Global Transcriptome Analysis of Porcine Oocyte, Granulosa, and Theca Cells to Identify Potential Secreted Factors and Receptors Involved in Cell-to-cell Interactions During Pre-Ovulatory Follicle Development.

Pre-ovulatory follicle development is a complex process involving extra-ovarian factors such as the gonadotrophins, and local growth factors produced by the oocyte, granulosa and theca cells. Although the role of the gonatrophins has been extensively studied during porcine pre-ovulatory follicle development, the intrafollicular cell-to-cell interactions necessary for proper follicle development remains somewhat elusive. Hence, a better understanding of the identity and the role of the many local growth factors and their corresponding receptors would greatly improve our understanding of the complex intrafollicular regulatory mechanisms necessary for porcine follicle development. Therefore, the objective of this study was to identify both the potential secreted factors produced by oocyte, granulosa and theca cells as well as the corresponding receptors expressed by these cell types during the period of preovulatory follicle growth in the pig. Oocyte, granulosa cells and theca cells were recovered by dissection from antral follicles (2-10 mm in diameter) obtained from first parity sows at day 1, 2 and 4 after weaning and at day 14, 16 and 20 of their subsequent estrous cycle. These time points correspond to specific phases of follicular growth; the recruitment of a growing cohort of follicles, the mid-selection phase and the final selection of the pre-ovulatory follicle population. Gene expression analysis was performed in duplicate on a pool of RNA from each cell type representing all the aforementioned stages using the Affymetrix Porcine Genome Array. The RNA amplification and labelling and the hybridization to the Affymetrix gene chip was performed at the Molecular Biology Core in the Centre for Reproductive Biology at Washington State University. The microarray image data were converted to numerical data with the GeneChip Operating Software (GCOS) using a global scaling strategy. In GCOS, a gene list was generated for each cell type and in order for a gene to be considered expressed, it had to be tagged as present in the GCOS absent/present call and have an expression level >50. Gene ontology annotation of the genes expressed in the different cell types was performed using GOTreePlus and a list of transcripts falling under the gene ontology term extracellular region, extracellular space and receptor activity was generated. Our preliminary observations reveal that gene encoding the TGF-β superfamily members, the EGF/EGF-like ligands, the IGF system as well as a variety of angiogenesis factors appears to be important for porcine follicle development. Interestingly, many of these factors appear to be localized to specific cell-types in the follicle. Future studies using real-time PCR will be carried out to confirm the expression of those candidate genes in each cell type and to determine their temporal expression patterns during follicle recruitment, mid-selection and final selection before and after the LH surge. (poster)

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