Gonadotropin releasing hormone (GnRH) reduces ligand-induced estrogen receptor (ER) dimerization in GT1-7 cells

DESIGN: In vitro, 3D culture of follicles in a steroid-depleted media with or without testosterone. MATERIALS AND METHODS: Secondary follicles (125-225 mm diameter; 100 follicles/group) from rhesus macaque (n¼4) ovaries were encapsulated in alginate beads and cultured for 40 days in aMEM media supplemented with insulin and hFSH. Media contained vehicle (control) or steroid synthesis inhibitor (trilostane, TRL; 250 ng/ml) with or without a low or higher dose of testosterone (T; 10 or 50 ng/ml). Follicle health, size, and media levels of estradiol (E), were monitored weekly. Oocytes were collected from antral follicles at the end of the study, 34 hr after addition of hCG. RESULTS: We previously reported that TRL exposure reduced survival and growth of macaque secondary follicles during 3D culture. Co-administration of low or high T increased follicle survival, compared to TRL (52 and 65 % of original follicles vs 25%; P¼0.09) comparable to controls (52%). Also, growth in low or high T was greater than the TRL group (568 and 466 um vs 330 mm; P<0.05), reaching similar sizes as control follicles at week 4 (501 mm). Many control and T-treated follicles achieved antrum formation by week 4. Media E levels were greater (P<0.05) in high (26413 pg/ml) or low T (4894 pg/ml), compared to TRL (215 pg/ml) or controls (1299 pg/ml). By week 5, low or high T promoted the recovery of healthy oocytes, compared to TRL (95 and 100% vs 50%; P¼0.06) to levels observed in controls (88%). Meiotically active (MII stage) oocytes were collected from T-treated and controls, but not TRL-treated follicles. CONCLUSION: Testosterone, either as an androgen and/or following its conversion to estrogen, acts locally to promote the survival and development of primate preantral follicles in vitro. Supported by: Oncofertility Consortium [NIH UL1 RR024926 (R01HD058294, PL1EB008542)], P51OD011092 and D43 TW/HD-00688-Fogarty (JKR). P-303 Wednesday, October 24, 2012 DETERMINATION OF 17b-HYDROXYSTEROID DEHYDROGENASE TYPES 1 AND 2 ACTIVITIES IN HUMAN PLACENTA BY ION TRAP GC/MS/MS. L. Dhariwal,a S. Arya,a M. Dalloul,a V. Nacharaju,a O. Muneyyirci-Delale,a J. G. Kral.b aObstetrics and Gynecology, SUNY Downstate Medical Center, Brooklyn, NY; bSurgery, SUNY Downstate Medical Center, Brooklyn, NY. OBJECTIVE: Androgens and estrogens are both substrates for 17b-hydroxysteroid dehydrogenase (17b-HSD) isozymes. 17b-HSD-type 1 (17bHSD-1) catalyzes the estrogenic reactions converting estone (E1) to estradiol (E2) and dehydroepiandrosterone (DHEA) into 5-androstenediol (5-AD); whereas 17b-hydroxysteroid dehydrogenase-type 2 (17b-HSD-2) regulates the appearance of E2 and 5-AD into fetal circulation through reverse reactions. Several methods exist to analyze 17b-HSD activity but Liquid Chromatography–Mass Spectrometry (LC/MS) and Gas Chromatography–Mass Spectrometry (GC/MS) are superior as they enable analysis of multiple hormones and metabolites in a single run. We developed a GC/MS method using androgens and estrogens with Ion Trap GC/MS/MS to evaluate 17b-HSD enzymatic activities. DESIGN: N/A MATERIALS AND METHODS: Placental homogenates were incubated with 20ug of androgen or estrogen with coenzyme (200 uM) at 37 degrees C for 60 minutes to assess activities of 17b-HSD types 1 and 2. pH 7.4 phosphate buffer was used for both oxidation and reduction reactions. 17b-HSD isozymes were measured using E1 and DHEA with NADH for type 1, and E2 and 5-AD with NAD+ for type 2 activity. Steroids were extracted using C-18 columns, derivatized with pentafluropropionic anhydride (PFPA) and analyzed by GC/MS (Varian 2200 Ô). Ion Trap GC/MS/MS was used to quantify steroids. 17bHSD-1 and 2 activities were expressed as the percent of steroid product. RESULTS: Distinct peaks of E2, 5-AD, DHEA and E1 were identified in all placentas and were eluted from the column at retention times: 7.96, Table 1: Columns 2 and 3 represent 17b-HSD-2 activity; columns 4 and 5 represent 17b-HSD-1 activity

17bHSD– and 2 activities

Mean % conversion

S202

E2 –> E1

5-AD –> DHEA

E1 –> E2

DHEA –> 5-AD

79.7

4.6

40.1

1.7

ASRM Abstracts

7.68, 8.77, and 9.2 minutes respectively. The enzyme activities are shown in Table 1. CONCLUSION: The Ion trap GC/MS/MS method measures placental 17b-HSD 1 and 2 activities using both androgens and estrogens as steroid substrates. P-304 Wednesday, October 24, 2012 GONADOTROPIN RELEASING HORMONE (GnRH) REDUCES LIGAND-INDUCED ESTROGEN RECEPTOR (ER) DIMERIZATION IN GT1-7 CELLS. R. J. Chason, P. K. Leung, A. H. DeCherney, J. H. Segars, K. J. Catt. NIH/NICHD, Bethesda, MD. OBJECTIVE: Nonclassical estrogen (E2) signaling in the hypothalamus may explain the differential effects of E2 that lead to the LH surge. Estrogen initiates rapid, membrane-initiated signaling pathways that alter GnRH secretion; and conversely, GnRH alters E2 binding to the ER. Given the critical role of GnRH and E2 signaling in the LH surge, we tested whether ERs interact with GnRH-Rs using bioluminescence resonance energy transfer (BRET1), which occurs if two proteins are < 10nm apart. DESIGN: Laboratory investigation. MATERIALS AND METHODS: Expression plasmids encoding BRET proteins were created by fusing GnRH-R, ERa, and ERb with Renilla luciferase (Rluc) or enhanced yellow fluorescent protein (eYFP). GT1-7 or HEK-293 cells were transfected with various combinations of BRET constructs. Quantitative BRET saturation assays were performed using a Mithras LB940 scanner. Specific BRET interactions were assumed to produce a hyperbolic curve and analyzed using a nonlinear regression curve assuming one-site binding. BRET ratios were plotted against quantitative levels of expression for the respective constructs. RESULTS: Expression of fusion proteins in HEK-293 cells was confirmed with immunoblot. In GT1-7 cells, fusion constructs exhibited dose-dependent fluorescence and luminescence, confirming expression and BRET functionality. Constitutive and agonist-activated increases in specific BRET1 ratios were observed in GT 1-7 cells expressing ERa-Rluc and ERa-YFP; ERa-Rluc and ERb-YFP; as well as GnRH-R-Rluc and GnRH-R-YFP. Notably, addition of GnRH reduced ERa dimerization in GT1-7 cells. CONCLUSION: The observation that GnRH-R activation reduced ERa dimerization within minutes strongly suggests there is rapid cross talk between estrogen and GnRH signaling pathways in GnRH neurons. Further studies will examine whether GnRH-R and ERs interact directly in GT1-7 cells. Supported by: This research was supported by the Intramural Research Program of NICHD, NIH.

OBESITY AND METABOLISM P-305 Wednesday, October 24, 2012 MAPPING OF EPIGASTRIC VESSELS IN FEMALE PATIENTS OF REPRODUCTIVE AGE STRATIFIED BY BODY MASS S. Lay,b J. Buedefeldt-Pollard,a INDEX. A. Garza-Cavazos,a K. Groesch,a,c M. E. McAsey,a S. A. Siddique.a aObstetrics and Gynecology, Southern Illinois University School of Medicine, Springfield, IL; bRadiology, Southern Illinois University School of Medicine, Springfield, IL; cCenter for Clinical Research, Southern Illinois University School of Medicine, Springfield, IL. OBJECTIVE: To determine if the course of epigastric vessels varies in female obese patients of reproductive age when using previously described safe zones for laparoscopic entry and to establish safe points of entry to avoid injury during laparoscopic surgery. DESIGN: Retrospective analyses of abdominal and pelvic computed tomography (CT) images with intravenous contrast were performed. MATERIALS AND METHODS: Abdominal and pelvic CT scans with intravenous contrast were randomly selected. Inclusion criteria: Female patients, 18–50 years of age who completed a CT scan. Exclusion criteria: females < 18 and >50 years of age and patients with any condition that may alter the location of the epigastric vessels. Location of the epigastric vessels from the midline were mapped at 5 levels: (1) xyphoid, (2) M1: midway between the xyphoid and anterior superior iliac spine (ASIS), (3) ASIS, (4) M2: midway between the ASIS and symphysis pubis (PS) and (5) PS. Safe zones were assessed using 4 different methods previously described. Patients were divided according to BMI values defined by WHO criteria. One-way

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