Infertility with defective spermatogenesis and steroidogenesis in male mice lacking androgen receptor in Leydig cells

Abstract

Androgen and the androgen receptor (AR) have been shown to play critical roles in male fertility. Our previous data demonstrated that mice lacking AR (AR(-/y)) revealed incomplete germ cell development and lowered serum testosterone levels, which resulted in azoospermia and infertility. However, the consequences of AR loss in Leydig cells remain largely unknown. Using a Cre-LoxP conditional knockout strategy, we generated a tissue-specific knockout mouse (L-AR(-/y)) with the AR gene deleted by the anti-Müllerian hormone receptor-2 (Amhr2) promoter driven Cre expressed in Leydig cells. Phenotype analyses show that the outside appearance of L-AR(-/y) mice was indistinguishable from wild type mice (AR(+/y)), but with atrophied testes and epididymis. L-AR(-/y) mice were infertile, with spermatogenic arrest predominately at the round spermatid stage and no sperm could be detected in the epididymis. L-AR(-/y) mice also have lower serum testosterone concentrations and higher serum leuteinizing hormone and follicle-stimulating hormone concentrations than AR(+/y) mice. Further mechanistic studies demonstrated that hypotestosteronemia in L-AR(-/y) mice is not caused by reducing numbers of Leydig cells, but instead by the alterations of several key steroidogenic enzymes, including 17beta-HSD3, 3beta-HSD6, and P450c17. Together, L-AR(-/y) mice provide in vivo evidence that functional AR in Leydig cells is essential to maintain normal spermatogenesis, testosterone production, and required for normal male fertility.

Effects of luteinizing hormone and androgen on the development of rat progenitor Leydig cells in vitro and in vivo

“…The separated cells were filtered through two layers of nylon mesh, centrifuged at 250 g and resuspended in 55% isotonic Percoll. Followed by density gradient centrifugation at 25 000g for 45 min at 4 uC, the progenitor Leydig cell fraction was collected between densities of 1.064 and 1.070 g ml 21 . The cells were washed with Hank's buffered salt solution, centrifuged at 250 g and then resuspended in phenol red-free Dulbecco's modified Eagle medium: Nutrient mixture F-12 (DMEM-Ham's F-12) medium (D-2906; Sigma Chemical Co., St Louis, MO, USA) supplemented with 1 mg ml 21 bovine serum albumin.…”

“…Three replicates of the isolation were performed. The purity of the progenitor Leydig cells was determined by performing immunohistochemical staining of 3bHSD1, using 0.4 mmol l 21 etiocholanolone as substrate and NAD1 as cofactor according to a previously described method. 13 Enrichment of the progenitor Leydig cells was typically up to 95% purity.…”

“…Progenitor Leydig cells obtained from normal 14-day-old rat testes were also purified according to the previously described method. Progenitor Leydig cells were cultured for 48 h in phenol red-free medium (DMEM-Ham's F12) supplemented with 1 mg ml 21 bovine serum albumin, 1 mg ml 21 bovine lipoprotein and 25 m mol l 21 HEPES (pH 7.2) in a 34 uC, 5% O 2 in a 5% CO 2 humidified incubator. In the animal groups that received in vitro hormonal treatment, Leydig cells were first cultured for 24 h in hormone-free medium.…”

“…Although androgen may exert autocrine regulation of progenitor Leydig cell differentiation (Figure 6), we previously demonstrated that androgen did not stimulate progenitor Leydig cell proliferation but stimulated immature Leydig cell proliferation in vitro. 5 Xu et al 21 also showed that androgen receptors expressed on Leydig cells were not required for Leydig cell proliferation because the number of Leydig cells was normal in Leydig cellspecific androgen acceptor knockout (L-AR-/y) mice. Furthermore, our present study also showed that MENT partially maintained progenitor Leydig cell proliferation in vivo (Figure 3), indicating that this action may exert indirectly via other testicular cell types.…”

Therapeutic Potential of Gene Transfer to Testis; Myth or Reality?

“…There is also evidence of Leydig cell impairment in a proportion of men treated with cytotoxic chemotherapy for malignant disease (Howell, 2001). Endocrine disruptors can disrupt not only spermatogenesis but also normal endocrine function of Leydig cells, acting directly on Leydig cells to diminish testosterone production by interfering with the expression of specific genes in the steroidogenic pathway (Skakkebaek, 2001 -1 [134,146,147] are expressed in Leydig cells (Weiss, 1992, Morohashi, 1993Luo, 1994;Xu, 2007;Kojima, 2006c,], which are implicated in steroidogenesis or spermatogenesis. They may also be candidate genes for gene therapy in the future.…”