Androgenic potential of human fetal adrenals at the end of the first trimester

Abstract

The onset of steroidogenesis in human fetal adrenal glands (HFA) during the first trimester is poorly investigated. An unresolved question is the capacity of the HFA to produce potent androgen DHT via conventional and/or the backdoor pathway(s) at the end of first trimester, when androgen-responsive organs are developed. Our aim was to explore steroidogenesis and the expression of steroidogenic enzymes and transcription factors in HFA at gestational weeks (GW) 9-12 with focus on their androgenic potential. Steroids in the HFA were analyzed by gas chromatography/mass spectrometry. The expression of steroidogenic enzymes and transcription factors in the HFA at GW9-12 was investigated by qPCR, automated Western blotting and immunohistochemistry. We demonstrated that during GW9-12 HFA produced steroids of the ∆⁵, ∆⁴ and the backdoor pathways of the biosynthesis of DHT, though the latter was limited to production of 17α-OH-dihydroprogesterone, androsterone and androstanedione without further conversion to DHT. The only androgens identified in the HFA were testosterone and androsterone, a precursor in the biosynthesis of DHT. We also observed higher levels of CYP17A1 but low expression of 3βHSD2 at GW11-12 in the HFA. Elevated levels of CYP17A1 were associated with an increased expression of SF-1 and GATA-6. Altogether, our data demonstrate that of those steroids analyzed, the only potent androgen directly produced by the HFA at GW9-12 was testosterone. The onset of steroidogenesis in the HFA is a complex process that is regulated by the coordinated action of related transcription factors.

Keywords: androgens; human fetal adrenals; steroid profiles; steroidogenic enzyme expression.

Figure 1

The tissue levels (ng/g) of steroids in the HFA at the end of the first trimester. Concentrations of pregnenolone (5P), 17-OH pregnenolone (17-OHP5), progesterone (P4), 17-OH progesterone (17-OHP4), DHEA, androstenedione (A),17β-testosterone (17β-T), 17α-testosterone (17α-T), androstenediol, androsterone, 17α-OH-dihydroprogesterone (17OHDHP), 5-androstene-3β,17α-diol (epi5-diol), epiandrosterone, androstenedione, etiocholanolone, DHT and 17β-estradiol in the HFA were measured by GC–MS/MS as described in the ‘Materials and methods’ section. The values shown are means ± s.d. for five and seven HFA isolated from individual fetuses at GW9–10 and GW11–12, respectively. **P < 0.01, ***P < 0.001 compared to GW9–10. ND, not detectable.

Figure 2

Levels of mRNAs encoding steroidogenic genes in the HFA during GW9–12. The mRNA levels were normalized to GAPDH as a housekeeping gene. The values presented are means ± s.e. for eight and six HFA at GW9–10 and GW11–12, respectively. *P < 0.05 compared to GW9–10.

Figure 3

Expression of steroidogenic enzyme proteins by the HFA during GW9–12. (A). Protein levels were analyzed by Wes-automated Western blotting and representative blots are shown to the left. To the right, band densities are expressed as a percentage of the corresponding density of GW9. The values shown are means ± s.e. for three (GW9–11) and two (GW12) HFA isolated from individual fetuses. *P < 0.05 compared to GW9. (B) Expression of androgen-metabolizing enzymes in the HFA at GW10 identified by immunohistochemistry. Inserts show the absence of immunostaining in the presence of non-immune serum instead of primary antibody. Scale bar: 100 μm.

Figure 4

Levels of transcription factors involved in the regulation of steroidogenic enzyme expression in the HFA during GW9–12. (A) The mRNA and (B) protein levels were measured by qPCR and Wes-automated Western blotting, respectively. The values presented are means ± s.e. for the same number of samples as indicated in Figs 2 and 3. (A) *P < 0.05 compared to GW9–10, (B) *P < 0.05, **P < 0.01, ***P < 0.001 compared to GW9.