IMD/ADM21-47, a factor that improves embryo quality.

Starting in vitro fertilization process with competent oocytes that may endure first cellular divisions is a critical step for obtaining an embryo. To obtain in vitro competent oocytes, culture conditions should emulate the in vivo microenvironment as close as possible. With the aim of improving the in vitro culture medium, the present study evaluated the IMD/ADM21-47 peptide as a factor that promotes oocyte competence and improves embryo quality in bovine systems. The culture supplemented with 153 µg/mL of IMD/ADM21-47 was correlated with the production of healthy oocytes in metaphase II (MII) stage in compacted cumulus-oocyte complexes (COC) with a decrease of BAX/BCL-2 to mRNA ratio and a reduction of late apoptosis by TUNEL in MII oocytes. In addition to this, treatment with IMD/ADM21-47 caused cAMPi level to be constant over time, and the cAMPi level kept increasing until 6 h. COC supplementation with 153 µg/mL of IMD/ADM21-47 increased the blastocyst production rate two-fold in comparison with control conditions. Only embryos from COC treatment with this peptide were capable of developing blastocysts in stage-6 grade I; compared with the control culture, it was the treatment with the greater number of blastocysts stage-5; these are characteristics of good quality blastocysts.

New insights into the pathogenesis of cystic follicles in cattle: microarray analysis of gene expression in granulosa cells.

Ovarian follicular growth and development are regulated by extraovarian and intraovarian factors, which influence granulosa cell proliferation and differentiation. However, the molecular mechanisms that drive follicular growth are not completely understood. Ovarian follicular cysts are one of the most common causes of reproductive failure in dairy cattle. Nevertheless, the primary cause of cyst formation has not been clearly established. A gene expression comparison may aid in elucidating the causes of ovarian cyst disease. Our objective was to identify differentially expressed genes in ovarian granulosa cells between normal dominant and cystic follicles of cattle. Granulosa cells and follicular fluid were isolated from dominant and cystic follicles collected via either ultrasound-guided aspiration from dairy cows (n = 24) or slaughterhouse ovaries from beef cows (n = 23). Hormonal analysis for progesterone, estradiol, and androstenedione in follicular fluid was performed by RIA. Total RNA was extracted and hybridized to 6 Affymetrix GeneChip Bovine Genome Arrays (Affymetrix, Santa Clara, CA). Abundance of mRNA for differentially expressed selected genes was determined through quantitative real-time reverse-transcription PCR. Follicular cysts showed greater (P < 0.05) progesterone, lesser (P < 0.05) estradiol, and no differences (P > 0.10) in androstenedione concentrations compared with noncystic follicles. A total of 163 gene sequences were differentially expressed (P < 0.01), with 19 upregulated and 144 downregulated. From selected target genes, quantitative real-time reverse-transcription PCR confirmed angiogenin, PGE(2) receptor 4, and G-protein coupled receptor 34 genes as upregulated in cystic follicles, and Indian hedgehog protein precursor and secreted frizzled-related protein 4 genes as downregulated in cystic follicles. Further research is required to elucidate the role of these factors in follicular development and cyst formation.

Microarray analysis of insulin-like growth factor-I-induced changes in messenger ribonucleic acid expression in cultured porcine granulosa cells: possible role of insulin-like growth factor-I in angiogenesis.

Insulin-like growth factor-I in conjunction with gonadotropins are important stimulators of mitosis and ovarian steroid production by granulosa and thecal cells, which are required for normal oocyte development and hormonal feedback signaling to the hypothalamus and pituitary. However, a comprehensive evaluation of the changes in gene expression induced by IGF-I has not been conducted. Our objective was to characterize granulosa cell gene expression in response to IGF-I treatment. Porcine granulosa cells were pooled in 4 biological replicates and treated with FSH (baseline) or FSH+IGF-I for 24 h in vitro. The RNA was collected and hybridized to 8 Affymetrix Porcine GeneChips (Affymetrix, Santa Clara, CA) in a paired design. Differentially regulated gene sequence element sets (P < 0.01) were used as queries in the UniGene database searching for annotated genes. Abundance of messenger RNA (mRNA) for genes differentially expressed in the microarray analysis was determined through multiplex assays of one-step real-time reverse transcription-PCR and further analyzed under a statistical model including the fixed effect of treatment. A total of 388 gene sequence element sets were differentially expressed, and 42 matched annotated genes in the UniGene database. Of the 3 upregulated target genes selected for further quantitative reverse transcription-PCR analysis, only FGF receptor 2 III c (FGFR2IIIc) mRNA abundance was significantly increased by IGF-I. Of the 3 downregulated target genes selected for further analysis, only thrombospondin-1 (THBS1) mRNA abundance was significantly decreased by IGF-I. Further study revealed that neither FSH nor estradiol affected the IGF-I-induced suppression of THBS1 mRNA abundance. These results provide the first comprehensive assessment of IGF-I-induced gene expression in granulosa cells and will contribute to a better understanding of the molecular mechanisms of IGF-I regulation of follicular development. Involvement of FGFR2IIIc and THBS1 in mediating IGF-I-induced granulosa cell steroidogenesis and proliferation during follicular development is novel, but their specific roles will require further elucidation.