Western analysis using the antiLgr4 antibody showed a specific band around 70 kDa in the bovine follicular fluid. This signal did not overlap with prominent albumin bands as compared to the protein migration positions visualized by Coomassie blue staining, suggesting that the Lgr4 antibody was specific. In homogenates prepared from immature or mature rat ovaries and immature or mature mouse testes, signals of a similar molecular size were also detected. Taken together, these findings suggest that the Lgr4-ED protein is naturally secreted by both types of gonads of various mammals. To determine the testicular cell types that express Lgr4, the testes from mice at 7 days of age were digested and the cell types were further separated using magnetic activated cell sorting based on antibodies against Thy1 antigens OTX015 Epigenetic Reader Domain inhibitor expressed in spermatogonial stem cells. As shown in Fig. 4A, the Lgr4 mRNA is mainly detected in the Thy12 somatic cell-enriched fraction but not in the Thy1 + spermatogonial stem cell-enriched germ cell fraction. To determine the expression of Lgr4 in the ovary, the ovaries were collected from superovulated immature rats primed with PMSG followed by hCG for different intervals. Quantitative realtime PCR analyses showed that the Lgr4 mRNA expression was elevated and remained at high levels 24 h after hCG injection. Subsequent analyses of the Lgr4 transcript in various ovarian SAR131675 VEGFR/PDGFR inhibitor compartments, including granulosa cells, theca shells, cumulus-oocyte complexes and corpora lutea, indicated that Lgr4 is widely expressed in these different ovarian cell types with corpora lutea showing the highest expression level. In addition, immunohistochemical analyses were also carried out to confirm the distribution of the Lgr4 protein. In mouse testes, the Lgr4 positive cells were mainly located around the periphery of the seminiferous tubules. Considering the Lgr4 mRNA profile above and the cell morphology shown in the immunohistochemical staining, this data suggests that the Lgr4 protein is probably expressed in peritubular myoid cells but not in spermatogonia or Sertoli cells.