Essential Roles of Interstitial Cells in Testicular Development and Function

Abstract

Background

Testicular architecture and sperm production are supported by a complex network of communication between various cell types. These signals ensure fertility by: regulating spermatogonial stem/progenitor cells; promoting steroidogenesis; and driving male-specific differentiation of the gonad. Sertoli cells have long been assumed to be the major cellular player in testis organogenesis and spermatogenesis. However, cells in the interstitial compartment, such as Leydig, vascular, immune, and peritubular cells, also play prominent roles in the testis but are less well-understood.

Objectives

Here we aim to outline our current knowledge of the cellular and molecular mechanisms by which interstitial cell types contribute to spermatogenesis and testicular development, and how these diverse constituents of the testis play essential roles in ensuring male sexual differentiation and fertility.

Methods

We surveyed scientific literature and summarized findings in the field that address how interstitial cells interact with other interstitial cell populations and seminiferous tubules (i.e., Sertoli and germ cells) to support spermatogenesis, male-specific differentiation, and testicular function. These studies focused on 4 major cell types: Leydig cells; vascular cells; immune cells; and peritubular cells.

Results and Discussion

A growing number of studies have demonstrated that interstitial cells play a wide range of functions in the fetal and adult testis. Leydig cells, through secretion of hormones and growth factors, are responsible for steroidogenesis and progression of spermatogenesis. Vascular, immune, and peritubular cells, apart from their traditionally acknowledged physiological roles, have a broader importance than previously appreciated and are emerging as essential players in stem/progenitor cell biology.

Conclusion

Interstitial cells take part in complex signaling interactions with both interstitial and tubular cell populations, which are required for several biological processes, such as steroidogenesis, Sertoli cell function, spermatogenesis, and immune regulation. These various processes are essential for testicular function and demonstrate how interstitial cells are indispensable for male fertility.

INTRODUCTION

The mammalian testis develops in utero when the expression of the Y-linked gene Sry is expressed in a subset of bipotential progenitor cells that initially have the ability to become testicular or ovarian cells (Albrecht & Eicher 2001; Koopman et al., 1990; Sinclair et al., 1990). Under the influence of Sry and its target gene Sox9, during E10.5-E11.5 in the mouse and 4–6 weeks in the human, gonadal progenitor cells become Sertoli cells, which are the first male-specific cell type specified in the gonad. Sertoli cells intimately associate with germ cells and ensure proper germline development throughout life. While Sertoli cells are necessary for testicular differentiation, recent studies have revealed that other cell types are critical contributors to testicular development and function.

The interstitial compartment of the testis, which constitutes the area of the organ outside of the testis cords/seminiferous tubules, is comprised of several cell types. The tubule compartment is maintained separately from the interstitium via a collagen- and laminin-containing basement membrane, which is overlaid by peritubular cells (often called peritubular myoid cells) that physically separate the two compartments. While the vast majority of studies described in this review are based on rodent models, some research has also been done on human testicular cells. Although the basic architecture of the testis among mammals is fairly similar, there are some differences, such as the fact that human testes have a multi-cell-layered peritubular wall, in contrast to a single-cell-layered wall in rodents (Mayerhofer 2013). Additionally, human testes contain mast cells (Meineke et al., 2000), which are rarely found in rodent testes. In spite of some species-specific differences, lessons can be learned from each species regarding the diverse functions of testicular cell types.

Within the interstitial compartment of both human and rodent testes, there are various cell types, including: peritubular cells; vascular endothelial cells; vascular smooth muscle and other perivascular cells; steroidogenic Leydig cells and their undifferentiated mesenchymal progenitors; and immune cells, which under normal conditions is mostly comprised of testicular macrophages. Recent work has shown that these cell types are critical for morphogenesis of the fetal testis (Bott et al., 2006; Combes et al., 2009; Cool et al., 2011; DeFalco et al., 2014), as well as promoting spermatogenesis to ensure male fertility (DeFalco et al., 2015; Potter & DeFalco 2017).

Male fertility is dependent on the continual production of sperm, starting at puberty and continuing throughout adult life. Constant sperm production necessitates the maintenance and regulation of the spermatogonial stem cell (SSC) population in the testis, in which SSCs must balance self-renewal and differentiation to promote fertility (Oatley & Brinster 2012); therefore, a strict microenvironment must be established and maintained for the SSC population. A stem cell niche is a highly specialized microenvironment that, through the contribution of extrinsic growth factors and/or architecture, maintains a stem cell population and regulates its continual self-renewal and differentiation. Undifferentiated SSCs (A_Undiff) are located along the basal compartment of the seminiferous epithelium, with Sertoli cells serving as a barrier between A_undiff and their more differentiated progeny through tight junctions forming the blood testis barrier (BTB). Sertoli cells are intermixed with the A_undiff spermatogonial population and provide extrinsic signaling that contributes to the maintenance of the SSC niche, and have long been understood to be an important contributor to SSC niche regulation (Oatley et al., 2011). Due to their intimate interactions, the communication between Sertoli cells and germ cells has been thoroughly investigated. In particular, glial-cell derived neurotrophic factor (GDNF), a growth factor secreted by Sertoli cells, is critical for SSC self-renewal (Meng et al., 2000); however, recent studies have revealed that GDNF production by peritubular cells is also essential for SSC maintenance in mice (Chen et al., 2014; Chen et al., 2016) and likely also in human testes (Spinnler et al., 2010). Therefore, a greater appreciation is being gained for the role interstitial cells play in the mammalian testis, as a growing number of studies have revealed that Leydig cells, macrophages, vasculature-associated cells, and peritubular cells are also necessary for regulation of the SSC niche (Figure 1), fetal testicular differentiation (Figure 2), and for male fertility as a whole. Here we will discuss the diverse and wide-ranging roles for interstitial cells during testicular organogenesis in utero and during spermatogenesis throughout the male reproductive lifespan.

Figure 1. Role of interstitial cells in adult spermatogenesis.

Illustration of a cross section of a seminiferous tubule and surrounding interstitium of a rodent testis, highlighting our current knowledge of the mechanisms through which testicular interstitial cells influence adult spermatogenesis. Text and receptors shown in red indicate interactions needing further study or are currently unclear. Arrows indicate a positive influence, T-shaped lines indicate an inhibitory effect. Abbreviations are as follows: Vasopressin (ADH); Androgen receptor (AR); colony stimulating factor 1/receptor (CSF1/CSF1R); glial-cell derived neurotrophic factor/receptor 1 (GDNF/GFRA1); insulin-like growth factor 1/receptor (IGF1/IGF1R); interleukin-1 (IL-1); prostaglandin (PG); retinoic acid (RA); reactive oxygen species (ROS); Testosterone (T); transforming growth factor alpha (TGFa); vascular endothelial growth factor/receptor (VEGFA/VEGFR).

Figure 2. Role of interstitial cells in fetal testis morphogenesis and differentiation.

Illustration of E13.5 fetal mouse testis depicting current knowledge of the mechanisms through which interstitial cells influence early fetal testis development. Text and receptors shown in red indicate areas needing further study. Arrows indicate a positive influence, T-shaped lines indicate an inhibitory effect. Abbreviations are as follows: Androgen receptor (AR); Cadherin 5/VE-Cadherin (CDH5); desert hedgehog (DHH); platelet derived growth factor (PDGF); Testosterone (T); vascular endothelial growth factor (VEGF).

Leydig Cells

Testosterone

Leydig cells are the major steroidogenic cell population of the testicular interstitium. Their most well-known role in male reproductive function is their production of testosterone, a necessary component for the progression of spermatogenesis (Walker 2011). Through its binding to the androgen receptor (AR), testosterone has many effects on sexual behavior, fertility, and other aspects of adult health, such as bone development (Khosla & Monroe 2018; Tsujimura 2013). While testosterone’s role in differentiation of the reproductive tract is well-known, not as much is known about testosterone’s role directly within the fetal gonad, in particular with respect to germ cells. Several studies suggested that testosterone does not act directly on germ cells, as Ar conditional deletion in germ cells had no impact on spermatogenesis (Tsai et al., 2006) and Ar-mutant SSCs can effectively reconstitute spermatogenesis in transplantation assays (Johnston et al., 2001). Contrary to these findings, one study suggested that fetal germ cells do express Ar and can be direct targets for androgens, which were proposed to regulate germ cell proliferation (Merlet et al., 2007). Yet another study indicated that germ cell deficiency during fetal stages affected Leydig cell gene expression (Rios-Rojas et al., 2016). Therefore, it appears that more research is needed into understanding the direct (and indirect) communication between Leydig and germ cells during development.

Genetic studies of Ar revealed that testosterone signaling does not act directly on germ cells in the adult testis and, instead acts through testicular somatic cells. Sertoli-specific Ar function is required for completion of meiosis, as Sertoli-specific Ar (SCARKO) mutants exhibit spermatogenic arrest at the primary spermatocyte stage (Chang et al., 2004; De Gendt et al., 2004). Leydig Ar function is also required for spermatogenesis, as conditionally deleting Ar in Leydig cells led to spermatogenic arrest, although mainly at the round spermatid stage (Tsai et al., 2006). A peritubular-cell-specific knockout of Ar also showed severe disruptions in spermatogenesis, with broad loss of germ cells and virtually no sperm present in the epididymis of mutant mice (Welsh et al., 2009). Cell culture assays of human peritubular cells also revealed that testosterone acts on peritubular cells to influence their phenotype, such as modulating AR levels, smooth muscle contractility, and cytokine secretion (Mayer et al., 2018). Studies of androgen receptor mutant mouse models, in conjunction with cell culture studies of human testicular cells, indicate that testosterone signaling is required in multiple somatic cell types for spermatogenesis to proceed and for male fertility to be maintained. Overall, it is clear that testosterone released from Leydig cells acts directly upon other somatic cell types, thus indirectly affecting germ cells and promoting spermatogenesis.

In addition to effects on spermatogenesis during adulthood, testosterone from Leydig cells also may act upon their own progenitors within the fetal interstitial mesenchyme. Two studies of fetal Leydig cell development have revealed that there is feedback between testosterone from differentiated Leydig cells and undifferentiated progenitor cells, the latter of which strongly express AR (Defalco et al., 2013; Kilcoyne et al., 2014). In one report, it was shown that testosterone administration during early postnatal stages reduces Notch signaling and increases the number of Notch-active progenitor cells in the interstitium (Defalco et al., 2013). Another study demonstrated that testosterone reduction via Ar mutation in mice or dibutyl phthalate-induced reduction in intratesticular testosterone in rats significantly reduced adult Leydig progenitor numbers, and proposed that progenitor cells were affected by epigenetic mechanisms that could be perpetuated throughout life (Kilcoyne et al., 2014).

Igf1

A critical mitogen that has multiple developmental roles, including in male reproduction, is insulin-like growth factor 1 (IGF1) (Cannarella et al., 2018; Griffeth et al., 2014). While the source of locally secreted IGF1 was up until recently unclear, single-cell RNA-Seq analyses of mouse and human testes have shed light on testicular Igf1-expressing cells. Igf1 was mainly expressed by interstitial and Leydig cells, whereas Igf1r, which encodes the receptor for IGF1, was expressed in most testicular cell types earlier in development, but mainly in spermatogonia among adult germ cells (Green et al., 2018; Guo et al., 2018; Hermann et al., 2018; Neirijnck et al., 2019). Mice with a conditional deletion of Igf1r and the related insulin receptor (Insr) in germ cells, using a Neurogenin3-Cre (Ngn3-Cre) had normal testicular histology, size, and sperm production (Pitetti et al., 2013), indicating that the insulin/IGF signaling pathway is not required in differentiating, meiotic, and postmeiotic germ cells; however, it is currently unclear whether IGF signaling is required for the spermatogonial stem cell phase of spermatogenesis. Loss of IGF signaling in somatic cells, in contrast, had major negative impacts on male fertility; Sertoli-specific loss of Igf1r and Insr resulted in a reduction of testis size (likely due to reduced spermatogenesis) and significantly disrupted sperm production (Pitetti et al., 2013), while loss of Igf1r and Insr in differentiated Leydig cells resulted in reduced steroidogenic activity in adult Leydig cells (but not in fetal Leydig cells) and drastically reduced sperm production (Neirijnck et al., 2018).

Csf1

Another potential major contributor to the germline stem cell niche is colony stimulating factor 1 (CSF1). Although CSF1 is traditionally known for promoting differentiation of myeloid immune cells such as macrophages (Jones & Ricardo 2013), CSF1 signaling likely has more diverse roles in development. Microarray studies on mouse postnatal testes, at a stage in which germ cells are enriched for undifferentiated spermatogonia and spermatogonial stem cells, found that Csf1r (the gene encoding the CSF1 receptor) was highly enriched in GFRA1+ or THY1+ cells (Kokkinaki et al., 2009; Oatley et al., 2009). Immunofluorescence analyses of adult mouse testes revealed that CSF1 is expressed by Leydig cells, peritubular cells, and macrophages (DeFalco et al., 2015; Oatley et al., 2009), although recent single-cell RNA-Seq analyses suggest that Csf1 mRNA is mostly expressed by Leydig and peritubular cells in the adult mouse and human testis (Green et al., 2018; Hermann et al., 2018). Addition of CSF1 in vitro to primary cultures of undifferentiated spermatogonia or SSC cell lines resulted in increased SSC proliferation or self-renewal (Kokkinaki et al., 2009; Oatley et al., 2009), revealing a potential contribution of CSF1 to the SSC niche. If and when CSF1 signaling is functionally required during germ cell development in vivo, however, remains an open question.

Localization of undifferentiated spermatogonia

A major outstanding question in the spermatogenesis field is how to define the SSC niche. In the mouse adult testis, there are potentially several thousand to 35,000 SSC niches (Shinohara et al., 2001; Tegelenbosch & de Rooij 1993), so it has proved difficult to determine where/how these niches are physically located in the testis. Upon initial examination of the localization of SSCs, which are numerous in the testis and spread throughout the organ, it might seem as if SSCs are randomly distributed within seminiferous tubules. However, systematic and morphometric analyses by Chiarini-Garcia et al. revealed a non-random distribution of undifferentiated (type A) spermatogonia within seminiferous tubules in both mice and rats (Chiarini-Garcia et al., 2001; Chiarini-Garcia et al., 2003). Specifically, these studies demonstrated that undifferentiated spermatogonia preferentially localized to regions of the tubules that directly contact interstitial cells on the other side of the tubule basement membrane versus direct contact with other tubules without intervening interstitial tissue; in the same manner, hamster undifferentiated spermatogonia also localize adjacent to interstitial regions, during the active mating season in a photoperiod-dependent manner (do Nascimento et al., 2009). Interestingly, this bias in spermatogonial localization toward interstitial regions in all 3 rodent species was dependent on the stage of spermatogenesis of the seminiferous tubule. Leydig cells could be involved in this localization, as several studies have reported that Leydig cell structure is dependent on the spermatogenic stage of neighboring seminiferous tubules in human and rat (Bergh 1982; Bergh 1983; Paniagua et al., 1988), suggesting that there could be potential paracrine communication between germ cells and Leydig cells. Further research is needed in this area, although other cell types (such as vasculature) are likely also involved in this process and are discussed below.

Feedback between Leydig cells and Sertoli cells

In addition to impacts on spermatogenesis, Leydig cells and their secreted factors, such as testosterone and IGF1, also have direct impacts on Sertoli cell development. Both SCARKO mice and mice with Sertoli-specific deletion of Igf1r/Insr have disrupted Sertoli function and/or reduced Sertoli cell number (Chang et al., 2004; De Gendt et al., 2004; Pitetti et al., 2013), indicating that properly functioning Leydig cells are critical for Sertoli cell development and male fertility. Conversely, Sertoli cells are required for Leydig cell development, not only in fetal stages, such as through Sertoli-derived secretion of PDGF and DHH ligands (Brennan et al., 2003; Yao et al., 2002), but even in adult stages. A cell-specific genetic ablation of adult Sertoli cells or fetal Sertoli cells resulted in reduced Leydig cell numbers (Rebourcet et al., 2017; Rebourcet et al., 2014a; Rebourcet et al., 2014b), suggesting that there is an ongoing interdependence between Leydig and Sertoli cells throughout life.

Influence on testicular macrophages

Leydig cells and testicular macrophages share the interstitial compartment of the testis, and several observations suggest that there is extensive and intimate crosstalk between the two cell types. Ultrastructural analyses in rat testes demonstrated that testicular macrophages and Leydig cells share unique intercytoplasmic digitations that physically connect the two cell types, which only arise upon puberty (Hutson 1992). CSF1, which is at least partly produced in the testis by Leydig cells, is also known to regulate cells of the phagocytic mononuclear lineage, and is directly correlated to macrophage number (Stanley et al., 1983; Wiktor-Jedrzejczak et al., 1990; Wiktor-Jedrzejczak et al., 1982), and thus may indirectly influence male fertility through this macrophage interaction.

Along with Leydig-produced CSF1, testosterone also regulates testicular macrophage function. Administration of testosterone in vitro to macrophages results in diminished production of pro-inflammatory cytokines by macrophages (Rettew et al., 2008), and maintains a negative feedback loop by reducing the production of 25-hydroxycholesterol, a precursor to testosterone and stimulant for LC steroidogenesis, by macrophages (Lukyanenko et al., 2002). This indicates that in addition to their androgen production, Leydig cells may also contribute to maintained fertility by regulating an immunoprivileged niche for spermatogenesis via regulation of testicular macrophages. Consistent with this intimate relationship, the depletion of either testicular macrophages or Leydig cells has negative consequences on the development of the other cell type, during both developmental and regenerative contexts (Gaytan et al., 1994a; Gaytan et al., 1994b; Gaytan et al., 1994c; Wang et al., 1994). Therefore, there is likely a direct or indirect regulation of testicular macrophages by Leydig cells and vice versa.

Influence on peritubular cells

In addition to promoting peritubular cell differentiation via production of testosterone, Leydig cells also influence seminiferous tubule contractions by regulating peritubular cell muscular function. Leydig cells produce the peptide hormone vasopressin (Ivell et al., 1992), which has been demonstrated to induce contractions in rat peritubular cells, along with endothelin 1 (Tripiciano et al., 1996). Leydig cells also produce oxytocin (Nicholson & Hardy 1992; Nicholson et al., 1987; Yeung et al., 1988), which is a potent inducer of contraction in multiple tissues within the male reproductive tract, including seminiferous tubules (Thackare et al., 2006). Oxytocin, through its contraction-inducing activity on peritubular cells, is required for effective spermiation and sperm transfer from the testis to the epididymis in mice (Assinder et al., 2002). Although oxytocin is also produced in the human testis (Frayne & Nicholson 1998), comparative studies suggest that there are variable requirements among different mammals for testicular oxytocin and tubule contraction during sperm transport (Ellis et al., 1978; Ellis et al., 1981). Additionally, prostaglandins (in particular prostaglandins E2 and F2 alpha), which are also produced by Leydig cells in rats (Haour et al., 1979), may play a role in peritubular cell contractions to move sperm and testicular fluid to the epididymis in both rodents and humans (Ellis et al., 1981; Maekawa et al., 1996; Rey-Ares et al., 2018; Tripiciano et al., 1998; Welter et al., 2013; Yamamoto et al., 1987). In general, it seems that Leydig-produced extrinsic factors help drive peritubular cell contractility to promote sperm transfer and ensure fertility.

Macrophages and other immune cells

Direct influence on testicular function

Initial studies using Csf1-mutant mice, in which macrophages throughout the body are severely depleted, revealed numerous defects in male fertility (Cohen et al., 1996), suggesting that testicular macrophages are important for male reproductive functions. However, it was unclear whether these impacts on fertility were due to local impacts in the testis or whether it was largely due to disruptions in the hypothalamic-pituitary-gonadal hormonal axis. Local depletion of testicular macrophages in a single testis via clodronate liposomes resulted in reduced Leydig cell testosterone secretion (Bergh et al., 1993), indicating that testicular macrophages regulate Leydig cell function locally.

In addition to regulating Leydig cell function, testicular macrophages may also locally regulate other aspects of spermatogenesis. Upon genetic macrophage ablation in the adult mouse testis, spermatogonial number was reduced, likely due to changes in proliferation or defects in differentiation, while spermatogonial stem cells were unaffected (DeFalco et al., 2015), suggesting that macrophages may have critical roles in spermatogonial development. However, it still remains unclear if these defects were due to direct macrophage-germ cell crosstalk or are mediated indirectly through Sertoli or other cells. As a proposed mechanism for how testicular macrophages regulate spermatogonial proliferation or differentiation, it was reported that CSF1 and retinoic acid (RA) synthesis enzyme expression were disrupted after macrophage ablation (DeFalco et al., 2015). Although macrophages likely do not express Csf1 mRNA, they may regulate or sequester CSF1 at the protein level via CSF1R and release it in a specific manner to regulate germ cell behavior; it is known that macrophages can regulate levels of CSF1 in circulation through CSF1R-mediated endocytosis (Bartocci et al., 1987).

RA induces the differentiation of spermatogonia, as well as entry into meiosis (Hogarth & Griswold 2010), which are necessary steps in spermatogenesis, and testicular macrophages express the RA synthesis enzymes ALDH1A2 and RDH10 (DeFalco et al., 2015). The disruption of spermatogenesis at the differentiating spermatogonia stage in macrophage-ablated testes is similar to RA-depleted animals, suggesting that RA synthesis may be impaired in the absence of testicular macrophages. Whether or not macrophages are functionally required for proper RA synthesis in the testis is currently unclear. While the molecular mechanisms need to be elucidated, testicular macrophages appear to contribute to the regulation of adult spermatogenesis and testicular homeostasis.

Influence on Leydig cells

As previously mentioned, macrophages have an intimate relationship with Leydig cells, and they interact with each to maintain a spermatogenic environment. Their close communication is easily observed in their physical interaction, with intracytoplasmic digitations linking the two cell populations. While the influence of Leydig cells on testicular macrophages has been previously discussed here, there is reciprocal regulation of Leydig cell function by macrophages. Testicular macrophages have been shown to produce 25-hydroxycholesterol, which stimulates Leydig cell differentiation and steroidogenesis (Chen et al., 2002; Lukyanenko et al., 2001; Nes et al., 2000). Other factors secreted by testicular macrophages, such as interleukin-1 (IL-1) and transforming growth factor alpha (TGFa), promote Leydig cell proliferation (Khan et al., 1992a; Khan et al., 1992b). Conversely, testicular macrophages are also capable of negatively regulating steroidogenesis. Production of pro-inflammatory cytokines and reactive oxygen species (ROS) by testicular macrophages can inhibit Leydig cell function and reduce testosterone production (Calkins et al., 1988; Kostic et al., 1998).

Specialized immune regulation

Though testicular macrophages are similar to other tissue-resident macrophages in their chemotactic and phagocytic roles, testicular macrophages have a unique need to balance this immune role with a tolerance to germ cells in order to maintain an immunoprivileged niche in which spermatogenesis can occur. Indeed, there have been several documented occurrences of male infertility resulting from testicular macrophages acquiring immunity to germ cells (McLachlan 2002). Consistent with these findings, testicular macrophages have an altered cytokine profile and immune-responsiveness compared to other macrophage classes (Kern et al., 1995), such as reduced expression of pro-inflammatory cytokines. Recently, studies in rat have revealed that testicular macrophages produce corticosterone, the major corticosteroid produced in rodents, and corticosterone from testicular interstitial fluid has the ability to polarize macrophages to an M2-like phenotype (Wang et al., 2017).

Meiotic cells arise in the testis after immune tolerance is established; therefore, neo-antigens that appear in meiotic and post-meiotic cells are potentially auto-immunogenic. The previous consensus in the field proposed that all meiotic neo-antigens are uniformly immunogenic are sequestered from the immune system via the Sertoli cell blood-testis barrier in seminiferous tubules. However, a recent study demonstrated that a select group of neo-antigens egressed from the seminiferous tubules, and were able interact with systemically injected antibodies and form local immune complexes outside blood-testis barrier (Tung et al., 2017). It was proposed that these select antigens were derived from cell fragments discarded by spermatids during the spermiation process and exited Sertoli cells as cargo in residual bodies. These antigens maintained T-regulatory (Treg)-cell-dependent physiological tolerance of these antigens. This Treg-mediated tolerance may be maintained by macrophages, a subset of which resemble antigen-presenting cells due to strong expression of major histocompatibility complex II (MHCII) (DeFalco et al., 2015). These complex mechanisms help the testis maintain a balance between tolerance for potentially auto-immunogenic germ cells and the ability to fight infection.

Fetal testis vascularization and morphogenesis

While the roles of adult testicular macrophages in Leydig function and immune regulation have been appreciated for decades, until recently not much was known about the role of macrophages in the fetal testis. Yolk-sac derived macrophages are observed throughout the mouse embryo as early as E9.5-E10.5 (Schulz et al., 2012), as well as in the human gonad and genital tract as early as 4 weeks (Gurevich et al., 2001), suggesting they may be important in early gonad development. An analysis in mouse revealed that yolk-sac-derived macrophages were already present in the fetal gonad at E10.5 and were preferentially localized near nascent mesonephric vasculature (DeFalco et al., 2014). A functional analysis was performed, using a genetic cell-ablation technique, to assess any requirements for macrophages in the fetal testis. In the absence of macrophages, the fetal testis showed disruptions in male-specific vascular remodeling (see below) and aberrant testis cord architecture (DeFalco et al., 2014). Consistent with findings in other tissues and organs (Stefater et al., 2011), these studies revealed that macrophages are a critical player in fetal organogenesis and are required for proper testicular morphogenesis.

Vasculature-associated Cells

Endothelial cells

The vascular system is traditionally known for its essential roles in supplying oxygen and metabolites for tissue growth and maintenance. However, studies over the past several decades have provided new and significant insights into the developmental roles of blood vessels in organogenesis (Crivellato et al., 2007). Seminal studies by several labs revealed that endothelial cells have instructive roles, particularly in the differentiation of progenitor populations and the maintenance of those cells within niches in tissues such as liver, pancreas, and nervous system (Lammert et al., 2001; Matsumoto et al., 2001; Shen et al., 2004). In the testis it has also been shown that blood vessels likely play key roles in organogenesis and organ homeostasis.

Localization of undifferentiated spermatogonia

A three-dimensional reconstruction of adult mouse seminiferous tubules by Yoshida and colleagues showed that Ngn3-GFP-expressing cells, which represent a wide range of undifferentiated spermatogonia, were localized almost exclusively in regions of the tubule that bordered vascularized interstitial regions (as opposed to bordering other tubules without any intervening interstitium) (Yoshida et al., 2007). These findings suggested that there was an interstitial/vascular niche for undifferentiated spermatogonia, but the specific cell type that was responsible for localization of undifferentiated spermatogonia was not determined in that study. As an apparent follow-up to this study, it was proposed that lymphatic CD34+ endothelial cells are critical for stem cell homeostasis through their secretion of FGF ligands (which are in a limited supply), in a “mitogen competition” paradigm (Kitadate et al., 2019). Recently, a study proposed that testicular endothelial cells are an essential part of the SSC niche, whereby endothelial cells produce GDNF and other secreted factors that promote SSC maintenance (Bhang et al., 2018).

One apparent paradox between an interstitial-associated niche and maintenance of stem cells is the observation that tissue stem cells in vivo are normally maintained in a hypoxic environment (Mohyeldin et al., 2010), but testicular interstitial regions are vascularized and are likely the most highly oxygenated regions of the organ. Id4-GFP-positive cells in the adult testis, which are enriched for stem cell activity and represent a large subset of SSCs, were found to be localized in regions of tubules that are not adjacent to interstitial vascularized tissue (Chan et al., 2014), which suggests that the SSC population is distributed among avascular niches. One possible explanation is that the “true” SSC niche is avascular and hypoxic, but once SSCs transition into transit-amplifying undifferentiated spermatogonia, they home towards vascularized regions, where they can gain increased access to essential secreted factors released from the interstitium and vasculature (e.g., growth factors, cytokines, and hormones) that are required once cellular metabolism increases. Consistent with this idea, recent RNA-Seq studies of human and mouse spermatogenic cells indicate that the most naïve SSC-like cells in the adult testis are not highly proliferative and only enter active cell cycle upon transitioning into more differentiated cell states (Guo et al., 2018; Hermann et al., 2018).

Vegf and vascular development in utero

The vascular endothelial growth factor (VEGF) pathway is a critical developmental pathway for angiogenesis and vascular development (Koch & Claesson-Welsh 2012). Vascularization is one of the first hallmark dimorphisms of the testis, in which only XY gonads undergo vascular remodeling to form a new coelomic arterial network (Brennan et al., 2002; Coveney et al., 2008). This new arterial network is generated via breakdown of the existing mesonephric vascular plexus, which allows freed individual endothelial cells from the plexus to migrate into the testis and reassemble into new testicular arteries. VEGF signaling is required for this testis-specific neo-vascularization event, as VEGF signaling inhibitors result in a hypo-vascularized testis (Bott et al., 2006; Cool et al., 2011; DeFalco et al., 2014). As a result of blocking VEGF-mediated vascular remodeling, testis cords do not form, and instead Sertoli cells remain as a disorganized group of cells (Bott et al., 2006; Cool et al., 2011), although Sertoli cells maintain their proper fate identity and Leydig cells still develop (Cool et al., 2011). Additionally, endothelial cell-cell junctions are critical for this vascular-mediated morphogenesis process, as CDH5 (VE-Cadherin) blocking antibody treatment ex vivo also resulted in disrupted testis cord morphogenesis (Combes et al., 2009). Therefore, blood vessel remodeling and vascularization are required for cord morphogenesis in the fetal testis.

Blood vessels, perivascular cells, and Leydig cell development

In addition to testicular blood vessels being required for testis cord morphogenesis, they are also presumably a critical conduit for hormone export into the rest of the body, which is required for virilization of the brain and reproductive tract. Blood vessels are located in the interstitial compartment adjacent to developing Leydig cells, but any links between vascular and Leydig development were not examined until recently. In particular, Leydig progenitors are especially important in male sexual development, since differentiated Leydig cells do not divide (Miyabayashi et al., 2013; Orth 1982); therefore, the increase in Leydig cell numbers during fetal and pubertal development is dependent on an interstitial stem/progenitor pool that must be maintained (Chen et al., 2010). Kumar and DeFalco recently found that blood vessels are required to maintain Leydig progenitors in the fetal testis (Kumar & DeFalco 2018). They describe perivascular mesenchymal cells that are Nestin-positive, similar to stem/progenitor cells in other organs (Bernal & Arranz 2018), and they used lineage-tracing techniques to determine that fetal testicular perivascular cells are a multipotent progenitor population. Nestin-positive perivascular cells failed to be maintained in the absence of testicular vasculature, as they precociously and excessively differentiated into Leydig cells. It was further shown that perivascular cells undergo active Notch signaling and Notch ligands from blood vessels are likely the critical signals that were required to maintain perivascular progenitors in the fetal testis (Defalco et al., 2013; Kumar & DeFalco 2018). Therefore, blood vessels form a critical cellular constituent of the fetal Leydig/interstitial progenitor niche.

Vegf in adult testicular function

While it is normally associated with vascular development and angiogenesis, the VEGF pathway has been recently implicated in adult testicular function; however, its role in the adult testis is likely not mediated via vascular mechanisms. Expression of VEGF receptors was reported in adult germ cells, and Cupp and colleagues assessed the role of VEGF signaling in spermatogenesis (Caires et al., 2012; Lu et al., 2013; Sargent et al., 2016). In stem cell transplantation assays, the treatment of perinatal germ cell donors with pro- or anti-angiogenic affected stem cell activity, whereby seminiferous tubules showed less SSC colonization following transplantation with cells from VEGFA-165b-treated donors versus VEGFA-164-treated donors. The findings suggested that anti-angiogenic isoforms of VEGFA reduced SSC number or activity either by promoting premature differentiation, causing apoptosis, or by inhibiting SSC establishment, while pro-angiogenic VEGFA promoted SSC self-renewal. The authors proposed that the different isoforms, and their counteracting activities on germ cells, balance each another out to maintain SSC number and homeostasis. In these studies, there were no reported impacts on vascular development, suggesting that VEGF may act directly on germ cells and not through angiogenic mechanisms (Caires et al., 2012; Lu et al., 2013; Sargent et al., 2016).

Feedback between vasculature and other testicular somatic cells

In addition to providing a link between systemic and local factors in the testis, testicular blood vessels may also be important intercellular mediators for communication between different somatic cell types. A recent study proposed that there is feedback between Sertoli and vascular cells that is important for testicular hormonal function. When Sertoli cells, and not germ cells, were specifically ablated in the adult testis, there was a reduction in total testicular vascular volume, vascular branch number, and small microvessel number. The authors observed that the resultant disturbed testicular vasculature led to reduced fluid exchange between the vasculature and the interstitial compartment. The reduced fluid exchange subsequently reduced circulating testosterone levels and suggested that there was impaired Leydig cell stimulation and/or reduced release of testosterone into the vasculature. This study proposed a new idea in which the transport of interstitial secreted factors may be influenced by Sertoli cells and that there is an intimate endocrine relationship between vascular cells and other somatic cells (Rebourcet et al., 2016).

Peritubular cells

Physical support of spermatogenesis

Peritubular cells, often referred to as peritubular myoid cells, are smooth-muscle-like cells that, in laboratory rodents, form a single layer surrounding the seminiferous tubules (Maekawa et al., 1996). Peritubular cells are functionally important for fertility, as they provide structural support for the seminiferous tubules wherein spermatogenesis takes place. Given their smooth-muscle-like identity, e.g., expression of smooth muscle alpha actin, they are responsible for peristaltic action during copulation and movement of sperm through the tubules (Clermont 1958; Maekawa et al., 1996; Tung & Fritz 1990). In addition to regulation of contractility via Leydig-produced hormones (see above), there is also intrinsic regulation of contractile function, as it was proposed that the transcription factor GATA4 has an inhibitory influence on peritubular cell contraction, mediated via regulation of genes belonging to the smooth muscle contraction pathway (Wang et al., 2018). In this physical (contractile) aspect of testicular function, peritubular cells are essential for male fertility, and regulating this feature of peritubular cells was even once considered as a male contraceptive strategy (Romano et al., 2005). However, as described below, peritubular cells have additional roles in the testis that are critical for male fertility apart from their smooth-muscle-like contractile function.

Androgenic regulation of Sertoli and Leydig cells

The involvement of peritubular cells in spermatogonial regulation is less well-understood than the other interstitial populations. However, several studies have uncovered their importance for maintaining male fertility. The loss of peritubular cells, in a drug-induced rat model, has been reported to be detrimental to normal spermatogenesis (Franca et al., 2000), revealing a critical presence for peritubular cells in the testis. Since peritubular cells exhibit expression of AR, it was proposed that they influence spermatogenesis through this molecular pathway. Indeed, peritubular cells in culture secrete a yet-unidentified protein in response to androgen stimulation that was capable of regulating Sertoli cell function, which was discovered several decades ago (Skinner & Fritz 1985). Studies investigating conditional knockout models of Ar in peritubular cells show that adult mutant mice exhibit reduced testis size, smaller sperm count, and infertility. Further examination revealed aberrant seminiferous epithelium formation and reduced numbers of all germ cell types. Sertoli cell function is also seemingly influenced by the androgen pathway in peritubular cells, as seminal fluid production, tubule diameter, and Sertoli-specific gene expression are all reduced in mutant mice, and individual Sertoli cells are capable of supporting fewer germ cells. Sertoli cells also displayed aberrant localization, with many nuclei distant from the basement membrane, indicating that androgen signaling may be one way in which peritubular cells mediate seminiferous tubule structure (Welsh et al., 2009; Zhang et al., 2006).

In addition to regulation of Sertoli function by peritubular cells, mouse studies have demonstrated that AR signaling by peritubular cells is also critical for Leydig cell function. Peritubular-specific Ar mutant testes showed defective Leydig differentiation and ultrastructure (Welsh et al., 2012), indicating that there is paracrine regulation of Leydig cells by peritubular cells. Defects in peritubular-specific Ar mutants, which are likely in multiple cell types, lead to poor testosterone regulation, suggestive of decreased Leydig cell activity, and which ultimately leads to azoospermia and infertility (Welsh et al., 2009).

Gdnf

GDNF is a critical factor for maintenance of spermatogonial stem cells, as Gdnf mutants lose fertility with age due to loss of undifferentiated spermatogonial populations (Meng et al., 2000). It was assumed that Sertoli cells were the main producer of GDNF in the adult testis, but studies proposed that peritubular cells also produced GDNF, first in humans (Spinnler et al., 2010), and then in mice (Chen et al., 2014). This additional source of GDNF was critical for maintenance of SSCs in mice, as peritubular-specific conditional deletion of Gdnf resulted in an age-dependent loss of spermatogenesis and fertility, consistent with a failure to maintain spermatogonial populations (Chen et al., 2016). These findings indicate that peritubular cells are a critical cellular component of the SSC niche, whereby they produce GDNF in response to testosterone and can support mouse SSC self-renewal in culture (Chen et al., 2014) and in vivo (Chen et al., 2016).

Pedf/Serpinf1

A major role of peritubular cells is to physically separate the tubular compartment from the interstitial compartment. One specific aspect of this role is to maintain avascularity of the seminiferous tubule, which is likely critical for maintenance of testicular structure, the BTB, and spermatogenesis. It was shown in the human testis that peritubular cells express pigment epithelium derived factor (PEDF; also called SERPINF1) (Flenkenthaler et al., 2014; Windschuttl et al., 2015), which is a potent anti-angiogenic factor (Dawson et al., 1999; Fernandez-Garcia et al., 2007). Secretome analyses revealed that human peritubular cells expressed a wide array of angiogenic and anti-angiogenic factors, and pathway analyses indicated that the peritubular secretome was significantly enriched for proteins related to vasculature development (Flenkenthaler et al., 2014). Co-culture assays using human peritubular cells and human umbilical vein endothelial cells (HUVECs) revealed that HUVECs were repulsed by HTPCs, and this repulsive activity was mediated via a PEDF-dependent mechanism (Windschuttl et al., 2015). These findings in the human testis suggest that peritubular cells are a critical regulator of testicular blood vessels by preventing vascularization within seminiferous tubules.

Mediators of testicular inflammation and infertility?

Given that peritubular cells are located at the interface of the interstitial and tubular compartments of the testis, lying immediately atop the basement membrane of the seminiferous tubules, they are uniquely positioned to play critical roles in testicular function. This unique aspect of peritubular cells is likely accentuated even more in humans, which have a multi-cell-layered peritubular wall (Mayerhofer 2013), as opposed to the single-cell-layered wall in rodents. Interestingly, there are unique cellular phenotypes between human peritubular inner and outer layers, in which inner layers had a smooth muscle phenotype, while the outer layers presented more of a connective tissue or fibroblast-like identity (Davidoff et al., 1990). Studies of human infertile patients have revealed a consistent observation of altered peritubular cell structure and identity, in which peritubular cells display increased ECM and fibrosis, as well as a decrease in expression of smooth muscle contractility markers (Haider et al., 1999; Schell et al., 2010; Welter et al., 2013). Additionally, there are increased numbers of immune cells, such as mast cells and macrophages, in the peritubular wall of testes from infertile men (Frungieri et al., 2002; Meineke et al., 2000). In particular, the number of tryptase-positive mast cells was positively correlated with the amount of fibrosis and thickness of peritubular walls of seminiferous tubules in infertile men (Meineke et al., 2000). In all, these findings suggest that peritubular cells may represent an intermediary between the seminiferous tubule, inflammatory immune cells in the interstitium, and deleterious fibrosis of the tubule wall. Further research into the role of peritubular cells and their interactions with other testicular cell types is warranted and will likely lead to new insights regarding the etiology of male infertility.

CONCLUSIONS

Most studies of sex determination in the early post-Sry era focused on Sertoli cells as the central player in testicular development and function. While Sertoli cells are undoubtedly critical for testicular differentiation, studies in the last decade have revealed that interstitial cells are not merely passive players in male reproductive biology, but rather have active and essential roles in promoting organ function and male fertility. The interstitial compartment of the testis contains several different cell types, and recent research has provided new insights into how these cell types interact with one another to drive spermatogenesis and male sexual differentiation. Vasculature, Leydig cells, immune cells, and peritubular cells are all responsible for orchestrating testicular activities both in the interstitial compartment (steroidogenesis and immunity) and tubular compartment (gametogenesis). At both the local and systemic level, interstitial cells are critical links between the immune system, the reproductive system, and overall health.

There are several areas of research that will likely be fruitful in the future, including: defining the molecular signals and cell types responsible for SSC niche localization and function in the adult testis; identifying how vasculature promotes male-specific differentiation both in utero and throughout life; elucidating how the testis balances tolerance and immune response in a unique immunological environment that must sustain spermatogenesis; and examining the diverse roles of peritubular cells in adult testis biology. Efforts into these areas of research will help us understand the complex interactions that underpin male sexual development and fertility, and will, ultimately, inform ongoing efforts for male contraception and for treatments for male factor infertility.