Padmasana Singh, Ph. D. , Amitabh Krishna, Ph. D

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Effects of gonadotropin-inhibitory hormone on folliculogenesis and steroidogenesis of cyclic mice Padmasana Singh, Ph.D., Amitabh Krishna, Ph.D., Kazuyoshi Tsutsui, Ph.D. Fertility and Sterility Volume 95, Issue 4, Pages 1397-1404 (March 2011) DOI: 10.1016/j.fertnstert.2010.03.052 Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Transverse sections of the ovaries (stained with hematoxylin-eosin) of cyclic mice showing histological changes following administration of gonadotropin-inhibitory hormone (GnIH). Ovary of control mouse: (A) section of ovary of vehicle-treated normal control mouse showing antral follicle containing healthy granulosa cells (GC) and oocyte; (B) section of ovary of vehicle-treated normal control mouse showing healthy corpus luteum (CL) having normal luteal cells. Ovary of mouse treated with 100 ng/d GnIH: (C) normal healthy antral follicle; (D) note the presence of subnormal corpus luteum (CL). Ovary of mouse treated with 500 ng/d GnIH: (E) section of ovary showing abnormal follicle having condensed (black arrows) and hypertrophied (white arrows) nuclei of granulosa cells; (F) regressed corpus luteum (CL) having loosely arranged (white arrows) and vacuolated (black arrows) luteal cells, Ovary of mouse treated with 2 μg/d GnIH: (G) section of ovary showing abnormal follicle having condensed and hypertrophied granulosa cells and abnormal oocyte (Oo); (H) high magnification of the abnormal follicle showing condensed (black arrows) nuclei of the granulosa cells (GC); (I) hypertrophied and vacuolated theca cell (Tc); (J) regressed corpus luteum (CL) having loosely arranged (white arrows) and vacuolated luteal cells (black arrows). Fertility and Sterility 2011 95, 1397-1404DOI: (10.1016/j.fertnstert.2010.03.052) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effect of in vivo treatment of different doses of gonadotropin-inhibitory hormone (GnIH) on circulating (A) P4 and (B) E2 concentration (n = 6) and (C) Western blot analyses of LH receptor (LH-R), (D) steroidogenic acute regulatory (StAR), and (E) 3β-hydroxysteroid dehydrogenase (3β-HSD) proteins in the ovaries of mice. Densitometric analyses of the blots are shown in bar graphs (n = 3). Mice treated with normal saline solution served as control. Values are mean ± SEM. *P<.05 versus control. **P<.01 versus control. Fertility and Sterility 2011 95, 1397-1404DOI: (10.1016/j.fertnstert.2010.03.052) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Western blot analyses of different doses of gonadotropin-inhibitory hormone (GnIH) treatment in vivo, (A) on caspase-3 and (B) poly–adenosine diphosphate–ribose polymerase (PARP) proteins in the ovaries of cyclic mice. Mice treated with normal saline served as control. Values are mean ± SEM obtained from three immunoblots. *P<.05 versus control. **P<.01 versus control. #P<.01 versus control. Fertility and Sterility 2011 95, 1397-1404DOI: (10.1016/j.fertnstert.2010.03.052) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Effect of in vitro treatment of different doses of gonadotropin-inhibitory hormone (GnIH) on (A) ovarian progesterone synthesis and (B) Western blot analyses of steroidogenic acute regulatory (StAR), and (C) 3β-hydroxysteroid dehydrogenase (3β-HSD) proteins in the ovaries of mice. Densitometric analyses of the blots are shown in bar graphs (n = 3). Values are mean ± SEM. **P<.01 versus control. Fertility and Sterility 2011 95, 1397-1404DOI: (10.1016/j.fertnstert.2010.03.052) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions