At all investigated stages in the abdominal region, the cortical anlages of the adrenal glands were already present as oval-shaped conglomerates of large cells with abundant oxyphilic cytoplasm (Fig. 1A, Fig. 2A). Nearby, in the paraaortic region adjacent to the aorta, large conglomerates of cells representing the developing ganglia and OZ were observed, along with crossing nerve bundles. In the early stages, the boundaries between the ganglionic anlages and the developing OZ were indistinct. However, at later stages, a prominent thin capsule surrounding the OZ became apparent.
At early stages of development (from 8 g.w. to 12 g.w.), the developing ganglia and OZ consisted of two morphological cell types. The first and the most common cell type was the small cell type (SC). These cells had scarce, sometimes inconspicuous cytoplasm and hyperchromatic nuclei appearing as small blue cells that formed rounded or oval solid structures. At later stages (from about 12 g.w.), numerous neuron-like cells with processes and conspicuous large nuclei with nucleoli began to appear among the SC conglomerates, indicating differentiation along the neuronal pathway. By the latest investigated age (25–26 g.w.), well-formed ganglia were present in the paraaortic region and no more SCs were found extraadrenally.
The second cell type was the large cell type (LC). LCs had clear oxyphilic cytoplasm and clear prominent nuclei. At early stages, they formed small rounded clusters (Fig. 1C, E), which increased rapidly with age. From the age of 12 g.w., these LCs already formed well-developed, well-shaped, and functionally active OZ (Fig. 2A, B, D). Such rapid growth in size of these structures could be explained by the proliferative activity of LCs themselves or it may be due to the differentiation of a part of SCs along the chromaffin lineage.
LCs and SCs correspond to the “large” and “small” cells described by Molenaar et al. (Molenaar et al. 1990). The description of LCs also aligns with the characteristics of AM chromaffin cells, which have large nuclei with “open chromatin,” as reported by Cooper et al. (Cooper et al. 1990).
LCs and SCs were present at all stages of development within the cortical anlage. Unlike in the ganglia and OZ, SCs persisted in the adrenal anlage even at the latest investigated age (25–26 g.w.).
During the investigated stages of human ontogenesis, the structures formed by these cells underwent distinct morphological and immunohistochemical changes as follows. Results of immunohistochemical reactions are shown in Supplementary Table S2.
8–9 g.w. At 8–9 g.w., paraaortic conglomerations consisted of rounded to oval clusters of SCs and LCs. Numerous mitotic figures were present among SCs with occasional mitoses observed in LC clusters. The majority of SCs were found in contact with nerve bundles running along the aorta or medially toward the adrenal anlage. LC clusters were located close to the SC conglomerations and also connected to nerve bundles. Immunohistochemically, LCs displayed strong cytoplasmic reactivity with antibodies to TH, DBH, and βIII-tubulin (Fig. 1D, F). In contrast, SCs, showed significantly weaker reactivity with antibodies to TH and DBH, with some SCs being negative for DBH. SCs did, however, exhibit marked cytoplasmic expression of βIII-tubulin. Neither LCs nor SCs expressed PNMT at this developmental stage, suggesting that extra-adrenal LCs may already be producing norepinephrine, but not epinephrine. S100 was weakly positive in the nuclei and cytoplasm of occasional cells associated with nerves. In specimen No. 3, S100+ cells were also observed at the periphery of LC and SC clusters.
In the cortical anlages of the adrenal glands, there were individual cells and small groups of cells that were morphologically and immunohistochemically similar to the LCs in the extraadrenal conglomerations. These cells were dispersed among the oxyphilic cells of the cortical anlage. Notably, a marked gradient of these LCs was observed, decreasing with distance from the extraadrenal LC groups. This pattern strongly suggests a migratory process, indicating that large TH+DBH+βIII+ cells may migrate from the extraadrenal chromaffin tissue to the cortical anlages. This observation aligns with the findings of Molenaar et al. (Molenaar et al. 1990) and Hervonen (Hervonen 1972). βIII-tubulin positive nerve fibers were also seen transitioning adrenal anlage, with some of these fibers closely associated with LCs interspersed within the cortical anlage.
11–12 g.w. At 11–12 g.w., both large cells (LCs) and small cells (SCs) remained present, with a more distinct pattern of LCs migrating from the extraadrenal developing OZ to the cortical anlage becoming apparent (Fig. 1A, B, C, E; Fig. 2A, B). In some areas, these migrating LCs appeared to travel along βIII-tubulin+ nerves, while in others, they were freely interspersed among cortical cells. Intraadrenally, LCs were strongly positive for TH, DBH, with slightly weaker positivity for βIII-tubulin. Clusters of SCs were also present intraadrenally, but they were less abundant, and their migratory path was less distinct compared to that of LCs. SCs were strongly positive for βIII-tubulin and weakly positive for TH and DBH.
Extraadrenally, at the level of the developing kidneys, large rounded to oval masses of the developing OZ, composed primarily of LCs, were surrounded by clusters of SCs, which were mostly located peripherally. The boundaries between these SC and LC clusters were more distinct in one 12 g.w. specimen (No. 6) compared to earlier specimens (Nos. 4 and 5), with a thin capsule already forming around the developing OZ. However, the peripheral location of SCs was not consistent, as in other sections and specimens, SCs and LCs formed independent round structures composed of only one cell type.
As in previous stages, extraadrenal LCs and, to a lesser extent, SCs were positive for TH and DBH. Given the large size of the developing OZ formed by LCs, these organs were likely the main source of norepinephrine in the developing human organism.
Among the SCs, a few scattered, larger, triangle-shaped cells with more prominent cytoplasm and large nuclei with conspicuous nucleoli were observed, likely representing developing neurons. S100-positive cells with marked nuclear and cytoplasmic expression were found along the nerves, at the periphery of LC conglomerations, and among SCs (Fig. 3A). These S100-positive cells were round, oval, or spindle-shaped, with no morphological differences based on their location along the nerve or at the periphery of LC structures.
In one of two specimens at 12 g.w. (No. 6), intracortical chromaffin cells exhibited strong cytoplasmic positivity for PNMT (Fig. 2C), while in extraadrenal chromaffin tissue and in the other specimen of the similar age (No. 5), PNMT was negative.
16 g.w. At 16 g.w. (1 specimen), no significant differences were observed compared to the 12 g.w. prefetus. However, LCs were negative for PNMT. This may be due to the poorer preservation of this specimen compared to others, as PNMT may be highly sensitive to autolysis. Other cytological and immunohistochemical characteristics of large and small intra- and extraadrenal cells remained consistent with those observed at earlier stages.
20–23 g.w. At 20–23 g.w. intraadrenally, numerous large, rounded, and oval groups of SCs were observed, situated within the cortical tissue and occasionally in the walls of vessel-like structures, directly adjacent to the lumens. Many of these lumens contained numerous erythrocytes (Fig. 3B, C, D). This pattern of SC location within true vessel walls or vessel-like structures may be the result of artificial changes during specimen preparation, or it could represent a true anatomical feature. Regardless, it was a consistent morphological characteristic observed in all specimens at this stage. Such vessel-like spaces have been described in previous studies as cell-free cavities (Iwanaga and Fujita 1984; Golub 1936). Iwanaga and Fujita suggested that these structures might result from the degeneration of so-called primitive sympathetic cells, which we refer to as SCs here. However, no evidence of necrosis or apoptosis was observed.
LCs were organized in small nests, primarily located at the periphery of SC groups and among cortical cells. LCs were strongly TH+DBH+ and to a lesser extent βIII+, which is similar to earlier ages (Fig. 2E, F). This weaker positivity of LCs for βIII-tubulin aligns with the findings of Katsetos et al. (Katsetos et al. 1998), who demonstrated focal distribution of βIII positivity in chromaffin cells in human fetuses at 20 g.w. and later. LCs were also strongly positive for PNMT, except for three of seven specimens, which appeared to be negative. SCs were strongly βIII+, and, to a lesser extent, TH+DBH+. S100+ cells were present in vessel walls, at the periphery of SC clusters, and in a single cell pattern among LC groups. These S100+ cells were spindled or oval in shape.
Extraadrenally, well-formed rounded or oval shaped ganglia composed of large triangle-shaped stellated neurons with prominent processes and nucleoli were observed. Extraadrenal SCs were no longer present. Neurons in the ganglia were βIII+ and, to a lesser extent, TH+DBH+. In close proximity to ganglia, large OZ represented by LCs were located. They were strongly TH+DBH+ and, to a lesser extent, βIII+. Nerve fibers, connecting ganglia and paraganglia, were also βIII+. S100+ cells were present along nerves and among large cells in OZ.
25–26 g.w. At 25–26 g.w. intraadrenally, the ratio of LCs to SCs seemed to increase, and occasional neuron-appearing cells with processes and prominent nucleoli appeared among SCs. Other morphological and immunohistochemical features remained similar to those at earlier stages.
Our results suggest that during embryogenesis, there are two possible pathways for the recruitment of AM cell populations. The first and most prevalent pathway involves the migration of LCs from the developing OZ. The second, less histologically apparent pathway is associated with the migration of SCs from extraadrenal conglomerations.
At the earliest investigated stage (8–9 gestational weeks), chromaffin cells, indicated here by the LC morphology and marked cytoplasmic TH- and DBH- positivity, were already present in the extraadrenal paraaortic region. These developing chromaffin cells were also observed in the medial part of the cortical anlage, either freely interspersed among cortical cells or in connection with nerves, resembling a migration pattern. This suggests that the developing OZ play a crucial role in distributing cells that are partially differentiated along the chromaffin lineage, contributing to the formation of the AM.
At the earliest investigated stages, SCs were primarily located extraadrenally, in close association with nerve bundles, with only occasional SCs observed among cortical cells. As a result, their migration was less apparent compared to the more evident migration of LCs.
At later stages, we can see the LC migratory pattern becoming more obvious with age. Alongside LCs, small groups of SCs are also observed intraadrenally. These SCs are typically found in close contact with nerves, whereas LCs are often seen within the cortical anlage, independent of nerve structures. This suggests that LCs may migrate not only along nerves but also freely between cortical cells, while SCs primarily migrate to the adrenal anlage along nerve pathways.
The specific roles of these two cell types in AM morphogenesis remain uncertain. In the early stages, the developing AM was primarily composed of migrating LCs, with only occasional SCs present in the cortical anlage. However, at later stages, large clusters of SCs were observed within the adrenal gland. This may be attributed to the high proliferative activity of LCs, which likely contributes to the significant increase in the overall cell population.
At the same time, some LCs were located at the periphery of SC clusters, closely associated with them. This suggests that a portion of SCs may differentiate into LCs. Moreover, at the age of 25–26 g.w. the ratio of LCs to SCs increased significantly, likely indicating that the majority of SCs had differentiated into LCs and, to a lesser extent, into neurons.
This raises questions about the role of the initially migrating LCs, which are not connected to the differentiation of intraadrenal SC clusters. One possibility is that these primarily migrating LCs play a provisional role in producing catecholamines and facilitating the migration of SCs into the cortical anlage. Following this, the primarily migrated LCs may undergo apoptosis. Another possibility is that both the initially migrating LCs and those differentiated from SCs collectively contribute to the formation of the AM. However, our methods did not allow us to distinguish between these LC subpopulations.
At the age of 12 g.w., we observed strong cytoplasmic positivity for PNMT in intraadrenal LCs, both those associated with SCs and those lying freely. This finding is intriguing in light of ultrastructural results reported by Hervonen, who identified the first ultrastructural features of epinephrine-containing granules at 16 g.w. in the human fetus, approximately four weeks after the appearance of PNMT in our study (Hervonen 1971). Hervonen also noted that typical synaptic profiles were present on medullary chromaffin cells in a 12-week-old fetus but stated that mature synapses on these cells did not appear until 14 weeks. He also demonstrated that extraadrenal paraganglia remained uninnervated, as no synapses were found on their chromaffin cells, and they lacked epinephrine-containing granules. These observations suggest a correlation between the presence of synapses on chromaffin cells and the presence of adrenaline-containing granules, and consequently, the presence of PNMT, the enzyme responsible for epinephrine synthesis. Therefore, it is possible that PNMT synthesis in humans is connected to the process of synaptogenesis. Moreover, it is likely that the establishment of synapses triggers the further differentiation of chromaffin cells from norepinephrine-synthesizing to epinephrine-synthesizing cells. This idea, that synaptogenesis can induce differentiation, dates back to an early study by Golub (Golub 1936).
In contrast, the presence of such a correlation in rats is less clear. Ultrastructural features of mature synapses on chromaffin cells in rats appear at 15.5 days of gestation, suggesting that neural control over medullary chromaffin cells could be established from this point (Daikoku et al. 1977). However, it has been shown that in 1-day-old newborn rats, neural regulation of catecholamine release is absent until about 8 days of postnatal life (Seidler and Slotkin 1985), even though adrenaline is already present in medullary chromaffin cells. This discrepancy indicates that the relationship between synaptogenesis and catecholamine regulation in rats requires further investigation.
The appearance of PNMT at 12 gestational weeks (g.w.) in a human prefetus raises questions about the role of glucocorticoids in the expression of PNMT. The adrenal cortex in human fetuses is distinct from that in other species, and cortisol is believed to be produced transiently early in gestation (around 7–10 g.w.). However, due to the lack of hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2 (HSD3B2) activity—the enzyme essential for de novo cortisol synthesis—cortisol biosynthesis appears to be suppressed until 23–24 g.w. (Ishimoto and Jaffe 2011). Consequently, at 12 g.w., the hypothetical level of cortisol would be low, making it unlikely to stimulate PNMT expression. This suggests that glucocorticoid induction of PNMT synthesis in humans may not be the primary mechanism. However, further investigation into the adrenal cortex, adrenal medulla, and extraadrenal paraganglia as a complex is necessary to better understand this issue in humans.