Oestrogen and Progesterone Receptor Gene Expression in Canine Oocytes and Cumulus Cells Throughout the Oestrous Cycle

The aim of this research was to analyze oestrogen receptor-alpha (ER alpha), ER beta and progesterone receptor (PR) gene expression in the canine oocyte and cumulus cells throughout the oestrous cycle. Ovaries from 38 bitches were recovered after ovariohysterectomy and sliced. The phase of the oestrous cycle was determined by vaginal cytology, vaginoscopy and serum hormonal measurements. Oocytes were mechanically denuded by repeated pipetting. For each phase of the cycle, a sample was composed by a pool of 50 oocytes (sample number: prooestrus = 3, oestrus = 8, dioestrus = 5 and anoestrus = 5) or a pool of cumulus cells (prooestrus = 4, oestrus = 7, dioestrus = 4 and anoestrus = 6). Oocyte and cumulus cells` total RNA was isolated and reverse transcription was conducted to perform real-time PCR. Oestrogen receptor-alpha was expressed throughout the cycle in the oocyte (33.33%, 25.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively) and cumulus cells (50.0%, 47.14%, 25.0% and 66.67% for prooestrus, oestrus, dioestrus and anoestrus, respectively). In the oocyte, the ER beta was also expressed in all phases of the cycle (33.33%, 50.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively), whereas in cumulus cells, ER beta was only expressed during prooestrus (50%) and oestrus (14.29%). Interestingly, while the oocyte PR was not detected in any phase of the cycle, this receptor was expressed during prooestrus (50%), oestrus (42.86%) and anoestrus (16.67%) in cumulus cells. In conclusion, canine oocytes express ER alpha and ER beta throughout the oestrous cycle, however, there is a lack of PR expression in all these phases. Moreover, in cumulus cells, only ER alpha was expressed throughout the oestrous cycle.

Effect of glycine and alanine supplementation on development of cattle embryo cultured in CR1aa medium with or without cumulus cells

Selostus: Glysiinin ja alaniinin vaikutus CR1aa-liuoksessa viljeltyyn kumulussolullisen ja -soluttoman naudanalkion kehitykseen

Steroidogenesis in cumulus cells of bovine cumulus-oocyte-complexes matured in vitro with BSA and different concentrations of steroids

The present in vitro experiments were designed to evaluate the ability of bovine cumulus-oocyte-complexes (COCs)to produce steroids and also to evaluate the modulatory effects of added estradiol, progesterone and testosterone on the steroidogenic activity of COCs. Considerable estradiol accumulation was observed in the control maturation medium for in vitro maturation of bovine COCs during the 24h of maturation (P < 0.05). When testosterone was added to the medium at various concentrations, a slight estradiol accumulation occurred, which, however, was lower (P < 0.05) than that observed in the control medium. Slight estradiol accumulation was observed in maturation medium containing progesterone at concentrations of 2.5, 5.0 and 10.0 mug/ml, but these increases were less (P < 0.05) than those observed in the control medium. However, in the presence of 1.0 mug/ml progesterone, estradiol accumulation was equal to that of the control medium (P > 0.05). Progesterone accumulation (P < 0.05) was observed in the control medium for in vitro maturation of bovine COCs. When estradiol was added to the maturation medium, progesterone accumulation was observed, but was significant (P < 0.05) only when the medium was supplemented with the lesser concentrations of estradiol utilized in the experiment (1.0 mug/ml). The results demonstrated that (1) cumulus cells of bovine COCs are able to secrete estradiol and progesterone in culture systems for in vitro maturation, and this steroidogenesis is modulated by the steroids progesterone, testosterone and estradiol, and (2) the addition of estradiol to the in vitro maturation medium of bovine oocytes should be reviewed, since cumulus cells of COCs have been demonstrated to secrete estradiol in the maturation medium. (C) 2002 Elsevier B.V. B.V. All rights reserved.

Effect of melatonin on DNA damage of bovine cumulus cells during in vitro maturation (IVM) and on in vitro embryo development

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of follicle size on mRNA expression in cumulus cells and oocytes of Bos indicus: an approach to identify marker genes for developmental competence

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Characterisation of the methylation pattern in the intragenic CpG island of the IGF2 gene in Bos taurus indicus cumulus cells during in vitro maturation

The aim of this study was to characterise the methylation pattern in a CpG island of the IGF2 gene in cumulus cells from 1-3 mm and a parts per thousand yenaEuro parts per thousand 8.0 mm follicles and to evaluate the effects of in vitro maturation on this pattern.Genomic DNA was treatment with sodium bisulphite. Nested PCR using bisulphite-treated DNA was performed, and DNA methylation patterns have been characterised.There were no differences in the methylation pattern among groups (P > 0.05). Cells of pre-IVM and post-IVM from small follicles showed methylation levels of 78.17 +/- 14.11 % and 82.93 +/- 5.86 %, respectively, and those from large follicles showed methylation levels of 81.81 +/- 10.40 % and 79.64 +/- 13.04 %, respectively. Evaluating only the effect of in vitro maturation, cells of pre-IVM and post-IVM COCs showed methylation levels of 80.17 +/- 12.01 % and 81.19 +/- 10.15 %.In conclusion, the methylation levels of the cumulus cells of all groups were higher than that expected from the imprinted pattern of somatic cells. As the cumulus cells from the pre-IVM follicles were not subjected to any in vitro manipulation, the hypermethylated pattern that was observed may be the actual physiological methylation pattern for this particular locus in these cells. Due the importance of DNA methylation in oogenesis, and to be a non-invasive method for determining oocyte quality, the identification of new epigenetic markers in cumulus cells has great potential to be used to support reproductive biotechniques in humans and other mammals.

Gene Expression in Bovine Oocytes and Cumulus Cells After Meiotic Inhibition with the Cyclin-Dependent Kinase Inhibitor Butyrolactone I

Contents The aim of this study was to determine the effect of temporary inhibition of meiosis using the cyclin-dependent kinase inhibitor butyrolactone I (BLI) on gene expression in bovine oocytes and cumulus cells. Immature bovine cumulusoocyte complexes (COCs) were assigned to groups: (i) Control COCs collected immediately after recovery from the ovary or (ii) after in vitro maturation (IVM) for 24 h, (iii) Inhibited COCs collected 24 h after incubation with 100 mu m BLI or (iv) after meiotic inhibition for 24 h followed by IVM for a further 22 h. For mRNA relative abundance analysis, pools of 10 denuded oocytes and respective cumulus cells were collected. Transcripts related to cell cycle regulation and oocyte competence were evaluated in oocytes and cumulus cells by quantitative real-time PCR (qPCR). Most of the examined transcripts were downregulated (p < 0.05) after IVM in control and inhibited oocytes (19 of 35). Nine transcripts remained stable (p > 0.05) after IVM in control oocytes; only INHBA did not show this pattern in inhibited oocytes. Seven genes were upregulated after IVM in control oocytes (p < 0.05), and only PLAT, RBP1 and INHBB were not upregulated in inhibited oocytes after IVM. In cumulus cells, six genes were upregulated (p < 0.05) after IVM and eight were downregulated (p < 0.05). Cells from inhibited oocytes showed the same pattern of expression regarding maturation profile, but were affected by the temporary meiosis inhibition of the oocyte when the same maturation stages were compared between inhibited and control groups. In conclusion, changes in transcript abundance in oocytes and cumulus cells during maturation in vitro were mostly mirrored after meiotic inhibition followed by maturation.