Comparative Proteome Profile Of Immature Rat Ovary During Primordial Follicle Assembly And Development

The assembly of primordial follicles early in ovarian development and subsequent transition to primary follicles are critical processes in ovarian biology. These processes directly affect the number of oocytes available to a female throughout her reproductive life. Once the pool of primordial follicles is depleted a series of physiological changes known as menopause occur. Inappropriate coordination of these processes contributes to ovarian pathologies such as premature ovarian failure and infertility.To better understand the molecular mechanisms involved in primordial follicle assembly and development, 2-D PAGE and MALDI-TOF/TOF technologies were used to construct a comparative proteome profile of the immature rat ovary at specific time-points (0, 24, 48, and 72h postpartum). A total of 154 differential protein spots corresponding to 134 different proteins were definitively identified between any two time-points. Further cluster analysis showed four expression patterns, and each pattern correlated with specific cell processes that occur during early ovarian development. Six proteins were randomly selected to verify expression patterns using Western blotting, and subsequently immunohistochemistry was performed to further investigate their cellular localization. Additionally, detailed functional analyses of these differentially expressed proteins were performed via bioinformatics. Elucidation of how these changes in protein expression level coordinate primordial follicles assembly and development is intended to provide a better understanding of these critical biological processes early in ovarian development and will provide potential therapeutic molecular targets to regulate ovarian function and treat ovarian disease.In order to furher explore the molecular mechanisms involved in the early development of follicles, we selected a transcription factor Hnrnpk from comparative proteome profile of immature rat ovary during primordial follicle assembly and development for further study. Hnrnpk is a protein differentially expressed early in the neonatal rat ovary with maximal abundance at 24 hours after birth, i.e. during the initiation of follicular assembly. We examined the functional significance of Hnrnpk in primordial folliculogenesis in the rat ovary using RNA interference knockdown of Hnrnpk mRNA and protein expression. Reducing Hnrnpk mRNA levels via Hnrnpk siRNAs to neonatal ovaries resulted in substantial loss of naked oocytes, primordial and primary follicles, and structural disorganization of the ovary characterized by groups of oocytes arranged in nests, clusters of somatic cells not associated with any oocytes and many oocyte nucleus highly condensed. Tunel assay demonstrated that these abnormalities may be partly attributable to apoptosis. Furthermore, Rat Genome Array was utilized to grossly screen differential expressed genes between the ovaries treated with Hnrnpk siRNAs and the controls. Microarray results showed that the expression of 63 genes changed similaritily (≥2 fold or≤0.5 fold) by Hnrnpk siRNAs, with 22 up-regulation and 41 down-regulation. Bioinformatics analysis revealed that such 63 differential genes in common involved in several critical biological processes in ovarian early development. The above results suggest that transcription factor Hnrnpk is required for primordial follicle assembly and initial growth by regulating expression of several downstream genes important for ovarian early development which provides a new therapeutic target to regulate ovarian function and treat ovarian disease.