Dose-dependent testis infiltration of peritubular macrophages after MEHP exposure of Peri-pubertal rats and their role in the recovery of spermatogenesis

Abstract

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.

Introduction

The testis is acknowledged as an “immune-privileged” organ due to its immune-suppressed microenvironment. This environment serves as a shield for developing germ cells, safeguarding them from the immune system’s scrutiny. The immunosuppressive conditions are orchestrated by testicular cells, which release signals to dampen immune responses, and by structural elements like the blood-testis barrier (BTB) that limit the exposure of adluminal germ cells to factors emanating from the blood.¹

Despite the prevailing immunosuppression, various immune cells are discernible within the interstitial space of the testis. Notably, macrophages constitute a substantial portion of this interstitial population.² These macrophages can be further classified into resident macrophages and those that infiltrate the testicular tissue during episodes of inflammation.

Resident macrophages are pivotal in shaping and sustaining the immune-suppressed milieu within the testis. Their concerted efforts safeguard mitotic spermatogonia, thereby contributing to this unique immunological landscape’s overall integrity and functionality.^3,4

Previous research in our laboratory elucidated a transient infiltration of macrophages and neutrophils into the testes of peripubertal rats. This was followed by a notable increase in spermatocyte apoptosis after acute exposure to MEHP.⁵ However, subsequent investigations revealed that these macrophages did not directly contribute to spermatocyte apoptosis following acute MEHP exposure.⁶ This discovery prompted a paradigm shift from focusing solely on the destructive role of macrophages to considering their potential involvement in the recovery and restoration of spermatogenesis after toxicant-induced injury.

Previous reports have proposed the existence of a newly identified subset of macrophages in the testis, located on the surface of seminiferous tubules, hence termed peritubular macrophages.⁷ We hypothesize that this distinctive macrophage subtype is involved in supporting and/or stimulating spermatogonial proliferation. Our previous study unveiled a significant augmentation in the numbers of peritubular macrophages alongside an increase in differentiating spermatogonia after acute exposure to MEHP.⁸ Furthermore, recent publications from our laboratory report that this elevation in peritubular macrophage numbers is contingent upon the age and species of the rodent.⁹

In response to MEHP exposure, we observed a surge in peritubular macrophages in peripubertal rats but not in adult rats.⁹ Interestingly, we also noted that basal levels of peritubular macrophage numbers are elevated in mice compared to rats, and MEHP exposure does not significantly affect their numbers. This age and species-related effect poses an intriguing avenue for further exploration. The present study explored the acute or chronic dose-dependent escalation of peritubular macrophages following MEHP exposure. Our investigation unveiled a threshold dose of MEHP necessary for peritubular macrophage infiltration in peripubertal rats. Surprisingly, we found no correlation between spermatocyte apoptosis and the increase in peritubular macrophages. However, doses of MEHP that induced disruption of the BTB coincided with the rise in peritubular macrophages. This intriguing observation prompted a recent publication from our laboratory, elucidating that the disruption of the BTB is a prerequisite for the infiltration of peritubular macrophages.¹⁰ Notably, only toxicants that disrupted the BTB (such as MEHP and CdCl2) led to a significant increase in peritubular macrophages. In contrast, a toxicant causing germ cell loss (MAA) did not exhibit this effect.¹⁰

Furthermore, our exploration extends to unraveling the functional role of peritubular macrophages. We posit that toxicant-induced testicular injury triggers an upsurge in peritubular macrophages, which, in turn, contribute to the restoration of spermatogenesis by stimulating the division kinetics of differentiating spermatogonia. Additionally, it shows a correlation between the disruption of the BTB and the augmentation of peritubular macrophages, shedding light on the intricate interplay between testicular microenvironment dynamics and macrophage response.

Materials and methods

Animals and treatments

All animal work was conducted using humane procedures preapproved by the Institutional Animal Care and Use Committee at The University of Texas at Austin and according to NIH guidelines (AUP-2023-00060). Postnatal day (PND) 25, male Fischer CDF344 rats were purchased from Charles River. Fischer CDF344 rats are an outbred strain that more closely represents genetic variation as is found in the human general population. Animals were maintained in a temperature-controlled housing facility on a 12:12 L: D cycle with ad libitum access to food and standard rat chow. MEHP (CAS no. 4376-20-9) MEHP (Mono(2-ethylhexyl) phthalate) is an organic compound classified as a phthalate monoester. It is a metabolite of di(2-ethylhexyl) phthalate (DEHP), a commonly used plasticizer in polyvinyl chloride (PVC) plastics. MEHP is formed in the body when DEHP is hydrolyzed by enzymes such as lipases. MEHP belongs to Chemical class of Phthalate Monoester and in functional group of Ester, Aromatic (Benzene Ring). After 3 days of acclimatization, on PND 28, males were randomly assigned to one of two treatment exposure groups: vehicle control (VEH) or MEHP.

For acute treatment, rats were exposed via oral gavage to either 250 mg/kg or 500 mg/kg MEHP dissolved in corn oil or an equivalent volume of corn oil (VEH). MEHP or corn oil was given by oral gavage in a 90–110 μL volume, depending on the animal’s body weight. Animals were euthanized by CO₂ asphyxiation followed by thoracotomy after 48 h of treatment.

For multiple doses of exposure, animals were exposed via oral gavage to either 250 mg/kg/day for 3 days or 100 mg/kg/day for 7 days MEHP dissolved in corn oil or an equivalent volume of corn oil (VEH). Animals were euthanized by CO₂ asphyxiation followed by thoracotomy after 24 h of last treatment. Our previous publication, Gillette et al., 2021 reported infiltration of PTMO after an acute dose of 700 mg/kg exposure of MEHP. These doses were chosen to evaluate if a low-dose repeated exposure would elicit a similar peritubular macrophage response to a high single dose of MEHP.

For the macrophage depletion experiment, an i.p. injection of 0.4 mL clodronate liposomes (Liposoma B.V., Amsterdam, the Netherlands) or PBS liposomes (Liposoma B.V.) was administered 48 h immediately before MEHP treatment and 48 h after MEHP treatment. Animals were euthanized 48 h after the MEHP treatment.

All exposed animals were monitored carefully for adverse effects. The testes were rapidly removed and weighed. One testis was randomly selected for whole tubule immunofluorescence and was immediately placed in cold PBS (Gibco, Cat# 10010–023) on ice. The opposite testis used for paraffin embedding was fixed in Bouin’s fixative for TUNEL and cross-section staining of ZO-1 and Occludin.

Whole seminiferous tubule staining and confocal microscopy

Seminiferous tubule staining for MHC II and PLZF was followed as described previously.⁸ Briefly, tubules were fixed in 4% paraformaldehyde and incubated with MHC II and PLZF antibodies. Hoechst 33342 was used as a DNA/nuclei counter stain. Stained tubules were mounted whole on super frost slides and stored at −20 °C until imaging. Slides were imaged on a Zeiss scanning confocal microscope (Zeiss, LSM 710).

Immunofluorescence staining

Immunofluorescent staining was performed as described previously. Briefly, the paraffin-embedded tissue block is sliced into a 5-micrometer section and mounted on glass slides. Following heat treatment in a sodium citrate-based antigen retrieval solution, tissue sections were incubated with 3% hydrogen peroxidase (Thermo Fisher Scientific) to block endogenous peroxidase activity and then incubated in blocking buffer (10% normal horse serum (Sigma-Aldrich) and 0.3% Triton X-100 (Sigma-Aldrich) in PBS). The primary antibodies used for immunofluorescent staining include PLZF (rabbit polyclonal, Cat# sc-28,319) (1:200 concentration), which was purchased from Santa Cruz Biotechnology (Dallas, TX), MHCII (mouse anti-rat, Cat# 205401) (1:200 concentration), which was purchased from BioLegend (San Diego, CA).

Sections were probed with Foxo-1 (1:200 concentration), Occludin (Cat # E6134R; Cell Signaling) (1:200 concentration), or ZO-1 (Cat # 617300; Invitrogen) (1:200 concentration) antibody. Images were acquired using a Nikon Eclipse microscope with a 20x objective and captured with a Nikon DS-Fi1 camera and NIS Elements software. Fluorescence intensity was quantified using ImageJ software. Fluorescence intensity was normalized to the tubule area. For each animal, at least 30 seminiferous tubule images were analyzed.

TUNEL assay

The presence of apoptotic fragmentation of DNA was determined by the TUNEL (terminal deoxynucleotidyl Transferase-Mediated Digoxigenin-dUTP Nick End Labeling) assay as described previously.⁶ The apoptotic index (AI) was determined by the percentage of seminiferous tubules containing three or more than three TUNEL-positive germ cells in each cross-section. For each animal, at least 100 seminiferous tubules were analyzed.

Statistical analysis

Results from experiments are presented as individual data points and means ± S.E.M. Each replicate represents a biological replicate, and the number of replicates is indicated in each figure. The data were subjected to a Student t-test or parametric 1- or 2-way ANOVA followed by the Tukey test for post hoc comparisons. GraphPad Prism Software Version 5.0 (GraphPad Software Inc., La Jolla, CA) was utilized for all calculations. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001 were considered statistically significant.

Results

Acute MEHP dose-dependent response to macrophage recruitment and apoptosis

Previous studies conducted in our laboratory demonstrated the disruption of the BTB at a dosage of 700 mg/kg of MEHP,¹¹ along with a concurrent rise in interstitial macrophages and the number of apoptotic spermatocytes.⁵ To delve deeper into this correlation, we sought to investigate whether there exists a dose-dependent response of MEHP in recruiting peritubular macrophages, influencing the population of differentiating spermatogonia, inducing apoptosis of spermatocytes, and disrupting the BTB.

Peripubertal rats PND 28 were given an acute dose of either 250 mg/kg (n = 7) or 500 mg/kg (n = 7) of MEHP or an equal volume of corn oil (Veh) (n = 7) and sacrificed 48 h later. Peritubular macrophages are verified by MHC II staining and their localization on the seminiferous tubules as visualized via confocal microscopy. Seminiferous tubules were stained with MHC II (peritubular macrophage) and PLZF (differentiating spermatogonia) and mounted on a slide for analysis on a confocal microscope. A significant increase in MHC II positive and PLZF cells was observed in the 500 mg/kg MEHP group compared to the control group (Fig. 1A and B).

Fig. 1.

Acute MEHP dose-dependent response to macrophage recruitment. PND 28 rats were treated with either Veh or 250 mg/kg or 500 mg/kg MEHP for 48 hrs (n = 7 for each group). A) Representative micrographs of MHCII⁺ (green) peritubular macrophages and PLZF⁺ (red) spermatogonia are shown on the surface of seminiferous tubules in both Veh and MEHP groups. All images in this figure were taken at 20x. The scale bar (50 μm) represented in the top left panel applies to all images in this fig. B) Quantification of peritubular macrophages and differentiating spermatogonia population from Veh and MEHP (250 mg/kg and 500 mg/kg). The number of MHCII⁺ cells and PLZF+ is normalized to the tubule area. The data were subjected to a Student t-test or parametric one-way ANOVA followed by a Tukey test for post hoc comparisons. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. C) Representative TUNEL images. Dark brown dots are considered apoptotic germ cell. D) Quantification of apoptotic cells from Veh and MEHP (250 mg/kg and 500 mg/kg). E) Representative images of tight junctions Occludin (green) ZO-1 (red) and quantification of fluorescence intensity using ImageJ.

The 250 mg/kg group did not see a significant increase in the MHC II-positive or PLZF cells (Fig. 1A and B).

Quantification of peritubular macrophage numbers at different doses of MEHP 250 mg/kg, 500 mg/kg, and 700 mg/kg. We report a dose-dependent increase in peritubular macrophage numbers with different MEHP dose exposures (Fig. 2).

Fig. 2.

Increase in peritubular macrophage number with increase in MEHP dose.

MEHP dose-dependent response to spermatocytes apoptosis and disruption of BTB junction

To assess if MEHP induces a dose-dependent increase in spermatocyte apoptosis, we stained cross-sections of fixed testes for the TUNEL assay. We report a significant increase in apoptotic spermatocytes observed in both the 250 mg/kg and 500 mg/kg groups (Fig. 1C and D).To visualize the disruption of BTB, we stained a cross-section of fixed testes with occludin and ZO-1, both of which are tight junction proteins that constitute BTB. We observed a decreased expression of both occludin and ZO-1(Fig. 1E) in the 500 mg/kg group but not in the 250 mg/kg group.

Multiple MEHP dose-dependent response to macrophage recruitment and germ cell apoptosis

To further explore if peritubular macrophage recruitment is specific to a high acute dose of MEHP, peripubertal rats PND 28 were given 3 consecutive doses of either 250 mg/kg/day (n = 7) MEHP or an equal volume of corn oil (Veh) (n = 7) and sacrificed 24 h later.

In a separate experiment, peripubertal rats PND 28 were given 7 consecutive doses of either 100 mg/kg/day (n = 7) MEHP or an equal volume of corn oil (Veh) (n = 7) for 7 days and sacrificed 24 h later.

Seminiferous tubules were stained with MHC II and PLZF and mounted on a slide for analysis on a confocal microscope. We observed a significant increase in the MHC II-positive and PLZF-positive population in the 250 mg/kg/day MEHP for 3 days treatment group compared to the Veh control group (Fig. 3A and B).

Fig. 3.

Chronic high MEHP dose-dependent response to macrophage recruitment. PND 28 rats were treated with either Veh or 250 mg/kg for 3 days (n = 7 for each group). A) Representative micrographs of MHCII⁺ (green) peritubular macrophages and DAPI⁺ (blue) cells are shown on the surface of seminiferous tubules in both Veh and MEHP groups. All images in this figure were taken at 20x. The scale bar (50 μm) represented in the top left panel applies to all images in this fig. B) Quantification of peritubular macrophages and differentiating spermatogonia population from Veh and MEHP groups. The number of MHCII⁺ and PLZF+ cells normalized to tubule area. The data were subjected to a Student’s t-test or parametric one-way ANOVA followed by the Tukey test for post hoc comparisons. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. C) Representative TUNEL images. Dark brown dots are considered apoptotic germ cells. D) Quantification apoptotic cells from Veh and MEHP (250 mg/kg for 3 days). E) Representative images of tight junctions Occludin (green) ZO-1 (red) and quantification of fluorescence intensity using ImageJ.

In the 100 mg/kg/day for 7 days group, no significant increase in the MHC II-positive (peritubular macrophage) or PLZF-positive population was seen (Fig. 4A and B).

Fig. 4.

Chronic low MEHP dose-dependent response to macrophage recruitment. PND 28 rats were treated with either Veh or 100 mg/kg for 7 days (n = 7 for each group). A) Representative micrographs of MHCII⁺ (green) peritubular macrophages and PLZF⁺ (red) spermatogonia are shown on the surface of seminiferous tubules in both Veh and MEHP groups. All images in this figure were taken at 20x. Scale bar =50 μm. B) Quantification of peritubular macrophages and differentiating spermatogonia population from Veh and MEHP groups. The number of MHCII⁺ and PLZF+ cells normalized to tubule area. The data were subjected to student t-test or parametric one-way ANOVA followed by Tukey test for post hoc comparisons. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. C) Representative TUNEL images. Dark brown dots are considered apoptotic germ cell. Scale bar =50 μm. D) Quantification of apoptotic cells from Veh and MEHP (100 mg/kg for 7 days). E) Representative images of tight junctions Occludin (green) ZO-1 (red). Scale bar =50 μm and quantification of fluorescence intensity using ImageJ.

Multiple MEHP dose-dependent response to spermatocytes apoptosis and disruption of BTB junction

The TUNEL assay data revealed a significant loss of germ cells, specifically apoptosis of spermatocytes, in the 250 mg/kg/day MEHP for the 3-day treatment group compared to the Veh control group (Fig. 3C and D). BTB disruption was visualized by decreased expression of both Occludin and ZO-1(Fig. 3E) in the MEHP treatment group.

No significant spermatocyte loss was seen in 100 mg/kg/day for 7 days group (Fig. 4C and D). As expected, there was no change in the expression of both occludin and ZO-1(Fig. 4E) in the MEHP treatment group.

Depletion of MEHP-induced increase in peritubular macrophages causes a decrease in differentiating spermatogonia

To establish the role of peritubular macrophages in stimulating differentiating spermatogonia.

(PLZF+), clodronate liposomes were used to deplete macrophages in the testis before MEHP treatment. As expected, chlodronate liposomes inhibited the MEHP-induced increase in peritubular macrophages. This correlated with a significant decrease in PLZF-positive differentiating spermatogonia (Fig. 5A and B). We used Foxo-1 to stain for a small subset of spermatogonia that have stem cell potential. Increased Foxo-1 positive cells were found in the MEHP group, and clodronate liposomes inhibited that increase (Fig. 5C and D).

Fig. 5.

Depletion of MEHP-induced increase in peritubular macrophages causes a decrease in differentiating spermatogonia. PND 28 rats treated with MEHP pretreated with clodronate liposomes to deplete monocytes from circulation, or control PBS liposomes. A) Representative images of MHCII⁺ (green) peritubular macrophages and PLZF⁺ (red) spermatogonia are shown on the surface of seminiferous tubules. Cells nuclei DAPI (blue). Scale bar =50 μm. B) Quantification of peritubular macrophages and PLZF⁺ spermatogonia. C) Foxo-1 (green) in cross sections of Bouin fixed testis. Scale bar =50 μm. D) Quantification of Foxo-1 positive cells. Results from experiments are presented as individual data points and means ± S.E.M. each replicate represents an individual biological sample and the number of replicates is indicated in each figure. The data were subjected to Student’s t test or parametric 1- or 2-way ANOVA followed by the Tukey test for post hoc comparisons. All calculations were carried out using GraphPad prism software version 5.0 (GraphPad software Inc., La Jolla, California). Values are considered significant with P < 0.05.

Discussion

Peritubular macrophages (PTMφ) have recently garnered considerable interest as a promising area of exploration. This specific subset of macrophages possesses unique characteristics, particularly their substantial presence near undifferentiated spermatogonia and their expression of specific molecules that are thought to impact the proliferation and differentiation of spermatogonia in mice.⁷ A recent investigation by our lab noted that acute MEHP exposure of peripubertal male rats led to an amplified increase in PTMφs, accompanied by a higher count of spermatogonia.⁸ However, the precise mechanism behind the rise in PTMφs within the testicular environment remains poorly elucidated.

Our previous study demonstrated that exposure to acute high doses of 700 mg/kg MEHP significantly enhanced PTMφs. Here, we focus on the dose-dependent effect of MEHP on peritubular macrophages and differentiating spermatogonia population. A low dose of MEHP (100 mg/kg) for 7 days did not increase PTMφs or the differentiating spermatogonia population. Similarly, a single dose of MEHP (250 mg/kg) for 1 day didn’t incite a PTMφ increase either. However, 3 doses of MEHP (250 mg/kg) led to a significant increase in PTMφs as well as differentiating spermatogonia population similar to the acute dose of MEHP (500 mg/kg). This data suggests a threshold dose is required to stimulate the peritubular macrophage number. Oishi et al. reported that the half-life of MEHP in the testes is 49.9 h.¹² Utilizing this data, we plotted a graph assuming metabolism follows a linear degradation pattern. From our observations regarding the infiltration of peritubular macrophages at various MEHP doses, we estimate that the threshold dose is approximately 500 mg/kg.

Previous studies from our lab have reported that an acute high dose of 700 mg/kg MEHP resulted in BTB disruption, which occurs only in peri-pubertal and not adult rats.¹¹ We observed a significant germ cell loss in peri-pubertal and not adult rats after an acute 750 mg/kg dose of MEHP.⁵ Furthermore, a recent publication from our lab demonstrated that significant infiltration of PTMφs occurs in peri-pubertal and not adult rats after a high acute dose of MEHP.⁹ These findings led to the hypothesis that disruption of BTB and germ cell loss in peri-pubertal rats is a prerequisite for increased PTMφs numbers after MEHP exposure.

To challenge this hypothesis, we exposed peri-pubertal rats to doses of MEHP that caused germ cell loss but not disruption of BTB (single dose of 250 mg/kg) and dose that resulted in neither BTB disruption nor germ cell loss (100 mg/kg for 7 days) as well as a dose that disrupted BTB and resulted in significant germ cell loss (250 mg/kg for 3 days and 500 mg/kg for 1 day). We were intrigued to see a gain in peritubular macrophages when BTB is disrupted, as depicted by the reduced expression of occludin and ZO-1. The doses that disrupted BTB led to a gain in peritubular macrophage numbers. Therefore, this increase in peritubular macrophage number depends on BTB status and not germ loss. A recent publication by our lab has depicted that BTB disruption is a prerequisite to inciting peritubular macrophages.¹⁰ This publication also reported that cadmium chloride, a toxicant disrupting BTB, also significantly increased peritubular macrophages.¹⁰ Our previous publication has demonstrated that the BTB is fully recovered in 5 wk after an acute exposure of 700 mg/kg MEHP.¹¹ Ongoing studies in our lab are designed to challenge the hypothesis that the peritubular macrophages play a key role in this BTB recovery process.”

Lately, peritubular macrophages in the testis have garnered a lot of attention. However, the functional significance of these peritubular macrophages remains unknown. We know that an increase in peritubular macrophage numbers occurs after BTB disruption and massive germ cell loss caused by various toxicants.¹⁰ Previous studies reported that an increase in peritubular macrophage numbers is always associated with a significant increase in differentiating spermatogonia (PLZF+ cells).⁸ Considering that peritubular macrophages are understood solely for their role in encouraging spermatogonia development, and given that a depletion of spermatocytes would require replenishment through the initiation of spermatogonial differentiation, it is plausible to propose that the observed rise in peritubular macrophages aims to prompt spermatogonial differentiation for the replacement of lost spermatocytes. Therefore, it is hypothesized that this increase in peritubular macrophage numbers assists in the repair and recovery of spermatogenesis after testis injury.

We utilized clodronate liposomes to eradicate peritubular macrophages temporarily after MEHP exposure. This depletion of peritubular macrophages led to a significant decrease in differentiating spermatogonia. We have previously reported that the depletion of macrophages by clodronate does not impact germ cell loss.⁶ Our data shows that exposure to MEHP caused an increase in Foxo-1 expression. It has been well established that Foxo-1 expression is required to maintain spermatogonial stem cells and spermatogenesis in mice.¹³

Here, we report that depletion of peritubular macrophages caused a decrease in Foxo-1 expression after MEHP exposure. These data establish that eliminating peritubular macrophages reduces Foxo-1 expression and the number of differentiating spermatogonia. Therefore, we hypothesize that one of the significant functions of peritubular macrophages is to stimulate the recovery of spermatogenesis after toxicant injury.

We can gain further insight into the possible function of peritubular macrophages by their location and morphology. Sertoli cells exhibit macrophage-like functionalities, thus assuming an immunological role within the seminiferous epithelium, including engulfing apoptotic spermatocytes.¹⁴ Typically, Sertoli cells induce germ cell apoptosis and remove them, retaining meiotic and immunogenic remnants behind BTB.¹⁵ However, Sertoli cells may struggle to handle apoptotic bodies efficiently in cases of severe germ loss, like acute MEHP exposure. This could compromise the BTB, potentially triggering an autoantigenic immune response against “nonself” antigens and exacerbating damage.^11,16 Sertoli cells also act as antigen-presenting cells, dampening inflammatory immune responses.¹⁷ Peritubular macrophages, situated proximally to Sertoli cells and capable of responding to widespread apoptosis events, may aid in tolerizing inflammatory immune responses during catastrophic scenarios. Direct communication between macrophages and testicular cells is documented, as interstitial macrophages interact with Leydig cells to support steroidogenesis.¹⁸ Given their proximity, peritubular macrophages potentially interact with Sertoli cells. However, further investigation, such as localizing peritubular macrophages and assessing their penetration of the PTMC layer, is necessary to confirm this hypothesis.

Conclusions

In conclusion, we report a dose-dependent increase of peritubular macrophages as well as differentiating spermatogonia population in response to various doses of MEHP. A threshold dose of MEHP is required to incite this mobilization of peritubular macrophages. More importantly, the disruption of BTB correlates to an increase in the number of peritubular macrophages rather than germ cell loss. Finally, peritubular macrophages aid in spermatogenesis by stimulating the proliferation of Foxo-1 and differentiating spermatogonia, thereby facilitating repair and recovery after toxicant-induced testicular injury.

Author contributions

Conception and design: R.T and JHR; Data collection: R.T., X.F., V.P.N.; Data analysis and interpretation: R.T., approved of the final version.: R.T., J.H.R.

Funding

Supported by NIH/NIEHS R01ES016591 and The Center for Molecular Carcinogenesis and Toxicology.

Conflicts of interest: The authors declare no conflict of interest.