Análise do mRNA da proteína BMP-15 e IGF-2 em oócitos e células do cumulus de ovelhas do semiárido pernambucano. / Analysis of mRNA expression of BMP-15 and IGF-2 proteins in oocytes and cumulus cells of sheep in semi-arid state of Pernambuco.

AUTOR(ES)
FONTE

IBICT - Instituto Brasileiro de Informação em Ciência e Tecnologia

DATA DE PUBLICAÇÃO

29/02/2012

RESUMO

Two experiments were conducted to analyze the expression of genes related to follicular development and subsequent reproductive efficiency in oocytes and cumulus cells in two distinct phases of folliculogenesis. Ovaries of native breeds of sheep slaughtered in the abattoir in Caruaru/PE were collected and transported to the Laboratory of Applied Molecular Animal Physiology (FAMA)/UFRPE. The first group of samples was composed of cumulus cells and oocytes from follicles in size between 3.1 and 6 mm, classified morphologically as antral follicles. The follicles were simply punctured/aspirated using a needle and syringe (10 ml with needle 0.7 x 25 mm) and analyzed using a magnifier stereomicroscope for localization of the oocytes. The oocytes which were surrounded by cumulus cells were denuded using multiple pipetting and stored separately in phosphate buffer (PBS 1x). In this advanced stage of follicular development, oocytes were collected and distributed in "pools" of 10 cells (n = 10), and 10 samples with "pools" of cumulus cells (n = 10). For collection of follicles smaller than 3 mm, that formed the second group of samples, morphologically classified as preantral follicles (primary and secondary), the ovaries were sliced with a blade and the tissue kept in PBS 1x while the oocytes were separated and removed from the ovarian tissue using a magnifier. Oocytes were grouped in 10 samples with "pools" of 10 cells (n = 10), and 10 samples with "pools" of cumulus cells (n = 10) for this initial phase of follicular development. RNA was extracted from the oocytes and the cumulus cells and the cDNA was obtained by RT-PCR using random oligo dT to the mRNA. In the first study, the expression analysis was performed by qPCR (real time PCR) for BMP15 gene expression. Oocytes, but not cumulus cells, showed amplification for BMP15, and there was an increase in the relative expression of BMP15 mRNA during the later stage of follicular development and differences were considered significant (p <0.05). In the second study aimed to analyze the expression of IGF-II gene by PCR in real time in both groups of oocytes and cumulus cells according to the follicular phase. The expression analysis confirmed the expression of IGF-2 gene for groups of oocyte and the cumulus cells. There was an increase in the relative expression of mRNA for IGF-2 for groups of oocytes during the later stage of follicular development and differences were considered significant (p <0.05). There was no significant variation in the expression of IGF2 between groups of cumulus cells. It follows that the BMP-15 and IGF-2 have higher levels of expression in sheep oocytes in the second stage of follicular development in the conditions adopted. However, BMP-15 was not expressed in cumulus cells, but IGF-2 presented the same pattern during the stages of follicular development studied.

ASSUNTO(S)

biotecnologia gene ovino reprodução medicina veterinaria biotechnology gene ovine reproduction

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