| The process of follicular development associated with the degradation of a large number of follicles before ovulation, which known as follicular atresia (atresia). Follicular atresia is the main form of germ cell degeneration after the formation of follicles in all vertebrates, and the vast majority of follicular disappeared with atresia. Follicular atresia is a multi-factor involved process, animal experiments have confirmed the regulation role of certain factors, but the mechanism of follicular atresia is subject to further research, it haves important guiding significance in human reproduction control and livestock breeding rate improvement.This study aimed to analyze the role of steroidogenic enzymes and related receptors in the process of follicular atresia in the pigs, understand the regulation mechanism follicular atresia and provides theoretical support for the reproduction research; Then examined the changes of gene expression of steroidgenesis enzymes and related hormone receptors during the atresia process; last analysis the correlation ship among these genes, and the physiological situation of follicles. The results are as follows:1 Established the comprehensive method to classify different stages of atresia of pig antral follicles. We successfully separated and examined 239 antral follicles 3-5mm in diameter. Obtained totally 60 follicles in health, early atresia and progressed atresia stages use the "appearance plus granulosa cells density plus progesterone/estrogen ratio" method.Specific judging criteria include:1) The apparent criteria:healthy follicles is clear, pink coloured, cumulus oocyte complex is visible, granulosa cell layer is integrity; early atretic follicles is turbid, dark orange coloured, cumulus oocyte complex is illegible, granulosa cell layer is partially detachment and crinkled; progressed atresia follicles is gray coloured, cumulus oocyte complex fall off, granulosa cell layer seriously lost and deposited. 2) The granulosa cell density standards:health group contains less than 2500 perμL; early atresia group contains 2500-10000 perμL; progressed atresia group contains more than 10,000 perμL.3) The hormone ratio criteria:health group P4/E2<5; early atresia Group 5 <P4/E2<20; late atresia group P4/E2> 20.2 Detected the changes of mRNA expression during follicular atresia process. Extracted total RNA from healthy, early and progressed atresia porcine follicles, detected changes in gene expression by Real Time-PCR for STAR, CYP11, CYP 17, HSD3p,CYP 19, FSHR, LHCGR, AR, PGR and GAPDH genes.1)STAR gene expression in a high volume in health group, decreased gradually with atresia process; 2) CYP 11 gene expression is low in the health group, gradually increased with atresia process, there is significant difference between the health, early atresia groups and the progressed group; 3) CYP 17 gene expression is high in the health group, decreased gradually with atresia process; 4) HSD3B gene is low in the health group, lower in early atresia group and rises a little in progressed atresia group; 5) CYP 19 gene expression is high in the health group, decreased with atresia process,;6) FSHR gene expression is high in the health group, decreased much in early atresia group and rises a little in progressed atresia group, there is significant difference between the health and the other two groups; 7) LHCGR gene expression is high in the health group, decreased in early atresia group and rises a little in progressed atresia group; 8) PGR gene expression is low in the health and early atresia group, rises a lot in progressed atresia group, there is significant difference between the progressed atresia and the other two groups; 9) AR gene expression is low in the health and early atresia group, gradually increased with atresia process, there is significant difference between the health, early atresia groups and the progressed group.3 Illustrated the correlationship of the follicular physiological indicators, genes of steroidogenesis enzymes and related hormone receptors.The correlation coefficient of granulosa cell density and P4/E2 is 0.438, (P=0.047); The correlation coefficient of CYP 19 expression and granulosa cell density is-0.461, (P =0.036); The correlation coefficient of CYP 11 gene expression and granulosa cell density is 0.690, (P=0.001); The correlation coefficient of CYP 11 gene expression and P4 content is 0.467, reached a very significant positive correlation (P=0.009); The correlation coefficient of HSD3B quantity and P4 content is 0.539, (P=0.002); The correlation coefficient of FSHR expression and P4 content is-0.385, (P=0.035); The correlation coefficient of PGR expression and P4 content is 0.368, (P=0.031); Other genes and P4 showed no significant correlation. The correlation coefficient of STAR gene expression and E2 is 0.456, (P=0.011); CYP19 expression and E2 content is 0.446, (P=0.013); The correlation coefficient of LHR gene and E2 is 0.684, (P=0.000);The expression of other genes and E showed no significant correlation. The correlation coefficient of STAR gene and LHR gene expression is 0.575, (P=0.001); The correlation coefficient of CYP11 and HSD3B gene expression is 0.386, (P=0.035); The correlation coefficient of CYP11 and PGR gene expression is 0.525, (P=0.003); The correlation coefficient of CYP11 and AR gene expression is 0.369, (P=0.045); The correlation coefficient of HSD3B and PGR gene expression is 0.706, (P=0.000); The correlation coefficient of CYP19 and FSHR gene expression is 0.361, (P=0.038); The correlation coefficient of CYP19 and LHR gene expression is 0.434, (P=0.017); other gene expression levels had no significant correlation. The correlation coefficient of AR and PGR gene expression is 0.575, (P=0.001).4 Analysis the consistency among different standards used to classify the follicles.There is a high degree of consistency between morphological standard and density of granulosa cells; there is also a significant correlation between single follicular estradiol content standard and morphological standard; there is no direct relationship between follicular progesterone level and morphological standard; P/E standard acts as a combination of two factors of progesterone and estradiol, there is a significant correlation between it and the morphological standard. |
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