| Molecular mechanisms involved in terminal follicular growth, ovulation and luteinization are poorly understood. This project targets a better understanding of the molecular determinants involved during these physiological processes in cattle. The underlying hypothesis is that the growth of the dominant follicle followed by its development into an ovulatory follicle results from spatio-temporal variation in the expression profile of specific genes. The objectives were: (1) to identify differentially expressed genes in granulosa cells (GC) of dominant follicles (DF) at day 5 (D5) of the estrous cycle compared to ovulatory follicles (OF) following hCG injection using the suppression subtractive hybridization (SSH) technology; and (2) to characterize new cDNAs isolated from a GC library from DF at D5. A subtracted cDNA library was generated using SSH, from which 940 clones were randomly selected and used to generate identical macro-arrays. These macro-arrays were differentially screened using complex radioactives probes generated from unsubtracted (DF, OF) and subtracted (DF-OF, OF-DF) cDNAs. The differentially expressed cDNAs were characterized by sequencing, and compared in GenBank data bases revealing 34 non-redundant cDNAs classified as following: 23 cDNAs with known sequences and functions; 8 cDNAs with partially or completely known sequences but unknown function; and 3 cDNAs with novel sequences. The cDNAs including ApoER2, CPD, CYP11A, CYP19, FSHr, and SERPINE2 were identified and previously known to be down-regulated by hCG. Differential mRNA expression of ARFGAP3, CX43, CYP19, FSHr, FST, INHBA, LHr, LHr without exon 10, PRG1, RPA2, and TRB2 were determined by comparing their expression profiles in GC of follicles at different developmental stages and CL at D5. The cDNA of TRB2 was characterized (3571 bp) and encodes an atypical serine/threonine kinase protein with an incomplete kinase domain. Temporal analysis of TRB2 mRNA expression showed a strong and rapid reduction in OF compared to DF 6 h after hCG injection. Western blot analysis using anti-TRB2 antibody revealed the expression of TRB2 in GC of small follicles (2--4 mm) and DF but its absence in hCG-induced follicles and CL. The expression of TRB2 in GC appears to be crucial for the control of cellular proliferation during follicular growth. (Abstract shortened by UMI.)... |
