The Possible Roles Of Four Main Categories Of Proto-oncogenes During The Initiation Growth Of Rat Primordial Follicles

Objective:The aim of this study is to preliminary explore the effects of four categories of major proto-oncogenes, which are involved in cell proliferation, apoptosis and signal transduction, during the initiation growth of primordial follicles.Methods:(1) Ovaries were collected from 2-day-old SD rats, after 8d cultured in the Waymouth culture system which was treated with either 50ng/ml epidermal growth factor (EGF), 200ng/ml growth differentiation factor-9 (GDF-9), 10-5mol/ml estradiol (E2), 10-5mol/ml progesterone (P4), or nothing. The morphous and quantity of every stage follicles of ovaries cultured for 8d were observed by histological section stained with hematoxylin after ovaries were cultured.(2) The gene expression levels were studied using Illumina rat RatRef-12 whole genome expression chips. RNA from these ovaries was hybridized to rat microarray gene chips, and the gene expression (i.e., ovarian transcriptome) was compared between the developmental stages, in order to explore the change of four major categories of proto-oncogenes during the initiation growth of primordial follicles, and analyze whether the regulations of EGF, GDF-9, E2 and P4 during the initiation growth of primordial follicles are connected with four major categories of proto-oncogenes.Results:(1) The quantities of the primary follicles and second follicles in the EGF and GDF-9 groups were higher than those in the control groups (P<0.01). The quantities of the primordial follicles in the E2 and P4 groups were higher than those in the control groups (P<0.01). 50ng/ml EGF and 200ng/ml GDF-9 could further promote the initiation growth of primordial follicles in the Waymouth culture system, but 10-5mol/ml E2 and 10-5mol/ml P4 could inhibit the initiation growth of primordial follicles in the Waymouth culture system. (2) Analysis of the ovarian genes demonstrated 3540 differentially expressed genes were identified between the neonatal groups and 8d control groups. Among these genes, 1638 up-regulated genes (Diffscore>+20), including 7 proto-oncogenes A2m, Egfr, Fgf7, Fgr, Myc, Neu1, Thrb, 1902 down-regulated genes (Diffscore<-20), including 5 proto-oncogenes Braf, Kit, Mycl1, Ntrk3, Reln. There were 88 up-regulated genes, including proto-oncogene A2m, and 79 down-regulated genes, including proto-oncogene Mycn between EGF and control groups. The expression array analysis revealed that 178 and 121 differentially expressed genes in GDF-9 and P4 groups respectively, but no known proto-oncogene among them. Compared with 8d control groups, there were 235 differential expressed genes in E2 groups. Interestingly, proto-oncogene Mycn was down-regulated in E2 groups.Conclusion:(1) We find a number of proto-oncogenes, such as A2m, Egfr, Fgf7, Myc, Reln and Kit, potentially involve in primordial follicle development using the microarray approach.(2) The promotion of EGF during the initiation growth of primordial follicles may have relation to the proto-oncogene A2m. The regulation of E2, GDF-9 and P4 may have nothing to do with four categories of major proto-oncogenes.