The influence of prorenin/(pro)renin receptor on progesterone secretion by the bovine corpus luteum.

The aim of this study was to evaluate the role of prorenin/(pro)renin receptor activation on luteal progesterone (P4) secretion. Our hypothesis was that the nonproteolytic activation of (pro)renin receptor [P(RR)] is part of the regulatory mechanism responsible for corpus luteum (CL) function. In the first three experiments, prorenin was found to stimulate the production of P4, which is not inhibited by an angiotensin receptor antagonist (saralasin), but rather by a renin/prorenin inhibitor (aliskiren), a MAPK1/3 inhibitor (PD325901) or an EGFR inhibitor (AG1478), which are evidence of nonproteolytic activation of prorenin. Moreover, prorenin induced phosphorylation of MAPK1/3 in luteal cells. Following these in vitro experiments, a sequence of in vivo experiments was performed demonstrating that the intrafollicular injection of aliskiren in preovulatory follicles impaired P4 secretion in cows that ovulated. Furthermore, all profibrotic genes studied were present in the CL and TGFB1 and FN1 mRNA were upregulated from day 5-10 post-ovulation. During luteolysis, REN was downregulated at 48 h, whereas TGFB1 and SERPINE1 were dramatically upregulated in luteal tissue at 12 h after PGF. In summary, these data are evidence that nonproteolytic activation of (P)RR is involved in luteal function.

Bovine ovarian cells have (pro)renin receptors and prorenin induces resumption of meiosis in vitro.

The discovery of a receptor that binds prorenin and renin in human endothelial and mesangial cells highlights the possible effect of renin-independent prorenin in the resumption of meiosis in oocytes that was postulated in the 1980s.This study aimed to identify the (pro)renin receptor in the ovary and to assess the effect of prorenin on meiotic resumption. The (pro)renin receptor protein was detected in bovine cumulus-oocyte complexes, theca cells, granulosa cells, and in the corpus luteum. Abundant (pro)renin receptor messenger ribonucleic acid (mRNA) was detected in the oocytes and cumulus cells, while prorenin mRNA was identified in the cumulus cells only. Prorenin at concentrations of 10(-10), 10(-9), and 10(-8)M incubated with oocytes co-cultured with follicular hemisections for 15h caused the resumption of oocyte meiosis. Aliskiren, which inhibits free renin and receptor-bound renin/prorenin, at concentrations of 10(-7), 10(-5), and 10(-3)M blocked this effect (P<0.05). To determine the involvement of angiotensin II in prorenin-induced meiosis resumption, cumulus-oocyte complexes and follicular hemisections were treated with prorenin and with angiotensin II or saralasin (angiotensin II antagonist). Prorenin induced the resumption of meiosis independently of angiotensin II. Furthermore, cumulus-oocyte complexes cultured with forskolin (200µM) and treated with prorenin and aliskiren did not exhibit a prorenin-induced resumption of meiosis (P<0.05). Only the oocytes' cyclic adenosine monophosphate levels seemed to be regulated by prorenin and/or forskolin treatment after incubation for 6h. To the best of our knowledge, this is the first study to identify the (pro)renin receptor in ovarian cells and to demonstrate the independent role of prorenin in the resumption of oocyte meiosis in cattle.

Steroidogenic enzymes mRNA expression profile and steroids production in bovine theca cells cultured in vitro and stimulated with angiotensin II / Perfil de expressão de RNAm de enzimas esteroidogênicas e produção de esteroides a partir de células da teca bovina cultivadas in vitro e estimuladas por Angiotensina II

The main objective of this study was to detect the steroidogenic effects of Ang II in bovine theca cells in vitro. Bovine theca cells were obtained from follicles (larger than 10mm of diameter) collected from a local abattoir and submitted to different treatments in a sequence of experiments. In experiment 1, CYP17A1 mRNA profile was evaluated in LH- (10ng ml-1) and Ang II-treated (0.1µM) theca cells. In experiment 2, a dose-response effect of Ang II (0.001; 0.1 e 10µM) plus insulin (100ng ml-1) and LH (100ng ml-1) was evaluated on steroidogenesis of bovine theca cells. Experiment 3 explored the effects of saralasin (an antagonist of Ang II receptors) on steroid production and steroidogenic enzymes regulation in theca cells. After 24 hours, culture media from experiments 2 and 3 was collected to evaluate testosterone and androstenedione levels by High-Performance Liquid Chromatography. In parallel, mRNA levels of key steroidogenic enzymes (HSD3B2, CYP11A1, CYP17A1) and STAR were assessed by RT-PCR. There was no difference in testosterone and androstenedione production between treated and controls groups, as well as in mRNA levels of the evaluated genes. In conclusion, the results suggest that Ang II does not regulate steroidogenesis in bovine theca cells.

O objetivo deste trabalho foi verificar o efeito da Angiotensina II (Ang II) sobre a esteroidogenese nas células da teca bovina, cultivadas in vitro. Para isso, células da teca bovina foram obtidas de folículos maiores que 10 mm de diâmetro de ovários oriundos de abatedouro e submetidas a diferentes tratamentos em uma sequência de experimentos. No experimento 1, o perfil de expressão do RNAm de CYP17A1 foi avaliado nas células da teca em resposta ao LH (10ng ml-1) e/ou Ang II (0,1µM) em diferentes momentos de tratamento. No experimento 2, foi investigado o efeito dose-resposta de Ang II (0,001; 0,1 e 10µM), acrescido de insulina (100ng ml-1) e LH (100ng ̸ml) sobre a esteroidogênese nas células da teca bovina. O experimento 3 explorou os possíveis efeitos da Ang II por meio do tratamento de células da teca com saralasina (antagonista dos receptores da Ang II). Após 24 horas, nos experimentos 2 e 3, o meio de cultura foi coletado e avaliado quanto aos níveis de testosterona e androstenediona pela técnica de HPLC. Em paralelo, a expressão gênica de enzimas-chave da esteroidogênese (HSD3B2, CYP11A1, CYP17A1) e STAR foi avaliada por qRT-PCR. Não se observou diferença na produção de testosterona e androstenediona entre controle e grupos tratados, bem como, na expressão do RNAm para os genes estudados. Em conclusão, nossos resultados não demonstraram um papel da Ang II sobre a esteroidogenese nas células da teca bovina.