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Letrozole increases growth of rat theca-interstitial cells and CYP17A1 gene expression in the rat ovary
FEMALE REPRODUCTIVE ENDOCRINOLOGY P-16 Tuesday, October 18, 2011 EFFECT OF RESVERATROL ON PROLIFERATION AND STEROIDOGENESIS OF RAT OVARIAN THECA-INTERSTITIAL CELLS. I. Ortega, D. H. Wong, A. B. Cress, A. Sokalska, S. D. Stanley, A. J. Duleba. Department of Obstetrics and Gynecology, University of California, Davis, Davis, CA; IVI Madrid, Madrid, Spain; Department of Gynecology, Obstetrics and Gynecological Oncology, Karol Marcinkowski, Poznan, Poland; Department of Molecular Biosciences, University of California, Davis, Davis, CA. OBJECTIVE: Previously, we reported that the bioflavonoid resveratrol (RES), inhibits proliferation of rat theca-interstitial cells. This study evaluated the mechanisms of actions of RES on rat theca-interstitial cell proliferation and steroidogenesis. Since actions of RES may be due to inhibition of the mevalonate pathway, we investigated whether effects of RES were related to a decrease of isoprenylation substrates: farnesyl-pyrophosphate (FPP) and geranylgeranyl-pyrophosphate (GGPP). DESIGN: In-vitro study. MATERIALS AND METHODS: Rat theca-interstitial cells were cultured for up to 48 hrs in the absence (control) or presence of RES (10-30mM), GGPP (30mM) and/or FPP (30mM). Proliferation was determined by thymidine incorporation assay. Expression of key genes regulating steroidogenesis (StAR, CYP11A1, CYP17A1) was evaluated by qrt-PCR. Androstenedione and androsterone levels were determined by LC-MS. Statistical analysis was performed by ANOVA followed by the Tukey test. RESULTS: RES (30mM) inhibited cell proliferation by 56% (P<0.001). FPP and GGPP partially restored proliferation, by 32% and 42% (P<0.001) respectively, above the level of proliferation observed in the presence of RES alone. RES decreased androgen levels in culture media in concentration-dependent fashion: androstenedione by 78-89% (P<0.001) and androsterone by 74-89% (P<0.001). Furthermore, RES decreased CYP11A1 and CYP17A1 gene expression, respectively, by 38-46% (P<0.001) and 73-86% (P<0.001). RES (30mM) induced a modest increase of StAR expression by 19% (P<0.05). GGPP and FPP did not alter the inhibitory effect of RES on CYP17A1 gene expression. CONCLUSION: RES decreases proliferation of theca-interstitial cells, at least in part, by reducing isoprenylation. In contrast, RES inhibits androgen production by selectively decreasing expression of CYP11A1 and CYP17A1 by mechanisms independent of isoprenylation. We hypothesize that RES may reduce hyperandrogenism in conditions such as PCOS. Supported by: Eunice Kennedy Shriver NICHD Grant RO1 HD050656. P-17 Tuesday, October 18, 2011 LETROZOLE INCREASES GROWTH OF RAT THECA-INTERSTITIAL CELLS AND CYP17A1 GENE EXPRESSION IN THE RAT OVARY. I. Ortega, E. Stener-Victorin, J. A. Villanueva, A. Sokalska, S. D. Stanley, A. J. Duleba. Department of Obstetrics and Gynecology, University of California, Davis, Davis, CA; IVI Madrid, Madrid, Spain; Department of Physiology/Endocrinology, Institute of Neuroscience and Physiology, Gothenburg, Sweden; Department of Gynecology, Obstetrics and Gynecological Oncology, Karol Marcinkowski University of Medical Sciences, Poznan, Poland; Department of Molecular Biosciences, University of California, Davis, Davis, CA. OBJECTIVE: Letrozole (LET) is a non-steroidal aromatase inhibitor, blocking the conversion of androgens to estrogens in granulosa cells. However, little is known regarding the effect of LET on theca-interstitial cell function. This study evaluated the effect of LET in vivo and in cultures of theca-interstitial cells. DESIGN: In-vitro and in-vivo studies. MATERIALS AND METHODS: Effects of LET were studied in intact rats receiving either LET (90-day-continuous-release sc pellets, 400 mg/day) or placebo pellets (control group). After 10 weeks, the animals were sacrificed, ovaries were isolated and blood samples collected. In vitro experiments evaluated theca-interstitial cells isolated from 30 day-old female rats and cultured for up to 48 hrs in the absence (control) or in the presence of LET (0.1-1mM). Cell proliferation was evaluated by quantification of DNA synthesis as determined by radiolabelled thymidine incorporation assay. CYP17A1 gene expression was evaluated by qrt-PCR using HPRT as a reference gene. Androstenedione levels in plasma were determined by liquid chromatography-mass spectrometry. Comparisons between groups were performed by ANOVA followed by post-hoc pairwise testing.
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Abstracts
RESULTS: LET, in vivo, induced an increase in ovarian size compared to the control group: 179.1 9.46 mg vs. 115 5.19 mg (P<0.05). LET also induced a 57-fold (P<0.001) increase in CYP17A1 gene expression and a 52-fold (P<0.001) increase in plasma androstenedione level. In vitro, LET stimulated theca-interstitial cell proliferation in a concentrationdependent fashion by up to 65% (P<0.01) at the highest concentration (1 mM). CONCLUSION: LET stimulates growth of the theca-interstitial cell compartment and increases expression of the key enzyme (CYP17A1) regulating the androgen biosynthesis pathway in the rat. The present findings reveal novel mechanisms of action of LET in the rat ovary. Supported by: Eunice Kennedy Shriver NICHD Grant RO1 HD050656.
P-18 Tuesday, October 18, 2011 EFFECTS OF RESVERATROL ON RAT OVARIAN GRANULOSA CELLS. I. Ortega, D. H. Wong, A. B. Cress, J. A. Villanueva, S. D. Stanley, A. J. Duleba. Department of Obstetrics and Gynecology, University of California, Davis, Davis, CA; IVI Madrid, Madrid, Spain; Department of Molecular Sciences, University of California, Davis, Davis, CA. OBJECTIVE: Polycystic ovary syndrome (PCOS) is associated with theca-interstitial hyperplasia and impaired folliculogenesis. Previously, we reported that resveratrol (RES), a natural polyphenol with anti-proliferative and anti-oxidant properties, reduces proliferation and induces apoptosis in rat theca-interstitial cells. The present study evaluated the effect of RES on growth and function of granulosa cells. DESIGN: In-vitro study. MATERIALS AND METHODS: Rat granulosa cells were cultured for up to 48 hrs without (control) or with RES (10-50mM). DNA synthesis was determined by thymidine incorporation assay. The total viable cell number was estimated by MTS assay while apoptosis was determined by Caspase-3/7 activity and TUNEL. Cell morphology was evaluated by DAPI/F-actin staining. Expression of AMH, VEGF and aromatase (CYP19) was evaluated by qrt-PCR. Estrogen levels were determined by LC-MS. Comparisons between groups were performed using ANOVA and Tukey test. RESULTS: RES induced a biphasic effect on DNA synthesis, whereby a lower dose (10 mM) stimulated thymidine incorporation by 54% (P<0.05), and a higher dose (30mM) decreased cell proliferation by 49% (P<0.05). RES (10mM) induced a slight increase of the cell number by 19% (P<0.05). At this dose, RES protected granulosa cells from caspase3/7 activation by 11-14% (P<0.05). RES had no effect on DNA fragmentation or granulosa cell morphology. RES decreased CYP19 gene expression and estrogen levels in a dose-dependent manner, respectively, by 29-45% (P<0.001) and 56-71% (P<0.001). RES also decreased VEGF expression by 42-18% (P<0.001), but induced a 3.4-fold (P<0.05) increase in AMH gene expression. CONCLUSION: RES induces cytostatic, but not cytotoxic effects and promotes an undifferentiated phenotype of granulosa cells. We hypothesize that RES may alter the ratio of theca-to-granulosa cells and hence may be of potential therapeutic use in conditions associated with theca-interstitial hyperplasia, such as that seen in PCOS. Supported by: Eunice Kennedy Shriver NICHD Grant RO1 HD050656.
P-19 Tuesday, October 18, 2011 TESTOSTERONE INHIBITS SUBCUTANEOUS ABDOMINAL ADIPOGENESIS DURING ADIPOSE STEM CELL DIFFERENTIATION TO PREADIPOCYTES. G. Chazenbalk, D. Irge, A. Shah, D. A. Dumesic. Department of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA, Los Angeles, CA; Department of Obstetrics and Gynecology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel. OBJECTIVE: Androgen affects body fat distribution through subcutaneous (SC) abdominal adipogenesis, defined by events whereby adipose stem cells (ASCs) become preadipocytes (early stage of cell differentiation), followed by preadipocyte differentiation to adipocytes (late stage of cell differentiation). In female conditions of androgen excess, such as PCOS, abnormal SC abdominal adipocyte size and lipolysis accompany insulin resistance in nonobese individuals. The present study examines the in vitro effects of testosterone (T) at early and late stages of adipogenesis in SC abdominal adipose of nonobese women. DESIGN: Prospective.
Vol. 96., No. 3, Supplement, Sepetmber 2011
S114
Abstracts
RESULTS: LET, in vivo, induced an increase in ovarian size compared to the control group: 179.1 9.46 mg vs. 115 5.19 mg (P<0.05). LET also induced a 57-fold (P<0.001) increase in CYP17A1 gene expression and a 52-fold (P<0.001) increase in plasma androstenedione level. In vitro, LET stimulated theca-interstitial cell proliferation in a concentrationdependent fashion by up to 65% (P<0.01) at the highest concentration (1 mM). CONCLUSION: LET stimulates growth of the theca-interstitial cell compartment and increases expression of the key enzyme (CYP17A1) regulating the androgen biosynthesis pathway in the rat. The present findings reveal novel mechanisms of action of LET in the rat ovary. Supported by: Eunice Kennedy Shriver NICHD Grant RO1 HD050656.
P-18 Tuesday, October 18, 2011 EFFECTS OF RESVERATROL ON RAT OVARIAN GRANULOSA CELLS. I. Ortega, D. H. Wong, A. B. Cress, J. A. Villanueva, S. D. Stanley, A. J. Duleba. Department of Obstetrics and Gynecology, University of California, Davis, Davis, CA; IVI Madrid, Madrid, Spain; Department of Molecular Sciences, University of California, Davis, Davis, CA. OBJECTIVE: Polycystic ovary syndrome (PCOS) is associated with theca-interstitial hyperplasia and impaired folliculogenesis. Previously, we reported that resveratrol (RES), a natural polyphenol with anti-proliferative and anti-oxidant properties, reduces proliferation and induces apoptosis in rat theca-interstitial cells. The present study evaluated the effect of RES on growth and function of granulosa cells. DESIGN: In-vitro study. MATERIALS AND METHODS: Rat granulosa cells were cultured for up to 48 hrs without (control) or with RES (10-50mM). DNA synthesis was determined by thymidine incorporation assay. The total viable cell number was estimated by MTS assay while apoptosis was determined by Caspase-3/7 activity and TUNEL. Cell morphology was evaluated by DAPI/F-actin staining. Expression of AMH, VEGF and aromatase (CYP19) was evaluated by qrt-PCR. Estrogen levels were determined by LC-MS. Comparisons between groups were performed using ANOVA and Tukey test. RESULTS: RES induced a biphasic effect on DNA synthesis, whereby a lower dose (10 mM) stimulated thymidine incorporation by 54% (P<0.05), and a higher dose (30mM) decreased cell proliferation by 49% (P<0.05). RES (10mM) induced a slight increase of the cell number by 19% (P<0.05). At this dose, RES protected granulosa cells from caspase3/7 activation by 11-14% (P<0.05). RES had no effect on DNA fragmentation or granulosa cell morphology. RES decreased CYP19 gene expression and estrogen levels in a dose-dependent manner, respectively, by 29-45% (P<0.001) and 56-71% (P<0.001). RES also decreased VEGF expression by 42-18% (P<0.001), but induced a 3.4-fold (P<0.05) increase in AMH gene expression. CONCLUSION: RES induces cytostatic, but not cytotoxic effects and promotes an undifferentiated phenotype of granulosa cells. We hypothesize that RES may alter the ratio of theca-to-granulosa cells and hence may be of potential therapeutic use in conditions associated with theca-interstitial hyperplasia, such as that seen in PCOS. Supported by: Eunice Kennedy Shriver NICHD Grant RO1 HD050656.
P-19 Tuesday, October 18, 2011 TESTOSTERONE INHIBITS SUBCUTANEOUS ABDOMINAL ADIPOGENESIS DURING ADIPOSE STEM CELL DIFFERENTIATION TO PREADIPOCYTES. G. Chazenbalk, D. Irge, A. Shah, D. A. Dumesic. Department of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA, Los Angeles, CA; Department of Obstetrics and Gynecology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel. OBJECTIVE: Androgen affects body fat distribution through subcutaneous (SC) abdominal adipogenesis, defined by events whereby adipose stem cells (ASCs) become preadipocytes (early stage of cell differentiation), followed by preadipocyte differentiation to adipocytes (late stage of cell differentiation). In female conditions of androgen excess, such as PCOS, abnormal SC abdominal adipocyte size and lipolysis accompany insulin resistance in nonobese individuals. The present study examines the in vitro effects of testosterone (T) at early and late stages of adipogenesis in SC abdominal adipose of nonobese women. DESIGN: Prospective.
Vol. 96., No. 3, Supplement, Sepetmber 2011