Kayoko Kida

Canine oocyte maturation in culture: Significance of estrogen and EGF receptor gene expression in cumulus cells

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.

Epidermal growth factor, transforming growth factor-α, and epidermal growth factor receptor expression and localization in the canine endometrium during the estrous cycle and in bitches with pyometra.

Gene expression and immunohistochemical localization of epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and epidermal growth factor receptor (EGF-R) were compared between the endometrium of bitches (Canis familiaris) with pyometra accompanied by cystic endometrial hyperplasia (CEH) and that of healthy bitches at similar stages of the estrous cycle. In normal bitches, endometrial TGF-alpha mRNA levels were highest at proestrus and gradually decreased as the cycle progressed to anestrus. Epidermal growth factor receptor mRNA levels were not significantly affected by the stage of the estrous cycle. Epidermal growth factor mRNA levels were higher at Day 35 of diestrus than at other stages of the estrous cycle (P<0.05). In bitches with pyometra, endometrial TGF-alpha and EGF-R mRNA levels did not differ significantly from those at diestrus in normal bitches, but EGF mRNA levels were lower than those at Day 35 of diestrus in normal bitches (P<0.05). In normal bitches, positive immunohistochemical staining for TGF-alpha, EGF, and EGF-R was mainly present in the glandular and luminal epithelial cells of the endometrium. In contrast, in bitches with pyometra, immunoreactivity for EGF was clearly present in endometrial stromal cells. Inflammatory cells that had infiltrated the endometrial stroma stained strongly for TGF-alpha and EGF-R. Luminal and glandular epithelial cells also stained positive for EGF-R. In conclusion, expression of TGF-alpha by inflammatory cells and a low level of expression and differential localization of EGF may be involved in aberrant growth of endometrial glands and development of CEH.

Factors associated with patency of the uterine cervix in bitches with pyometra.

This study examined factors involved in the patency of uterine cervices in the bitch with pyometra. The uterine cervices were obtained from the bitches with pyometra at the time of ovariohysterectomy. Cervical patency was measured by inserting the stainless steel rods with different diameter into cervical canals. Collagen concentration and collagenase activity (for type I collagen) in the tissue were determined and the number of neutrophils, which contain the enzymes related to collagen metabolism, and morphological changes in collagenous fibers were studied by histological examination. Levels of mRNA expressions for hormonal factors, estrogen receptor-α (ER-α), progesterone receptor (PR), relaxin (Rlx) and an attractant of neutrophils, interleukin-8 (IL-8), were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). In the statistical analysis, the cervical patency positively correlated with the collagenase activity, and negative correlation was found between the cervical patency and collagen concentration. Histological examination indicated distinct positive correlation between the cervical patency and the number of neutrophils in the cervical stroma and that the collagenous fiber in the uterine cervix became thinner and degraded with increase of the cervical patency. Although there was no relationship between the cervical patency and the level of mRNA for ER-α, PR or Rlx, IL-8 mRNA level has significant positive correlation with the cervical patency and the number of neutrophils in the cervical stroma. These results suggest that the increased number of neutrophils in the uterine cervix, which could be related to the local expression of IL-8, may be involved in collagen degradation and connective tissue remodeling to increase cervical patency in the bitch with pyometra.