Characterization of Human Adrenal Steroidogenesis During Fetal Development

Abstract

Context: The endocrine function of human fetal adrenals (HFAs) is activated already during first trimester, but adrenal steroidogenesis during fetal life is not well characterized.

Objective: This study aimed to investigate HFA steroidogenesis by analyzing adrenal glands from first and second trimesters.

Design and setting: Male and female HFA from gestational weeks (GWs) 8 to 19 were examined, including a total of 101 samples from 83 fetuses.

Main outcome measure(s): Expression level of steroidogenic genes and protein expression/localization were determined by quantitative PCR and immunohistochemistry, respectively, and intra-adrenal steroid levels were quantified by LC-MS/MS.

Results: Transcriptional levels of StAR, CYP11A1, CYP17A1, CYP21A2, CYP11B1/2, and SULT2A1 were significantly higher in second trimester compared to first trimester (P < 0.05), whereas expression levels of 3β-HSD2 and ARK1C3 were unaltered between GWs 8 and 19. All investigated steroidogenic proteins were expressed in a distinct pattern throughout the investigated period, with most enzymes expressed primarily in the fetal zone, except 3β-HSD1/2, which was expressed mainly in the definitive zone. Abundant steroidogenic enzyme expression was reflected in overall high intra-adrenal tissue concentrations of mineralocorticoids, glucocorticoids, and androgens; cortisol was the most abundant (1071 to 2723 ng/g tissue), and testosterone levels were the lowest (2 to 14 ng/g tissue).

Conclusions: The expression profiles of HFA steroidogenic enzymes are distinct from first to second trimester, with no major differences between male and female samples. Intra-adrenal steroid hormone concentrations confirm that cortisol is produced throughout first and second trimesters, suggesting continued regulation of the hypothalamus-pituitary-adrenal axis during this entire period.

Figure 1.

Gene expression level of human fetal adrenal steroidogenic enzymes during the first and second trimesters. (a‒i) Quantitative reverse transcription polymerase chain reaction analysis of a range of steroidogenic-associated enzymes and receptors in male and female human fetal adrenal samples divided into four age groups: GWs 8 to 9, GWs 10 to 12, GWs 14 to 16, and GWs 17 to 19. Expression is relative to the reference gene RPS20. The expression level is set to 1 in male GWs 8 to 9 samples. In total, 39 adrenal samples were used. Bars represent mean ± SEM with individual data points shown as blue triangles and red circles for male and female samples, respectively; n = 3 to 6. Differences in age compared with male GWs 8 to 9 are indicated as significantly different: *P < 0.05; **P < 0.01. (j) Overall human fetal adrenal transcript levels from first- and second-trimester samples; n = 39. Data points represent −ΔCt values (relative to the housekeeping gene RPS20) of investigated gene transcript levels in individual adrenal samples (male and female). Error bars represent mean ± SD. ARK1C3, aldo-keto reductase family 1 member C3.

Figure 2.

Human fetal adrenal gland morphology and zonation. Schematic illustrations of the distinct morphological zonation in (a) first-trimester human fetal adrenals (GWs 8 to 12) and (b) second-trimester human fetal adrenals (GWs 14 to 19) viewed as a cross section of the gland. (c) Hematoxylin and eosin (HE) and Mayer stainings of a first-trimester human fetal adrenal gland. (d) HE and Mayer stainings of a second-trimester human fetal adrenal gland. Scale bar = 100 µm.

Figure 3.

Expression of steroidogenic enzymes in first- and second-trimester human adrenal glands. Immunohistochemical staining of serial sections for CYP11A1, CYP17A1, 3β-HSD1/2, CYP21A2, CYP11B1, and SULT2A1 (steroidogenic proteins) as well as MC2R (ACTH receptor) in male samples are shown. No differences were observed between male and female samples within a given gestational group; therefore, only male samples are shown. Scale bar = 100 µm.

Figure 4.

Tissue levels of steroids in first- and second-trimester human adrenals. (a‒c) LC-MS/MS measurements (ng/g, wet tissue) of adrenal steroid hormones were determined from male and female adrenal tissue extracts. Bars represent mean ± SEM; individual data points shown as blue triangles and red circles represent male and female samples, respectively; n = 3 to 5. Differences in age compared with male GWs 8 to 9 are indicated as significantly different: *P < 0.05. Differences between sexes within the same age group are indicated as significantly different ¤P < 0.05.

Figure 5.

Overview of human fetal adrenal steroidogenic development. (a) Schematic overview of adrenal steroidogenesis. Gene transcripts of enzymes significantly upregulated in the second trimester are shown in green boxes, with unaltered gene transcripts shown in red boxes. Measured steroid hormones are shown in boldface font. (b) Overview of steroid hormone levels relative to progesterone levels throughout the investigated developmental period. Data are shown as mean values of fetal adrenal samples (for SEM values, see data in Fig. 4). DHEA, dehydroepiandrosterone.