John Henry Richburg

The basement membrane (BM) is a crucial support structure of the seminiferous epithelium, mainly made up of collagens, fibronectin, laminins, and proteoglycans. Along with the collagen fiber network, the peritubular myoid cell (PTMC) forms the tunica propria, which acts as a protective barrier for the blood-testis barrier (BTB). Here, we examined how PTMCs respond to mono-2-ethylhexyl phthalate (MEHP) exposure by altering the transcriptomic and proteomic profiles of extracellular matrix proteins, which are essential structural components of the BM. Bulk RNA was used for transcriptomics, and protein lysates were prepared for proteomics from primary PTMC isolates. Interstitial fluid (IF) was utilized to assess the baseline microenvironment of the testis, and the in vivo relevance of the protein changes was confirmed through immunofluorescence of testis cross-sections. Gene Set Enrichment Analysis (GSEA) revealed a suppression of genes in the basement membrane-linked pathway after MEHP exposure. These findings are further supported by proteomics analysis of PTMC lysates. Notably, downregulation of key basement membrane components—such as collagen IV, fibronectin, laminin, and others—was observed alongside the classical complement activation pathway in the testis microenvironment, specifically in the interstitial fluid. The data indicate that MEHP exposure downregulated the expression of BM component proteins in PTMC, underscoring the interconnections among BM degradation, BTB disruption, spermatogenesis, and disorganization of seminiferous tubule architecture during MEHP-induced testicular injury.

We report the presence of a previously undocumented cell type in the spermatogonial stem cell niche expressing CD2 protein, using whole-mount immunofluorescence of rat seminiferous tubule preparations. Confocal imaging revealed that CD2+ cells reside interspersed within the peritubular myoid cell (PTMC) layer surrounding the seminiferous tubules. Three-dimensional reconstruction and surface rendering models revealed cellular interaction between peritubular macrophages (PTMφs) and CD2+ cells. The mono-(2-ethylhexyl) phthalate (MEHP)-induced testicular injury model was utilized to investigate the dynamics of PTMφ-CD2+ cell interaction. Fischer CDF344 male rats were exposed to 700 mg/kg-bw MEHP or corn oil via oral gavage on postnatal day (PND) 28 and euthanized at 48 h or 2 weeks post-exposure. In MEHP-exposed animals, a significant number of PTMφs encounter CD2+ cells at 48 h post-exposure, suggesting it to be an adaptive response against the MEHP-induced toxicity. Moreover, neither the vehicle group (corn oil) nor the MEHP-exposed animals show a change in the temporal division dynamics of the CD2+ cells when followed along the acute (48 h) and recovery (2 weeks) phases of post-injury, suggesting these cells as dormant cell population lacking susceptibility against MEHP-induced toxicity. Nevertheless, an increase in PTMφ-CD2+ cell interactions in response to testicular toxicity indicates a CD2+ cell-mediated link for dynamic intercellular communication between the innate immune cells and niche cells of the testis. This first report of the existence of a unique CD2+ cell population in the rat seminiferous tubules and their direct cellular interaction with PTMφs is anticipated to stimulate further research in the field of testicular immunobiology.

We have previously reported that acute MEHP exposure resulted in a significant increase in peritubular macrophages along with differentiating spermatogonia. Here, we hypothesize that the recruitment of peritubular macrophages is MEHP dose-dependent and that the peritubular macrophages stimulate spermatogonial differentiation in response to MEHP-induced testicular injury. Peripubertal rats were exposed to a single dose of either 250 mg/kg or 500 mg/kg MEHP or 250 mg/kg MEHP for 3 consecutive days or 100 mg/kg for 7 consecutive days to study chronic exposure. Here, we report that an acute exposure to 500 mg/kg and a repeated 250 mg/kg exposure to MEHP resulted in significant loss of spermatocytes as well as increased numbers of peritubular macrophages and that this increase in peritubular macrophages corresponded closely with an observed increase in the numbers of differentiating (PLZF+) spermatogonia. Interestingly, a disruption of the blood-testis barrier (BTB) also corresponded closely with increased peritubular macrophage numbers after MEHP exposure. To delineate if the peritubular macrophages play a role in the repair and recovery of spermatogenesis after MEHP exposure, depletion of MEHP-induced increase in the numbers of peritubular macrophages via pre-treatment with clodronate resulted in a consequent decrease in number of PLZF-positive differentiating spermatogonia. Our findings are significant as it is the first demonstration of the infiltration of peritubular macrophages by a toxicant and is dose-dependent. Additionally, this recruitment of PTMO depends on the disruption of BTB and not germ cell loss. Furthermore, depletion of peritubular macrophages resulted in a decrease in the number of differentiating spermatogonia.

Peritubular macrophages (PTMφ) are predominantly localized near spermatogonial stem cells in the testis. We previously revealed that exposure of peripubertal male Fischer rats to mono-(2-ethylhexyl) phthalate (MEHP) leads to increased PTMφs in the testis. The mechanisms that trigger increases in PTMφs in the testis are poorly understood. However, MEHP exposure is known to both induce spermatocyte apoptosis and to perturb the blood-testis barrier (BTB). This study aims to elucidate the association between the disruption of BTB and the increases of PTMφs in the testis by comparing the effects observed with MEHP to 2 other testicular toxicants with variable effects on the BTB and subtype of germ cell undergoing apoptosis. Methoxyacetic acid (MAA) acts directly on spermatocytes and does not affect BTB function, whereas cadmium chloride (CdCl₂) induces profound injury to BTB. The results indicated that MAA exposure significantly increased spermatocyte apoptosis, whereas no significant changes in the numbers of PTMφs in the testis occurred. In contrast, CdCl₂ exposure disrupted BTB function and increased the abundance of PTMφs in the testis. To further investigate whether MEHP-induced changes in BTB integrity accounted for the increase in PTMφs, a plasmid for LG3/4/5, the functional component of laminin-alpha 2, was overexpressed in the testis to stabilize BTB integrity before MEHP exposure. The results showed that LG3/4/5 overexpression substantially reduced the ability of MEHP to compromise BTB integrity and prevented the increase in PTMφ numbers after MEHP exposure. These results indicate that BTB disruption is necessary to increase PTMφs in the testis induced by toxicants.

Exposure of rodents to mono-(2-ethylhexyl) phthalate (MEHP) is known to disrupt the blood-testis barrier and cause testicular germ cell apoptosis. Peritubular macrophages (PTMφ) are a newly identified type of testicular macrophage that aggregates near the spermatogonial stem cell niche. We have previously reported that MEHP exposure increased the numbers of PTMφs by 6-fold within the testis of peripubertal rats. The underlying mechanism(s) accounting for this change in PTMφs and its biological significance is unknown. This study investigates if MEHP-induced alterations in PTMφs occur in rodents (PND 75 adult rats and PND 26 peripubertal mice) that are known to be less sensitive to MEHP-induced testicular toxicity. Results show that adult rats have a 2-fold higher basal level of PTMφ numbers than species-matched peripubertal animals, but there was no significant increase in PTMφ numbers after MEHP exposure. Peripubertal mice have a 5-fold higher basal level of PTMφ compared with peripubertal rats but did not exhibit increases in number after MEHP exposure. Further, the interrogation of the testis transcriptome was profiled from both the MEHP-responsive peripubertal rats and the less sensitive rodents via 3′ Tag sequencing. Significant changes in gene expression were observed in peripubertal rats after MEHP exposure. However, adult rats showed lesser changes in gene expression, and peripubertal mice showed only minor changes. Collectively, the data show that PTMφ numbers are associated with the sensitivity of rodents to MEHP in an age- and species-dependent manner.

The blood-testis barrier (BTB) is constituted by tight junctions between adjacent Sertoli cells (SCs) that create a specialized adluminal microenvironment to foster the development of spermatocytes and spermatids. The BTB is a well-studied target of numerous environmental toxicants, including di-(2-ethylhexyl) phthalate (DEHP), a compound widely used in various consumer products. Mono-(2-ethylhexyl) phthalate (MEHP) is the active toxic metabolite of DEHP that has long been recognized in postnatal rodents to disrupt SC function. This study evaluates the impact of MEHP on the integrity of the BTB in both pubertal and adult rats and the signal transduction pathways known to be involved in the disruption of the BTB. Treatment of prepubertal rats with 700 mg/kg MEHP for 24 h functionally disrupted the BTB integrity. A similar treatment of adult rats with MEHP did not disrupt the integrity of the BTB. The observed disruption of the BTB integrity in the MEHP-treated prepubertal rats occurred concomitantly with a decreased expression and mislocalization of both the ZO1 and occludin tight junction-associated proteins, as well as sloughing of spermatocytes and spermatids. At this same time, MEHP treatment induced a transient surge of p44/42 mitogen-activated protein kinase (MAPK) pathway. Interestingly, after a recovery period of 5 weeks, the BTB recovered and was functionally intact. This is the first report to indicate that acute MEHP exposure of prepubertal rats, but not adult rats, disrupts the functional integrity of the BTB and that this effect on the BTB is reversible.

Peripubertal exposure of male rodents to the phthalate metabolite mono-(2-ethylhexyl) phthalate (MEHP) causes testicular inflammation, spermatocyte apoptosis, and disruption of the blood-testis barrier. The MEHP-induced inflammatory response in the testis includes an infiltration of macrophages and neutrophils, although the cause and purpose of this response is unknown. Recently, a population of testicular macrophages known as peritubular macrophages that are phenotypically distinct from those resident in interstitium was described in mice. Peritubular macrophages aggregate near the spermatogonial stem cell niche and are believed to stimulate their differentiation. We hypothesized that if testicular peritubular macrophages do indeed stimulate spermatogonial differentiation, MEHP exposure would result in an increase of peritubular macrophages to stimulate the replacement of lost spermatocytes. Male rats were exposed to 700 mg/kg MEHP or corn oil (vehicle control) via oral gavage at postnatal day 28 and euthanized at 48 h, 1 or 2 weeks later. Seminiferous tubules were stained with immunofluorescent markers for macrophages (major histocompatibility complex class II [MHC-II⁺]) and undifferentiated spermatogonia (PLZF). Peritubular macrophages were observed in rat testis: MHC-II⁺ cells on the surface of seminiferous tubules with heterogeneous morphology. Quantification of MHC-II⁺ cells revealed that, unlike in the mouse, their numbers did not increase through puberty (2-week period). MEHP increased macrophage presence by 6-fold 48 h after exposure and remained elevated by 2-fold 2 weeks after exposure. An increase of differentiating spermatogonia occurred 2 weeks after MEHP exposure. Taken together, our results suggest that peritubular macrophages play a crucial role in the testis response to acute injury and the subsequent recovery of spermatogenesis.

Exposure to the chemotherapeutic agent cis-diamminedichloroplatinum(ii) (cDDP) is well known to instigate acute and prolonged testicular injury in male patients. Many investigators have hypothesized that cDDP-induced dysfunction of Sertoli cells (SCs) may, in part, account for the cDDP-induced lasting testicular injury. Nevertheless, the relative contribution of cDDP-induced SC injury versus direct effects on germ cells (GCs) to the pathogenesis of GC loss remains to be elucidated. The expression of the copper transporter 1 (CTR1) protein in cells directly corresponds with cDDP uptake and its cellular toxicity. Therefore, to discern the role of SCs in the pathogenic mechanism, mice were developed with a SC-specific disruption of the Ctr1 gene (SCΔCtr1) as a strategy to prevent their exposure to cDDP. Adult mice at postnatal day (PND) 60 were treated with 5 mg kg−1 cDDP and then testis collected at 48 hours. A two-fold increase in GC-apoptosis occurred in the testis of cDDP-treated wildtype (WT) mice as compared to saline-treated WT mice. In contrast, cDDP-treated SCΔCtr1 mice exhibited only a half-fold increase in GC-apoptosis as compared to the saline-treated SCΔCtr1 mice. This reduced incidence of GC apoptosis in the SCΔCtr1 mice corresponded to a significantly lower level of platinum within the testis. Taken together, these findings reveal that the uptake of cDDP by CTR1 in SCs accounts for the accumulation of cDDP in the testis and plays a pivotal role in the pathogenic sequence of events leading to the loss of germ cells via apoptosis.