Molecular cloning, expression analysis, and function of decorin in goat ovarian granulosa cells.

Decorin (DCN), a component of the extracellular matrix (ECM), participates in ECM assembly and influences cell proliferation and apoptosis in many mammalian tissues and cells. However, expression and function of DCN in the ovary remain unclear. This study cloned the full-length cDNA of goat DCN obtained from the ovary of an adult goat. Sequence analysis revealed that the putative DCN protein shared a highly conserved amino acid sequence with known mammalian homologs. The tissue distribution of DCN mRNA expression was evaluated by real-time PCR, and the results showed that DCN was widely expressed in the tissues of adult goat. Immunohistochemistry results suggested that DCN protein existed in the granulosa cells and oocytes from all types of follicles and theca cells of antral follicles. Moreover, hCG-induced DCN mRNA expression was significantly reduced by the inhibitors of protein kinase A, PI3K, or p38 kinase (P < 0.05), which are key mediators involved in hCG-induced DCN expression. Overexpression of DCN significantly increased apoptosis and blocked cell cycle progression in cultured granulosa cells (P < 0.05). Western blot analysis also showed that overexpression of DCN upregulated the expression levels of p21 protein (P < 0.05), whereas no effects were observed on the expression of Bax and Bcl-2 and on Bcl-2/Bax ratio (P > 0.05). These findings suggested that DCN regulates the apoptosis and cell cycle of granulosa cells.

MicroRNA-10b suppresses goat granulosa cell proliferation by targeting brain-derived neurotropic factor.

Brain-derived neurotropic factor (BDNF) and its high-affinity receptor, tyrosine kinase receptor B, have been assumed to be involved in female reproduction and have recently shown to play an essential role in follicle activation and oocyte maturation. In this study, we analyzed the expression of miR-10b and BDNF in the ovary and discovered that the expression of miR-10b was higher in monotocous goat ovaries than in polytocous goat ovaries, whereas the expression pattern of BDNF in ovary was opposite. Moreover, human chorionic gonadotropin induced rapid and transient expression of BDNF messenger RNA and protein. In contrast, human chorionic gonadotropin upregulated miR-10b expression in a time-dependent manner. The BDNF gene was identified as a direct target of miR-10b using a dual-luciferase reporter assay. Transfection of granulosa cells with miR-10b decreased BDNF messenger RNA and protein levels. MiR-10b overexpression inhibited cell proliferation, whereas BDNF promoted cell proliferation. However, a combined treatment with miR-10b and BDNF promoted cell proliferation, indicating that the reintroduction of BDNF reversed the suppressive effect of miR-10b. These results demonstrate that miR-10b downregulates BDNF expression in granulosa cells by directly targeting the 3' untranslated regions and plays an important role in inhibiting granulosa cell proliferation by targeting BDNF.

Molecular characterization and hormonal regulation of tissue inhibitor of metalloproteinase 1 in goat ovarian granulosa cells.

Tissue inhibitor of metalloproteinase 1 (TIMP1) belongs to a group of endogenous inhibitors that control the activity of matrix metalloproteinases and other metalloproteinases. TIMP1 is ubiquitously expressed and implicated in many physiological and pathologic processes. In this study, the full-length complementary DNA of goat (Capra hircus) Timp1 was cloned from adult goat ovary for the first time to better understand the regulatory role of TIMP1. The putative TIMP1 protein shared a high amino acid sequence identity with other species. Real-time polymerase chain reaction results showed that Timp1 was widely expressed in adult goat tissues, and messenger RNA expression was higher in the ovary than in other tissues; meanwhile, increasing expression of Timp1 was also discovered during the process of follicle growth and corpus luteum. We then investigated Timp1 expression patterns in different types of ovarian follicular cells from goats. In small or large antral follicles, Timp1 expression was higher (P < 0.05) in theca cells than in granulosa cells, cumulus cells, and oocytes. Increasing expression of Timp1 in theca and granulosa cells was observed as the variation of the follicle size. Immunohistochemical analyses further revealed the presence of the TIMP1 proteins in follicles at all antral stages of development. The most intense staining for TIMP1 was observed in the theca cells and granulosa cells of large antral follicles and corpus luteum. Timp1 was highly (P < 0.05) induced in granulosa cells in vitro after treatment with the luteinizing hormone agonist, human chorionic gonadotropin. Treatments with forskolin, phorbol 12-myristate 13-acetate, or phorbol 12-myristate 13-acetate + forskolin could also stimulate Timp1 messenger RNA expression. The effects of human chorionic gonadotropin were reduced (P < 0.05) by the inhibitors of protein kinase A, protein kinase C, MAPK kinase, or p38 kinase, indicating that Timp1 expression could be adjusted by luteinizing hormone-initiated activation of these signaling mediators. Our results suggested that TIMP1 may be involved in regulating ovarian follicle development and ovulation.

[The realigation of hospital computer network system based on Intranet/Internet].

An introduction to the realization of the Intranet/Internet hospital computer network system is given here in the paper.