The thyroid-stimulating hormone receptor (TSHR), activated by either TSH or the newly discovered glycoprotein hormone thyrostimulin, plays a central role in the control of body metabolism. Interestingly, in addition to its thyroid expression, we discovered that the mRNA level of TSHR is periodically regulated in rat ovary by gonadotropins. Ovarian microdissection followed by real-time PCR analysis indicated that granulosa cells show the highest level of TSHR expression. Cultures of follicles and primary granulosa cells demonstrated that the level of TSHR is up-regulated and decreased by the gonadotropindriven cAMP cascade and estradiol production, respectively. Furthermore, in contrast to the negligible expression of TSH in the ovary, we also found by real-time PCR and immunohistochemical analysis that thyrostimulin is expressed mainly in oocytes. Evolving before the appearance of gonadotropins, thyrostimulin is considered the most ancestral glycoprotein hormone. Therefore, the presence of thyrostimulin in the ovary suggests that it may have a primitive function in reproduction when it activates ovarian TSHR. Next, we generated recombinant thyrostimulin protein and characterized its non-covalent heterodimeric nature. Using purified recombinant thyrostimulin, we show that the human ovarian cell line NIH:OVCAR-3 also expresses endogenous and functional TSHR. Using cultured rat granulosa cells isolated from different ovarian stages, we found that treatments with thyrostimulin significantly increase cAMP production and the c-fos gene response in the presence of gonadotropins. Thus, this study demonstrates that oocyte-derived thyrostimulin and granulosa cell-expressed TSHR compose a novel paracrine system in the ovary, where the activity is tightly controlled by gonadotropins.

Leucine-rich repeat containing G protein-coupled receptor 4 (LGR4) promotes the Wnt signaling through interaction with R-spondins or norrin. Using PCR amplification from rat ovarian cDNAs, we identified a naturally occurring Lgr4 splice variant encoding only the ectodomain of Lgr4, which was named Lgr4-ED. Lgr4-ED can be detected as a secreted protein in the extracts from rodent and bovine postnatal gonads, suggesting conservation of Lgr4-ED in mammals. Recombinant Lgr4-ED purified from the conditioned media of transfected 293T cells was found to dose-dependently inhibit the LGR4-mediated Wnt signaling induced by RSPO2 or norrin, suggesting that it is capable of ligand absorption and could have a potential role as an antagonist. Intraperitoneal injection of purified recombinant Lgr4-ED into newborn mice was found to significantly decrease the testicular expression of estrogen receptor alpha and aquaporin 1, which is similar to the phenotype found in Lgr4-null mice. Administration of recombinant Lgr4-ED to superovulated female rats can also decrease the expression of estrogen receptor alpha, aquaporin 1, LH receptor and other key steroidogenic genes as well as bring about the suppression of progesterone production. Thus, these findings suggest that endogenously expressed Lgr4-ED may act as an antagonist molecule and help to fine-tune the R-spondin/norrin-mediated Lgr4-Wnt signaling during gonadal development.