The small heterodimer partner is a gonadal gatekeeper of sexual maturation in male mice

Abstract

The small heterodimer partner (SHP) is an atypical nuclear receptor known mainly for its role in bile acid homeostasis in the enterohepatic tract. We explore here the role of SHP in the testis. SHP is expressed in the interstitial compartment of the adult testes, which contain the Leydig cells. SHP there inhibits the expression of steroidogenic genes, on the one hand by inhibiting the expression of the nuclear receptors steroidogenic factor-1 and liver receptor homolog-1 (lrh-1), and on the other hand by directly repressing the transcriptional activity of LRH-1. Consequently, in SHP knockout mice, testicular testosterone synthesis is increased independently of the hypothalamus-pituitary axis. Independent of its action on androgen synthesis, SHP also determines the timing of germ cell differentiation by controlling testicular retinoic acid metabolism. Through the inhibition of the transcriptional activity of retinoic acid receptors, SHP controls the expression of stimulated by retinoic acid gene 8 (stra8) - a gene that is indispensable for germ cell meiosis and differentiation. Together, our data demonstrate new roles for SHP in testicular androgen and retinoic acid metabolism, making SHP a testicular gatekeeper of the timing of male sexual maturation.

Figure 1.

SHP deficiency leads to hypertestosteronemia. (A) Plasma testosterone levels in SHP^L2/L2 and SHP^L−/L− mice (n = 10–15 per group). (B) Testicular mRNA expression of star, cyp11a1, 3β-hsd, cyp17, sf-1, and lrh-1 in whole testis of SHP^L2/L2 and SHP^L−/L− mice. (C) Weights of liver, spleen, testis, epididymides, and seminal vesicles normalized to body weight in SHP^L2/L2 and SHP^L−/L− mice. (*) p < 0.05. (D) Plasma LH and pituitary mRNA expression of lhb in SHP^L2/L2 and SHP^L−/L− mice. (*) p < 0.05. (E) Quantitative RT–PCR analysis of lhcgr, fshr, lrh-1, shp, sf-1, and fxrα in extracts of liver, adrenals, whole testis, and laser-microdissected interstitial or tubular compartments of testis of 12-wk-old C57BL/6J mice (n = 3). (*) p < 0.05.

Figure 2.

LRH-1^+/− and SHP^L−/L− mice show opposite testicular phenotypes. (A) Plasma testosterone concentrations in LRH-1^+/+ and LRH-1^+/− mice (n = 10–15 per group). (*) p < 0.05. (B) Plasma LH and pituitary mRNA expression of lhb in LRH-1^+/+ and LRH-1^+/− mice. (C) Testicular mRNA expression of lrh-1, star, cyp11a1, 3β-hsd, and cyp17 in whole testis of LRH-1^+/+ and LRH-1^+/− mice. (*) p < 0.05. (D) Weights of liver, spleen, testis, epididymides, and seminal vesicles normalized to body weight in LRH-1^+/+ and LRH-1^+/− mice. (*) p < 0.05. (E) Transient transfection assay of COS-1 cells cotransfected with a mouse star promoter luciferase reporter plasmid together with either empty pCMX or the pCMX-mLRH-1 expression plasmid. An increasing dose of the pCMX-mSHP expression plasmid was cotransfected (0–40 ng). Normalized luciferase activity was expressed as relative light units (RLU) of triplicate assays (mean ± SD). (*) p < 0.05. (F) ChIP assay performed to detect LRH-1 on genomic sequences of the star and cyp11a1 genes in TM3 Leydig cells transfected either with pCMX or pCMX-LRH-1 using an anti-LRH-1 antibody. A sequence of ±100 bp covering either the LRH-1-RE (A) or a sequence 4.0–2.4 kb upstream of the LRH-1-RE (B) was amplified. Results are expressed as fold enrichment over cells transfected with pCMX and represent amplification variability (n = 4). (G) ChIP to detect SHP on genomic DNA isolated from TM3 Leydig cells transfected either with pCMX and pCMV2, pCMX and pCMV2-Flag-SHP or pCMX-LRH-1 and pCMV2-Flag-SHP using an anti-Flag antibody. DNA amplification of the sequences of the star and cyp11a1 genes was performed as described in F.

Figure 3.

Role of FXRα in testicular testosterone production. (A) FXRα^+/+ and FXRα^−/− mice (n = 4–5 per group) were injected with GW4064 (50 mg/kg, intraperitoneally) or vehicle (DMSO) for 6 h. Testicular SHP mRNA expression was analyzed by quantitative RT–PCR. (B) Plasma testosterone levels in FXRα^+/+ and FXRα^−/− mice (n = 5). (C) Testicular mRNA expression of shp, star, cyp11a1, 3β-hsd, and cyp17 in whole testis of SHP^L2/L2 and SHP^L−/L− mice injected with GW4064 (50 mg/kg, intraperitoneally) or vehicle (DMSO) for 6 or 12 h (n = 6). All data are expressed as relative values with the nontreated samples assigned a value of 1. (*) p < 0.05. (D) Plasma testosterone levels in SHP^L2/L2 and SHP^L−/L− mice treated with GW4064 for 12 h (n = 6). (*) p < 0.05.

Figure 4.

Early fertility and reproductive tract maturation in male SHP^L−/L− mice. (A) Weights of testis (T), epididymides (E), and seminal vesicles (SV) normalized for body weight in SHP^L2/L2 and SHP^L−/L− mice (n = 6). (*) p < 0.05. (B) Expression of shp, lhcgr, sf-1, and lrh-1 mRNA in the interstitial testicular compartment in samples obtained at different stages of the postnatal development by LMD (n = 3). Asterisk denotes difference from previous time point (p < 0.05). (C) Plasma testosterone concentrations from C57BL/6J mice between 25 and 56 d (n = 6). (D) Testicular mRNA expression of lhcgr, star, and cyp11a1 in whole testis of SHP^L2/L2 and SHP^L−/L− mice during postnatal development (n = 6). Asterisk denotes significant difference between genotypes (p < 0.05), and number sign (#) denotes difference in the same genotype with the previous time point. (E) Two representative micrographs of eosin-hematoxylin-stained testis of 28-d-old SHP^L2/L2 and SHP^L−/L−. Arrows indicate elongated spermatids. (F) Testes from 20- to 56-d-old SHP^L2/L2 and SHP^L−/L− mice were collected, processed for histology, and stained with eosin-hematoxylin. For each testis, the number of seminiferous tubules showing elongated spermatids were counted, and the results are expressed as the number of positive tubules per 100 tubules (n = 6). (*) p < 0.05. (G) Mean mRNA expression of shp in seminiferous tubular cells of the testes of C57BL/6J mice of different ages, purified by LMD. (*) p < 0.05.

Figure 5.

Earlier germ cell differentiation in male SHP^L−/L− mice. (A) Testicular mRNA expression of cyclin a1, cyclin b2, cyclin a2, cyclin d2, stard6, and gcnf in whole testis of 8-, 11-, 15-, 20-, 25-, 28-, and 30-d-old SHP^L2/L2 and SHP^L−/L− mice (n = 6). (*) p < 0.05. (B) Testicular mRNA expression of rarα, rarβ, rarγ, cyp26b1, stra8, scp3, cdx1, hoxa1, and dmc1 in whole testis of 8-, 11- and 15-d-old SHP^L2/L2 and SHP^L−/L− mice (n = 6). (*) p < 0.05.

Figure 6.

Lack of SHP in the tubular testis compartment alters expression of genes involved in RA signaling. (A) Intratesticular concentrations of all-trans RA, all-trans retinol, and all-trans retinylester in 9-d-old SHP^L2/L2 and SHP^L−/L− mice. Each sample represents a pool of the testis of 20 mice. (B) mRNA expression of shp, stra8, hoxa1, and cdx1 in F9 cells transfected with increasing amounts of pCMX-SHP (0–1 μg) (n = 3). Asterisk denotes difference from the previous amount of transfected pCMX-SHP (p < 0.05), and number sign (#) denotes difference between vehicle and atRA-treated cells. (C) ChIP of cross-linked DNA for RARγ from F9-RARγ^+/+ and F9-RARγ^−/− using an anti-RARγ antibody. A sequence of ±100 bp covering either the RARE (A) or a sequence 4.0–2.4 kb upstream of the RARE (B) was amplified. Results are expressed as fold enrichment over F9-RARγ^−/− cells and represent amplification variability (n = 4). (D) ChIP from F9-RARγ^+/+ transfected with either pCMV2 or pCMV2-Flag-SHP using an anti-Flag antibody. DNA amplification and expression of results were as in C. (*) p < 0.05. (E) mRNA expression of stra8, dmc1, and scp3 in whole testis of SHP^L2/L2 and SHP^L−/L− mice injected with GW4064 (50 mg/kg, intraperitoneally) or vehicle (DMSO) for 12 h (n = 6). (*) p < 0.05.

Figure 7.

Proposed model for the role of SHP in the onset of fertility. Our results indicate that SHP regulates the timing of fertility in the male via its repressive actions on testicular testosterone biosynthesis and on the entry of germ cells into meiosis. As shp expression in the seminiferous tubules increases during the late prepubertal period, its absence leads to the increased expression of meiosis-inducing genes like stra8. This effect is in part explained by the higher testicular RA levels and in part by the lack of the repressive activity of SHP on RARs. The increase in testicular RA could be explained by the decreased expression of the RA-degrading enzyme cyp26b1. SHP expression also increases in the interstitial cells during the prepubertal period, with its absence leading to a precocious increase in testosterone biosynthesis. SHP inhibits testicular steroidogenesis on the one hand by inhibiting the expression of sf-1 and lrh-1 that control the expression of the steroidogenic genes, and on the other hand by repressing the transcriptional activity of LRH-1.