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Progesterone stimulates pituitary adenylate cyclase-activating polypeptide (PACAP) expression in pituitary gonadotropes
DESIGN: A prospective controlled study. MATERIALS AND METHODS: Sperm was induced for capacitation with Krebs-Ringer solution containing calcium and BSA or in normal saline. The sperm was assessed for routine markers and for the expression of a2V-ATPase (a2 isoform of vacuolar ATPase). Assessments were made by confocal microscopy and PCR. Immunolocalization to evaluate a2V protein expression, realtime PCR to evaluate gene expression of a2V and different cytokines, sandwich ELISA to evaluate the secretion of a2NTD, which is the secreted portion of a2V. RESULTS: The capacitation of sperm induced a2V transcription and upregulated a2V protein expression. The expression of a2V was only detected in the appropriate capacitation buffer. No transcription or translation products were detected in sperm incubated in normal saline or PBS. Minimal expression of a2V was measured in Krebs-Ringer buffer without calcium or BSA. Capacitated sperm released a2NTD which can induce IL-1b, TNFa and MCP-1 (Monocyte chemotactic protein 1) expression. The results are compared between fertile couples and those being seen for treatment for recurrent pregnancy loss. CONCLUSION: We have previously reported that sperm capacitation induces the expression of the immune regulatory molecule a2V, which in turn causes the induction of inflammatory cytokines in the uterus. Capacitated sperm initiates the immunity of pregnancy by inducing a2V that in turn induces inflammatory cytokine expression during onset of pregnancy. We demonstrate that the maternal-fetal immune dialogue begins in the male with the capacitation of sperm and could be an immune predictor of fertility in women. O-324 Wednesday, October 19, 2011 05:30 PM GENOME-WIDE IDENTIFICATION OF CHLAMYDIA TRACHOMATIS ANTIGENS ASSOCIATED WITH TUBAL FACTOR INFERTILITY. N. M. Budrys, A. K. Rodgers, S. Gong, A. Holden, R. S. Schenken, G. Zhong. Obstetrics and Gynecology, University of Texas Health Science Center, San Antonio, TX; Microbiology and Immunology, University of Texas Health Science Center, San Antonio, TX. OBJECTIVE: To identify Chlamydia trachomatis (CT) antigens associated with tubal factor infertility using a genome-wide proteome array. DESIGN: Subjects were assigned to the tubal factor infertility (TFI) or infertile control (IC)group based on results of pelvic laparoscopy. CT antibody response profiles were assessed to identify antigens associated with tubal pathology. MATERIALS AND METHODS: 31 TFI and 23 IFC patients from a university-based fertility clinic were enrolled after IRB approval. Patients with a history of appendicitis or endometriosis were excluded. Serum samples were analyzed for CT titre utilizing immunofluorescence assay. The antibody recognition of each of 933 CT antigens was independently confirmed using enzyme-linked immunosorbent assay. Log transformation of data was used for normalization prior to analysis with either Student T-test or Fisher’s Exact test. RESULTS: A greater percentage of patients in the TFI group had high antibody titers to CT (>1:10,000) compared to the IC group (61% vs 4.4%, p < 0.001). A total of 30 antigens were preferentially recognized by antibodies from the TFI group with a detection sensitivity and specificity of 80.6% and 56.5%, respectively, with 10 antigens showing 100% specificity. A combination of CT443 and CT381 yielded the highest sensitivity (67.7%) while maintaining 100% specificity, when compared with single antigens alone or in combination. CONCLUSION: Our findings show that antibodies to CT443 and CT381, when used in combination, have higher sensitivity and specificity in prediction of TFI than other indicators for TFI such as HSP60 (35.5%, 100%) or hysterosalpingogram (65%, 83%). In the future, utilization of a serum antibody panel may lead to a more reliable diagnosis of TFI which does not require expensive, invasive methods for diagnosis. Supported by: This work was supported in part by NIH grant R01AI64537 (to G. Zhong).
REPRODUCTIVE ENDOCRINOLOGY: RESEARCH O-325 Wednesday, October 19, 2011 03:45 PM WHAT IS THE MAJOR ESTROGEN BINDING PROTEIN IN THE FOLLICULAR FLUID? Y. Bentov, N. Esfandiari, R. F. Casper. Toronto Centre for Advanced Reproductive Technology, Torornto, ON, Canada; Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada; University of Toronto, Division of Reproductive Sciences, Department of Obstetrics and Gynecology, Toronto, ON, Canada. OBJECTIVE: Estrogens orchestrate multiple changes in gonadal and extragonadal tissues directly and indirectly important for reproduction. The main site of estrogen production is the granulosa cells of the ovarian follicle, with concentration of estrogen in follicular fluid (FF) 1000-fold higher than in serum. In se-
FERTILITY & STERILITYÒ
rum, almost all estrogen is bound to sex hormone binding globulin (SHBG) and albumin. We have previously shown that estrogen binding in the FF is maintained by the action of a yet unknown binding protein other than albumin or SHBG. The objective of this study was to determine if other estrogen binding proteins are responsible for maintaining the high level of estrogen in FF. DESIGN: Proteomic analysis of FF MATERIALS AND METHODS: Samples of FF were collected from women undergoing IVF treatment. FF was stripped using charcoal coated dextran and then incubated with biotinylated 17-b-estradiol. The FF was then passed through a Softlink Avidin column. We collected the flow-through (FT), and then washed the column with buffer to remove nonspecifically bound proteins, followed by a biotin solution wash (BW) to remove specifically bound proteins. The native TT, FT and BW were then analyzed with Mass-spectrometry. RESULTS: The protein with the highest rate of enhancement with biotin was basement membrane-specific heparan sulfate proteoglycan core protein (Perlecan). The concentrations in the native FF, FT and BW were 11, 0, and 325 nmol/L, respectively, representing a 30 fold enhancement. CONCLUSION: Perlecan was shown to be present in FF and is known to be synthesized by granulosa and cumulus cells. Its synthesis is stimulated by FSH and correlates with follicular maturation. It was previously speculated to have a physiological role in folliculogenesis and oocyte maturation. Our current finding suggests another role for Perlecan as the main estrogen binding protein in the follicle. The physiologic significance of enhanced estrogen binding in the FF is unknown but may protect the oocyte from excessive free estrogen. O-326 Wednesday, October 19, 2011 04:00 PM PROGESTERONE STIMULATES PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE (PACAP) EXPRESSION IN PITUITARY GONADOTROPES. J. L. Morgan, C. M. Grafer, C. Wang, L. M. Halvorson. Obstetrics and Gyencology, University of Texas Southwestern Medical Center, Dallas, TX. OBJECTIVE: PACAP has been shown to have widespread distribution and varying roles throughout the reproductive axis. In the anterior pituitary, PACAP secretion is well known to influence gonadotropin synthesis and release. Our objective was to investigate a potential role for progesterone in the regulation of pituitary PACAP expression. DESIGN: Progesterone-stimulated expression of the PACAP gene was analyzed in vitro. MATERIALS AND METHODS: 1) Gonadotrope-derived LbT2 cells were co-transfected with rat PACAP promoter-luciferase vectors and a progesterone receptor B (PR-B) expression construct and then treated with progesterone (100nM x 24h). A Renilla reporter vector controlled for transfection efficiency. 2) LbT2 cells were treated with progesterone (100nM x 6 or 24h) or vehicle followed by qPCR measurement of PACAP mRNA levels. RESULTS: Progesterone increased the full length -1906/+906 PACAP promoter activity in a dose-dependent manner with a peak of 14-fold at 300 nM. Progesterone-stimulated PACAP promoter activity was blunted with deletion to position -402 (52% of full length, P<0.001) and eliminated with further truncation to position -77 relative to the transcriptional start site (7% of full length, P<0.001). Further dissection of region -402 to -77 showed a significant decrease in PACAP responsiveness with deletion from position -308 to -242 (64% of the -402 construct, P<0.001) with further loss following deletion to position -171 (16% of the -402 construct, P<0.001). In addition to transcriptional activation, progesterone treatment increased endogenous PACAP mRNA levels by 3- to 5-fold at 6 and 24 hrs. CONCLUSION: Our results show that progesterone increases pituitary PACAP promoter activity and mRNA expression. These findings suggest a novel mechanism for ovarian steroid modulation of pituitary function, which ultimately influences gonadotopin gene expression and reproductive homeostasis. Supported by: R01HD054782
O-327 Wednesday, October 19, 2011 04:15 PM ANTI MULLERIAN HORMONE (AMH) LEVEL AND EXPRESSION IN MURAL AND CUMULUS CELLS IN RELATION TO AGE. A. Kedem, Y. Yung, H. Kanety, M. Hanochi, J. Dor, A. Hourvitz. Department of Obstetrics and Gynecology, In Vitro Fertilization Unit and Human Embryonic Stem Cell and Reproduction Lab, Sheba Medical Center, Israel. Sackler School of Medicine, Tel Aviv University, Tel Aviv Israel, Ranat-Gan, Israel; Endocrinology Institute, Ranat-Gan, Israel. OBJECTIVE: Anti-Mullerian hormone (AMH) has recently been shown to be one of the most important markers of ovarian reserve.
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REPRODUCTIVE ENDOCRINOLOGY: RESEARCH O-325 Wednesday, October 19, 2011 03:45 PM WHAT IS THE MAJOR ESTROGEN BINDING PROTEIN IN THE FOLLICULAR FLUID? Y. Bentov, N. Esfandiari, R. F. Casper. Toronto Centre for Advanced Reproductive Technology, Torornto, ON, Canada; Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada; University of Toronto, Division of Reproductive Sciences, Department of Obstetrics and Gynecology, Toronto, ON, Canada. OBJECTIVE: Estrogens orchestrate multiple changes in gonadal and extragonadal tissues directly and indirectly important for reproduction. The main site of estrogen production is the granulosa cells of the ovarian follicle, with concentration of estrogen in follicular fluid (FF) 1000-fold higher than in serum. In se-
FERTILITY & STERILITYÒ
rum, almost all estrogen is bound to sex hormone binding globulin (SHBG) and albumin. We have previously shown that estrogen binding in the FF is maintained by the action of a yet unknown binding protein other than albumin or SHBG. The objective of this study was to determine if other estrogen binding proteins are responsible for maintaining the high level of estrogen in FF. DESIGN: Proteomic analysis of FF MATERIALS AND METHODS: Samples of FF were collected from women undergoing IVF treatment. FF was stripped using charcoal coated dextran and then incubated with biotinylated 17-b-estradiol. The FF was then passed through a Softlink Avidin column. We collected the flow-through (FT), and then washed the column with buffer to remove nonspecifically bound proteins, followed by a biotin solution wash (BW) to remove specifically bound proteins. The native TT, FT and BW were then analyzed with Mass-spectrometry. RESULTS: The protein with the highest rate of enhancement with biotin was basement membrane-specific heparan sulfate proteoglycan core protein (Perlecan). The concentrations in the native FF, FT and BW were 11, 0, and 325 nmol/L, respectively, representing a 30 fold enhancement. CONCLUSION: Perlecan was shown to be present in FF and is known to be synthesized by granulosa and cumulus cells. Its synthesis is stimulated by FSH and correlates with follicular maturation. It was previously speculated to have a physiological role in folliculogenesis and oocyte maturation. Our current finding suggests another role for Perlecan as the main estrogen binding protein in the follicle. The physiologic significance of enhanced estrogen binding in the FF is unknown but may protect the oocyte from excessive free estrogen. O-326 Wednesday, October 19, 2011 04:00 PM PROGESTERONE STIMULATES PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE (PACAP) EXPRESSION IN PITUITARY GONADOTROPES. J. L. Morgan, C. M. Grafer, C. Wang, L. M. Halvorson. Obstetrics and Gyencology, University of Texas Southwestern Medical Center, Dallas, TX. OBJECTIVE: PACAP has been shown to have widespread distribution and varying roles throughout the reproductive axis. In the anterior pituitary, PACAP secretion is well known to influence gonadotropin synthesis and release. Our objective was to investigate a potential role for progesterone in the regulation of pituitary PACAP expression. DESIGN: Progesterone-stimulated expression of the PACAP gene was analyzed in vitro. MATERIALS AND METHODS: 1) Gonadotrope-derived LbT2 cells were co-transfected with rat PACAP promoter-luciferase vectors and a progesterone receptor B (PR-B) expression construct and then treated with progesterone (100nM x 24h). A Renilla reporter vector controlled for transfection efficiency. 2) LbT2 cells were treated with progesterone (100nM x 6 or 24h) or vehicle followed by qPCR measurement of PACAP mRNA levels. RESULTS: Progesterone increased the full length -1906/+906 PACAP promoter activity in a dose-dependent manner with a peak of 14-fold at 300 nM. Progesterone-stimulated PACAP promoter activity was blunted with deletion to position -402 (52% of full length, P<0.001) and eliminated with further truncation to position -77 relative to the transcriptional start site (7% of full length, P<0.001). Further dissection of region -402 to -77 showed a significant decrease in PACAP responsiveness with deletion from position -308 to -242 (64% of the -402 construct, P<0.001) with further loss following deletion to position -171 (16% of the -402 construct, P<0.001). In addition to transcriptional activation, progesterone treatment increased endogenous PACAP mRNA levels by 3- to 5-fold at 6 and 24 hrs. CONCLUSION: Our results show that progesterone increases pituitary PACAP promoter activity and mRNA expression. These findings suggest a novel mechanism for ovarian steroid modulation of pituitary function, which ultimately influences gonadotopin gene expression and reproductive homeostasis. Supported by: R01HD054782
O-327 Wednesday, October 19, 2011 04:15 PM ANTI MULLERIAN HORMONE (AMH) LEVEL AND EXPRESSION IN MURAL AND CUMULUS CELLS IN RELATION TO AGE. A. Kedem, Y. Yung, H. Kanety, M. Hanochi, J. Dor, A. Hourvitz. Department of Obstetrics and Gynecology, In Vitro Fertilization Unit and Human Embryonic Stem Cell and Reproduction Lab, Sheba Medical Center, Israel. Sackler School of Medicine, Tel Aviv University, Tel Aviv Israel, Ranat-Gan, Israel; Endocrinology Institute, Ranat-Gan, Israel. OBJECTIVE: Anti-Mullerian hormone (AMH) has recently been shown to be one of the most important markers of ovarian reserve.
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