Dependence of GnRH-induced CREB phosphorylation in hypothalamic neurons upon transactivation of the EGF receptor and activation of the phosphoinositide 3-kinase pathway

correlations of adiponectin to the hormonal and metabolic parameters including measures of insulin resistance (IR). DESIGN: Case-control study. MATERIALS AND METHODS: One hundred and eighty selected women were classified as follows: 45 obese (BMI ⬍30 kg/m2) with PCOS; 45 lean (BMI ⬍25 kg/m2) with PCOS; 45 obese (BMI ⬎30 kg/m2) without PCOS and 45 lean (BMI ⬍25 kg/m2) without PCOS. Blood samples were collected from all women with or without PCOS between 8:00⫺11:00 a.m., after an overnight fast. Serum levels of LH, FSH, PROL, TSH, FT4, T, 17-OHP, ⌬4-A, DHEA, DHEAS, SHBG, insulin and plasma levels of adiponectin and glucose were determined. Measures of IR included: fasting serum insulin, GIR and HOMA. RESULTS: Adiponectin concentrations were found to be significantly decreased in women with PCOS and in obese without PCOS as compared to lean women without PCOS. Adiponectin concentrations correlated inversely with body weight, BMI, fasting plasma glucose, serum insulin, ⌬4-A, DHEA, DHEAS, HOMA, but correlated positively with serum T, SHBG, FAI and GIR. Multiple regression analysis showed that BMI, HOMA, ⌬4-A, and insulin were independent determinants of adiponectin concentrations. CONCLUSION: Hypoadiponectinaemia is evident in obese and lean women with PCOS with variable degree of IR; and it is suggested that IR per se and/or other metabolic abnormalities of PCOS are involved in the regulation of adiponectin concentration in women with PCOS. Supported by: King Abdulaziz University Grant # 012/422

P-489 Relationship between GnRH, Bradykinin and Inhibin/Activin (beta A and beta B subunits) and apoptosis in testes of infertile and aging men. V. Tripathi, A. Krishna, U. S. Diwedi, R. Sridaran. Department of Zoology, Banaras Hindu Univaersity, Varanasi, India; Department of Urology, IMS, Banaras Hindu Univaersity, Varanasi, India; Department of Physiology, Morehouse School of Medicine, 720 Westview Dr., Atlanta-30310, GA. OBJECTIVE: The aim of present investigation was to examine the distribution of GnRH, Bradykinin and Inhibin/ Activin beta A (bA) and beta B (bB) subunits immunoreactivity and their relationship with the pattern of apoptosis in the testicular biopsy samples from ‘normal’, azoospermic and ‘aged’ men. DESIGN: Testes sections of normal, azoospermic and aged men were studied for GnRH, Bradykinin and Inhibin/Activin (bA & bB subunits) and apoptotic activity. MATERIALS AND METHODS: Testes samples were collected from patients advised for testicular biopsy (azoospermia) or bilateral orchiectomy (early metastasis of prostate) from Pathology Division and Urology Division, I.M.S., B.H.U. Prostate patients, with proven fertility, above 70 years were grouped as ‘aged’ while below 58 years as ‘normal’. Tissues were fixed in Bouin’s fluid and embedded in paraffin. GnRH, Bradykinin and Inhibin/ Activin (bA and bB subunits) were localized immunohistochemically using polyclonal antibodies and Vector ABC Kit. Apoptotic cells in testis sections were identified by in situ 3’-end labeling of DNA. (Intergen Co., USA). RESULTS: The results showed GnRH, Bradykinin and Inhibin/Activin (bA and bB subunits) immunoreactivity mainly in the Sertoli cells and Leydig cells of the testes. Further, GnRH immunoreactivity was intense in Leydig cells but mild in Sertoli cells. Whereas bA & bB immunoreactivity was more intense in the Sertoli cells but mild in Leydig cells. Bradykinin immunoreactivity was intense in both Leydig as well as in Sertoli cells. Comparison of GnRH, Bradykinin and Inhibin/Activin (bA & bB subunits) immunoreactivity in the testes sections of azoospermic, aged and normal men showed some marked variations. GnRH immunoreactivity was distinctly higher in the Leydig cells of azoospermic testes as compared to the aged and normal testes. Similarly, bA immunoreactivity was distinctly higher in the Sertoli cells of azoospermic testes as compared to the aged and normal testes. Immuno-fluorescein staining for apoptosis was observed in the Leydig cells of azoospermic testes. Aged and normal testes showed negligible staining for apoptosis. CONCLUSION: GnRH may be produced mainly in the Leydig cells, Inhibin/ Activin (bA & bB subunits) in the Sertoli cells and Bradykinin in both Leydig and Sertoli cells. Several apoptotic Leydig cells observed may be correlated with high GnRH immunoreactivity in the Leydig cells of

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azoospermic patients. Further studies are needed to establish the relationship of GnRH with apoptosis in the testes of infertile patients. Supported by: Supported by grants from UGC, New Delhi to VT & AK and GM08248 & HD41749 from NIH to RS.

P-490 Ovarian tissue cryopreservation did not compromise its potential to produce competent oocytes for nuclear transfer. H.-C. C. Liu, Z. Y. He, Z. Rosenwaks. Weill Medical College of Cornell University, New York, NY. OBJECTIVE: Cryopreservation of ovarian tissue may be of benefit to many areas of reproductive biology and medical research. Successful cryopreservation of ovarian tissue while maintaining the integrity of biological function would provide a valuable source for generating competent oocytes either for clinical use or for research purposes. In this study, we tested whether oocytes generated after in-vitro maturation of preantral follicles isolated from frozen-thawed ovarian tissue contain competent cytoplasts for nuclear transfer. DESIGN: Mouse GVs were transferred into cytoplasts of oocytes which were either matured in-vivo (control) or matured in-vitro. The competence of the reconstructed oocytes in terms of fertilization and embryogenesis were then compared. MATERIALS AND METHODS: Ovarian tissue from 14-day-old B6D2F1 mice were cryopreserved using standard slow-freezing protocol and kept in liquid nitrogen for more than 2 weeks. Thawed ovarian tissue were cultured in follicle cultured medium overnight, then subjected to mechanic isolation of preantral follicles. These preantral follicles underwent maturation in-vitro for 10 days to obtain competent oocytes. On the other hand, a group of oocytes were matured in-vivo in the superovulated female mice (stimulated with PMSG/hCG). Both oocytes matured in-vitro (cryopreserved group) and in-vivo (control group) were enucleated to obtain cytoplasts for study. Mouse GVs collected by micromanipulation from immature oocytes of adult female mice (stimulated with PMSG only) were fused with the cytoplasts of the cryoproserved or control groups. All reconstructed oocytes were matured in-vitro, fertilized by ICSI with the Piezo-actuated unit, and then cultured in-vitro for 5 days for comparison. RESULTS: Between the two study groups, no significant differences were found among the fusion, MII formation, fertilization, embryogenesis, and blatocyst formation rates. The outcome of the reconstructed oocytes are listed in the following table:

CONCLUSION: Our data clearly demonstrated that viable embryos can be generated following ovarian tissue cryopreservation and competent oocytes for nuclear transfer could result even after undergoing a long process of cryopreservation, in-vitro maturation and oocyte reconstruction. This may suggest that a tissue bank can be established to provide competent oocytes for clinical use (i.e. donor egg) and for basic science research (such as therapeutic cloning and stem cell study) as well. Therefore, we can predict that the combination of ovarian tissue cryopreservation and follicle in-vitro maturation will have a great impact in future advanced reproductive medicine. Supported by: N/A

P-491 Dependence of GnRH-induced CREB phosphorylation in hypothalamic neurons upon transactivation of the EGF receptor and activation of the phosphoinositide 3-kinase pathway. A. B. Neithardt, B. H. Shah, K. J. Catt. National Institutes of Health, Bethesda, MD.

Vol. 82, Suppl. 2, September 2004

OBJECTIVE: Gonadotropin-releasing hormone (GnRH)-induced Gq-mediated stimulation of mitogen-activated protein kinases (MAPKs), including extracellularly regulated kinases 1 and 2 (ERK1/2), in GT1–7 neuronal cells, occurs via transactivation of the epidermal growth factor receptor (EGF-R) in a protein kinase C (PKC) dependent fashion. In this study, we determined whether other prosurvival proteins, including p90 ribosomal S6 kinase (RSK-1), cyclic AMP response element binding protein (CREB) and the antiapoptotic protein BAD, are also phosphorylated by GnRH in immortalized hypothalamic cells (GT1–7). Since the GnRH-R is coupled to Gs/adenylyl cyclase as well as Gq/PKC in these cells, and cAMP is known to stimulate MAPK signaling in neurons, we also examined the role of cAMP in mediating these effects. DESIGN: Cell culture and immunoblot analysis. METHODS: GT1–7 cells were grown in F12/DEMEM culture medium. Serum-deprived cells were stimulated with GnRH, EGF, forskolin, epinephrine and phorbol 12-myristate 12-acetate (PMA) in the presence and absence of selective inhibitors. After collection in Laemmeli buffer, cell lysates were subjected to immunoblot analysis to determine the phosphorylation of CREB, RSK-1 and BAD proteins. RESULTS: Stimulation of GT1–7 cells with GnRH caused rapid activation of ERK1/2, RSK-1, CREB and BAD. These actions of GnRH were attenuated by the selective EGF-R antagonist, AG1478, in a concentrationdependent manner. Activation of PKC by PMA, and of EGF-R by EGF, also caused rapid phosphorylation of these proteins. The selective phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, decreased GnRH-, PMA- and EGF-induced phosphorylation of ERK1/2, RSK-1, CREB and BAD in a concentration-dependent manner. Epinephrine-induced activation of Gscoupled adrenoceptors in GT1–7 cells also caused phosphorylation of ERK1/2, RSK-1 and CREB through activation of the EGF-R and PI3K. Blockade of cAMP/PKA with H-89 abolished the effects of epinephrine, and decreased those of GnRH, indicating partial involvement of Gs/cAMP/ PKA signaling in GnRH action in these cells. Agonist-induced stimulation of these prosurvival proteins was abolished by the MEK inhibitor, UO126, indicating the involvement of the MEK/ERK cascade in these responses. CONCLUSIONS: These data demonstrate that stimulation of Gq/PKC by GnRH, and of Gs/adenylyl cyclase/cAMP by epinephrine and forskolin, causes activation of ERK1/2, RSK-1 and CREB through transactivation of the EGF-R and PI3K in GT1–7 cells. The signals emanating from these agonists appear to converge at MEK in the ERK signaling cascade. Supported by: Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-4510.

P-492 The role of leptin in endometrial proliferation. A. K. Styer, R. R. Gonzalez, P. Ramos, M. P. Lynch, B. R. Rueda. Massachusetts General Hospital, Vincent Center for Reproductive Biology, Harvard Medical School, Boston, MA; Boston Biomedical Research Institute, Waltham, MA. OBJECTIVE: Leptin, a 16 kDa protein originally described as a key player in several hypothalamic pathways regulating metabolic balance, has recently been highlighted as an important cytokine in the implantation of the blastocyst. From a pathophysiological perspective, leptin levels are elevated in the peritoneal fluid of women with endometriosis, and the cytokine has been implicated in its establishment and early development. Leptin is mitogenic in breast, esophagus, and prostate cancer cells. Thus we hypothesized that Ishikawa cells (human epithelial adenocarcinoma cell line) would respond to leptin with increased phospho-STAT-3 levels in conjunction with increased cell proliferation. DESIGN: In vitro cell culture. MATERIALS AND METHODS: Ishikawa cells were grown to 70% confluence in DMEM 10% FBS (fetal bovine serum) medium. 3.0 ⫻ 105 cells/well were placed in 24 well plates and allowed to grow to 90% confluence. FBS was removed, and the appropriate wells were treated with or without leptin (10nM). Cells were collected and cellular/nuclear extracts and their immunoprecipitates were obtained for analysis. Western blot was performed to verify the expression of leptin receptor (OB-R) and activation of STAT-3 (phosphorylated STAT-3) in response to leptin treatment. Phosphorylated STAT-3 levels were normalized against non-phosphorylated STAT-3 and quantitated by Western blot analysis performed in triplicate. For cell proliferation studies, Ishikawa cells were treated in the absence or presence of leptin and collected at 0, 24, and 48 hr and counted. Statistical

FERTILITY & STERILITY威

analysis was performed utilizing Chi-squared tests and independent or paired t-tests as appropriate, with p ⬍0.05 considered statistically significant. RESULTS: Ishikawa cells express the OB-R receptor and respond to leptin with an increase in phosphorylated STAT-3 (p⬍0.05) suggesting OB-R activation. Cell proliferation was similar at 0 and 24 hr. However, cell proliferation rates of leptin treated cells was greater than controls at 48 hr (p⫽0.031). CONCLUSION: Collectively, these data suggest that leptin may well serve as a mitogenic agent in the endometrial epithelium in addition to stimulating proliferation of endometrial stroma. Further studies are necessary to verify that the increased cell number is not attributed to leptin induced cell survival. Regardless of whether or not the increase in cell number is attributed to leptin induced mitogenic activity or increased cell survival, leptin’s contribution to the pathology of endometriosis or endometrial adenocarcinoma is not known and is yet to be investigated. Supported by: This study was supported by the Vincent Memorial Research Funds (BRR) and the MGH Office of Multicultural Affairs (AKS)

P-493 Dopamine (D) agonists (A) administration diminishes increased vascular permeability (iVP) in ovarian hyperstimulation syndrome (OHSS) animals by blocking vascular endothelial growth factor receptor 2 (VEGFR2) phosporylation (ph). R. Gomez, M. Gonzalez, J. SanchezCriado, C. Simon, J. Remohi, A. Pellicer. IVI Foundation, Valencia, Spain; Hospital Dr Peset, Valencia, Spain; Universidad de Cordoba, Cordoba, Spain; IVI foundation and Department of Obstetrics and Gynaecology in the Faculty of Medicine, Universidad de Valencia, Valencia, Spain; IVI foundation, and Hospital Dr Peset and Department of Obstetrics and Gynaecology in the Faculty of Medicine, Universidad de Valencia, Valencia, Spain. OBJECTIVE: Blocking VEGFR2ph, the first step in VEGF downstream signaling, is able to avoid iVP in OHSS, but specific antiVEGFR2 drugs are toxic. D binding to its receptor 2 (R2)diminishes iVP in tumors and is thought to internalize VEGFR2 in close proximity to R2 in endothelial cells. Because our microarray analysis showed Tyrosine hydroxylase (TH) ( key enzyme in D synthesis) downregulation (dR) as a condition of OHSS ovaries, we aimed to 1) block iVP in OHSS bypassing the TH dR with the administration of DA) and 2) Elucidate if decreased (Dc)VP was debt to Dc VEGFR2ph or to luteolysis, related to OHSS resolution and (LX) induced in immature rats by DA inhibition of prolactin (Pr) secretion, which is needed to maintain corpus luteum. DESIGN: Immature wistar rats, 22 day(d)s old, were injected with PMSG 10 UI (d 22–25) and hCG 30 IU (d 26) to develop OHSS and treated, with different doses of the DA bromocriptine (Br) or cabergoline (Cb) 24 hours (h) later (d 27). The effects of DA on VP were measured (n⫽8 each dose) 48 h after hCG (maximal VP) (d 28). At the same time point serum samples (sp) were collected to quantify functional (f)LX by measuring P4 and Pr (n⫽8 each dose). Both ovaries were frozen too,one of them was used to quantify VEGFR2ph and mRNA expression (n⫽4 each dose) and the other for structural (st)LX studies (n⫽3 each dose). MATERIALS AND METHODS: VP was expressed as the extravasation of EB dye (␮g EB/100g weight ⫾SEM) previously injected by the vein. The housekeeping gene GAPDH and VEGFR2 were amplified with RT/QF-PCR and the ratio VEGFR2/GAPDH in arbitrary fluorescence units (AFU), used to compare VEGFR2mRNA expression between (bw)sp. VEGFR2ph was quantified by western blot analysis in which band signal densities of a phosporilated (pY)VEGFR2 antibody (ab) were normalized with those corresponding to a “whole” VEGFR2 ab and the ratio pYVEGFR2/ VEGFR2 (in AFU) used to compare VEGR2ph bw sp. Apoptosis and vascularization as parameters of stLX were detected by in situ TdT enzyme labelling and with CD31 ab staining respectively. We used image analysis software to quantify apoptotic cells per bright field and CD31area. Pr and P4 levels were quantified by RIA and ELISA respectively. ANOVA or MannWhitney tests were used, depending on variance homogeneity, for statiscal analysis. RESULTS: In a first set, Placebo (PL),Cb at 0.6, 3, 6,30 or Br at 0,75, 150, 300, 600 ␮g doses were administered in a single dose 24 h after hCG(d27). DA had no effect on stLX and VEGFR2mRNA expression. The minimal doses to avoid iVP compared to PL (30.03⫹4.07) were Br 150 ␮g (12.01⫾2.07; p⫽0.042) and Cb 3 ␮g (9.63⫾1.67, p⫽0.037)), and were related with a 60% Dc pYVEGFR2/VEGFR2 ratio but couldn’t

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