Hatoya S, Sugiyama Y, Nishida H, Okuno T, Torii R, Sugiura K, Kida K, Kawate N, Tamada H, Inaba T
Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.
Theriogenology. 2009 Mar 1;71(4):560-7. doi: 10.1016/j.theriogenology.2008.08.013. Epub 2008 Dec 19.
We examined the role of cumulus cells regarding in vitro maturation of canine oocytes, and investigated estrogen and epidermal growth factor (EGF) receptor gene expression and action on nuclear maturation. Canine cumulus-oocyte complexes (COC) were collected from anestrous and diestrous bitches; only COC with vitelline diameter >100 microm were used. In Experiment 1, expression of estrogen receptor (ER) alpha, ERbeta and EGF-receptor (EGF-R) were determined by reverse transcription-polymerase chain reaction (RT-PCR), using mRNA from the oocyte or cumulus cell. Transcripts for the ERbeta and EGF-R were detected in oocytes and cumulus cells, but no message was detected for ERalpha. In Experiment 2, intact COC and the denuded oocytes were cultured in TCM199 medium supplemented with various concentrations of estradiol-17beta (E(2); 0-10 microg/mL) or EGF (0-100 ng/mL) for 72 h; nuclear maturation was then evaluated. In oocytes cultured within intact COC, the rate of germinal vesicle breakdown (GVBD) was higher in the 1 microg/mL E(2) supplemented group (P<0.05), and the rate of metaphase I (MI) was higher in the 10 ng/mL EGF supplemented group, than in the non-supplemented group (P<0.05). However, supplementation of E(2) or EGF to denuded oocytes failed to promote nuclear maturation. In Experiment 3, intact COC were cultured in TCM199 supplemented with 1 microg/mL E(2), 10 ng/mL EGF, and 10% fetal bovine serum (FBS) for 72 h, and nuclear maturation was evaluated. There was no significant difference in the rate of metaphase II (MII) between the medium only, E(2)+EGF, and FBS supplement groups. When E(2) and EGF in combination with FBS were supplemented, the rate of MII was higher than in other groups (P<0.05). We inferred that cumulus cells were involved in mediating the stimulatory effects of E(2) and EGF on nuclear maturation of canine oocytes, and that E(2) and EGF in combination with FBS promoted the completion of oocyte meiotic maturation.
我们研究了卵丘细胞在犬类卵母细胞体外成熟过程中的作用，并调查了雌激素和表皮生长因子（EGF）受体基因的表达及其对核成熟的作用。从处于乏情期和发情后期的母犬收集犬类卵丘 - 卵母细胞复合体（COC）；仅使用卵黄直径>100微米的COC。在实验1中，利用来自卵母细胞或卵丘细胞的mRNA，通过逆转录 - 聚合酶链反应（RT-PCR）测定雌激素受体（ER）α、ERβ和EGF受体（EGF-R）的表达。在卵母细胞和卵丘细胞中检测到了ERβ和EGF-R的转录本，但未检测到ERα的信息。在实验2中，将完整的COC和裸卵在补充有不同浓度雌二醇-17β（E₂；0 - 10微克/毫升）或EGF（0 - 100纳克/毫升）的TCM199培养基中培养72小时；然后评估核成熟情况。在完整COC内培养的卵母细胞中，补充1微克/毫升E₂的组中，生发泡破裂（GVBD）率更高（P<0.05），补充10纳克/毫升EGF的组中，中期I（MI）率高于未补充组（P<0.05）。然而，向裸卵补充E₂或EGF未能促进核成熟。在实验3中，将完整的COC在补充有1微克/毫升E₂、10纳克/毫升EGF和10%胎牛血清（FBS）的TCM199中培养72小时，并评估核成熟情况。仅培养基组、E₂ + EGF组和FBS补充组之间的中期II（MII）率没有显著差异。当E₂和EGF与FBS联合补充时，MII率高于其他组（P<0.05）。我们推断卵丘细胞参与介导E₂和EGF对犬类卵母细胞核成熟的刺激作用，并且E₂和EGF与FBS联合促进了卵母细胞减数分裂成熟的完成。
