P-185 Wilms' tumor suppressor gene (WT1) is expressed in human term trophoblasts and regulated by cAMP

septum which was surgically resected. Her past medical history was entirely negative and there was no history of in-utero hormonal exposure. Within six months following surgery, the patient developed a profuse vaginal discharge. The discharge was studied on numerous occasions and demonstrated primarily mucus and pus cells. Topical treatment for vaginitis as well as hormonal modification were unsuccessful at controlling the discharge. After two years, the patient was taken to the operating room where she underwent vaginoscopy and vaginal biopsies. The vaginoscopy revealed glandular appearing upper vagina and the pathology was compatible with the diagnosis of vaginal adenosis. The patient subsequently underwent laser vaporisation of the upper vagina with some improvement of her vaginal discharge correlated with partial reversal of adenosis. Conclusions: This case illustrates that profuse vaginal discharge which may be associated with adenosis. A possible association between transverse vaginal septum and vaginal adenosis should be considered. Further investigations are warranted about this potential association.

T u e s d a y , October 21, 1997

Reproductive Biology P-185 Wilms' Tumor Suppressor Gene (WT1) is Expressed in H u m a n Term Trophoblasts and Regulated by cAMP. 1E. Hambartsoumian, 1M. Zilberstein, 1M. M. Seibel, 2M. Feingold, 1R. Srivastava. 1Faulkner Centre for Reproductive Medicine, Harvard Medical School, Boston, MA; 2Department of OB/GYN, Boston University, Boston, MA. Objectives: Human trophoblast differentiates by fusion of cytotrophoblast cells to form syncytiotrophoblasts. In culture this transformation coincides with copious production of cell specific hormones. However the regulation of trophoblast differentiation is not yet elucidated. Wilms' Tumor gene (WT1) encodes a transcription factor that is expressed in other tissues (i.e. kidney, gonads and uterus), where it is implicated in the induction of cell differentiation. We studied the effect of 8-bromo-cAMP on its expression. Design: Cytotrophoblasts were isolated from human placenta by trypsinization and purification on percoll gradient. Within 48 hours the majority of the culture transformed into syncytium. Cells were harvested for determination of WT1 gene expression at different time points in culture and after 8-bromo-cAMP treatment. Materials and Methods: Total RNA was isolated by gnanidine isothiocyanate and phenol/chloroform extraction. Reverse transcription and nested polymerase chain reaction (nested RT-PCR) ws used to quantitate WT1 gene expression, using WT1 specific primers. Results: WT1 mRNA is clearly expressed in both cytotrophoblast and syncytiotrophoblast cells. 8-bromo-cAMP

(1 mM) significantly increased the expression ofWT1 gene in trophoblast cells. Conclusion: This is the first time that WT1 gene expression was demonstrated in the functional components of the human placenta. Also it is the first time that cell specific WT1 gene expression has been shown to be regulated by a cAMP-dependent mechanism. Taken together these data suggest WT1 may regulate trophoblast differentiation. These data also implicate cAMP as a potential regulator of WT1 expression in human trophoblast.

P-186 Retinoic Acid Synthesis is Up Regulated in Concert With Matrix Metalloproteinase Suppression in a Human Model of Endometrial Stromal Cell Decidualization. 1E. Sierra-Rivera, 2W. L. Zheng, 2D. E. Ong, 1j. T. Hargrove, 1K. G. Osteen. 1Reproductive Medicine Research Laboratory, Dept of OB-GYN and 2Dept of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN. Objectives: In the human endometrium, members of the stromelysin family of matrix metalloproteinases (MMPs) are regulated by steroids during the dynamic events of the menstrual cycle. During the secretory phase, progesterone suppresses expression of stromal-specific MMP-3 at a time when endometrial tissue expression of transforming growth factor-/3 (TGF-fl) increases. However, stromal cell MMP-3 expression can be suppressed by retinoic acid (RA) in isolated stromal cells when these cells appear TGF/3 insensitive. In this study, we examined whether local synthesis of RA might occur in concert with TGF-fl secretion in endometrial stroma] cells during the progesteronedependent process of decidualization. Design: Endometria] tissues were acquired by biopsy on days 9 - 1 2 or days 17-21 of the menstrual cycle from a normal donor population. The proliferative phase tissues were utilized to examine the control of stromal-specific MMP regulation and the secretory samples were used to establish an in vitro model of stromal cell decidualization to determine RA synthesis. Materials and Methods: Organ cultures and isolated stromal cell cultures were established for analysis of MMP-3 secretion after treatments with estradiol (E; 10 -SM), E plus progesterone (P; 5 × 10 -7 M respectively), E plusTGF-~ (2ng/ml) or E plus RA (10 ~M). MMP-3 secretion was examined by electrophoresis and autoradiography following immunoprecipitation of labeled proteins. Secretory phase stromal cells were established to promote in vitro decidualization for RA synthesis analysis. The synthesis of RA from retinol precursor was determined by established HPLC methodology following extraction of culture media. Results: Exposure of short term endometrial organ cultures to E alone resulted in MMP-3 secretion while exposure to either E plus P, E plus TGF-~, or E plus RA resulted in suppression of MMP-3 in a time dependent manner. As opposed to the suppression of MMP-3 by either P, TGF-/3 or RA in organ culture, in isolated stromal cells only P and RA remain able to suppress this enzyme. In a Abstracts

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