Male fetal germ cells are targets for androgens that physiologically inhibit their proliferation

Abstract

In adulthood, the action of androgens on seminiferous tubules is essential for full quantitatively normal spermatogenesis and fertility. In contrast, their role in the fetal testis, and particularly in fetal germ cell development, remains largely unknown. Using testicular feminized (Tfm) mice, we investigated the effects of a lack of functional androgen receptor (AR) on fetal germ cells, also named gonocytes. We demonstrated that endogenous androgens/AR physiologically control normal gonocyte proliferation. We observed an increase in the number of gonocytes at 17.5 days postconception resulting from an increase in proliferative activity in Tfm mice. In a reciprocal manner, gonocyte proliferation is decreased by the addition of DHT in fetal testis organotypic culture. Furthermore, the AR coregulator Hsp90alpha (mRNA and protein) specifically expressed in gonocytes was down-regulated in Tfm mice at 15.5 days postconception. To investigate whether these effects could result from direct action of androgens on gonocytes, we collected pure gonocyte preparations and detected AR transcripts therein. We used an original model harboring a reporter gene that specifically reflects AR activity by androgens and clearly demonstrated the presence of a functional AR protein in fetal germ cells. These data provide in vivo and in vitro evidence of a new control of endogenous androgens on gonocytes identified as direct target cells for androgens. Finally, our results focus on a new pathway in the fetal testis during the embryonic period, which is the most sensitive to antiandrogenic endocrine disruptors.

Fig. 1.

Morphometric analysis of 17.5 dpc fetal testes. (A) Histologic appearance of the testis in control and testicular feminized (Tfm) fetuses. Sertoli cells (arrows) were immunostained with anti-mullerian hormone and located at the periphery of the seminiferous cord, whereas gonocytes (arrowheads) were the large unstained cells in the center of the cord. (Scale bars, 10 μm.) (B and C) Testicular volume (B) and number of gonocytes per testis (C) in Tfm mice and their normal littermates. All results are shown as the mean ± SEM; n = 5. ∗, P < 0.05 in Student's t test.

Fig. 2.

Effect of androgen receptor deficiency and dihydrotestosterone treatment on gonocyte proliferation. Pregnant females (15.5 or 16.5 dpc) were injected with BrdU 3 h before being killed. (A and B) The percentages of BrdU-positive gonocytes (A) and gonocyte mitotic figures (B) were determined from at least 1,000 counted gonocytes. (C) Testes were explanted at 13.5 dpc and treated (or not) for 30 h with dihydrotestosterone (10⁻⁶ M). The percentages of BrdU-positive gonocytes were determined from at least 1,000 counted gonocytes. Values are means ± SEM; ∗, P < 0.05 in Student's t test (A and B; n = 5) or in a paired Student's t test (C; n = 7).

Fig. 3.

Effect of androgen receptor deficiency on heat shock protein 90α (Hsp90α) and mRNA levels in 15.5-dpc testes. (A) immunohistochemical staining of Hsp90α in control and testicular feminized (Tfm) testes. (Scale bars, 10 μm.) (B and C) Oct-4 (B) and Hsp90α (C) mRNA expression in 15.5-dpc testis from control and Tfm fetuses was measured by Q-PCR using β-actin or Oct-4, respectively, as reference. Results are expressed in relative units with the control animals having a value of 1. n = 5; ∗, P < 0.05; ∗∗, P < 0.01 in Student's t test.

Fig. 4.

Detection of androgen receptor mRNA in testicular gonocytes at 15.5 dpc. mRNA expression of androgen receptor, Oct-4, AMH, P75, and 3β-HSD was examined by RT-PCR analysis in whole testicular tissue (T) and in magnetic-activated cell sorting isolated gonocytes (G). Results shown are representative of two independent experiments. −RT, control for genomic DNA contamination using Oct-4 primers on isolated gonocytes mRNA extracts.

Fig. 5.

Cell-specific expression of androgen receptor protein in 15.5-dpc testis. (A) Immunohistochemical staining of androgen receptor in control and testicular feminized (Tfm) testes. The arrowhead indicates an example of a stained gonocyte. No signal was detected in Tfm testes. (Scale bars, 10 μm.) (B) Enriched gonocyte cultures were transfected with the ARE₂-TATA-GFP-NLS vector and cultivated with or without dihydrotestosterone (DHT) (10⁻⁶ M) for 30 h. GCNA1, a gonocyte marker, and GFP were detected by immunostaining (red and green, respectively). Green gonocytes were present only after DHT treatment indicating the presence of a functional androgen receptor protein. (C) Five-day-old Sertoli cell cultures served as positive control. Cells were transfected with ARE₂-TATA-GFP-NLS vector and cultivated without or with DHT (10⁻⁶ M). Anti-mullerian hormone, a cytoplasmic marker of Sertoli cells, and GFP were detected by immunostaining (red and green, respectively). As expected, green Sertoli cells were present only after DHT treatment indicating the presence of a functional androgen receptor protein. All of the slides in B and C were counterstained with 4′-6 diaminido-2-phenylindole (DAPI) to visualize nuclei (blue). (Scale bars, 10 μm.)