Inhibition of ghrelin signaling improves the reproductive phenotype of male ob/ob mouse  Chu-Chao Zhu, B.Sc., Hua Zhang, M.D., Jin-Shan Zhang, M.D., Zhen Li, M.D., Jie Zhao, B.Sc., Wei Li, M.D., Ph.D., Yuan-Qiang Zhang, M.D.  Fertility and Sterility  Volume 99, Issue 3, Pages 918-926 (March 2013) DOI: 10.1016/j.fertnstert.2012.11.022 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Up-regulation of ghrelin signaling in ob/ob testis. (A) Quantitative analysis of ghrelin and GHS-R1α expression in adult ob/ob and age-matched WT testes by Western blotting (left panel). Densitometric scanning of immunoblots was then performed in which the level of a target protein was normalized against the protein level in WT testes, which was arbitrarily set at 1 (right panel). Each bar represents the mean ± SEM of results from three independent experiments. *P<.05, significantly different by analysis of variance. (B) Distribution of ghrelin and GHS-R1α protein in adult ob/ob and age-matched WT testes was demonstrated by immunohistochemistry. Replacement of the primary antibody with preabsorbed IgG abolished the immunostaining in the tissues, serving as the negative control (NC). Arrows indicate Leydig cells. SC = Sertoli cell; RS = round spermatid; PS = pachytene spermatocyte; ES = elongating/elongated spermatid; Bar = 25 μm. Fertility and Sterility 2013 99, 918-926DOI: (10.1016/j.fertnstert.2012.11.022) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Inhibition of ghrelin/GHS-R1α signaling resulted in improved steroidogenesis in ob/ob testis. (A) Expression levels of the testicular mRNAs encoding several key factors in the steroidogenic route were evaluated by RT-PCR at different time points after D-GHRP treatment. The data are expressed as the mean ± SEM from four independent experiments. Parallel amplification of GAPDH was used as the internal control. (B) PCR products were then quantified by the SYBR green intercalation method as described in Materials and Methods. Results are representative of three independent experiments with similar results. *P<.05 or **P<.01, significantly different when compared with the control group treated with saline. Plasma LH (C) and T (D) concentrations were determined using radioimmunoassay. The asterisks denote significant differences (*P<.05 or **P<.01) when compared with the control group treated with saline. Fertility and Sterility 2013 99, 918-926DOI: (10.1016/j.fertnstert.2012.11.022) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Suppression of ghrelin signaling attenuates germ cell apoptosis in ob/ob testis. (A) Testicular apoptotic status at the end of 3-day, 7-day, or 14-day D-GHRP treatment was assessed by TUNEL staining. Positive cells were indicated using arrows. (B) Quantification of relative apoptotic rate was carried out as described in Materials and Methods. Data are expressed as the mean ± SEM from three independent experiments. Statistically significant differences: *P<.05. (C) Expression level of antiapoptotic molecule bcl-2 and of proapoptotic molecule bax was evaluated by Western blotting. β-actin was used as the loading control. Fertility and Sterility 2013 99, 918-926DOI: (10.1016/j.fertnstert.2012.11.022) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Histological investigations were carried out to determine whether suppression of ghrelin signaling improves testicular morphology in ob/ob testis. (A) Morphological change of adult ob/ob testis after different durations of D-GHRP treatment was assessed by H&E staining. Bar = 25 μm. (B) Abnormal seminiferous tubules showing any sign of arrested spermatogenesis were quantified on the basis of H&E staining and expressed as the percentage of 100 total tubules. Statistically significant differences: *P<.05 or **P<.01 when compared with the control group treated only with saline. (C) Close histological examination of stage of seminiferous epithelium cycle in ob/ob testis was performed using PAS staining after different durations of D-GHRP treatment. PS = pachytene spermatocyte; SC = Sertoli cell; St 1 = stage 1 spermatid; St 2–3 = stage 2–3 spermatid; St 5–6 = stage 5–6 spermatid. (D) The percentage of tubules containing no germ cells beyond meiosis or of tubules containing no spermatid beyond stage 13 in 100 total tubules was calculated. Statistically significant differences: *P<.05 or **P<.01 when compared with the control group treated only with saline. UD = undetectable. Fertility and Sterility 2013 99, 918-926DOI: (10.1016/j.fertnstert.2012.11.022) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions