Expression of luteinizing hormone receptor (LH-R), follicle stimulating ...

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Oct 22, 2001 - Estrogen induces HOXA10 expression and HOXA10 upregulates p53 in human breast cancer. M. C. Chu, D. O. Cermik, H. S. Taylor. Dept of.
hormone is advantageous for studies focussing on the kinetics of testicular failures caused by spermatogenic hazards. Supported By: Technical University of Munich.

Monday, October 22, 2001 3:45 P.M. O-98 Estrogen induces HOXA10 expression and HOXA10 upregulates p53 in human breast cancer. M. C. Chu, D. O. Cermik, H. S. Taylor. Dept of Obstetrics and Gynecology, Yale Univ Sch of Medicine, New Haven, CT. Objective: The mechanism of estrogen action on breast cancer cell growth is poorly understood. Expression of the p53 gene protects cells against malignant transformation; cells that develop mutations in p53 demonstrate a diminished apoptotic potential, which may contribute to tumor growth and metastasis. The factors that regulate p53 expression are poorly characterized. HOXA10 is a homeobox gene that we have previously shown to be expressed in response to sex steroids in the uterus and is essential for implantation. Here we investigate the possibility that HOXA10 mediates estrogen action and p53 expression in breast cancer. Design: Northern analysis of RNA and western analysis of protein from normal and malignant human breast tissue and breast cancer cell lines. Materials/Methods: Eight samples of both benign and malignant human breast tissue were obtained at the time of surgery under an approved HIC protocol. MCF-7 breast cancer cells, which endogenously express estrogen receptor (ER) and HOXA10, were used to assay the effect of estradiol on HOXA10 expression. MCF-7 cells were cultured and treated with 10-6M estradiol. BT20 breast cancer cells, which do not express HOXA10, were used to assay the effect of treatment with HOXA10 on p53 expression. BT20 cells were cultured and treated with pcDNA/HOXA10, which constitutively expresses HOXA10, via liposome-mediated transfection. Breast tissue and MCF-7 cells were used for RNA extraction and northern analysis using a 32P-labeled HOXA10 riboprobe. BT20 cells were used for protein extraction and western analysis using p53 and progesterone receptor (PR) mouse anti-human monoclonal antibodies. Results: HOXA10 is expressed in both benign and malignant human breast tissue and in MCF-7 breast cancer cells. In MCF-7 cells, which are ER⫹, estradiol increased HOXA10 expression compared with control untreated cells. Constitutively expressing HOXA10 in BT20 cells, which do not normally express HOXA10, increased p53 expression but not PR expression. Conclusions: The mechanism by which estrogen influences both normal breast growth and development as well as breast cancer tumorigenesis may involve the regulation of developmental control genes such as HOXA10. HOXA10 is expressed in human breast cancer. Its expression is responsive to estrogen in cells that are ER⫹. Additionally, overexpression of HOXA10 induces expression of the tumor suppressor gene p53. Estrogen-mediated HOXA10 expression and subsequent p53 upregulation may be a molecular mechanism that controls breast cell growth, differentiation, and tumorigenesis. Estrogen therapy may thus alter p53 expression through a HOX gene-dependent pathway. Supported By: NIH HD36887.

Phosphorylated Smads translocate to the nucleus where they modulate transcription. In this study we evaluated the capacity of TGF␤ and activin to induce nuclear translocation of Smad2 and Smad3 in cultured granulosa cells. Design: Intracellular localization of Smad2 and Smad3 proteins was performed on cells treated with either TGF␤ or Activin. Materials/Methods: Granulosa cells obtained from 25-day-old rat ovaries were cultured on glass coverslips for 24 hours in medium containing 10% serum. The medium was then changed to serum-free medium containing FSH (10 ng/ml). After 24 hours of treatment with FSH, groups of cells were treated with either Activin (20 ng/ml) or TGF␤ (1 ng/ml) for 30 minutes. Cells were washed with PBS and fixed in 4% paraformadehyde prior to immunofluorescence staining with antibodies to either Smad2 or Smad3. Slides were visualized using a fluorescence microscope fitted with a 510 nm filter. 100 cells from each group were counted based on a semi-random grid with at least one field from each of the four quadrants. Virtually all of the cultured cells exhibited some level of staining for both proteins. Negative control slides did not have cellular staining. For each counted cell, the observer (who was blinded to the treatment group) determined visually whether the nuclear staining was greater than the cytoplasmic staining. This was defined as a nuclear stained cell. The counts were reported as a percent of all cells with staining based on this definition. The experiment was repeated with different cell isolations. Statistical significance between mean values was determined by analysis of variance followed by post-hoc testing and was accepted at 0.05 level. Results: Both Smad2 and Smad3 proteins are primarily expressed in cytoplasm of cultured granulosa cells in the absence of growth factors. In FSH-treated cells, Smad2 is localized to the nucleus in 14 ⫾ 1.2% of cells and Smad3 is localized to the nucleus in 22 ⫾ 1.7%. When FSH-treated cells are then exposed 30 minutes to either TGF␤ (1 ng/ml) or activin A (20 ng/ml), nuclear staining for both Smad2 and Smad3 is markedly increased. TGF␤ treatment results in 30 ⫾ 4.4% and 50 ⫾ 2.3% nuclear staining for Smad2 and Smad3, respectively (p ⬍ 0.05). But, activin treatment increases the level of nuclear staining for Smad2 to 53 ⫾ 4.6% and Smad3 to 36 ⫾ 4.1% (P ⬍ 0.05). % Nuclear localization.

Smad2 Smad3

FSH only

FSH ⫹ TGF␤

FSH ⫹ activin

14 22

30 50

53 36

Conclusions: In granulosa cells derived from large antral follicles, both Smad2 and Smad3 are activated and undergo nuclear transport in response to TGF␤ and activin treatment. However, Smad2 is more responsive to stimulation by activin and Smad3 is more responsive to stimulation by TGF␤. Supported By: The Ortho-MacNeil/ASRM Grant in Reproductive Medicine.

Monday, October 22, 2001 4:15 P.M. O-100

Monday, October 22, 2001 4:00 P.M. O-99 Transforming growth factor ␤ (TGF ␤) and activin stimulate nuclear translocation of Smad proteins in granulosa cells. E. A. McGee, J. I. Oakley, J. Xu. Magee Womens Research Institute, Pittsburgh, PA. Objective: The TGF␤ family of growth factors has long been thought to play a role in ovarian follicle development, however until recently the mechanisms of action of these growth factors have been unknown. A group of proteins that functions as intracellular mediators of TGF␤ family signaling has recently been identified and named Smad. Though there are 9 Smads so far, two of the receptor-specific smads: Smad2 and Smad3 are both associated with both TGF␤ and activin signaling. Receptor complexes that have bound ligand can activate receptor-specific Smads by phosphorylation.

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Abstracts

Expression of luteinizing hormone receptor (LH-R), follicle stimulating hormone receptor (FSH-R) and epidermal growth factor receptor (EGF-R) in cumulus cells of the oocytes collected from PCOS patients in hCG-priming IVM/F-ET program. S. Yang, W. Son, S. Lee, S. Yoon, Y. Ko, J. Lim. Maria Women’s Hosp, Seoul, South Korea; Dept of Animal Science, Korea Univ, Seoul, Korea, Seoul, South Korea. Objective: Recently, Chian et al. (2000) reported the high rates of oocyte maturation and pregnancy obtained from PCOS patients in IVM/F-ET program with priming hCG prior to oocyte collection. This study was undertaken to examine the correlation between oocytes maturation and their cumulus cell (CC) patterns in hCG-primed IVM/F-ET cycles of PCOS women and further to investigate mRNA expressions of LH-R, FSH-R, and EGF-R in the CCs of each pattern. Design: Cumulus-oocyte complexes (COCs) were collected after priming hCG to women with PCOS and divided into three groups according to CC pattern (compacted, dispersed, or sparse). The maturation rate and matura-

Vol. 76, No. 3, Suppl. 1, September 2001

tion time of the oocytes with dispersed CCs were compared with those with compacted CCs. In addition, the mRNA expressions of LH-R, FSH-R, and EGF-R were compared in both groups. Materials/Methods: A total of 40 IVM/F-ETs were performed in 34 patients. All patients were primed with 10,000 IU hCG 36 hours before oocyte aspiration and follicular aspirates except for COCs were filtered through 70-␮m (in hole size) mesh. The isolated COCs were then washed with fresh medium and divided into two groups according to the CC pattern: group 1, the oocytes with dispersed CCs and group 2, the oocytes with compacted CCs (the oocytes with sparse CCs were not alloted to any group). Immature oocytes were cultured in maturation YS medium supplemented with 30% hFF, 1 IU/ml rFSH, and 10 IU/ml hCG. The nuclear maturity of the oocytes was determined at 0, 24, 48 and 72 hours. The CCs were isolated from each COC using two pieces of needles and then washed with dPBS. Total RNA from CCs of each group was isolated using RNeasy pre kit. The expressions of the FSH-R, LH-R, and EGF-R mRNA were analyzed by semi-quantitative RT-PCR using glyceraldehyde-3-phosphate dehydrogenase as an endogenous internal control and the PCR products were detected by Gel Doc 2000 (Bio-Rad Laboratories, USA). Results: 1) In the cultures for 24 hours, the maturation rates in group 1 and group 2 were 80.9% (251/310) and 30.6% (96/313), respectively. After 48 hours of culture, the maturation rates were 90.6% (281/310) and 58.46% (183/313), respectively. Finally, the maturation rate of 90.9% (282/310) until 72 hours of culture in group 1 was significantly higher than that in group 2 (61.6%, 193/313) (P ⬍ 0.001). 2) The mRNA expressions of FSH-R and EGF-R in group 1 were comparable to those in group 2. However, compared to compacted CCs (group 2), much abundant expression of LH-R mRNA was detected in the dispersed CCs (group 1). Conclusions: Collectively, these results showing that the ability of oocyte to mature is obtained from the dispersion of CCs suggest that the presence of dispersed CCs at the time of oocyte collection may be positively correlated with the maturation time of oocytes in hCG-priming IVM/F-ET program. In addition, the results also indicate that the expression of the LH-R in CCs may be correlated with the CC pattern at the time of oocyte collection. However, further studies are necessary to elucidate whether the expression of LH-R causes CC dispersion or whether the dispersion of CCs influences LH-R expression. Supported By: Maria Hospital.

Monday, October 22, 2001 4:30 P.M. O-101 Increasing levels of estradiol are deleterious to embryonic implantation due mainly to a direct effect on the embryo. D. Valbuena, J. C. Martı´n, J. L. De Pablo, J. Remohı´, A. Pellicer, C. Simo´n. Inst Valenciano de Infertilidad, Valencia, Spain; Inst Valenciano de Infertilidad, Dept of Pediatric, Obstetrics and Gynecology, Univ of Valencia, Valencia, Spain; Inst Valenciano de Infertilidad, FIVIER, Pediatric, Obstetrics and Gynecology Dept Valencia, Univ, Valencia, Spain. Objective: The influence of high levels of Estradiol (E2) achieved in controlled ovarian hyperstimulation (COH) used for IVF on embryonic implantation is controversial. The present in vitro study was conducted to investigate whether there is a deleterious effect of high E2 levels on embryonic implantation and if this was due to a direct effect on the endometrium, on the embryo, or both. Design: Prospective controlled in vitro study in which the impact of increasing concentrations of E2 on embryonic adhesion was studied by using an established embryo adhesion assay. Materials/Methods: E2 dose-response (0, 10-8, 10-7, 10-6, 10-5, and 10-4 M) and time course (day 2 versus 5) experiments were performed in an in vitro embryo adhesion assay composed of human polarized endometrial epithelial cells (pEEC) obtained from fertile patients in the luteal phase attending the infertility clinic as oocyte donors (n ⫽ 14) and mouse embryos (n ⫽ 2,589). Results: pEEC monolayers expressed ER␣ at the mRNA level. When blastocysts cultured in the presence of pEEC (n ⫽ 235) were treated with increasing dose of E2, there was a progressive reduction in embryonic adhesion, with results being significant with doses of E2 10-6 M. This pattern suggests an E2 dose-dependent toxic effect on the embryo and/or endometrium. When pEEC were treated alone with increasing doses of E2

FERTILITY & STERILITY威

for three days, and then E2 was removed and blastocysts added (n ⫽ 410), embryonic adhesion was not significant reduced, except in the group treated with 10-4 M E2. When 2-day mouse embryos (n ⫽ 609) were treated with increasing E2 concentrations until day 5, blastocyst rate significantly decreased when E2 was ⬎10-6 M. The embryonic adhesion rate was significantly decreased when blastocysts (n ⫽ 400) were obtained at E2 concentration ⬎10-7 M. Time course experiments further demonstrate that E2 induces a direct toxic effect on the embryo. When embryos (n ⫽ 426) were cultured for 2 days with pEEC, the adhesion rate was higher at E2 levels of 10-7, 10-6 and 10-5 M compared to embryos (n ⫽ 495) cultured for 5 days. Conclusions: High E2 levels are deleterious to embryo adhesion in vitro due mainly to a direct toxic effect on the embryo that may act at the cleavage stage. This finding, although apparently naive, is of paramount relevance not only for the adequate management of high responder patients but more important for the concept of minimal ovarian hyper stimulation and late embryo transfer versus the conventional aggressive COH and early transfer in IVF. Supported By: FIVIER.

Monday, October 22, 2001 4:45 P.M. O-102 Distribution of pinopods in the secretory phase: a prospective, randomized assessment in healthy, fertile women. R. S. Usadi, B. A. Lessey, R. C. Bagnell, W. R. Meyer, M. A. Fritz. Univ of North Carolina at Chapel Hill, Chapel Hill, NC. Objective: To reassess the distribution of pinopods on the surface epithelium of secretory phase endometrium in healthy, normally cycling, fertile women. Design: Prospective, randomized study. Materials/Methods: Sixty-six healthy, normally cycling fertile subjects were recruited from advertisements in local newspapers. None had received oral hormonal therapy in the past thirty days nor depot-provera in the past six months. Each subject was instructed in the use of a urinary LH surge detection kit and was instructed to call when the first urinary LH surge was detected. Based on a randomization schedule, each subject was assigned a day to return for endometrial biopsy. Each volunteer underwent one biopsy per cycle. Twenty subjects (23%) were invited for a repeat biopsy on the same LH-timed day in a subsequent month. At the time of endometrial biopsy, blood was obtained for serum estradiol and progesterone levels. Endometrial biopsies were viewed on a Cambridge Stereoscan S200 scanning electron microscope using an accelerating voltage of 20 kV, working distance of 25 mm, and specimen tilt of 40° (LEO Electron Microscopy, Inc., Thornwood, NY). Scanning photomicrographs were taken of 12 randomly selected areas and saved as a TIFF file for later viewing. Each photomicrograph was scored by a single blinded observer. The pinopod results were counted as none (0), few (1), some (2) or many (3), based on the most representative photomicrographs of that sample. The definition used to detect pinopods were apical protrusions from the surface epithelium that were smooth and without microvilli. All apical projections did not meet this criteria. Over 800 photomicrographs were examined but a single score was given to each biopsy based on the overall impression of all 12 images. The mean score for each LH day was calculated. In addition, each sample was read by a blinded gynecologic pathologist. This day was used to separately compare pinopod expression based on histologic dating criteria of Noyes et al. (1950). Results: After excluding samples with inadequate surface epithelium and those revealing obvious error in LH surge detection, 76 endometrial specimens from sixty-four subjects were examined. Pinopods were identified beginning on luteal day 5 and were observed across the remainder of the cycle, including as late as luteal day 14. Onset of pinopod expression corresponded to the mid-luteal rise in serum progesterone concentrations. Conclusions: Apical projections known as pinopods are a common feature of the mid- to late secretory phase endometrium. Whereas they may represent a specialized surface modification important to implantation, as has been suggested, their expression on surface epithelium clearly extends well beyond the putative implantation window. Supported By: The University of North Carolina at Chapel Hill, Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Fertility.

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