Temporal and spatial distribution of mast cells and steroidogenic enzymes in the human fetal adrenal

Highlights

• Mast cells are present in the human fetal adrenal cortex from 18 WG

• Spatio-temporal correlation between mast cells and 3βHSD and CYP11B2.

• CYP11B1 is detected from 18 WG in both the transitional and fetal zones.

• CYP11B2 is detected from 24 WG in the definitive zone.

Abstract

Mast cells are present in the human adult adrenal with a potential role in the regulation of aldosterone secretion in both normal cortex and adrenocortical adenomas. We have investigated the human developing adrenal gland for the presence of mast cells in parallel with steroidogenic enzymes profile and serotonin signaling pathway. RT-QPCR and immunohistochemical studies were performed on adrenals at 16–41 weeks of gestation (WG). Tryptase-immunopositive mast cells were found from 18 WG in the adrenal subcapsular layer, close to 3βHSD- and CYP11B2-immunoreactive cells, firstly detected at 18 and 24 WG, respectively. Tryptophan hydroxylase and serotonin receptor type 4 expression increased at 30 WG before the CYP11B2 expression surge. In addition, HDL and LDL cholesterol receptors were expressed in the subcapsular zone from 24 WG. Altogether, our findings suggest the implication of mast cells and serotonin in the establishment of the mineralocorticoid synthesizing pathway during fetal adrenal development.

Introduction

The human fetal adrenal (HFA) cortex, which, like gonads, derives from the adreno-genital primordium, is organized by 7–8 weeks of gestation (WG) into two distinct parts: an inner fetal zone (FZ) accounting for 70–80% of the whole tissue and a thinner outer definitive zone (DZ) (Ishimoto and Jaffe, 2010). In addition, a third zone, called the transitional zone (TZ), is classically observed between the FZ and DZ during the second trimester of pregnancy. Quantification of steroid production during pregnancy and analysis of steroidogenic enzyme expression profile have showed that active steroidogenesis occurs very early, as soon as 8 WG for androgen production (Goto et al., 2002, Mesiano and Jaffe, 1997). Steroidogenesis requires cholesterol either locally synthesized or retrieved from the circulating lipoproteins LDL and HDL via their specific receptors (Bochem et al., 2013, Liu et al., 2000). In adults, the HDL receptor, also named SRB1 (Scavenger receptor class B type I), is crucial for adrenal cholesterol supply as demonstrated by altered corticosteroidogenesis in cases of SRB1 mutations (Vergeer et al., 2011). Although some in vitro studies have indicated that cholesterol uptake mainly occurs via the LDL receptor pathway in the developing adrenal (Carr et al., 1980, Carr et al., 1982), the demonstration of high levels of SRB1 mRNA in fetal glands suggest that HDL cholesterol could also be an important source of substrate for steroidogenesis in these tissues (Liu et al., 1997). Androgens, (dehydroepiandrosterone [DHEA] and its sulfate [DHEAS]), are the first steroids detected, owing to early expression of 17α-hydroxylase (17α-OH) in both the FZ and TZ (Narasaka et al., 2001). This androgen production is responsible for prenatal virilization of female fetuses suffering from congenital adrenal hyperplasia (CAH) (Speiser and White, 2003).

Synthesis of mineralo- and glucocorticoids also requires 21-hydroxylase (21-OH) and 3β-hydroxysteroid dehydrogenase (3βHSD). 21-OH immunoreactivity is visualized in the fetal adrenal as soon as 14 WG (Coulter and Jaffe, 1998, Narasaka et al., 2001). However, the accurate timing of 3βHSD expression is still controversial, since Narasaka et al. observed the presence of the enzyme in the TZ from 22 WG (Narasaka et al., 2001) while Folligan et al. detected 3βHSD from 16 WG (Folligan et al., 2005). In addition, Goto et al. reported an earlier 3βHSD expression from the first trimester of pregnancy with transient cortisol secretion at 8–10 WG (Goto, 2006), a finding which also suggests expression of 11β-hydroxylase (11β-OH) during this period. However, to our knowledge, the timing of 11β-OH expression in the fetal human adrenal has never been studied. Similarly, the fetal expression profile of aldosterone synthase, the key enzyme for aldosterone synthesis encoded by CYP11B2, has not been analyzed so that the capacity of the fetal adrenal to produce aldosterone remains a matter of debate.

It is now considered that cell-to-cell interactions are critical for organogenesis in mammals (Durdu et al., 2014). Recent studies have emphasized the role of immune cells in testis morphogenesis during which macrophages have been demonstrated to play a major role in the establishment of organ vascularization and architecture (DeFalco et al., 2014, Zhang et al., 2014). However, the involvement of adrenocortical cell microenvironment in adrenal development has not yet been investigated. In the human adult adrenal, adrenocortical cells interact with various types of immune cells which control their mitogenic and secretory activities (Kanczkowski et al., 2015). We have previously demonstrated that mast cells, located in the subcapsular region of the gland, stimulate aldosterone secretion through a paracrine mechanism involving local release of serotonin (5-hydroxytryptamine; 5-HT) and activation of 5-HT₄ receptors (Lefebvre et al., 2001). Consistent with this observation, incubation of the human adrenocortical cell line H295R with human mast cell line (LAD2)-conditioned medium provokes an increase in CYP11B2 expression and aldosterone production (Duparc et al., 2015). Resident mast cells may also be involved in the pathophysiology of adrenal disorders. In fact, a positive correlation between adrenal mast cell density and plasma aldosterone levels was found in a subset of aldosterone-producing adenomas, arguing for a role of mast cells in aldosterone hypersecretion (Duparc et al., 2015). All these observations strongly suggest that mast cells may control mineralocorticoid synthesis and secretion in both physiological and pathophysiological conditions. As mast cells has been noticed in various fetal tissues (Chen et al., 2004, Noack et al., 2005, Raica et al., 2010), their presence in the human fetal adrenal gland may also be hypothesized with a particular role in the establishment of the mineralocorticoid production.

To test this hypothesis, we thus investigated the presence of mast cells in correlation with steroidogenic enzyme expression in the human developing adrenal gland.