Steroid hormones are mainly produced in the adrenal glands, gonads, and placenta, where they play endocrine roles in regulating target tissue or cell function depending on circulating steroid concentrations (1, 2). While specific hormone-producing cells in these tissues have received the major share of focus in the field, previous studies have shown that many peripheral tissues and cell types within the brain, kidney, lung, skeletal muscle, intestine, keratinocytes, adipocytes, astrocytes, and placental trophoblasts have the capacity of de novo steroidogenesis or steroid conversion (3–11). This diversity of tissues with steroidogenic capacity indicates that there are multiple cell types that can undertake or mediate steroid hormone production. One cell lineage that has been linked to steroidogenesis is the immune cell lineage, as local sex steroid production has been identified within immune cell populations such as macrophages and T lymphocytes (12–14). Within the testis, macrophages have been implicated in steroid production by Leydig cells (15, 16), although the mechanisms by which macrophages developmentally or functionally regulate Leydig cells are poorly understood. The unexpected and poorly understood steroidogenic capacity of immune cells and their roles in modulating glandular steroidogenesis is becoming an emerging area of research that is critical for a deeper understanding of the complex immunoregulatory roles of steroid hormones in normal and disease contexts. In this review we will discuss the various roles proposed for testicular macrophages in Leydig cell biology and we will highlight future areas of research that should be pursued to elucidate the mechanisms underlying regulatory functions of immune cells and their potential de novo steroidogenesis in the testis and, potentially, beyond.

Steroidogenesis is a process in which cholesterol is converted into steroid hormones by a series of steps mediated by steroidogenic enzymes. In this process, there are two key rate-limiting steps, which are 1) the transport of cholesterol from the cytoplasm into mitochondria and 2) the conversion of cholesterol into pregnenolone. Free cholesterol is derived from intracellular cholesterol that is synthesized either from acetate, from cholesterol ester stored in lipid droplets, or from uptake of cholesterol-containing low-density lipoproteins (LDLs). Plasma LDLs are the most important source of cholesterol when steroidogenic cells are chronically stimulated. Then steroidogenic acute regulatory protein (StAR) promotes the rapid flux of cholesterol into the mitochondria, where cholesterol is catalyzed to yield pregnenolone by side-chain cleavage enzyme cytochrome P450scc (also known as CYP11A1, encoded by the CYP11A1 gene) within the mitochondrial inner membrane. Pregnenolone, as an immediate precursor, requires further catalysis by two major families of enzymes, which are cytochrome P450 (CYP) and hydroxysteroid dehydrogenase (HSD) located in both mitochondria and the endoplasmic reticulum, to facilitate the biosynthesis of steroid hormones (2, 17, 18).

In many contexts, steroid hormones are classified based on the organs that produce them and the receptors to which they bind. The adrenal steroids, which consist of glucocorticoids and mineralocorticoids, are secreted by the adrenal cortex. Glucocorticoids such as cortisol in humans and corticosterone in rodents control many cell metabolic processes, including maintaining blood pressure and regulating immune cell function. Aldosterone is the most well-known mineralocorticoid, which maintains the body’s water and salt balance by acting primarily on the kidneys. Sex steroid hormones, which are composed of androgens (e.g., testosterone), estrogens (e.g., estradiol), and progestogens (e.g., progesterone), are produced by the gonads and placenta. These sex hormones are responsible for regulating sexual development and promoting fertility. Additionally, the adrenal cortex secretes sex hormones to a lesser extent than the gonads, and the gonads may produce adrenal steroids (1, 19). Aside from dedicated steroidogenic cells like Leydig cells, theca cells, or adrenocortical cells, future research should address the extent to which alternative glandular or extra-glandular cell types in the gonads and adrenal are involved in de novo steroidogenesis.

Early analyses of the immune cells in the testis revealed that macrophages are a large component of the testicular interstitial compartment, comprising approximately 20% of interstitial cells (20). Macrophages and Leydig cells, therefore, occupy the same compartment of the testis and are in intimate contact throughout development (21). Histological and ultrastructural studies of the postnatal and adult rat testis demonstrated that macrophages and Leydig cells form intercellular cytoplasmic digitations (21, 22), which only are observed between these 2 cell types and only upon puberty (22), indicating an intimate relationship linked to testicular maturation. Furthermore, macrophage-deficient osteopetrotic mice mutant for colony stimulating factor 1 (Csf1^op/op ) are infertile as a result of low testosterone, oligozoospermia, and decreased libido (15, 23, 24). Analyses of normal and cryptorchid testes revealed that there is a robust correlation between the volume density of Leydig cells and macrophages, as well as total mass of Leydig cells and macrophages per testis (25), leading to early ideas of functional coupling between the two cell types. These findings strongly suggest that testicular macrophages have trophic functions in Leydig cell differentiation and promote steroidogenesis, but the developmental and functional links between macrophages and Leydig cells are still open areas of investigation.

Multiple studies by Gaytan et al. in the 1990s revealed that there is an interdependent relationship between macrophages and Leydig cells in both developmental and regenerative contexts (26–28). Using dichloromethylene diphosphonate-containing liposome (Cl₂MDP-lp) injection to deplete testicular macrophages in prepubertal rats, they found that macrophages are required for the development of Leydig cells during postnatal testicular maturation (26). The authors concluded that, in the absence of macrophages, Leydig cell proliferation did not occur, nor were mesenchymal progenitor cells able to undergo differentiation into Leydig cells (26). They further speculated that macrophages were required for Leydig cell responsiveness to lutenizing hormone (LH) and human chorionic gonadotropin (hCG) (29, 30), as hCG-treated Leydig cells in Cl₂MDP-lp-injected testes did not increase in number as in contralateral intact testes. Regeneration of Leydig cells in testes that had selective Leydig cell depletion induced by ethylene dimethanesulfonate (EDS) treatment, which requires LH (31), was also hindered in the absence of macrophages (27, 28) (see next paragraph). These findings suggest that as-of-yet undefined macrophage factors are essential for Leydig cell responsiveness to LH/hCG.

Gaytan et al. demonstrated, again using a Cl₂MDP-lp-mediated ablation method (27, 28), that testicular macrophages are required for adult Leydig cell regeneration after specific depletion of Leydig cells via EDS treatment. In contrast, when macrophages were ablated in intact adult testes, there was no effect on Leydig cell numbers (28), indicating that macrophages are not as essential for steady-state maintenance of adult Leydig cell numbers; a more recent finding showed a similar result, in which a diphtheria-toxin-mediated ablation of adult macrophages did not result in a change in Leydig cell number (although there was a significant drop in testicular testosterone levels) (32).

Given the tight physical association between testicular macrophages and Leydig cells in the interstitial compartment, in the past 40 years most investigations into testicular macrophage functions focused on Leydig cell steroidogenesis (16, 33). Yee and Hutson in 1985 showed that testicular macrophage-conditioned medium (TMCM) in a dose-dependent manner increases testosterone production of Leydig cells (34). Consistent with this finding, bank vole Leydig cells from a long photoperiod in co-cultures with testicular macrophages or treated with TMCM produced more testosterone (35). However, some subsequent studies demonstrated that non-stimulated testicular macrophages have an inhibitory effect on the production of testosterone by Leydig cells (36–38), whereas TMCM obtained from lipopolysaccharide (LPS)-stimulated macrophages or macrophages isolated from autoimmune orchitis could promote testosterone production (36, 39). Therefore, the role of testicular macrophages in Leydig cell steroidogenesis under physiological conditions has been controversial. Furthermore, testicular macrophages isolated using different methods may have different phenotypes and metabolic properties in vitro due to the loss of their complex in vivo microenvironment. This could be one of the reasons why testicular macrophages need to be additionally activated in some circumstances in order to function properly. Our recent study found that the depletion of adult testicular macrophages in vivo decreases testicular testosterone levels (32), suggesting the beneficial effect of testicular macrophages on Leydig cell steroidogenesis.

Tissue immune cells, particularly macrophages and T lymphocytes, may be an important source of local steroid production by steroid conversion or de novo steroidogenesis. Intracrine and paracrine roles of immune-cell-derived steroids may be essential for cellular functions within various tissues. Therefore, immune cell-derived steroids and steroid metabolites potentially have biological effects within the tissue microenvironment, although their quantities in tissue fluids or blood are likely modest.

The presence of testicular macrophages and their potential roles in Leydig cell steroidogenesis have been investigated for several decades, but the mechanisms underlying their functional relationship is still unclear. One particular area that needs to be rigorously addressed is whether testicular macrophages merely promote steroidogenesis by Leydig cells or if they undergo de novo steroidogenesis in a meaningful way to promote spermatogenesis and fertility. Macrophages could impact Leydig cells through a number of mechanisms, such as regulating the cytokine environment, providing steroidogenic substrates, or through modulating Leydig cell ultrastructure via unique cell-cell junctions ( Figure 1 ). Given recent findings of de novo steroidogenesis by T cells in various contexts, the contributions of immune-cell-derived steroids should be addressed in the context of testicular function. Furthermore, as many studies have linked inflammation to infertility, it is also critical to study how macrophage polarization and the subsequent changes in their cellular activities cause or exacerbate testicular pathology. Reports in several fields indicate that immune cell steroid production is a broadly observed and evolutionarily conserved phenomenon; therefore, understanding the roles of immune cells in testicular steroidogenesis and Leydig cell function will likely provide new insights into endocrinology that will extend beyond the boundaries of the testis.

XG, S-YL, and SM performed literature searches and drafted the manuscript. TD conceptualized, drafted, and supervised the manuscript. All authors contributed to manuscript revision and approved the submitted version.

Work from the DeFalco laboratory is supported by National Institutes of Health (grants R35GM119458 and R01HD094698 to TD).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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