Kisspeptin and kisspeptin receptor are expressed in mouse oocytes and participate in meiosis resumption

enriched for kit oncogene (KIT), a marker of differentiating spermatogonia. Total RNA was isolated and real time RT-PCR was performed. Luciferase assays revealed direct binding of miR-146a to the 3’ untranslated region (3’UTR) of its targets. Overexpression of miR-146a in spermatogonial stem cells (SSCs) was achieved using mimics; knockdown of miR-146a was achieved using inhibitors. 1mM RA was added to SSCs to induce differentiation. RESULTS: miR-146a was downregulated 180-fold in KIT+ differentiating spermatogonia when compared to THY1+ undifferentiated spermatogonia. Luciferase assays revealed direct binding of miR-146a to the 3’UTR of Med1 (mediator complex subunit 1), a co-regulator of RA receptors (RARs). Overexpression of miR-146a in SSCs reduced transcript levels of Med1 and differentiation marker Kit. Conversely, knockdown of miR-146a increased transcript levels of these markers and reduced levels of SSC markers. 24h exposure of SSCs with RA significantly downregulated miR-146a and SSC marker Plzf, while upregulating Med1 and Kit. CONCLUSION: miR-146a is highly expressed in undifferentiated spermatogonia and directly inhibits Med1, a co-regulator of RARs. Because RA drives spermatogonial differentiation, we conclude that miR-146a promotes self-renewal in SSCs through Med1 inhibition. Supported by: CJP was funded by NIH grant HD055330. O-70 Monday, October 22, 2012 04:30 PM KISSPEPTIN AND KISSPEPTIN RECEPTOR ARE EXPRESSED IN MOUSE OOCYTES AND PARTICIPATE IN MEIOSIS J. Ding,b M. Lehman,b,c RESUMPTION. A. M. Rocha,a G. D. Smith.b,c,d,e aCell and Developmental Biology, University of Michigan, Ann Arbor, MI; bObstetrics and Gynecology, University of Michigan, Ann Arbor, MI; cUrology, University of Michigan, Ann Arbor, MI; dMolecular and integrative Physiology, University of Michigan, Ann Arbor, MI; eReproductive Sciences Program, University of Michigan, Ann Arbor, MI. OBJECTIVE: Expression of several neuropeptides and their receptors has been reported in mammalian ovary and oocytes. Kisspeptin (Kp) and its receptor (Kp-R) are present in the rat ovary and may be involved with ovulation. Objectives were to investigate presence and functional role of the Kp in mouse oocytes. DESIGN: Laboratory study. MATERIALS AND METHODS: Detection of Kp and Kp-R by Q-PCR was performed in meiotically-incompetent and -competent germinal vesicle intact (GVI), germinal vesicle breakdown (GVBD), meiosis I (MI) and meiosis II (MII) oocytes. The effect of pregnant mare serum gonadotropin (PMSG) on Kp/Kp-R expression was investigated in GVI collected with or without PMSG treatment. Role of Kp in meiotic resumption was investigated via the ability of Kp to overcome meiotic arrest induced by a phosphodiesterase inhibitor (IBMX). RESULTS: Kp mRNA increased 82.2 fold as oocytes gained meiotic competence (P<0.01). Expression of Kp was similar in GVI and GVBD, and decreased 5.4 (P<0.01) and 12 (P<0.05) fold as oocytes progressed to MI and MII. Expression Kp-R was steady during progression from GVI to MII. Expression of Kp was significantly up-regulated (1.8 fold; P<0.05) after in vivo treatment with PMSG. Kp significantly increased the proportion of IBMXarrested oocytes progressing to GVBD, but it did not affect percent of MII oocytes.

Proportions of GVBD and MII in IBMX and IBMX+Kp treated oocytes.

IBMX

IBMX+Kp

P-value

55 64

72 77

P<0.01 P>0.05

% of GVBD (2h) % of MII (18h)

CONCLUSION: Increased Kp expression occured with acquisition of meiotic competence and can influence resumption of meiosis yet does not impact progression to MII. Future experiments on external regulation of oocyte Kp/Kp-R may indicate new strategies for improved in vitro oocyte maturation. Supported by: NIH R01 HD 046768.

S22

ASRM Abstracts

O-71 Monday, October 22, 2012 04:45 PM THE HEPATOCYTE GROWTH FACTOR SYSTEM IN MENSTRUATION AND POST-MENSTRUAL ENDOMETRIAL C. Hergert,a J. Evans,b J. Yap,b REPAIR. O. D. Slayden,a L. A. Salamonsen.b aDivision of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Beaverton, OR; bEndometrial Remodelling, Prince Henry’s Institute, Monash Medical Centre, Clayton, VIC, Australia. OBJECTIVE: To characterize the HGF/c-MET paracrine system during menstruation and post menstrual endometrial repair. DESIGN: Studies of archived samples and in vitro studies on endometrial cell lines. MATERIALS AND METHODS: Ovariectomized rhesus macaques were treated with estradiol and progesterone to induce artificial 28-day menstrual cycles (Arch. Histol. Cytol. 67:393-409; 2004). Samples were collected on day 0, 1, 2, 3, 4, 5, 7 and 14 of the artificial cycle and analyzed for HGF and c-MET by real-time PCR, and immunohistochemistry (IHC). Human samples were obtained from women on menstrual cycle days 25-28 (late secretory phase), 1-4 (menstrual phase) and 5-6 (repair phase) and examined for HGF and c-MET by IHC. Human endometrial luminal epithelial cells were grown to over-confluence and then wounded. Wounded monolayers were treated with HGF neutralizing antibody (0.5mg/ml) or matched IgG. Repair of wounded areas was assessed daily by determining wound area vs day 0 (day of wounding). RESULTS: HGF and c-MET expression coincided with late menstruation and post menstrual repair. In macaques HGF mRNA was minimal in the late secretory and premenstrual phase and increased to maximum on d 5 (P<0.05). c-MET mRNA was minimal in the premenstrual phase and increased by d3 (P<0.05). IHC revealed stromal (S) localization of HGF whereas c-MET was localized to the glandular and repairing luminal epithelium (LE). IHC of human endometrium revealed minimal HGF in the late secretory phase. On day 1 of menses HGF localized to S only. During the repair phase HGF localized to S, glandular epithelium (GE) and repairing/repaired LE. c-MET localized to S on d 1 of menses and to GE/LE during repair. Treatment of wounded epithelial cell monolayers with anti-HGF IgG significantly reduced in vitro epithelial repair by > 50%. CONCLUSION: Our results support a functional role for the HGF/c-MET paracrine system during postmenstrual endometrial repair. Supported by: NIH P51OD011092-53, NHMRC #494802, Victorian Government’s Operational Infrastructure Support Program. O-72 Monday, October 22, 2012 05:00 PM LEUKEMIA INHIBITORY FACTOR IS NECESSARY FOR RHESUS MACAQUE OVULATION. P. A. Royer,a M. J. Murphy,b J. D. Hennebold.a,b aObstetrics and Gynecology, Oregon Health and Sciences University, Portland, OR; bReproductive and Developmental Sciences, Oregon National Primate Research Center, Beaverton, OR. OBJECTIVE: Determine the role leukemia inhibitory factor (LIF) plays in primate ovulation. DESIGN: In vivo studies using rhesus macaques. MATERIALS AND METHODS: A controlled ovulation protocol (Hum Reprod 18:2257, 2003) was employed that permits the development of a single, naturally-selected follicle in rhesus macaques. Individual follicles (n¼4-6/group) were obtained before (0 h) and after (12, 24, 36 h) an ovulatory bolus of human chorionic gonadotropin (hCG). RNA levels and cellular localization of proteins involved in LIF signaling were determined by quantitative real-time PCR (qPCR) and immunohistochemistry (IHC), respectively. Follicular LIF protein levels were assessed by enzyme linked immunosorbant assay (ELISA). Following intrafollicular injection (IFI) of soluble LIF receptor (sLIFR; a LIF antagonist; 10 mg, n¼3), follicle rupture was assessed by laparoscopy 72 h later. Data were analyzed for significance (P<0.05) by ANOVA and FLSD. RESULTS: As determined by qPCR, mRNA levels increased (P<0.05) 12 h post-hCG for LIF; glycoprotein 130 (gp130), the shared LIF receptor (LIFR) subunit; Janus kinase 1 (JAK1), the LIFR activated tyrosine kinase; and signal transducer and activator of transcription-3 (STAT3), the transcription factor responsible for regulating LIF responsive genes. A secondary increase in mRNA expression occurred after rupture. Follicular LIF protein levels were below the limit of detection prior to hCG administration, but were elevated at 12 and 24 h post-hCG (P<0.05). IHC revealed overlapping expression in theca, mural and cumulus granulosa cells for LIFR, gp130 and STAT3. IFI of sLIFR at the time of hCG administration blocked follicle rupture (3 of 3) as evidenced by the

Vol. 98, No. 3, Supplement, September 2012