title: Knockdown of Progesterone Receptor (PGR) in Macaque Granulosa Cells Disrupts Ovulation and Progesterone Production.
abstract: Adenoviral vectors (vectors) expressing short-hairpin RNAs complementary to macaque nuclear progesterone (P) receptor PGR mRNA (shPGR) or a nontargeting scrambled control (shScram) were used to determine the role PGR plays in ovulation/luteinization in rhesus monkeys. Nonluteinized granulosa cells collected from monkeys (n = 4) undergoing controlled ovarian stimulation protocols were exposed to either shPGR, shScram, or no virus for 24 h; human chorionic gonadotropin (hCG) was then added to half of the wells to induce luteinization (luteinized granulosa cells [LGCs]; n = 4-6 wells/treatment/monkey). Cells/media were collected 48, 72, and 120 h postvector for evaluation of PGR mRNA and P levels. Addition of hCG increased (P < 0.05) PGR mRNA and medium P levels in controls. However, a time-dependent decline (P < 0.05) in PGR mRNA and P occurred in shPGR vector groups. Injection of shPGR, but not shScram, vector into the preovulatory follicle 20 h before hCG administration during controlled ovulation protocols prevented follicle rupture in five of six monkeys as determined by laparoscopic evaluation, with a trapped oocyte confirmed in three of four follicles of excised ovaries. Injection of shPGR also prevented the rise in serum P levels following the hCG bolus compared to shScram (P < 0.05). Nuclear PGR immunostaining was undetectable in granulosa cells from shPGR-injected follicles, compared to intense staining in shScram controls. Thus, the nuclear PGR appears to mediate P action in the dominant follicle promoting ovulation in primates. In vitro and in vivo effects of PGR knockdown in LGCs also support the hypothesis that P enhances its own synthesis in the primate corpus luteum by promoting luteinization.
authors.affiliation: Division of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Beaverton, Oregon bishopc@ohsu.edu.
authors.forename: Cecily V
authors.initials: CV
authors.lastname: Bishop
authors.affiliation: Division of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Beaverton, Oregon Department of Obstetrics and Gynecology, Oregon Health & Science University, Portland, Oregon.
authors.forename: Jon D
authors.initials: JD
authors.lastname: Hennebold
authors.affiliation: Molecular Virology Support Core, Oregon National Primate Research Center, Beaverton, Oregon.
authors.forename: Christoph A
authors.initials: CA
authors.lastname: Kahl
authors.affiliation: Division of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Beaverton, Oregon Department of Obstetrics and Gynecology, Oregon Health & Science University, Portland, Oregon.
authors.forename: Richard L
authors.initials: RL
authors.lastname: Stouffer
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003338:Corpus Luteum / Q000187:drug effects / Q000378:metabolism; D005260:Female; D055785:Gene Knockdown Techniques; D006107:Granulosa Cells / Q000378:metabolism*; D008184:Luteal Cells / Q000187:drug effects / Q000378:metabolism; D034461:Luteinization / Q000187:drug effects / Q000235:genetics; D008253:Macaca mulatta; D010060:Ovulation / Q000235:genetics*; D011374:Progesterone / Q000378:metabolism* / Q000494:pharmacology; D011980:Receptors, Progesterone / Q000235:genetics*; D014162:Transfection
chemical_list: D011980:Receptors, Progesterone; D011374:Progesterone
keywords: PGR; luteinization; ovulation; progesterone; shRNA; siRNA