133
MOLECULAR CHARACTERIZATION OF THE OVULATORY CASCADE: THE GENERATION AND CHARACTERIZATION OF AN OVULATION-SELECTIVE cDNA LIBRARY .
Hourvitz, Ariel1, Hennebold, Jon1, Brendle, Chad1, Adashi, Eli1, 1
ABSTRACT- Ovulation-selective genes, i.e. genes the increased ovarian expression of which is delimited to the periovulatory interval, are of increasing interest. The critical importance of so-called ovulation-selective genes to murine ovarian function was unequivocally established through the generation of null mutants. It is these observations which underlie the hypothesis that ovulation-selective genes constitute critical molecular determinants of ovarian function. In the present work we identify and characterize novel ovulation-selective genes in a systematic manner via the differential screening technique Suppression Subtractive Hybridization (SSH). Resultant 485 target complementary DNA (cDNA) clones were sequenced and analyzed for homology to known genes using the Basic Local Alignment Tool (BLAST). From the above total number of clones sequenced, 262 were determined to be independent (non-redundant). 188 non-redundant clones were determined to be identical to previously-characterized genes. An additional 54 non-redundant clones proved significantly homologous to previously-characterized genes. Other independent clones, a total of 80, did not display significant homology to previously-characterized genes now listed in publicly-accessible non-redundant databases. As such, these latter genes were deemed novel. Inserts of 56 randomly chosen clones were PCR-amplified, {32P}-labeled, and used to probe blots containing preovulatory and ovulatory ovarian mRNAs to validate the efficiency of SSH. Of the 56 randomly chosen clones, analyzed in this manner, 16 clones (29%) failed to show any signal. Of the remaining 40 clones, fourteen clones were observed to hybridize equivalently to preovulatory and ovulatory cDNA populations thereby yielding a total false positive rate of 35%. Six (15%) clones displayed an ovulation-specific expression while 20 clones (40%) displayed an ovulation-selective expression. Taken together, the present study accomplished systematic identification of those ovarian genes that are upregulated by human chorionic gonadotropin (hCG). These ovulation-selective genes may have significant implications for the understanding of ovarian function in molecular terms and for the development of innovative strategies for the promotion of fertility or its control.
KEY WORDS: ovulation, cDNA library, mouse