Clone,Localization And Abundance Analysis Of GDF9 In Rabbit

According to the GDF 9 sequences of mouse and human in Genebank, two pair primers for PCR were designed. Using RT-PCR from rabbit ovary, two fragments of rabbit GDF9gene were isolated, then were cloned into pMD19-T-simple vector and were sequenced. The results showed that the obtained two fragments were 289bp and 299bp. The first sequence shared 86%,86%,86%,82%,87% nucleotide homology with the published mRNA of human, porcine, cow and mouse separately. The second one showed the homology of this gene sequence with pig, human, cow, mouse and goat were 86%,86%,86%,82%,87%,respectly.Using immunohistochemical method, the presence and distribution of GDF9 in rabbit ovary and testis were studied. The findings showed that the positive reaction was absent in primordial follicles. The GDF9 protein was seen in the oocytes'membrane of primary follicles. In the secondary follicles, the protein mainly located in the cytoplasm of oocytes and the level of it increased. The positive reactions were observed in oocytes'membrane and granulosa cells in antral follicles, but its expressed level declined. GDF9 protein were furthermore found in corpora lutea. In the testis, the protein was expressed in the primary spermatocytes and round spermatid.The expression of GDF-9 was studied by RT-PCR analysis using mRNAs derived from female rabbit tissues (hypothalamus, pituitary, ovary, uterus) and male tissues (hypothalamus, pituitary, testis, epididymis). GDF-9 was detected in all of these tissues.By fluorescence quantitative PCR (FQ-PCR), the mentioned tissues in the text were measured. The results showed in the male rabbit the hightest expression of GDF9mRNA was in epididymis that was3.0543-fold of the testis. The highest level were 0.1354 and 0.4394-fold of hypothalamus and pituitary separately. In the female, the level in ovary,hypothalamus and uterus were 13.7271-fold,5.035-fold and 2.0528- fold of the pituitary.