Identification of genes involved in follicular development by transcription gene profiling of ovaries from 5 and 15-day old mice

Abstract

Objective: Identification of the genes involved in the transition from secondary to antral follicles. Design: In this study we used the Affymetrix gene chip to differentially profile genes involved in follicular development from secondary to antral follicles present in 5 and 15-day old mouse ovaries, respectively. At day 5 after birth, folliculogenesis has progressed no further than the secondary follicle stage and low levels of circulating gonadotropins are detected in plasma. At day 15 of age, follicles are at all stages of differentiation (including antral follicles), and the plasma levels of FSH, LH, estradiol and estrone are similar to the normal circulating levels present in the mature cycling animal. FSH, LH and estradiol decline abruptly by day 21 of age until the animal reaches cycling maturity. Therefore, gene expression profiling of ovaries from these two populations may identify genes involved in the secondary to antral follicle transition. Materials/Methods: Total ovarian RNA from 5-day old and 15-day old mice was isolated using TRIzol, DNAse treated, and then submitted to two rounds of cDNA synthesis. First strand cDNA synthesis was achieved with a modified oligo-dT primer containing the promoter sequence for T7 RNA polymerase, followed by the synthesis of double stranded cDNA, and this was then followed by in-vitro transcription using biotin-labeled NTPs to produce biotinylated cRNA. Biotinylated cRNA was submitted to fragmentation and used to hybridize the Murine 11KA and 11KB GenechipTM arrays from Affymetrix. The arrays were scanned and raw data analyzed using Affymetrix MicroArraySuite 4.0. Genes displaying an absolute intensity difference greater than 4-fold between ovaries of the 5- and 15-day old mice were selected for further analysis. Selection of genes was performed only on present probesets. The data were normalized with eleven bacterial genes added to the hybridization solution. Results: All genes involved in the conversion of cholesterol to estradiol through the delta-4 and delta-5 pathways were up-regulated in the 15-day old animals. We also found an increase in expression at day 15 of other genes known to be involved in folliculogenesis such as inhibin alpha, adrenodoxin, LH hormone receptor, prostaglandin E receptor, TGF beta I receptor, FGF, estrogen receptor beta, and prolactin receptor 1, ARF-1, Thrombospondin, among others. A 4-fold change in expression was also found in some genes not yet described in the ovary. Among these genes we found an up-regulation at day 15 of GBX-2, Secretagranin III, RHAMM, PAF-3, G- alpha 12, FKBP-65, Neuronatin, Protein Kinase C Delta, NEK-2 and SEK-1. We selected RHAMM and Secretagranin III for follow-up studies using Taqman to confirm the up-regulation of these genes at day 15 of age. By in-situ hybridization, RHAMM gene expression was localized to the granulosa cells of late antral follicles and in the corpus luteum of 60-day old female mouse ovaries. Conclusions: The transcriptional gene data generated by this approach validates the benefit of using 5-day and 15-day old mouse ovaries to identify possible candidate genes such as RHAMM, involved in the transition from secondary to late antral follicles. Supported by: Wyeth.