The role of RXFP2 in mediating androgen-induced inguinoscrotal testis descent in LH receptor knockout mice.

LH receptor knockout (LhrKO) male mice exhibit a bilateral cryptorchidism resulting from a developmental defect in the gubernaculum during the inguinoscrotal phase of testis descent, which is corrected by testosterone replacement therapy (TRT). In vivo and in vitro experiments were conducted to investigate the roles of the androgen receptor (AR) and RXFP2 signals in regulation of gubernacular development in LhrKO animals. This study demonstrated that AR and RXFP2 proteins were expressed in the gubernaculum during the entire postnatal period. TRT normalized gubernacular RXFP2 protein levels inLhrKO mice. Organ and primary cell cultures of gubernacula showed that 5alpha-dihydrotestosterone (DHT) upregulated the expression of Rxfp2 which was abolished by the addition of an AR antagonist, flutamide. A single s.c. testosterone injection also led to a significant increase in Rxfp2 mRNA levels in a time-dependent fashion in LhrKO animals. DHT, natural and synthetic insulin-like peptide 3 (INSL3), or relaxin alone did not affect proliferation of gubernacular mesenchymal cells, while co-treatments of DHT with either INSL3 or relaxin resulted in an increase in cell proliferation, and they also enhanced the mesenchymal cell differentiation toward the myogenic pathway, which included a decrease in a mesenchymal cell marker, CD44 and the expression of troponin. These effects were attenuated by the addition of flutamide, siRNA-mediated Rxfp2 knockdown, or by an INSL3 antagonist. Co-administration of an INSL3 antagonist curtailed TRT-induced inguinoscrotal testis descent in LhrKO mice. Our findings indicate that the RXFP2 signaling pathway plays an important role in mediating androgen action to stimulate gubernaculum development during inguinoscrotal testis descent.

Cryptorchidism in LhrKO animals and the effect of testosterone-replacement therapy.

BACKGROUND: The objective of the current study was to characterize the morphological and genetic basis of cryptorchidism. METHODS AND RESULTS: We investigated cryptorchidism in LH receptor (Lhr) knockout (LhrKO) mice and how testosterone-replacement therapy (TRT) worked to correct the phenotype. The results revealed that while gubernacular development was indistinguishable between Lhr-null and wild-type animals until 7 days of age, it was subsequently severely impaired in null animals. This was due to a reduction in mesenchymal cell division, differentiation into cremaster muscle cells and their delayed maturation. While transcript levels of Hoxa10, Hoxa11, Desrt and Dll1 were indistinguishable, the levels of Notch1, Numb and Lgr8 in the gubernaculum and Insl3 in the testes were lower in Lhr-null than in wild-type siblings. The TRT, which completed testicular descent into the scrotum, corrected the morphological changes and the expression of Lgr8, Numb and Notch, but not Insl3, to wild-type levels. Transection of the genitofemoral nerve did not prevent the TRT effect. CONCLUSION: In summary, cryptorchidism in Lhr-null animals was caused by defects in the gubernacular development due to testosterone deficiency. TRT reversed all the morphological and gene expression changes except Insl3, suggesting that testosterone, not INSL3, secreted by Leydig cells, facilitates the completion of testicular descent.