Genetic ablation of androgen receptor signaling in fetal Leydig cell lineage affects Leydig cell functions in adult testis

Abstract

It is commonly accepted that androgen-producing fetal Leydig cells (FLC) are substituted by adult Leydig cells (ALC) during perinatal testis development. The mechanisms influencing this process are unclear. We used mice with a retinoid acid receptor 2 promoter-Cre recombinase transgene (Rarb-cre) expressed in embryonic FLC precursors, but not in postnatal testis, and a dual fluorescent Cre recombinase reporter to label FLC and ALC in vivo. All FLC in newborn testis had the recombinant, whereas the majority of LC in adult testis had the nonrecombinant reporter. Primary LC cultures from adult testis had either recombinant (20%) or nonrecombinant (80%) cells, demonstrating that the FLC survive in adult testis and their ontogeny is distinct from ALC. Conditional inactivation of androgen receptor (AR) allele using the Rarb-cre transgene resulted in a 50% increase of AR-negative LC in adult testis. The mutant males became infertile with age, with all LC in older testis showing signs of incomplete differentiation, such as a large number of big lipid droplets, an increase of finger-like protrusions, and a misexpression of steroidogenic or FLC- and ALC-specific genes. We propose that the antiandrogenic exposure during early development may similarly result in an increase of FLC in adult testis, leading to abnormal LC differentiation.

Development, function and fate of fetal Leydig cells

“…3). Although AR is not expressed in FLCs, the mesenchymal cells between and around FLCs strongly expressed AR in the interstitium of fetal testes [10]. These findings thus suggest that testosterone produced by FLCs and fetal SCs in fetal testes target progenitor ALCs through AR, and FLCs regulate the formation of progenitor ALCs via androgen/AR signal pathway.…”

“…3, Hypothesis 2). Studies using genetic mouse assays to trace FLCs from fetal to adult testes specifically have yielded new insights on the FLC fate [10,11,49]. The adrenal 4 binding protein/steroidogenic factor 1 (Ad4BP/SF1), also known as NR5A1, is expressed in both FLCs and ALCs and it is crucial for their development and steroidogenesis [50].…”

“…By using this transgenic mouse assay, the reporter genes are found to express in a small number of Ad4BP/SF-1 and 3β-hydroxysteroid dehydrogenase (3β-HSD) double positive FLCs, and are distributed throughout the interstitium in adult testes [11,49], confirming that FLCs, at least a small population, persist in adult testes. In another study in which a Cre/loxP recombination system was used to activate the expression of LacZ [51] or EGFP [52] reporter in FLCs or their precursors by the retinoic acid receptor isoform 2 gene - Cre transgene ( Rarb - Cre ) [53], FLCs identified by EGFP fluorescence, which marked the EGFP sites activated by the Rarb - Cre , were found to constitute ~20% of the total Leydig cell population in the interstitium of adult testes, illustrating FLCs and/or their precursors are part of the Leydig cell population in adult testis [10]. Collectively, these findings strongly suggest that FLCs indeed are found in adult testes and are integrated into the total Leydig cell population (Fig.…”

“…FLCs differentiate in the fetal testes by embryonic day 12.5 (E12.5) to E13.5 in rodents; after birth, FLCs undergo gradual atrophy, also known as involution or degeneration, and being replaced by ALCs in postnatal 2–3 weeks [7,8]. However, FLC atrophy is not an apoptotic process [9], and ALCs do not originate from FLCs [10,11], thus, the fate of FLCs remains controversial for years based on morphological analysis (for a review, see [7]). Recent studies using FLC specific lineage tracing methods have shown that FLCs persist in adult mouse testes as a subpopulation together with ALCs, constituted about ~20% of the total Leydig cell population [10,11].…”

“…However, FLC atrophy is not an apoptotic process [9], and ALCs do not originate from FLCs [10,11], thus, the fate of FLCs remains controversial for years based on morphological analysis (for a review, see [7]). Recent studies using FLC specific lineage tracing methods have shown that FLCs persist in adult mouse testes as a subpopulation together with ALCs, constituted about ~20% of the total Leydig cell population [10,11]. However, FLCs found in adult testes are HSD17B3 and HSD3B6 negative, and also androgen-independent [11], indicating ALCs are still the only steroidogenic cells capable of producing testosterone in adult testes.…”