Two waves of Sertoli cell proliferation are known to occur throughout its lifespan: firstly, during late fetal and early neonatal life dividing more than fivefold, and secondly just before puberty in which Sertoli cells grow more than twofold (44, 49). Androgen receptor (AR) expression in Sertoli cell signifies maturation of the cells after proliferation and their responsiveness to androgen during spermatogenesis (57). Sertoli cells acquire AR expression post-differentiation, at the onset of puberty (19). Willems et al. underlined the importance of AR in Sertoli cell using AR ablation models which caused abnormal Sertoli cell maturation and germ cell development (58). Sertoli cell androgen receptor knockout (SCARKO) model studies further showed AR importance in Sertoli cell maturation, demonstrated by increased permeability of the blood-testis-barrier (BTB), and evidence of a cell-autonomous activation of AR, important for normal Sertoli cell function (57, 58).

Sertoli cells undergoing differentiation from fetal into a more mature stage can be identified by specific markers and transcription factors such as FSH, AMH, SRY (male gonad), NR5A1 (SF1), GATA4, WT1, SOX9, and DMRT1. During Sertoli cell development, NR5A1, GATA4, and WT1 are seen as major transcription factors important in directing fetal somatic cells to fetal Sertoli cell fate (59, 60). SOX9 is known to be expressed throughout the lifetime of a male rat, albeit more strongly in the prenatal period, followed by a large decrease after birth (61). Nonetheless, SOX9 likely plays a significant role in adult germ cell differentiation as it is still expressed in adult Sertoli cells. DMRT1 has been shown to be important for testis differentiation in most mammalian species (51). Deleting murine DMRT1 causes Sertoli cells to over-proliferate in the testis as well as induces germ cell death, possibly related to a defect in Sertoli cells.

The role of AMH in Sertoli cell fate has been thoroughly investigated. Based on data collected from mice in the late perinatal period (PND 0-7), AMH was found to differentially regulate Sertoli cell development depending on its concentration (62). Indeed, AMH at low concentrations promoted Sertoli cell proliferation, whereas high concentrations of AMH induced apoptosis. The expression of proapoptotic proteins was upregulated, including cleaved caspase-3 and Bax in the presence of AMH with a decrease of the anti-apoptotic protein Bcl-2. In the presence of higher AMH expression, stem cell factor (Scf) which regulates Sertoli cell development, was also increased. The authors concluded that the timely expression of AMH and its receptor AMHRII in spermatogenesis may act in a paracrine or autocrine manner during testicular development. More recently, AMH secreted by Sertoli cells was shown to play a structural role in the formation of mouse testis (63). Mice positive for Amh-Cre and Diphtheria Toxin A (DTA) were used to generate offspring with Sertoli cell-ablated AMH, which were found to present disruption of testis cords and loss of germ cells. Mislocalization and reduction of α-smooth muscle actin (SMA)-labeled peritubular myoid (PTM) cells were also observed in Sertoli cell Amh-Cre; DTA mice. While it is known that there are markers to identify Sertoli cells at the mature and differentiated state, questions regarding the dynamic state of Sertoli cell and whether it is terminally differentiated have come up (17, 64).

Spermatogonia differentiation happens alongside the maturation and differentiation of Sertoli cells. The repression of c-KIT expression in gonocytes induces the transition to undifferentiated spermatogonia. Re-establishment of c-KIT expression in undifferentiated spermatogonia leads to transition into differentiated spermatogonia (45, 65). In the presence of KITL expression, a transition from spermatogonial stem cells (SSCs) to differentiated c-KIT-positive spermatogonia occurs. FGF2, glial cell derived neurotrophic factor (GDNF), and CXCL12 are secreted by Sertoli cells that induce a response in undifferentiated spermatogonia to 1) translationally repress c-KIT expression and 2) transition to differentiated spermatogonia (65). GDNF is negatively regulated by NOTCH1 signaling, likely to balance stimulation by FGF2 and FSH in Sertoli cells causing gonocytes exit from quiescence (66). RA secreted by Sertoli cells induces the progression to undifferentiated spermatogonia, a process initially observed in vitamin A-deficient rats and mice, with testes unable to proceed with normal spermatogenesis until RA was reintroduced (67). The concept of an RA intermediate produced by Sertoli cells during the transition from gonocytes to differentiating spermatogonia is becoming increasingly accepted as it was only confirmed within the last decade, based on paracrine communication between Sertoli cells and the transitioning gonocytes (45).

The mature Sertoli cell is known for its impact on tubular architecture, peritubular myoid cell fate, and adult LC number (68). Gap junctional protein (GJA1) which is also known as Connexin 43 (CX43) is vital in Sertoli cell differentiation, but not for spermatogonia cell maintenance/self-renewal (69). Recently, mTOR signaling was shown to be involved to during PI3K/Akt signaling of spermatogenesis and regulates GJA1 within Sertoli cells (70).

Sertoli cells and the formation of the BTB create a significant defense mechanism in the testis (71–73). The initial formation of the BTB occurs during puberty and two distinct sections form, based on a division of the seminiferous epithelium by tight junctions (TJs) along the basal region of Sertoli cells into adluminal and basal compartments (1). The BTB require CX43 (or GJA1) expression to maintain normal development and regulation of its dynamics (69, 74). Adult Sertoli cells lie at the basal lamina of seminiferous epithelium, in which BTB are comprised of not only TJs but also of basal ectoplasmic specializations (ES), desmosomes, and gap junctions (GJs) (72). Occludin, claudins-3, -5, -11, zonula occludens (ZO) -1, -2, and -3 and junction adhesion molecules A and B (JAM-A and JAM-B, respectively) are some of the major junction proteins which bridge communication across the BTB (75). Occludin and claudin-11 junction proteins are most important in the maintenance of barrier integrity (1). Androgens not only play a major role in Sertoli cell maturation but also mediate the formation of newly formed Sertoli cell TJs by regulating Claudin-3 expression and maintaining TJs structure, possibly involving Claudin-13, and noncanonical tight junction protein 2 isoform (Tjp2iso3) (76).

The BTB is absent in perinatal and juvenile periods, although some of its junctional proteins are already observed in low levels at these ages, but not organized as they become during puberty to form the BTB, which provides germ cell protection from immune cells (1). Studies elucidating the compartmentalization of immune cells from auto-immunogenic cells, ultimately showed that germ cells are sequestered from immune cells including macrophages, T cells, and dendritic cells via one (rodent) or more (human) layers of peritubular myoid cells (72, 77). Therefore, a tight control of keeping auto-immunogenic germ cells physically away from immune cells is established by the selective BTB between Sertoli cells and myoid cells (72). Altogether, the studies cited above depict the BTB as a plastic and highly dynamic structure, where the interaction between signaling molecules such as the focal adhesion kinase FAK and the SRC Proto-Oncogene, Non-Receptor Tyrosine Kinase (SRC) kinase, their regulation and interaction with TJ proteins and actin filaments, participate to the functional remodeling of the BTB, allowing the movement of germ cells, while insuring its essential barrier function.

Immune modulation by Sertoli cells contributes to the immune privilege of the adult testis (72). Recent review of Sertoli cell contributions to the immune privileged testis by Qu et al. (2020) include inter-Sertoli cell junctions, Fas ligand, Activin, IGF-1, epidermal growth factor, TGFβ, and lipocalin (78). Induction of regulatory T cells (Tregs) by Notch/JAGGED1 signaling and stimulation of peripheral conversion of CD4⁺FOXP3^- T cells to functional CD4⁺FOXP3⁺ Tregs in the presence of mouse Sertoli cell conditioned medium has been shown (79). Most recently, Kaur et al. (2020) used neonatal pig Sertoli cells to suggest a Sertoli cell immunosuppression mechanism via upregulated CD4 and CD8 Tregs and other immunoregulatory factors to improve survival of xenografts (80).

Due to their ability to produce immunomodulatory factors and maintaining the immune privilege status of the seminiferous tubules, Sertoli cells have recently been successfully used to provide immune protection to pancreatic islets xenotransplants and proposed as novel therapeutic tools in a number of diseases (81), broadening the scope of these cells beyond reproduction.

Cadmium is a chemical encountered by humans via occupational and environmental exposures. Cadmium-induced testicular injury was first observed in 1957 by Parizek and is known for its effects as an endocrine disruptor (85, 88). The effects of cadmium on Sertoli cell BTB integrity have been thoroughly investigated (84). Cadmium was found to induce human fetal germ cell apoptosis in organ cultures treated for 4 days with 1 and 10 μM Cadmium, likely via direct effect on germ cells, since neither fetal Leydig cell-produced testosterone nor the number of AMH-positive Sertoli cells were affected, suggesting that human fetal Leydig and Sertoli cell functions were not impaired by Cadmium (89). However, an in vivo study examining the effects of cadmium on fetal rats to a single low dose of Cadmium, showed that fetal Leydig cells and Sertoli cells were affected by Cadmium, as shown by reduced testosterone production, lower Leydig cell numbers, and decreased protein expression of several Sertoli cell markers, including desert hedgehog (DHH), AMH, and follicle-stimulating hormone receptor (FSHR) in fetal testes (90). These divergent data could be due to the difference between using in vivo vs organ culture models, or to the fact that fetal rat testis is more sensitive to Cadmium than fetal human testis. Such difference between species is reminiscent of a study comparing the effects of phthalates on human and rat fetal testis xenografts, in which rat Leydig cells were sensitive to phthalates, but not human Leydig cells (91).

The adverse effect of Cadmium on Sertoli cells is demonstrated by its disruptive effects on the BTB (84, 85). Several signaling molecules playing a role in BTB that are disrupted by Cadmium have been identified, including MAPK proteins i.e. ERK1/2 and MEK1/2 and focal adhesion kinase (FAK) proteins, and their phosphorylation status, important in regulating signaling to TJ proteins occludin and ZO-1 (84). More recently, Zhou et al. found that, in adult mouse testes, autophagy in Sertoli cells is crucial for mediating protection against Cadmium-triggered germ cell apoptosis (92). It is noticeable that most studies focus on adult exposure to Cadmium and the mechanisms altered within adult Sertoli cells by Cadmium, while there are minimal studies observing the impact of fetal and/or postnatal exposure to Cadmium on the formation and functioning of BTB later in life, which would suggest a Cadmium-driven injury of developing rather than mature Sertoli cells. Such delayed effect could be the result of either a direct effect of Cadmium on immature Sertoli cells, as suggested by the studies on fetal testis mentioned above, or the disruption of another testicular function affecting indirectly Sertoli cells.

BPA is an estrogenic EDC suggested to have detrimental reproductive effects following fetal exposure. Most human exposure to BPA occurs via housewares made of polycarbonate plastics, medical devices, and packaging. BPA exposure during fetal development has been shown to inhibit rat Sertoli and Leydig cell differentiation, as shown by the downregulation of AMH production by fetal Sertoli cells and lower testosterone production, indicating that BPA impaired Sertoli and Leydig cell functions, further jeopardizing the development of the male reproductive tract (93). In contrast, organ cultures of GD15.5 fetal mouse testes exposed to BPA for 5 days showed a slight increase in fetal Sertoli cell number and gene markers including Amh, Sox9, and Wt-1, leading the authors to propose that BPA might have positive effects on mouse fetal Sertoli cells. However, fetal germ cell number and gene markers, as well as Leydig cell markers were decreased. Thus, the positive effect on fetal mouse Sertoli cells postulated by the authors might have instead resulted in the disruption of the ratio between Sertoli and germ cell numbers, abnormal levels of Sertoli-produced signaling molecules involved in fetal germ and Leydig cell regulation. In such case, the toxic effect of BPA on mouse fetal testis could be driven in part by an unbalance in factors regulating the communication between the three cell types (94). Exposing explants of rat and human fetal testes to BPA showed that AMH production in fetal Sertoli cells was not affected by BPA exposure, indicating that fetal Sertoli cells were not targeted by BPA, whereas BPA exerted antiandrogenic effects resulting in reduced testosterone production in both species (95, 96). Thuillier and colleagues reported transient alterations of MAPK pathway in neonatal testes following in utero exposure to BPA or genistein, including increased expression of Raf1, and ERK1/2 proteins in Sertoli cells, but these changes were no longer observed in adult testes (97). All together, these data suggest that fetal exposure to BPA might increase mouse fetal Sertoli cell markers or signaling molecules in rat neonatal Sertoli cells but had no effect on human fetal Sertoli cells, suggesting species specific differences in BPA effects.

Several studies looked at BPA effects in immature Sertoli cells. Rossi and colleagues examined the effects of BPA on PND7 mouse primary Sertoli cells, reporting that exposure to 0.5 µM BPA increased the endocannabinoid (eCB) signaling, a family of lipid signal molecules that binds the cannabinoid receptors, previously shown to control spermatogenesis and sperm fertilizing ability (98). Because FSH modulates eCB signaling in immature Sertoli cells, the authors proposed a possible deregulation of FSH signaling by BPA. BPA also increased the production of the Sertoli cell functional marker inhibin B, but did not affect transferrin production, another marker of Sertoli cell function. Zhang and colleagues performed single-cell RNA sequencing (scRNA-seq) analysis of human juvenile testis determining germ cell-somatic cell interactions. They identified maturation markers expressed in Sertoli cells that regulate sperm maturation and reported that BPA affected the testis microenvironment that contributes to germ cell maturation, as a possible mechanism of BPA reproductive toxicity mechanisms (99).

The immature mouse Sertoli cell model TM4 was used to determine whether alternative structural analogs of BPA are safer than the legacy chemical BPA that they intend to replace, using high-content imaging analysis. Morphometric and functional parameters, including numbers of Hoechst-stained nuclei, lysosomes, lipid droplets, mitochondria and oxidative stress that were used to rank cytotoxicity, indicated that the alternative chemicals were more potent than the legacy chemical in inducing Sertoli cell toxicity (100). Another study performed on TM4 cells examined the impact of exposures to EDCs such as BPA, pesticides, and personal care product components, on the expression of TJ and GJ proteins. Using a scalpel loading-dye transfer technique to study gap junction morphological changes, they observed that BPA exerted a low inhibitory activity on testicular gap junctional intercellular communication that was increased with longer time of exposure, leading to altered phosphorylation status and translocation of the GJ protein Cx43, suggesting BPA ability to dysregulate testes development via its action on gap junctional intercellular communications in Sertoli cells (101). Another study using TM4 identified the androgen signaling pathway as a BPA target, based on a combination of end-points, showing that BPA treatment decreased TM4 cell proliferation, concurrently with inhibiting the interaction of the amino- and carboxyl-terminal regions (N/C) ends of androgen receptor (AR), and enhancing AR interactions with the co-repressors NCoR (Nuclear Receptor Co-Repressor) and SMRT (Silencing Mediator Of Retinoic Acid And Thyroid Hormone receptor) (102). The authors proposed a role of BPA as anti-androgenic, besides its well-established estrogenic effects, in TM4 Sertoli cells, resulting in the reduction of cell proliferation. However, these results suggest that the TM4 mouse cell line, generated from PND11-13 mice, might not represent a genuine immature Sertoli cell model, since the proliferation of immature Sertoli cells is inhibited by androgens (18).

In another study on TM4 Sertoli cells, BPA was found to have a biphasic effect, with micromolar BPA levels causing inhibition of cell proliferation while nanomolar BPA levels stimulated energy metabolism, such as higher ATP concentrations and greater mitochondria activity (103). Qi et al. (2014) attributed BPA-induced apoptosis of PND 18-22 rat Sertoli cells to the Fas/FasL and p38/JNK pathways (104). Additionally, in vitro study of primary Sertoli cells exposed to EDC mixture of phthalate MBP and BPA exerted disruptive effects on tight and gap junctions, reduced androgen receptor and junction protein expression, to result in overall disturbance of BTB (86). Having conducted phalloidin and hematoxylin eosin staining of Sertoli cells treated with MBP and BPA showed a loss of cell number and adhesion between Sertoli-Sertoli cell interaction.

Phthalates (phthalic acid esters) are another group of EDCs that causes disruption to male reproductive function, found in consumer products such as shampoo, cosmetics, hairspray, food packaging and other consumer products. Phthalates, also known for their use as plasticizers, include di(2-ethylhexyl) phthalate (DEHP), metabolized to bioactive MEHP, as well as di(n-butyl) phthalate (DBP) that breaks down into mono(n-butyl) phthalate (MBP) (105–107). The deleterious effects of these phthalates on fetal testis function are well documented, including decreased androgen production, and some effects on Sertoli cells, such as decreased secretion of Androgen Binding Protein (ABP) and induction of morphologic changes in prepubertal rat Sertoli cells by MEHP (106, 107). Additionally, FSH receptor signaling was altered in immature Sertoli cells exposed to MEHP (108). In cultured human fetal testes, Amh mRNA expression as well as germ cell number were reduced by MEHP treatment, with an increase in germ cell apoptosis detected by caspase-3-positive immunostaining (109).

Human studies, such as that conducted on the mother-child Hokkaido Study Sapporo Cohort, have also established a strong correlation between maternal phthalate exposure and abnormal Sertoli cell function in their infants (110). In this study, the concentration of MEHP, major bioactive metabolite of DEHP, was determined in the blood of pregnant women, while a panel of reproductive hormones were measured in cord blood to determine Sertoli and Leydig cell functions in the infants. The study showed that the Sertoli cell marker Inhibin B was decreased in a dose-dependent manner with increasing maternal levels on MEHP, identifying perinatal Sertoli cells as phthalate targets. However, since Leydig cell markers were also decreased in cord blood, one cannot conclude if Sertoli cells were primary or secondary targets of the phthalate in this case (110). Although such studies are essential for the validation and extrapolation of findings in animal models to humans, they also illustrate some of the limitations of in vivo, organ and organoid studies, which reflect better real exposure conditions at the organism or tissue levels but complicate the determination of primary vs secondary target cells of EDCs, based on physiological parameters. MEHP was found to induce oxidative stress in prepubertal rat Sertoli cells, an effect that was attenuated by combining MEHP with a low dose of genistein, suggesting a protective effect of genistein (111).

Elucidating the mechanisms by which phthalates cause dysfunction in testes is critical. Exposure of fetal and postnatal mice to DEHP at 2mg/kg/day and above, was found to prevent Sertoli cell differentiation by downregulating genes involved in Sertoli cell differentiation such as Sox9, FGF9, and DMRT1 (112). Furthermore, the sex determination pathway, Gadd45g → Gata4/Fog2 → Sry → Sox9 → FGF9 was altered in the same treatment conditions. A more recent study of MEHP exposure in mouse Sertoli cell line TM4 was performed to assess transcriptomic alterations (113). Based on high-throughput sequencing of RNA collected from TM4 cells, genes that were differentially expressed associated with the Gene Ontology (GO) term “extracellular region” when treated with high and low MEHP doses. Most recently, Ma et al. (2020) found that DBP causes abnormal proliferation of Sertoli cells in prenatal stage of male mice at low concentrations, via ubiquitination of interleukin-1 receptor-related kinase 1 (IRAK1) (key proliferation-related protein) by downregulating the E3 ubiquitin ligase Pellino 2 (Peli2) (105). They also suggested the activation of the intrinsic apoptotic pathway to increase apoptosis of TM4 cells when exposed to 10mM MBP.

Humans are exposed to chemical compounds that act as endocrine disruptors from agricultural farming practices such as applying pesticide/herbicide to crops, waste accumulation, and burning residues. The pesticide Methoxychlor, a chemical with estrogenic/antiandrogenic effects, given orally to rats during the perinatal/juvenile period was shown to reduce the spermatogenic potential via reduction of Sertoli cell number (114). Zearalenone is a mycotoxin with a dangerous secondary fungal metabolite that is found in food and animal feed and induces reproductive toxicity. Cell-cycle arrest in the G2/M phase of Sertoli cells isolated from Wistar rats exposed to the mycotoxins induced autophagy via PI3K/AKT/m-TOR signaling pathway (115).

Two plant-derived toxins, colchicine and carbendazim were shown to induce histopathology on adult Sertoli cell cytoskeleton (116). The seminiferous epithelium was targeted, causing germ cell sloughing off by either colchicine or carbendazim. This process has been proposed to occur due to a destabilization of Sertoli cell stalk by a lack of microtubule support. Other histopathologies included retained spermatids, seminiferous epithelium vacuolization, seminiferous epithelium atrophy, and enlarged seminiferous tubule lumen in relation to defects in Sertoli cell cytoskeleton and signaling.

Exposure of mice from GD7 to PND21 to the flame retardant deca-bromodiphenyl ether (BDE-209) was shown to disrupt BTB function by damaging the TJ proteins occludin, claudin-11, ZO-1, and F-actin via estrogen receptor α (ER-α) signaling (117). Another flame retardant, aroclor1254, impaired BTB by increasing caveolin-1 associated endocytosis of several junctional proteins and enhancing ubiquitination of occluding by E3 ligase Itch (118). TJ proteins were shown to be delocalized from the cell membrane to the cytoplasm in PND20 rat Sertoli cells exposed to glyphosate alone or in Roundup, a common pesticide used in agricultural practice (119). However, when glyphosate or Roundup 3 Plus (GBHs) was given to pregnant mice from GD10.5 to PND20, the deleterious effects of glyphosate and GBHs were to reduce the number of undifferentiated spermatogonia, while only glyphosate decreased testosterone levels, and perinatal Sertoli cells were rather unaffected (120). The two studies suggested different species susceptibility to glyphosate.

Furthermore, transgenerational changes in rat Sertoli cell epigenome and transcriptome were associated with the onset of testis disease (121). Gestating female rats were transiently exposed to the environmental toxicants dichlorophenyltrichloroethane (DDT) or fungicide vinclozolin at the period of fetal gonadal sex determination and later bred to analyze the F3 generation (i.e., great-grand-offspring) for possible effects on transgenerational Sertoli cell genetic profiles. DNA and RNA obtained from purified Sertoli cells in PND20 male testis of F3 generation rats showed transgenerational alterations in DNA methylation, noncoding RNA, and gene expression following the treatment of F0 females with DDT or vinclozolin and exposure of the F1 fetuses they carried, compared to control rats. These transgenerational alterations in Sertoli cells were suggested to be critical factors in developing transgenerational testis pathologies and male infertility.

Another well-known EDC class is that of per- and poly-fluoroalkyls substances (PFAS), which includes perfluorooctane sulfonate (PFOS) or perfluorooctanoic acid (PFOA). PFOS is commonly found in paints, textiles, general cleaning products, and other consumer products. In vitro studies showed that PFOS perturbs male rat BTB function by affecting F-actin organization via p-FAK-Tyr407 (122). PFAS are known to exert detrimental effects on the BTB of adult human and rat Sertoli cells, mechanistically similar to those of cadmium and BPA, described as inducing testis injury via action on Sertoli cell actin- and/or microtubule-based cytoskeletal support (123). Investigators proposed that these chemicals acted by disrupting the F-actin microfilaments of the BTB, leading to the loss of support in Sertoli cell adhesion mediated by tight junctions (TJ) and basal ectoplasmic specialization (ES). The affected junctional proteins were occludin, ZO-1, N-cadherin, and β-catenin that were either altered at the expression level or mislocalized (122, 124).

Actin filaments were found to be truncated in BTB due to PFOS exposure. MicroRNA miR-135b, specific to FAK protein, was transfected into Sertoli cells to knock down expression of FAK, which worsened PFOS-mediated Sertoli cell tight junction disruption. PFOS were proposed to mediate Sertoli cell injury by compromising BTB integrity or formation, whereas the effects of PFOA were less serious and mainly limited to the disruption of hormonal pathways in human and rat (125, 126). Moreover, a study comparing the effects of PFOA exposure in fetal and adult rat testis suggested that there were no major effects on the fetal, proliferating, and adult Sertoli cells. Rather, they found reduced steroidogenesis function, increased fetal Leydig cell apoptosis, and PFOA-induced germ cell apoptosis in adult rat seminiferous tubules (127). Taken together, these studies highlighted the difference between PFOS and PFOA in target cell types, with only PFOS acting primarily on Sertoli cells, despite their structural similarities.

The consequences in exposing adult Sertoli cells to EDCs could be different from those of exposing perinatal and juvenile Sertoli cells or targeting fetal development by reducing Sertoli cell proliferative capacity, Sertoli cell number, or spermatogonia population (based on reduced Sertoli cell number). Moffit et al. observed histopathological manifestations that vary in type and occur in a dose-dependent fashion when exposed to 2, 5-hexanedione, carbendazim, and MEHP in adult rat testis (128). Manifestations observed are not limited to but describe change in seminiferous tubule diameter, sloughing, vacuolization, retained spermatid heads, and germ cell apoptosis. Cadmium has also dangerous effects on the BTB in adult rat Sertoli cells, shown to target F-actin organization to impair adhesive Sertoli-Sertoli or Sertoli-germ cell interactions (124). Indeed, BPA and Cadmium in vitro treatment of adult human Sertoli cells showed that both EDCs exerted cytotoxic effects by disorganizing F-actin microfilaments, leading to their truncation and depolymerization. β-catenin, ZO-1, and N-cadherin expression levels were not altered, but these proteins were found to be mislocalized in immunostaining imaging with higher doses of Cadmium (>5µM) and BPA (>40µM) (124).

Another environmental toxicant that is prevalent in densely packed metropolitan areas is PM2.5 urban fine particulate matter, which mainly originates from coal combustion and vehicle exhaust. The testes of Sprague-Dawley rats exposed to PM2.5 particles, showed excessive ROS-mediated autophagy that damaged BTB integrity (129). The testes from rats exposed to PM2.5 had increased autophagosome based on transmission electron microscopic imaging. Sertoli cell dysfunction was proposed to be due to decreases in Occludin, Zo-1, and CX43 protein levels, as determined by immunoblot analysis following PM2.5-treatment compared to control, with reduced TJ and GJ integrity. A more recent study of PM2.5 exposure from automobile exhaust reported the induction of oxidative stress via MAPK pathway activation. BTB damage was observed in primary Sertoli cell culture, based on increased Sertoli cell apoptosis and decreased expression of Zo-1 and β-catenin, and F-actin disorganization under immunostaining. The authors concluded the BTB damage induced by PM2.5 exposure was regulated via a ROS-MAPK-Nrf2 pathway (130).

We are starting this section on pharmaceuticals with the example of non-hormonal male contraceptives that disrupt the integrity of the BTB and Sertoli cell junctions, because they represent an example where Sertoli cell functional disruption is desired rather than accidental. Indeed, the BTB is positioned both as therapeutic target to counter toxicant-induced infertility/subfertility, and as contraceptive target to disrupt spermatogenesis and block sperm formation in a reversible manner (87). The challenge remains to identify an agent that interferes only transiently with Sertoli cell functions and permits the recovery of spermatogenesis in a timely manner, while being also devoid of non-reproductive adverse effects. Currently, several non-hormonal male contraceptives have reached the stage of clinical phases, but none has yet been approved by the FDA, as summarized in the review of Arifuzzaman and colleagues (131). Male contraceptives such adjudin were shown to exert deleterious effects by damaging the BTB, resulting in infertility of adult rats (132, 133). mTORC1/rpS6 signaling complex, previously shown to be upregulated in BTB remodeling, was identified as mediating adjudin adverse effects on the BTB (134). The use of a constitutively active rpS6 mutant in combination with adjudin treatment was found to create a “leakier” BTB, promoting the entry of the nonhormonal male contraceptive into the adluminal compartment in the seminiferous epithelium to induce germ cell exfoliation. A recent article examined the effects of a single oral dose of adjudin at 50 mg/kg on rat testis, reporting that the adverse effect of adjudin on the BTB involved changes in actin dynamics and microtubule associated proteins (MAPs), in addition to disrupting mTORC1/rpS6 and p-FAK-Y407 signaling pathways (135). Although the study confirmed the reversibility of adjudin treatment, it reported long recovery times, from 163 days to recover full spermatogenesis, to 254 days to recover fertility in adjudin-treated rats. This long recovery time in rat, a species where a complete spermatogenic cycle takes around 54 days, and recovery periods of 8 weeks have been reported for toxicants affecting spermatogonia, suggests that the extend of damages afflicted on the Sertoli cell cytoskeletal and junctional proteins by adjudin was extensive (136). Similarly to adjudin, l-CDB-4022 acts on Sertoli-germ cell adherens junction proteins to effectively reduce spermatogenic activity (137). l-CDB-4022 also induces germ cell loss by activating MAPK pathway to suppress pro-survival factors such as stem cell factor, and inducing pro-apoptotic factor Fas ligand (131). Taken together, non-hormonal contraceptives like adjudin, l-CDB-4022, H2-gamendazole, and F5-peptide exemplify the possible therapeutic use of chemicals that target Sertoli-germ cell interactions at BTB junctions as a way to temporarily pause spermatogenesis and control male fertility.

A number of pharmaceuticals are known to inadvertently cause male infertility, including drugs exerting adverse effects on Sertoli cells (18). Some examples of drugs affecting Sertoli cells are described below, although in some instances, it is not clear whether Sertoli cells are primary or secondary targets.

Treatment of male infertility mentioned here include hormone human chorionic gonadotropin (hCG), selective estrogen receptor modulator clomiphene citrate (off-label drug for male infertility), and aromatase inhibitor Anastrazole (off-label drug for male infertility) to offset LH, FSH, and/or testosterone deficiencies. A review by Kilcoyne et al. reported effects of pharmaceutical and environmental exposure with pesticides and pharmaceuticals such as Valproate and Theophylline which highlights effects on testosterone production but minimally affected Sertoli cell population (96).

Ameli et al. highlighted a mechanism by which Clomiphene and hCG exposure of adult Wistar rats acted via cation channel sperm-associated protein 1 (CatSper1), cation channel sperm-associated protein 2 (CatSper2), luteinizing human chorionic gonadotropin receptor (LHCGR) and steroidogenic factor 1 (SF1) signaling. Based on treatment with either clomiphene (5mg/kg), hCG (100 IU/kg), or both (5mg/kg and 100 IU/kg respectively), the aforementioned genes were analyzed. CatSper1 and 2, LHCGR, and SF1 were upregulated in hCG-treated rats, while these genes were downregulated by Clomiphene treatment alone. Mechanism aside, the analysis of testicular tissue suggested a reduction in number of Sertoli cells, spermatogonial cells, primary spermatocytes, and spermatid cells with Clomiphene treatment. The authors proposed that while a reduction of these cell populations was observed, they did not see a significant reduction of Sertoli cells specifically. This study concludes that supplementation of hCG in treatment of clomiphene will be neutralized to maintain a normal spermatogenesis process which is otherwise hampered by Clomiphene alone.

Another off-label drug used to treat male infertility specifically in males with a low testosterone to estradiol (T/E₂) ratio is the aromatase inhibitor Anastrozole (Arimidex), based on the idea that blocking estrogen production may preserve higher levels of its androgen precursor (154). Guercio et al. have reviewed this aromatase inhibitor, which regulates inhibin B expression in Sertoli cells, and the production of testosterone with a focus on childhood and puberty development (155). Shoshany et al. provided some clinical relevance for using anastrazole in oligozoospermic hypoandrogenic men, since improved sperm parameters and T/E₂ ratio were observed in Anastrazole-treated men (156). They attributed these effects to the inhibition of estradiol formation by anastrozole, which normally stimulates the growth and development of Leydig cells, which in turn act as paracrine regulators of Sertoli cells. While studies regarding male contraception or infertility mention Sertoli cells as possible targets (157), the focus has often been on testosterone production by Leydig cells and spermatogenesis (158). Since Sertoli cells “nurse” the germ cells and interact with Leydig cells and other gonadal cell types, they likely play a role in successful treatments of male infertility.

The environmental metal Cadmium, to which people can be exposed via food, was studied alone or in combination with the organophosphate insecticide diazinon, commonly used on fruits and crops, which could also be present in food, by exposing 4-week-old rats to both chemicals in sub-chronic doses over a 90-day period via drinking water (161). In rats exposed to Cadmium or diazinon, germ cell sloughing, and cell necrosis were observed, and immunohistochemical analysis revealed the presence of seminiferous tubules displaying progressive damage, lacking germ cells and presenting Sertoli cell only phenotype. However, no synergy or additivity was found in rats exposed to the mixtures, which appeared to have lesser effects than the chemicals alone, suggesting that in this particular case, exposure to the mixture presented less reproductive health risk than exposure to each chemical alone.

Postnatal exposure of infants can happen in a clinical setting through soy-formula containing the phytoestrogen GEN, whilst receiving medical intervention with phthalate-containing medical tubing and equipment. DEHP which is not covalently bound, leaches onto the fluids and can reach out the infant via ingestion during medical intervention. Jones et al. performed in utero exposures studies in Sprague-Dawley rat with GEN and DEHP mixtures at 10mg/kg/day, observing morphological changes, as well as transcriptome changes and inflammatory responses in testes of offspring (162). The study identified Sertoli cells as one of the target testicular cell types, based on alteration of mRNA expression for Sertoli markers Amh and Wt1, in adult PND120 rats. Following this study, Jones et al. performed in utero exposure to GEN and DEHP at the same dosing as previously mentioned to further analyze the effects of GEN-DEHP mixtures in PND3 and PND6 rat testes, representing early gene responses. While various genes were altered, including antioxidant genes, it is interesting that the authors saw a decreased expression of ABP after exposure to either DEHP alone or GEN-DEHP mixture in PND6 rat testes. They also observed Wt-1 gene expression to be decreased significantly after exposure to DEHP alone but not GEN+DEHP mixture in PND3 rat testes. To complement these studies, Walker et al. analyzed the testes of adult rats exposed in utero to 0.1 and 10 mg/kg/day GEN+DEHP mixtures and observed an increased rate of abnormal testicular phenotypes characterized by Sertoli cells only in EDC-treated rats (164).

Another type of phthalate, DBP (and its metabolite MBP), was studied in mixture with nonyl phenol (NP) which has weak estrogenic activity, on Sertoli cells isolated from prepubertal rats (165). While they reported no remarkable changes on Sertoli cell morphology, a decreased cell viability was observed by Cell Counting Kit-8 (CCK-8) assay after 24-hour exposure to high concentrations of DBP (100 µM) and NP (10 µM) mixture. Additionally, the effects of NP and MBP were studied in vitro by treating 30-day-old primary rat Sertoli cells and analyzing the data via two mathematical models, Loewe additivity (LA) and Bliss Independence (BI) theory, used during risk assessments of EDCs (166). The authors concluded that these two mathematical models were able to indicate whether the combined effect of NP+DBP mixture had an antagonistic effect on cell viability and lactate dehydrogenase (LDH) leakage assay, which evaluates plasma membrane integrity. Furthermore, the authors extended their previous study of NP+MBP mixtures by performing an in vitro analysis of primary rat Sertoli cells simultaneously with an in vivo analysis of PND23-35 rats exposed to the same chemicals, using the BI model to quantify their data (167). The comparison of in vitro and in vivo Sertoli cell dysregulation by NP+MBP mixtures suggested that the BI model was able to predict interactions between estrogenic and antiandrogenic effects of the chemicals.

Kumar et al. performed a study to determine the effects of exposure to a mixture of three phthalates: diethyl phthalate (DEP), diphenyl phthalate (DPP), and dimethyl isophthalate (DMIP), using both in vivo and in vitro treatment of immature Sertoli cells (168). Data collected from in vitro treatment of 3-week-old primary mouse Sertoli cells exposed for 24 hours to the phthalate mixture showed some similar trends to the in vivo data on mRNA expression for various Sertoli cell markers. Additionally, in vitro exposure to the phthalate mixture was suggested to alter the structural and functional integrity of primary Sertoli cell culture, as seen in transmission electron microscopy (TEM) data.

Paracetamol/acetaminophen is an over-the-counter analgesic and antipyretic drug widely used by pregnant women and young children, that has been reported in epidemiological studies to correlate with an increased incidence of male reproductive disorders. Since these disorders are also associated with exposure to EDCs, Axelastad et al. examined the effects of in utero exposure to paracetamol alone and in combination with estrogenic and antiandrogenic EDCs, to explore the effects of these mixtures on male fertility (170). Mostly concerned with changes in sperm count post-exposure to mixtures, the authors found that the testes of rats exposed to estrogens, antiandrogens, and paracetamol mixtures presented seminiferous tubules with reduced germinal epithelium thickness and Sertoli-cell only phenotypes. Individual pesticides or insecticides are known to affect male reproduction, which can be further impacted by exposure to mixtures of pesticides. Patrick et al. used mixtures mimicking the exposure conditions of field workers in South Africa, where malaria as a public health threat (169). These areas have significant EDCs contaminants including the anti-malaria insecticides 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT), 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), and deltamethrin (DM). Additionally, people living in these areas are highly exposed to nonylphenol and cultural diets containing coumestrol, genistein, and zearalenone. Based on in utero, lactational, and direct exposure to these EDCs as mixtures, seminiferous tubules were found to have larger luminal sizes, suggesting Sertoli cell toxicity and altered fluid retention. In comparison, Durand et al. (2017) studied the mixed effects of two common fungicides carbendazim (CBZ) and iprodione (IPR), as androgenic and anti-androgenic prototypes, on pubertal rat seminiferous tubules (STs) in culture (171). Ex vivo treatment of cultured STs from prepubertal rats that were exposed to CBZ, IPR, or the mixture, showed altered ratios of germ to somatic cell (Sertoli) populations and reduced levels of key junctional proteins Cx43 and Claudin 11 in samples exposed to the mixture. Lastly, Petricca et al. reported the dysregulation of TM4 Sertoli cell proliferation, cell cycle arrest, and mitochondria dysfunction in response to treatments with fungicides triazoles, ketoconazole and miconazole and imidazole, prochloraz as mixtures, showing synergistic effects (172).