Gene expression

Total RNA was extracted from one frozen HFA gland in samples from GWs 8 to 12 (11 male and 11 female, collected from 22 fetuses), whereas half of a frozen HFA gland was used in samples from GWs 14 to 19 (9 male and 8 female, collected from 17 fetuses) and isolated using the NucleoSpin RNA II purification kit according to the manufacturer’s instructions (Macherey-Nagel, Düren, Germany). cDNA was synthesized using a dT20 primer and random hexamers. Real-time polymerase chain reaction (RT-PCR) was performed using specific primers targeting preselected mRNAs. All primers were designed to span intron-exon boundaries with optimal annealing temperatures of ~62°C, comparable primer length, and CG contents (Table 1). All amplicons were initially verified by sequencing (Eurofins MWG GmbH, Ebersberg, Germany), and primer amplification efficiency and detectable dynamic range of all primer sets were validated before the analysis of the HFA samples. RT-PCR cycle conditions were as follows: one cycle of 3 minutes at 95°C, 40 cycles of 30 seconds at 95°C, 1 minute at 62°C, 1 minute at 72°C, and one cycle of 5 minutes at 72°C. Quantitative RT-PCR analysis was performed in triplicate using Brilliant II SYBR Green qPCR Master Mix (Agilent Technologies, Santa Clara, CA). Changes in gene expression were quantified using the 2^−ΔΔCt method (19). Expression levels were normalized to the reference gene, RPS20, and calculated as a ratio with male samples GWs 8 to 9 set to 1 in respective samples.

Immunohistochemistry

Adrenal tissue from GWs 8 to 12 (13 male and 13 female, collected from 26 fetuses) and GWs 14 to 19 (10 male and 6 female, collected from 16 fetuses) were used for immunohistochemistry (IHC). Formalin-fixed adrenal tissues were dehydrated, paraffin embedded, and sectioned (4 µm) using standard procedures. Primary antibodies, dilutions, and retrieval buffers are listed in Table 2. IHC staining was initially conducted according to a standard protocol as described previously (20). Subsequently, the protocol was modified to include antigen retrieval by pressure cooker as previous described (21). In brief, tissue sections were subjected to heat-induced antigen retrieval buffer in a pressure cooker, and endogenous peroxidase was blocked with 3% (v/v) H₂O₂ in MeOH for 30 minutes. Between each step, sections were washed in Tris-buffered saline. Sections were incubated in 5% BSA (w/v) in horse serum (20% v/v) ImmPRESS (Vector Laboratories, Burlingame, CA) and Tris-buffered saline (80% v/v) or 0.5% milk powder for 30 minutes depending on optimization for each antibody. Sections were subsequently incubated overnight with primary antibody diluted in serum at 4°C in a humidified chamber followed by 1 hour at room temperature. Sections were then incubated for 30 minutes with the appropriate ImmPRESS HRP (peroxidase, Vector Laboratories) secondary antibody diluted in normal serum. Visualization was performed using ImmPACT AEC peroxidase substrate (Vector Laboratories, Burlingame, CA). Included negative controls replaced the primary antibody with dilution buffer only, none of which showed any staining. All sections were counterstained with Mayer hematoxylin before mounting with Aquatex (Merck, Damstadt, Germany).

Quantification of stained cells

Two independent investigators evaluated all stainings. Sections were first investigated manually on a Nikon Microphot-FXA microscope; subsequently, slides were scanned on a NanoZoomer 2.0 HT (Hamamatsu Photonics, Herrsching am Ammersee, Germany) and analyzed using NDP view software, version 1.2.36 (Hamamatsu Photonics). The intensity of immunoreactivity was classified according to a predefined scoring system: ++, strong staining in all cells of a given type in the sample; ++/+, strong staining prevalent, but some weakly stained cells also visible; ++/−, strong staining present, but negative cells also present; +/++, majority of cells weakly stained, but some strong staining present; +/++/−, heterogeneous pattern with a mixture of strongly positive, weakly stained, and negative cells; + (1), weak staining overall or (2) strong staining in a small number of cells; +/−, weak staining in limited areas; and −/+, weak staining in single cells.

Quantification of steroid hormones

Tissue samples from intact HFA glands from GWs 8 to 12 (10 male and 10 female, collected from 20 fetuses) and halved HFA glands from GWs 14 to 19 (9 male and 8 female, collected from 17 fetuses) were included for the intra-adrenal steroid analysis by LC-MS/MS measurements. Tissue samples were weighed (1.7 to 43 mg wet weight) and ground in 1 mL 80% (v/v) MeOH. The homogenate was transferred to glass tubes evaporated to almost dryness under an N₂ stream. Internal standard stock solution (5 to 500 ng/mL in 100 µL) was added to each sample pellet as previously described (22), and 375 µL 1M ammonium acetate buffer (pH, 5.5) was subsequently added. Next, 2 mL Heptan/Ethylacetat 3:2 (v/v) was added, and samples were transferred to Eppendorf tubes. Samples were then shaken for 15 minutes followed by 10 minutes of centrifugation at 2000g (4°C). Each tube was transferred to a dry ice bath (dry ice pills in ethanol, 99%) for a few minutes to freeze the aqueous phase, followed by decantation of the organic phase to a new glass tube. The organic phase was evaporated to dryness under a stream of N₂, and finally, the steroids were resolved in an appropriate amount of 50% (v/v) MeOH (tissue GWs 8 to 12: 100 µL; tissue GWs 14 to 19: 200 µL) for LC-MS/MS analysis as previously described (22). All samples were measured in one single batch, which included standards for calibration curves, unknown samples, and two blanks and for method control; three unspiked human serum pool samples and three serum pool samples spiked with low and high levels, respectively.

Distribution of steroidogenic enzymes in the HFA cortex

The morphology of fixed adrenal samples included in the study was thoroughly examined. All samples appeared morphologically normal, containing distinct zones in accordance with the gestational age of the developing HFA gland (Fig. 2). Sections were stained with hematoxylin and eosin as well as Mayer hematoxylin. The DZ consisted of small cells, whereas FZ cells were larger, with a high content of cytoplasm (Fig. 2c). As expected, the TZ was detected in second-trimester samples and consisted of a mixture of small distinct DZ cells and larger distinct FZ cells (Fig. 2d), as previous described (1).

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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.

The expression profiles of steroidogenic proteins were analyzed using IHC on fixed adrenal samples. No differences between male and female samples were observed regarding the level of expression and the cell type‒specific enzyme localization. Therefore, only male samples are shown for the age groups GWs 8 to 9, GWs 10 to 12, GWs 14 to 16, and GWs 17 to 19 in Fig. 3. Adrenal zone‒specific protein expression, localization, and level of expression were evaluated according to a predefined scale and are summarized in Table 3.

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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.

Most of the investigated steroidogenic enzymes were abundantly expressed in the FZ throughout development of both the first and second trimesters (Fig. 3). More specifically, CYP11A1, CYP21A2, and SULT2A1 were abundantly expressed in the cytoplasm of first-trimester FZ cells and weakly expressed in a limited number of DZ cells toward the late first trimester and throughout the second trimester. CYP17A1 showed a similar expression and localization pattern, although CYP17A1 was expressed specifically in the FZ except for a few strongly positive cells scattered within the DZ in both first- and second-trimester samples. During the second trimester, protein expression of CYP17A1 increased around the TZ as it decreased in the cells of the inner FZ. In contrast, 3β-HSD1/2 was the only observed steroidogenic enzyme frequently expressed in a limited number of DZ cells and in only a very few FZ cells. During the second trimester, 3β-HSD1/2 was still abundantly expressed, though only in a subpopulation of DZ and FZ cells compared with first-trimester samples.

Like the steroidogenic enzymes, the ACTH receptor MC2R was also detected throughout the investigated period of fetal development. More specifically, weak cytoplasmic MC2R expression was detected throughout the first and second trimesters, but nuclear MC2R expression was also occasionally found in the late first trimester and throughout the second trimester. Therefore, the MC2R expression patterns observed in this study should be interpreted with caution because the unexpected nuclear expression (23) is most likely an artifact of the antibody.

The authors thank staff members at the Departments of Gynecology (Rigshospitalet and Hvidovre Hospital) and Growth and Reproduction (Rigshospitalet) for help with the collection of fetal tissue. Furthermore, the excellent technical assistance of Ana Ricci Nielsen, Lene Andersen, Brian Vendelboe Hansen, and Camilla Tang Thomsen is gratefully acknowledged. We appreciate the kind gift of 3β-HDS2 antibody from Professor Ian Mason (University of Edinburgh, Center for Reproductive Biology, Scotland). Also, we acknowledge the Human Developmental Biology Resource (www.hdbr.org) that provided second-trimester tissue for these studies (Joint MRC/Wellcome Trust, Grant No: MR/R006237/1).

Financial Support:This work was supported by the International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), the Lundbeck Foundation (PhD scholarship grant no. R249-2017-1484; to C.M.) and Wellcome Trust (Intermediate Clinical Fellowship grant no. 098522; to R.T.M.).

Author Contributions:C.M., A. Juul, and A. Jørgensen conceived and designed the experiments. C.M., J.E.N., and H.F. performed the experiments. K.K., S.P., L.L., L.L.T., and K.J.H. provided study material. C.M., J.E.N., H.F., A.-M.A., R.T.M., A. Juul, and A. Jørgensen analyzed the data. C.M. and A. Jørgensen wrote the manuscript. All authors read and approved the submitted version of the manuscript.

Disclosure Summary: The authors have nothing to disclose.