The blood-testis barrier disruption is a prerequisite for toxicant-induced peritubular macrophage increases in the testis of peripubertal rats

Abstract

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.

Graphical Abstract

Graphical abstract.

Peritubular macrophages (PTMφ) have gained significant attention in recent years as a promising area of investigation. This subset of macrophages exhibits distinct characteristics, primarily their abundant presence in the vicinity of undifferentiated spermatogonia and their expression of specific molecules hypothesized to influence spermatogonia proliferation and differentiation in mice (DeFalco et al., 2015). Notably, PTMφs possess an irregular morphology and are situated on the surface of seminiferous tubules, distinguishing them from other types of testicular macrophages. A recent study conducted by our group (Gillette et al., 2021) observed that exposure of peripubertal male Fischer rats to mono-(2-ethylhexyl) phthalate (MEHP) resulted in an augmented increase of PTMφs, accompanied by an increased number of spermatogonia. However, the underlying mechanism driving the increase of PTMφs in the testis remains poorly understood.

The blood-testis barrier (BTB) is a crucial physiological structure in the testis first discovered in the 1960s. It is primarily composed of tight junctions between adjacent Sertoli cells, which contain specific proteins such as members of the claudin family (Chakraborty et al., 2014; Dietze et al., 2015; Haverfield et al., 2014; Komljenovic et al., 2009; Mazaud-Guittot et al., 2010), junctional adhesion molecule family (Aurrand-Lions et al., 2001), tricellulin and coxsackievirus, and adenovirus receptor (Steed et al., 2010). Functioning as a physical and biochemical barrier, the BTB restricts the movement of molecules and germ cells between the basal and the adluminal compartment, thereby maintaining immune privilege within the testis and safeguarding germ cells from immunoregulatory factors and harmful substances (Kaur et al., 2014; Mita et al., 2011). Numerous studies have examined the impact of environmental contaminants on BTB integrity, including cadmium (Cd), phthalates, and bisphenol A (BPA) (Siu et al., 2009). For instance, bisphenol AF (BPAF) has been shown to disrupt the BTB integrity and influence cytoskeletal architecture and tight junction permeability in Sertoli cells (Wu et al., 2019a). Similarly, di-(2-ethylhexyl) phthalate (DEHP) exposure has been found to impair the BTB in rats, manifested by decreased expression of occludin and connexin43 (Shen et al., 2017). MEHP, the active metabolite of DEHP, is well-recognized to target Sertoli cells, causing functional damage in their capability to maintain the specialized testicular environment for spermatogenesis (Yao et al., 2009). Intriguingly, our recent study reported that a single acute dose of MEHP exposure reversibly disrupted the BTB in peripubertal rats, which provides clues as to whether BTB plays a functional role in the PTMφs process.

The current study explores the interplay between disruption of the BTB and the increase in PTMφs in peripubertal rats by exposing animals to toxicants acting by different mechanisms and comparing the resulting PTMφ responses with our previous reports in MEHP-treated animals (Tiwary and Richburg, 2023; Voss et al., 2018). Specifically, we compared the response of PTMφs in Fischer CDF344 rats at postnatal day/PND 26 following the administration of 3 known testicular toxicants: MEHP causes spermatocyte apoptosis and BTB disruption (Gillette et al., 2021; Tiwary and Richburg, 2023; Voss et al., 2018), methoxyacetic acid (MAA) is a direct pachytene spermatocyte toxicant (Brinkworth et al., 1995; Singh and Gupta, 2019), and cadmium chloride (CdCl₂) a well-established BTB disruptor (Bekheet, 2010; Setchell and Waites, 1970) resulting in the disruption to the BTB and spermatogenesis. Intriguingly, the findings reveal that the mere occurrence of spermatocyte apoptosis is insufficient to increase the number of PTMφs in the testis. Furthermore, we employed LG3/4/5, the active domain of laminin-alpha 2 in the testis to safeguard the BTB from MEHP-induced disruption, providing evidence that BTB disruption is a prerequisite for increasing PTMφs after exposure to MEHP.

Materials and methods

Male Fischer CDF344 rats were purchased from Charles River (Wilmington, Massachusetts). The animals were housed in a regulated environment with controlled temperature (22°C ± 0.5°C) and lighting (12 L:12D) for 3 days to adapt to the experimental challenges. They were supplied with standard lab chow (Purina Mills Lab Diet No. 5LL2, St Louis, Missouri) and tap water without any restrictions. All animal procedures were performed by the guidelines and approval of The University of Texas at Austin’s Institutional Animal Care and Use Committee.

MEHP with a purity of 97.3% was procured from Wako Chemicals (Richmond, Virginia), whereas MAA with a purity of 98% and CdCl₂ were obtained from Sigma-Aldrich, USA. Hematoxylin was purchased from VWR North American (Austin, Texas), and Eosin Yellowish solution was obtained from Fischer Diagnostics (Middletown, Virginia).

The primary antibodies used for immunofluorescent staining include PLZF (rabbit polyclonal, Cat No. sc-28319), which was acquired from Santa Cruz Biotechnology (Dallas, Texas), MHCII (mouse antirat, Cat No. 205401), which was purchased from BioLegend (San Diego, California), laminin alpha2 (mouse antirat, Cat No. MAB1922) was obtained from Millipore Sigma. The secondary antibodies, including Alexa Fluor 488 goat antimouse and 568 goat antirabbit, were procured from Invitrogen (Waltham, Massachusetts). The antimouse HRP secondary antibody was purchased from Bio-Rad for the Western blotting.

Male Fischer F344 rats at exact PND 26 were administered a single oral dose of MEHP (700 mg/kg in corn oil) or an equivalent volume of control (corn oil, 2 ml/kg, p.o.), MAA (600 mg/kg in 0.9% NaCl) or an equivalent volume of control (0.9% NaCl) via intraperitoneal (i.p.) injection, or received a single dose of CdCl₂ (1.25 mg/kg or 2.5 mg/kg in 0.9% NaCl) or an equivalent volume of control (0.9% NaCl) through intraperitoneal (i.p.) injection with a 23-gauge 0.5 cc insulin syringe. Based on our previous reports, these doses were selected that 700 mg/kg incited a significant increase in PTMφ numbers in peripubertal male Fischer rats (Gillette et al., 2021). The animals were periodically monitored for general health following the treatments. After 48 h, EZ-link Sulfo-NHS-Biotin (Thermo Scientific, Waltham, Massachusetts) was injected into the testis at 0.5 mg/g body weight using a 30-gauge 0.5 cc insulin syringe, and the heartbeat was continuously monitored for 5 min. Following 40 min of biotin circulation, the rats were euthanized via CO₂ asphyxiation, and cervical dislocation was used as the secondary method of euthanasia. One testis was collected for whole seminiferous tubule staining, whereas the other was paraffin-embedded for further use. In brief, testis for paraffin embedding was stored overnight on a shaker in Bouin’s Fixative solution (RICCA, Texas). Bouin’s solution was replaced with lithium-saturated ethanol the following days until the solution and the testis were clear. Subsequently, the tissue is processed through a series of alcohol gradient steps using an automatic tissue processor with a vacuum (Leica TP 1020) following the manufacturer’s instructions. Tissues were embedded in paraffin using the embedding station (Leica EG1150H and EG1150C) for further experimental downstream procedures required for microscopic evaluation.

The pCl_neo mammalian expression vectors, containing LG3/4/5 cDNA or without, were procured from GenScript (Piscataway, New Jersey) following the method detailed by Dr Linxi Li (Li et al., 2020). The LG3/4 and LG4/5 cDNA constructs were obtained through polymerase chain reaction (PCR) using primers specific to LG3, LG4, or LG5 based on the Genbank Accession Number XM_017590489.1 for rat laminin alpha 2 chain. The PCR products of LG3/4 and LG4/5 were denatured, reannealed, and served as templates for a second PCR using primers LG3 (sense) and LG5 (antisense) to obtain the LG3/4/5 construct. The LG3/4/5 cDNA construct was then cloned into the pCI-neo vector (Promega) at the XhoI/SalI restriction sites, purified, and amplified for subsequent use, used primer sequences are cited from Li et al. (2020) as listed below in Table 1. For in vivo transfection, the testes were transfected with either pCl_neo empty vector or pCl_neo LG3/4/5 on day 0 (PND 26), 2 (PND 28), and 4 (PND 30), followed by MEHP administration on day 6 (700 mg/kg oral gavage) (PND 32). Each testis was transfected with 10 µg plasmid DNA (either pCI_neo/LG3/4/5 or pCI_neo empty) and 1.5 µl in vivo-JetPEI reagent (PolyPlus-transfection, Illkirch-Graffenstaden, France) suspended in sterile 5% glucose (wt/vol) to a final transfection mixture of 30 µl per testis. Eight animals were injected with the transfection mixture containing pCI_neo empty vector, and another 8 animals were injected with the transfection mixture containing pCI_neo LG3/4/5 plasmid DNA, which was administered to each testis via a 30-gauge insulin syringe. To prevent a sudden increase in intratesticular hydrostatic pressure while administering the transfection mixture, we inserted the needle vertically from the apical to the basal end of the testis. As the needle was withdrawn, the transfection solution was gently released, evenly filling the entire testis, following the described procedure (Gao et al., 2016, 2017b; Li et al., 2020).

Table 1.

Primers used for PCR

Gene fragment	Primer sequences	Nucleotide position
(1) LG3 (sense)	5′-CCGCTCGAGATGGTCGGAACGGAAATCAACCTG-3′	7741-7761
(2) LG3 (antisense)	5′-ACGCGTCGACTTAAGCTATAGGCTGTGCAAAGTC-3′	8188-8208
(3) LG4 (sense)	5′-CCGCTCGAGATGAAAAACCGCCTCACCATTGAG-3′	8455-8475
(4) LG4 (antisense)	5′-ACGCGTCGACTTAGGTAGGCTGGTCCAGATCAAC-3′	8863-8883
(5) LG5 (sense)	5′-CCGCTCGAGATGGTTGGATTGGACCTTCTTGTA-3′	8977-8997
(6) LG5 (antisense)	5′-ACGCGTCGACTTACAGAGCCTTGGCAAAATTAAC-3′	9412-9432
(7) LG3/4 (sense)	5′-CCGCTCGAGATGGTCGGAACGGAAATCAACCTG-3′	7741-7761
(8) LG3/4 (antisense)	5′-ACGCGTCGACTTAGGTAGGCTGGTCCAGATCAAC-3′	8863-8883
(9) LG4/5 (sense)	5′-CCGCTCGAGATGAAAAACCGCCTCACCATTGAG-3′	8455-8475
(10) LG4/5 (antisense)	5′-ACGCGTCGACTTACAGAGCCTTGGCAAAATTAAC-3′	9412-9432
(11) LG3/4/5 (sense)	5′-CCGCTCGAGATGGTCGGAACGGAAATCAACCTG-3′	7741-7761
(12) LG3/4/5 (antisense)	5′-ACGCGTCGACTTACAGAGCCTTGGCAAAATTAAC-3′	9412-9432

The successful overexpression of LG3/4/5 plasmid was verified via Western blotting. In detail, proteins from animals with control and LG3/4/5 plasmid injection groups were isolated using RIPA buffer as described (Gao et al., 2017b) and quantified via BCA assay using Pierce BCA protein assay kits from ThermoFisher Scientific. An equal amount of protein lysate of around 80 μg was resolved by 10% SDS-PAGE and transferred to nitrocellulose membrane (ThermoFisher Scientific) for immunoblot analysis using the laminin alpha 2 primary and antimouse HRP secondary antibody. Target proteins were visualized by chemiluminescence using a SuperSignal kit prepared according to the manufacturer’s instructions (ThermoFisher Scientific). Images were acquired by using a Amersham Imager 600 with adjustable manual exposure time. ImageJ and Graphpad Prism were further employed to analyze the Western-blot images.

The functional integrity of BTB was identified via biotin tracer assay. Specifically, EZ-link Sulfo-NHS-Biotin (Thermo Scientific, Waltham, Massachusetts) was administered as described above, followed by the collection and fixation of the testis. The BTB permeability was assessed through fluorescent staining by using streptavidin Alexa Fluor 488 conjugate (Life Technologies, Eugene, Ore).

Briefly, the testis was decapsulated by puncturing and tearing the tunica, and the tubules were evacuated into cold PBS. The tubules were gently teased apart, then the interstitial tissue and blood vessels were removed with blunt forceps. The tubules were then washed 4 times in ice-cold PBS to further remove interstitial tissue and cells. The tubules were then fixed in 4% paraformaldehyde (Electron Microscopy Sciences, 15710-S) overnight at 4°C, washed an additional 4 times in cold PBS, and then start staining with MHCII and PLZF antibodies for imaging. Stained seminiferous tubules were imaged on a Zeiss scanning confocal microscope (Zeiss, LSM 710). Images that were used to count PTMφ and spermatogonia were taken at 20× (425.10 μm by 425.10 μm) and with a depth of 15 µm in 3-µm increments to ensure the surface of the seminiferous epithelium and spermatogonia were captured. Z-stacks were flattened in ImageJ (version 1.49 T) using the ‘Max Intensity’ method. Positive cells were then counted with ImageJ as previously published (Gillette et al., 2021); specifically, PTMφs have been identified based on their seminiferous tubule surface localization as well as the MHCII positive signal and further checked via an artificial intelligence-based nonbiased quantification technique called NIS-elements NIS.ai General Analysis 3 developed by NIKON. Cell counts were normalized to tubule area and are represented as cell counts per 10⁵ pixel area. For each animal, at least 30 seminiferous tubule images were analyzed.

The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis using the ApopTag kit (EMD Millipore, Billerica, Massachusetts) was used to indicate the presence of apoptotic fragmentation of DNA in paraffin-embedded testis cross-sections. In accordance with previous studies (Voss et al., 2018), the apoptotic index (AI) was calculated as the percentage of essentially round seminiferous tubules containing more than 3 TUNEL-positive germ cells in each cross-section. For each animal, at least 3 cross sections and at least 100 seminiferous tubules were analyzed.

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 < .05.

Results

 

MAA caused severe spermatocyte apoptosis but did not disrupt the integrity of the BTB or instigate increases of PTMφs in the testis

Figures 1A and 1B show severe spermatocyte apoptosis in peripubertal rats 48 h after exposure to 600 mg/kg MAA. Quantifying these results (Figure 1C) suggests that the AI increased from 6 to 35 tubules among the total of 100 tubules being quantified; the MAA-treated group shows significantly higher apoptosis than the vehicle control group. Biotin tracer assay result shows that in both the control and the MAA exposure groups, there is no diffusion of biotin molecules to the adluminal compartment or lumen area (Figs. 1D and 1E), which suggests that MAA administration did not disrupt the BTB functional integrity. It can be seen from the representative confocal image that the MAA group does not have a higher level of PTMφ number in their seminiferous tubules (Figs. 1F and 1G). It is also obvious from the quantification result that there is no significant difference in the number of PTMφ between control and MAA-treated animals (Figure 1H).

Figure 1.

Methoxyacetic acid (MAA) exposure did not increase peritubular macrophage numbers. A and B, TUNEL assay results of PND 26 F344 rats exposed to the vehicle control (A) or 600 mg/kg MAA (B). Black arrows indicate apoptotic spermatocytes, scale bar=100 µm. C, The apoptotic index (AI) was calculated with N=6 rats per treatment group, counting 3 cross-sections and 100 tubules per animal. D and E, Biotin tracer assay results of vehicle control (D) and MAA-treated rats (E), scale bar=50 µm. F and G, Representative figures of whole seminiferous tubule staining results of vehicle control (F) and MAA-treated rats (G), peritubular macrophages are stained with MHCII. The green color represents positively stained peritubular macrophages (white arrow). The red color represents PLZF staining for undifferentiated and differentiating spermatogonial cells, scale bar=50 µm. (H) Quantification of changes in peritubular macrophage levels with N=5 rats per treatment group.

CaCl₂ led to spermatocyte apoptosis, disruption of the integrity of the BTB, and an increased number of PTMφs in the testis

It is apparent from the TUNEL assay result that 1.25 mg/kg CdCl₂ did not cause severe germ cell apoptosis in the cross-section (Figs. 2A and 2B). However, 2.5 mg/kg CdCl₂ led to noticeable cell apoptosis (Figure 2C). Quantification of results (Figure 2D) validates the observation that a higher dose of CdCl₂ indeed caused significantly severer germ cell apoptosis compared with the control group or low dose CdCl₂ group. As Figures 2E and 2F show, no biotin diffusion was observed in the seminiferous tubules in both the control group and the 1.25 mg/kg low-dose CdCl₂ group. This observation suggested that the BTB in these 2 groups was intact upon treatment. In contrast, in the 2.5 mg/kg CdCl₂ group, all the tubules were obviously damaged, and the biotin molecules were widely dispersed (Figure 2G), which suggests that the BTB was permeable after high-dose CdCl₂ exposure. Figures 2H–J present the seminiferous tubule staining result of control group, 1.25 mg/kg CdCl₂ and 2.5 mg/kg CdCl₂ group. A trend of PTMφ increase exists in the 2.5 mg/kg CdCl₂ animals. Quantification results (Figure 2K) suggested that a low dose of CdCl₂ did not incite an increase in the number of PTMφ, whereas the high dose did significantly increase the PTMφ numbers.

Figure 2.

Cadmium chloride (CdCl₂) treatment increased peritubular macrophage numbers. A–C, TUNEL assay results of PND 26 rats exposed to the vehicle control (A) or 1.25 mg/kg (B) and 2.5mg/kg CdCl₂ (C). Positively stained apoptotic germ cells are suggested by black arrows, scale bar=100 µm. D, The apoptotic index (AI) was calculated with N=6 rats per treatment group, 3 cross-sections, and 100 tubules per animal. E–G, Biotin tracer assay results of vehicle control (E) and CdCl₂ treated animals (F and G). White arrowheads indicate the biotin tracer within the adluminal compartment, scale bar=50 µm. H–J, Representative figures of whole seminiferous tubule staining results of vehicle control (H) and CdCl₂ exposure animals (I–J), peritubular macrophages are stained by MHCII (green), positively stained peritubular macrophages are indicated by white arrow. PLZF (red) stain indicates undifferentiated and differentiating spermatogonial cells, scale bar=50 µm. (K) Quantification of changes in peritubular macrophage numbers with N=5 rats per treatment group.

Tightening of BTB integrity via LG3/4/5 overexpression protects against MEHP-induced testis injury and blocks MEHP-induced increase of PTMφs in the testis

To test whether the overexpression of LG3/4/5 was successful, Western blot and immunofluorescent staining were employed to verify the protein level of laminin alpha 2. As depicted in the Figure 3 upper panel, in the pCl_neo LG3/4/5 groups the level of laminin alpha 2 protein is robustly elevated compared with pCl_neo empty control groups, with or without MEHP exposure. Similarly, in the immunofluorescent staining result, a similar protein level trend was observed (Figure 3 lower panel). The pCl_neo empty plasmid + MEHP treatment has very dim to none signals, whereas pCl_neo LG3/4/5 + CO treatment has noticeably enhanced laminin alph2 signals in its cross-section.

Figure 3.

LG3/4/5 overexpression in the animals’ testes. The upper panel represents the representative Western-blot result of laminin alpha2 protein and the control beta action reference protein level in the animals, the 83- and 40-kDa markers are shown on the most right lane. The protein densitometry result is adjusted with beta-actin. The bottom panel shows the immunofluorescent staining result of laminin alpha 2 protein among empty plasmid injection + corn oil treatment (A), empty plasmid injection + MEHP treatment (B), LG3/4/5 overexpression plasmid + corn oil treatment (C), and LG3/4/5 overexpression plasmid + MEHP treatment (D), scale bar=50 µm.

As shown in Figure 4, in the pCl_neo empty + CO and pCl_neo LG3/4/5 + CO treatment groups (Figs. 4B and 4D), the functional BTB was capable of blocking the entry of biotin molecule into the seminiferous tubules but restrained biotin in the interstitial space. In contrast, from the pCI_neo empty + MEHP group, it shows that biotin was able to enter the seminiferous epithelium (Figure 4C). However, overexpression of LG3/4/5 was effectively blocked the BTB disruption effect caused by MEHP, maintained the BTB functional integrity (Figure 4E). Whole seminiferous tubule staining results suggest that in pCl_neo empty + MEHP group, rats showed a significant increase of PTMφ numbers compared with the corn oil-treated group. However, with overexpression of LG3/4/5, when the BTB disruption was successfully blocked, and no significant increase of PTMφ was observed in the pCl_neo LG3/4/5 + MEHP group (Figs. 4F–J).

Figure 4.

LG3/4/5 overexpression protected BTB from MEHP and blocked the increase of peritubular macrophages. A, Schematic design of LG 3/4/5 overexpression stabilizing the MEHP-induced BTB disruption and peritubular macrophage increase. B–E, Biotin tracer assay results of animal (B) with empty plasmid injection + corn oil treatment, (C) empty plasmid injection + MEHP treatment, (D) LG3/4/5 overexpression plasmid + corn oil treatment and (E) LG3/4/5 overexpression plasmid + MEHP treatment, white arrowheads indicate the biotin tracer within the adluminal compartment, scale bar=50 µm. F–I, Representative figures of whole seminiferous tubule staining results of empty and LG3/4/5 overexpression plasmid with corn oil vehicle control or MEHP exposure animals, peritubular macrophage is stained by MHCII, positively stained peritubular macrophages are indicated by white arrows, PLZF-staining indicates undifferentiated and differentiating spermatogonial cells, scale bar=50 µm. (J) Quantification of changes in the numbers of peritubular macrophages with N=5 rats per treatment group.

Discussion

The primary aim of this study was to investigate the mechanistic basis underlying the observed increase in PTMφs and to elucidate the requisite significance of BTB disruption for PTMφ increases in the testis. Our previous study demonstrated an elevated presence of PTMφs in peripubertal rats following exposure to an acute dose of MEHP (Gillette et al., 2021). However, the precise molecular mechanisms underlying the observed increase of PTMφs in the testis are unknown. The Defalco group previously suggested that Sertoli cells play an essential role in the testis microenvironment for testicular monocyte recruitment and macrophage differentiation (Gu et al., 2023). In addition, our prior work also observed the reversible disruption of BTB integrity in peripubertal rats by MEHP, potentially involving the mitogen-activated protein kinase(MAPK)pathway (Tiwary and Richburg, 2023). These notable findings prompted an inquiry into the correlation between BTB disruption and the observed increase in PTMφs.

BTB is a vital physiological structure within the testes that separates the adluminal and luminal compartments. Comprising tight junctions between adjacent Sertoli cells, the BTB functions as an intricate and selective barrier, regulating the passage of substances between the bloodstream and the seminiferous epithelium (Dym, 1994; Siu and Cheng, 2004). The BTB is critical in establishing a unique microenvironment required for functional spermatogenesis and safeguarding developing germ cells against potentially harmful substances. Nonetheless, the perturbation of the BTB can have significant repercussions on male reproductive health. Prior investigations have highlighted various factors, including infections, hormonal imbalances, and toxicants, as potential disruptors of BTB integrity (Boekelheid and Hall, 1991; Kolasa et al., 2011; Meistrich et al., 2003; Wu et al., 2019b). Previous findings from our laboratory have indicated a concurrent increase in PTMφs and BTB disruption (Tiwary and Richburg, 2023). Specifically, our observations have revealed a significant elevation in PTMφ numbers within the testes of peripubertal rats 24 h after exposure to a single acute dose of MEHP. Intriguingly, structural and functional impairments were observed in the BTB during the same timeframe.

The primary objective of this study was to compare the responses of PTMφs under different conditions, specifically after exposure to MEHP, the direct germ cell toxicant MAA, and the well-known BTB disruptor agent CdCl₂ (Krishnamurthy et al., 1998; Nemetallah and Ellis, 1985). This experimental strategy will allow for the distinction if increases in PTMφs occur solely in response to BTB disruption or occur more generally to toxicant-induced testicular injury. To further challenge the critical significance of the BTB disruption in this process, we employed the approach to tighten the functional integrity of BTB via the overexpression of LG3/4/5 (Li et al., 2020), before challenging the animals with MEHP.

MAA exposure for 48 h resulted in significant germ cell apoptosis without BTB disruption (Figs. 1A–C), which is consistent with previous literature reporting extensive germ cell apoptosis following MAA exposure (Stermer et al., 2017; Tirado et al., 2004). Interestingly, we observed nonspermatocyte cell death and empty tubules in the MAA group, which can be possibly attributed to the extended exposure time compared with previous studies, where MAA exposure was limited to 24 h. However, in our study, animals were sacrificed, and testes tissue was collected 48-h post-MAA exposure. Additionally, quantification of PTMφ levels through whole seminiferous tubule staining revealed no increase in PTMφ numbers following MAA treatment (Figure 1F). These observations indicate that germ cell apoptosis alone is insufficient to trigger an increase in PTMφs in the testes of peripubertal rats. To further explore the relationship between PTMφ increase and BTB disruption, we utilized the BTB disruptor CdCl₂. We found that a low dose of 1.25 mg/kg CdCl₂ did not induce significant germ cell apoptosis, whereas a high dose of 2.5 mg/kg CdCl₂ led to pronounced germ cell apoptosis (Figs. 2A–D). Similarly, the low dose of 1.25 mg/kg CdCl₂ did not disrupt BTB functional integrity, whereas the high dose of 2.5 mg/kg CdCl₂ resulted in BTB permeability disruption (Figs. 2E–G). These observations align with previous literature where 3 mg/kg CdCl₂ caused an evident loss of BTB integrity (Elkin et al., 2010). Consistent with our hypothesis, we observed a significant increase in PTMφ levels only with the BTB disrupting dose of 2.5 mg/kg CdCl₂ (Figs. 2H–K). Accordingly, our group has previously reported that a single acute dose of MEHP leads to a significant increase of PTMφs (Gillette et al., 2021) and compromised BTB integrity in peripubertal rats (Tiwary and Richburg, 2023). These findings clearly demonstrate the critical role of BTB disruption in the increase of PTMφs. Although the actual function of the PTMφs is not yet fully understood. Previous literature reported that PTMφs express spermatogonial proliferation- and differentiation-including factors, such as colony-stimulating factor 1 and enzymes involved in retinoic acid biosynthesis (DeFalco et al., 2015). Additionally, our group previously observed the association between the increase in PTMφs and the differentiating spermatogonia cell upon rodent testis becoming injured (Gillette et al., 2021). These findings led to the hypothesis that PTMφs could contribute to the recovery of the injured rat testes from the effects of toxicants. Therefore, in the current study, we observed that MAA, CdCl₂, and MEHP induced different PTMφs responses, it is plausible this would cause a distinct recovery of spermatogenesis among these toxicant-treated animals.

To further substantiate our hypothesis regarding the necessity of BTB disruption for the increase in PTMφ numbers, we employed LG3/4/5, an active functional domain of laminin-alpha 2, to prevent BTB disruption induced by MEHP. Laminin-alpha 2, an extensively studied extracellular matrix protein, is primarily expressed in the basement membrane surrounding the seminiferous tubules. It plays a pivotal role in maintaining the structural integrity and function of the BTB through interactions with other extracellular matrix molecules and cell adhesion proteins (Gao et al., 2017a,b). Thus, it is instrumental in stabilizing the tight junctions between Sertoli cells and preserving the selective permeability of the barrier. Previous studies have demonstrated that LG3/4/5 overexpression can effectively block or rescue BTB disruption induced by the well-recognized BTB disruptor CdCl₂ (Li et al., 2020). In this study, we aimed to test the hypothesis that LG3/4/5 overexpression would also prevent BTB disruption caused by MEHP and consequently impede the increase of PTMφ (Figure 4A). We first verified that the LG3/4/5 plasmid overexpression was successful in the rodents’ testes via Western blotting and immunofluorescent staining (Figure 3). Our results revealed that LG3/4/5 overexpression successfully blocked MEHP-induced BTB disruption (Figs. 4B–E). Furthermore, in the LG3/4/5 overexpression group, no significant increase in PTMφ was observed (Figs. 4F–J), providing evidence that BTB disruption is a prerequisite for the increase in PTMφ. These findings advanced our understanding of the relationship between BTB integrity and PTMφ dynamics in the context of MEHP exposure.

This is the first report to reveal that BTB disruption instigates an increase of PTMφs in the peripubertal rodents’ testis. This assertion is based on the ability of LG3/4/5 overexpression to block BTB disruption induced by MEHP and prevent MEHP-induced increases in PTMφs. Nevertheless, the specific cellular mechanisms accounting for increased PTMφs in the testis after MEHP exposure remain unresolved. Maturation of the BTB occurs in the early stages of postnatal development in rodents before the establishment of the immunotolerance mechanisms (Mruk and Cheng, 2015). Therefore, it is likely that a disruption of the BTB integrity has the potential to allow germ cell antigens to ‘leak’ into the basal compartment, thereby rendering them susceptible to detection by immune cells and subsequently triggering the expression of chemokine secretion, MCP-1, by peritubular myoid cells as we have previously described (Murphy et al., 2014). This could recruit PTMφs precursors originating from circulating bone marrow-derived progenitors into the testis (Mossadegh-Keller et al., 2017). However, some investigators have indicated utilizing single-cell RNA techniques that PTMφs are exclusively seeded in the testis from embryonic precursors before birth under normal conditions (Lokka et al., 2020). The current study found that BTB functional integrity is a requisite for the PTMφ increases in the testis after toxicant exposure. Expanding the PTMφs from embryonic precursors could account for the observed increases in PTMφs under toxicants-treated conditions. Studies are underway to understand the cellular origin of the PTMφs in response to toxicants and the functional significance of increases in PTMφs.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

Grant support 2R01ES016591.

Author contributions

The authors’ contribution to the paper is as follows: study conception and design: X.F., R.T.; data collection: X.F., R.T., V.P.N.; data analysis and interpretation: X.F., R.T., J.H.R.; draft manuscript preparation: X.F., J.H.R. All authors reviewed the results and approved the final version of the manuscript.

Data availability

The data underlying this article are available in the article and in its online supplementary material.