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

Abstract

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.

Figure 1

RT-PCR (A) and western blot (B) demonstrate that neonatal (D1), pubertal (D30) and adult (D60) gubernacula (Gub), similar to adult epididymis (Epi), contain Ar mRNA (A) and 110 kDa protein (B). Rpl19 and β-actin were used as loading controls for RT-PCR and western blot, respectively. Immunohistochemistry shows that AR protein was localized in the nuclei of gubernacular mesenchymal and cremaster muscle cells (D–F) and epididymal epithelium (C).Representative pictures show one of three mice in each age group. Mag. C–F=×150, Inset in F=×600.

Figure 2

RXFP2 immunostaining in neonatal (D1), premature (D7 and D14), pubertal (D30), and adult (D60) gubernacula of wild-type (+/+) and LhrKO (−/−) mice, and LhrKO mice placed on testosterone replacement therapy (−/−, TRT). Immunohistochemistry shows that RXFP2 protein was localized in gubernacular mesenchymal and cremaster muscle cells. Panel (L) is a procedural control, in which the primary antibody was pre-adsorbed with RXFP2 peptide. All sections were counterstained with Gill’s hematoxylin. Representative pictures show one of three mice in each group. Mag. A–L=×160.

Figure 3

Gubernacular RXFP2 protein levels in 60-day-old animals (n=6) were significantly lower in LhrKO (−/−, *P<0.05) than the age-matched WT (+/+) as determined by western blotting. TRT (−/−, TRT) of LhrKO mice restored RXFP2 to WT levels. Results are the mean±S.E.M.

Figure 4

Effect of DHT on Rxfp2 mRNA levels in organ cultures of gubernacula from 22-day-old WT mice as determined by semi-quantitative RT-PCR. (A) Time-dependent upregulation of Rxfp2 expression by DHT. (B) Flutamide (Flut) blocked the DHTeffect. Results are the mean±S.E.M. for five independent experiments, and the gubernacular tissues used in each experiment were pooled from two to three mice. *P<0.05, **P<0.001 compared with controls.

Figure 5

Effect of a single s.c. injection of testosterone propionate (TP) on gubernacular Rxfp2 mRNA levels at 16 and 24 h in 30-day-old LhrKO mice (n=5) as determined by semi-quantitative RT-PCR. Results are the mean±S.E.M. *P<0.05 compared with control.

Figure 6

Effects of DHT and MA10 cell-conditioned medium (MACM) on the cell proliferation in organ cultures of gubernacula from 30-day-old LhrKO mice. (A) Immunohistochemical detection of BrdU incorporation in control (a and b), DHT (c and d), MACM (e and f), and combined treatment of DHT and MACM (g and h). (B) Quantitative analysis of the percentage of BrdU-positive nuclei in the gubernacular tissues. Results are the mean±S.E.M. for five independent experiments, and the gubernacular tissues used in each experiment were pooled from two to three mice. *P<0.05 compared with control. Mag. a, c, e and g=×160, b, d, f and h=×600.

Figure 7

Effect of relaxin on the cell proliferation in organ cultures of gubernacula from 30-day-old LhrKO mice in the presence of DHT. (A) Immunohistochemical detection of BrdU incorporation in control (a and b), 0.2 (c and d), 1.0 (e and f), 1.5 (g and h), and 2.0 (i and j) μg/ml relaxin. (B) Quantitative analysis of the percentage of BrdU-positive nuclei in the gubernacular tissues. Results are the mean±S.E.M. for five independent experiments, and the gubernacular tissues used in each experiment were pooled from two to three mice. *P<0.05 compared with control. Mag. a, c, e, g and i=×160, b, d, f, h and j=×600.

Figure 8

Effects of DHT and INSL3 on cell proliferation in the primary cultures of mesenchymal cells isolated from the gubernacula of neonatal WT mice. Cell proliferation was determined by the BrdU incorporation using a cell proliferation ELISA kit. Results are the mean±S.E.M. for five independent experiments, and the gubernacular tissues used for the preparation of mesenchymal cells in each experiment were pooled from three mice. *P<0.05 and *P<0.01 compared with DMEM (control).

Figure 9

Effect of Rxfp2 siRNA on cell proliferation in the primary cultures of mesenchymal cells isolated from the gubernacula of neonatal WT mice. Representative pictures of RT-PCR and western blot from three replicates show that Rxfp2 siRNA but not scramble siRNA effectively knocks down Rxfp2 expression (A). Cell proliferation was determined by the BrdU incorporation using a cell proliferation ELISA kit (B). Results are the mean±S.E.M. for five independent experiments, and the gubernacular tissues used for the preparation of mesenchymal cells in each experiment were pooled from three mice. *P<0.01 compared with control.

Figure 10

Effects of daily injections of testosterone propionate (TP) and RXFP2 inhibitor (INSL3i) for 3 days on BrdU incorporation in the gubernacula of 30-day-old LhrKO mice (n=3). Results are the mean ±S.E.M. ^aP<0.01 compared with control; ^bP<0.05 compared with TP/INSL3i.

Figure 11

Effects of DHT and INSL3 on cell differentiation in the primary cultures of mesenchymal cells isolated from the gubernacula of neonatal WT mice. Immunofluorescent staining of a mesenchymal cell marker, CD44 (A–E) and a muscle cell marker, troponin (F–J) showed that the combined treatment of DHT and INSL3 led to a reduction of CD44 (C) and appearance of troponin (H). Addition of either flutamide (Flut) or INSL3i abolished DHT and INSL3 effects (D, E, I and J). The nuclei are labeled with blue color by Hochest dye. Representative pictures show one of three independent experiments, and the gubernacular tissues used for the preparation of mesenchymal cells in each experiment were pooled from three mice. Mag. All=×300.