Human Fetal Ovary MiRNA Profiling And Study Of MiR-376a In Primordial Follicle Assembly

In mammals, the primordial follicle pool, providing all oocytes available to a female throughout her reproductive life, is established perinatally. Establishment of this source of oocytes is absolutely essential for fertility. For human being, dys-regulation of primordial follicle assembly results in female reproductive diseases, such as premature ovarian insufficiency (POI) and infertility.An increasing number of studies indicate that primordial follicle assembly is strictly regulated transcriptionally and post-transcriptionally. As a well-understood post-transcriptional regulator, miRNAs have been studied in various cellular processes (including reproduction). Female mice lacking Dicer, a gene required for biogenesis of miRNAs, show abnormal morphology of follicles and infertility. However, the contribution of individual miRNAs to primordial follicle assembly remains largely unknown. Tissue specific miRNAs identification is considered the key step towards understanding the role of miRNAs in biological functions.The present study reported the first systematic study on miRNAs profiling in human fetal ovaries of19-21weeks of gestation when primordial follicle formation occurred actively in the ovaries. The Solexa sequencing identified733known and5novel miRNAs. Of the mappable sequences, the majority small RNAs were19-23nt in size. The22nt sequences were the dominant small RNAs (26.23%). RT-PCR was used to validate the expression of9known miRNAs. Results were consistent with the Solexa sequencing data. Furthermore, the GO term annotation and the KEGG pathway analysis indicated that the predicted target genes of the top abundant known and novel miRNAs were involved in the cell adhesion molecules assotiated pathway, mTOR signaling pathway, and Notch signaling pathway, which are importat pathways involved in primordial follicles assembly. Our data provides a useful resource for further study on the complicated regulation network of these important signaling pathways involved in primordial follicle formation and growth.In the miRNA profile of fetal ovaries, one of miR-376a predicted target genes was Pcna (Proliferating cell nuclear antigen, Pcna). PCNA has been reported to regulate primordial follicle assembly by regulating oocyte apoptosis in mouse ovaries. So, we speculate that miR-376a is likely to be involved in the formation of primordial follicles, through targeting Pcna mRNA. RT-PCR was performed to detect the expression level of miR-376a and Pcna mRNA. miR-376a was shown to be negatively correlated with Pcna mRNA expression during primordial follicle formation. Dual luciferase assay confirmed the direct binding of miR-376a to Pcna mRNA3’UTR. Then, an in-vitro culture system of18.5dpc fetal ovaries was used to study how miR-376a and Pcna was involved in the primordial follicle formation. Cultured18.5dpc mouse ovaries transfected with miR-376a mimics exhibited decreased Pcna expression both in protein and mRNA levels. Moreover, miR-376a overexpression significantly increased primordial follicles and reduced apoptosis of oocytes, which was very similar to those in ovaries co-transfected with miR-376a and Pcna siRNAs. Taken together, the present study demonstrate that miR-376a regulates primordial follicle assembly by targeting Pcna. To our knowledge, this is the first miRNA-target mRNA pairs that has been reported to regulate mammalian primordial follicle assembly and further our understanding of the regulation of primordial follicle assembly.