TCM-199 containing 10% fetal calf serum, 0.02 U/ml follicular stimulating hormone and 1 μg/ml estradiol-17 β) with or without insulin supplementation (10 μg/ml). The maturation and fertilization rates of oocytes and subsequent embryonic development to the blastocyst stage were not affected by the treatment with insulin in the presence of serum and the hormones during the maturation period. In Experiment 2,to avoid the effects of serum and the hormones, a serum- and hormone-free maturation medium (TCM-199 containing 1 mg/ml polyvinyl alcohol) was used. In the absence of serum and hormones during the maturation period, the maturation rate was not affected by treatment with insulin, but the fertilization rate was improved. In Experiment 3,when denuded oocytes were inseminated together with cumulus cells cultured in serum- and hormone-free maturation medium supplemented with insulin, the fertilization rate was increased. These results demonstrate that the addition of insulin to the serum- and hormone-free maturation medium improves the fertilization rate of bovine oocytes in vitro, and suggest that insulin may stimulate the secretion of sperm capacitating agent (s) from cumulus cells

INFLUENCE OF TIME AFTER THE REMOVAL OF NOCODAZOLE FROM NUCLEAR DONORS ON THE DEVELOPMENT OF RECONSTITUTED EMBRYOS IN BOVINE NUCLEAR TRANSPLANTATION

The present study examined the influence of post-cleavage time of nuclear donors on the development of reconstituted embryos in bovine nuclear transfer. Blastomeres of 16-cell stage embryos derived from in vitro-maturation, fertilization and culture were used as nuclear donor source. They were treated with 10 μM nocodazole for 12 hr. Blastomeres that cleaved within 3 hr after the removal of nocodazole were used for the study. Metaphase II (M-II) oocytes were used as recipient cytoplasm. In experiment 1, donor blastomeres at 6, 11 and 15 hr after the removal of nocodazole and donor blastomeres not treated with nocodazole were transferred into ethanol-exposed and enucleated oocytes. The reconstituted embryos produced by donor blastomeres at 6 hr after the removal of nocodazole had a significantly higher developmental rate to the blastocyst stage than those at 15 hr and the untreated groups (P<0.01). In experiment 2, blastomeres at 6 hr after the removal of nocodazole used as nuclear donors were transferred into ethanol-exposed and enucleated M-II oocytes. The reconstituted embryos with ethanol-exposed and enucleated oocytes as recipient cytoplasm had a significantly higher rate of initial-cleavage (P<0.05) and development to the blastocyst stage (P<0.01) than non ethanol-exposed and enucleated M-II oocytes. These results demonstrate that the development of reconstituted embryos was improved when cleaved donor blastomeres after the removal of nocodazole were immediately transferred (at 3-6 hr post-cleavage) into activated enucleated oocytes by exposure to ethanol