Identification Of Differentially Expressed Genes In Follicular Granulosa Cells And Effects Of Antioxidant On Expression Of GSTA1 In Hu Sheep

The present study was to investigate expression parttens of receptor genes for BMP 15 and GDF-9, identify the differently espression genes of ovine follicular granulosa cells, and examine effects of hormone and green tea polyphenols on these genes mRNA expression in Hu sheep. It is of momentous theoretical significance and practical significance for effective use of Hu Sheep in the breeding, revealing moleculal mechanism of follicle development, and optimizing embryo industry protocol.1. Stage-specific of BMP 15 and GDF-9 receptor genes expression:effects of follicle-stimulating hormone on ovine antral follicleOvaries were collected and granulose cells were harvested from two diameter size follicles (3-5mm,>5mm). For in vitro studies, granulosa cells were obtained from follicles of 2-5mm diameter and cultured in serum-free McCoy'5A medium supplemented with different doses of FSH (0,1,5,10 ng/ml) or combination 5ng/ml FSH with 1ng/ml E2. Total mRNA expression of BMPRII, BMPRIB and ALK-5 was estimated by the quantitative real-time PCR. Our results demonstrated that BMPRII, BMPRIB and ALK-5 expression was significantly higher in the granulosa cells of large follicles than in those of small follicles. Treatment of granulose cells with FSH (1-10ng/ml) alone down-regulated the expression of BMPRIB (P<0.05). BMPRII and ALK-5 mRNA expression were no significantly differentiation by the concentration of FSH (5ng/ml), compared to control. A further increase FSH (10ng/ml) down-regulated the expression of BMPRII and ALK-5 (P<0.05). Combination FSH (5 ng/ml) with E2 (1 ng/ml) up-regulated the expression of the BMPRII, BMPRIB and ALK-5 in the granulosa cells (P<0.05). Therefore, the present study provided the expression levels of receptor genes of BMP15 and GDF-9 and suggested the expression of BMPRII, BMPRIB and ALK-5 might be regulated by FSH and E2 in ovine granulose cells.2. Analysis of gene expression in granulosa cells of ovine antral follicles from Hu sheep using suppression-subtractive hybridizationIn the present study, suppression subtractive hybridization(SSH) was performed to screen genes that were differentially expressed in the granulosa cells between large follicles(LF,>5mm) and small follicles(SF,3-5mm) in Hu sheep during antral follicle phase, and a subtractive cDNA library was constructed. Furthermore, with dot blot analysis and quantitative reverse transcription-polymerase chain reaction, a total of 92 clones randomly selected from the library were proven to be differentially expressed in the granulosa cells. Among of these,38 exhibited high homology to known genes, 14 identified as new EST (Accession No. EY202207, FD480266-FD480278). Four ESTs, LAPTM4A, SERPINE2, GSTA1, and INHBA, were further examined the reproducibility of the SSH data by the real-time quantitative PCR, and the results of confirmed an increase expression of their mRNA in granulosa cells of large follicles compared with that of small follicles. We conclude that we have identified several genes (known or unknown) that may effect follicular growth, dominance or ovulation in ewes.3. Cloning of GSTA1 and regulation of FSH on expression of GSTA1 of ovine granulosa cellsAccording to the identified ESTs, electic cloning and RT-PCR were used to obtained cDNA of the GSTA1 gene (Genbank Accession No, EU399787). The nucleotide sequence of cDNA of GSTA1 was 840bp and contained open reading frame of 666 nucleotides, encoding a predicted protein of 222 amino acides. The deduced molecular weight was 25.4kD and the theoretical pI was 8.66. Ovine GSTA1 was 82% to Homo sapiens,88% identical to Sus scrofa, and 98% identical to Bos taurus. The mRNA was detected in corpus luteum, testis, liver, lung, and kidney. By using cultured ovine granulosa cells, this study was conducted to analyze the effects of GSTA1 on expression in granulosa cells. Results showed that concomitant to the increase in glutathione S-transferase activity were significantly enhanced when cultured ovine granulosa cells were treated with FSH. Furthermore, relative expression of GSTA1 mRNA in cultured granulosa cells sensitized with lOng/ml FSH has increased significantly (P＜0.05). It was concluded that GSTA1 may have a functional role in ovine ovarian follicular development under endocrine regulation.4. Effects of green tea polyphenols on the granulosa cells against oxidative stress induced apoptosisWe sought to investigate the induction effect of the reactive oxygen species (ROS)-producing system hypoxanthine/xanthenes oxidize (HX/XO) on the expression of GSTA1 in vitro cultured ovine granulosa cells. This study showed that HX/XO (1mM; 1mU/ml) exerted toxic effects on ovine granulosa cells induced oxidative damage and cell death. Granulosa cells GST activity and GSH levels decreased after HX/XO exposure (P<0.05). HX/XO obviously induced the expression of GSTA1 in cultured granulosa cells (P＜0.05). The expression of GSTA1 mRNA was lowest when XO concentration is at 1mU/ml. GTPs obviously attenuated the oxidative stress induced by HX/XO system, increased the cell number, and restored HX/XO-induced decrease in glutathione (GSH) level and HX/XO-induced increase in malondialdehyde (MDA) formation. GTPs also counteracted the HX/XO-induced GST activity decrease and down expression of GSTA1. Taken together; these studies provide insights into the action of GTPs and suggest the GTPs-mediated increased GST may be a mechanism contributing to prevention against reactive oxygen species injury in the follicular development of sheep. Ovine GSTA1 protect against granulosa cells apoptosis caused by HX/XO, suggesting an important role of GSTA1 in protection against oxidative stress in follicular development, selection, and ovulation.