The midcycle gonadotropin surge initiates ovulation and luteinization, but little is known about the gonadotropin-stimulated processes in follicular cells which are involved in follicle rupture and the formation of the primate corpus luteum. To identify specific gene products expressed in follicular cells after, but not before, the midcycle gonadotropin surge, a novel subtractive hybridization strategy was used. Granulosa cells were obtained from monkeys receiving gonadotropins to stimulate the development of multiple follicles. Cells were obtained by follicle aspiration before (nonluteinized) or 27 hours after (luteinized) the administration of the ovulatory gonadotropin bolus. Messenger RNA was obtained using oligo-dT linked magnetic beads, and reusable, bead-linked cDNA libraries were prepared from both cell groups using reverse transcriptase. Oligonucleotide probes prepared by random priming of the luteinizing granulosa cell cDNA library were allowed to hybridize with the nonluteinized granulosa cell cDNA library; unhybridized fragments, which are unique to or more highly expressed in luteinized granulosa cells, were used to screen a monkey luteal cDNA library. The initial screen yielded 13 clones, and the monkey cDNA sequences obtained were compared to available databases. Two cDNAs, identified as thymosin b-10 and ubiquitin, were selected for further study. Reverse transcriptase-PCR with primers based on these sequences will be used to amplify fragments from RNA obtained from granulosa cells as well as luteal tissue in order to confirm the expression of identified gene products in luteinized, but not nonluteinized, granulosa cells and to characterize their expression in the corpus luteum throughout the luteal phase. Identification of these factors will allow further study of their role in the processes of follicular rupture and luteinization in the monkey ovary.
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