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. 2023 Dec 19;198(1):76–85. doi: 10.1093/toxsci/kfad128

Responses of peritubular macrophages and the testis transcriptome profiles of peripubertal and adult rodents exposed to an acute dose of MEHP

Xin Fang 1,2, Richa Tiwary 3, Vivian P Nguyen 4, John H Richburg 5,✉
PMCID: PMC10901151  PMID: 38113427

Abstract

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.

Keywords: peritubular macrophage, mono-(2-ethylhexyl) phthalate (MEHP), testis, age, species, sensitive life stage

The use of phthalates as plasticizers has been increasing since their introduction in the 1930s due to their cost-effectiveness and favorable properties as an additive to plastic materials (Thomas et al., 1984). Di-(2-ethylhexyl) phthalate (DEHP), the most commonly used plasticizer globally, is found in numerous consumer goods such as construction materials, furniture, clothing, food packages, children’s toys, and even in pharmaceutical products (Bekö et al., 2013). The lack of a covalent bond between phthalate polymers and the product matrix enables them to leach into the environment and subsequently enter human bodies through ingestion, inhalation, absorption, or injection (Gong et al., 2016; Hoppin et al., 2013), leading to significant exposure. In fact, all human urine samples analyzed contained phthalates (Blount et al., 2000; Toppari et al., 1996). The proximate toxic metabolite of DEHP is mono-(2-ethylhexyl) phthalate (MEHP) and it has been linked to a number of adverse health effects in experimental animals, depending on the life period of exposure, including endocrine disruption, reproductive toxicity, and developmental toxicity (Li et al., 1998).

MEHP’s detrimental effects on the reproductive system have long been identified with studies indicating that MEHP primarily affects testis somatic cells in postnatal animals such as Sertoli and Leydig cells (Dostal et al., 1988; Richburg and Boekelheide, 1996; Yao et al., 2010). In addition, MEHP has been shown to induce dramatic changes in germ cells, leading to spermatocyte apoptosis through the FasL/Fas signaling system (Richburg, 2006). Studies have revealed that exposure to MEHP may also lead to oxidative stress, mitochondrial dysfunction, and DNA damage in germ cells, ultimately leading to the activation of apoptotic pathways (Kasahara et al., 2002; Onorato et al., 2008; Richburg and Boekelheide, 1996). Furthermore, MEHP disrupts the balance between pro- and anti-apoptotic signals, leading to an increase in germ cell apoptosis (Richburg et al., 1999; Rogers et al., 2008).

Peritubular macrophages (PTMφs) are a recently described subset of macrophages that are found explicitly in the testis and as such, have garnered interest among researchers. PTMφs are abundant in areas with close proximity to undifferentiated spermatogonia. PTMφs have an irregular shape and reside on the surface of seminiferous tubules, differentiating them from other testicular macrophages found in the interstitial space (DeFalco et al., 2015). Previous studies investigating the origin of PTMφs have proposed that PTMφs are exclusively seeded postnatally from bone marrow-derived progenitors during puberty (Mossadegh-Keller et al., 2017). However, more recent research has claimed that PTMφs originate from embryonic precursors before birth (Lokka et al., 2020). A 2021 study from our group (Gillette et al., 2021) reported that exposure to MEHP in peripubertal rats increased the recruitment of PTMφs coincident with an increase in the number of undifferentiated spermatogonia, which led to the hypothesis that PTMφs increase occurs in response to germ cell apoptosis and/or BTB disruption. Although investigations into the origins of PTMφs have been conducted, our understanding of their responsiveness to toxicant-induced testicular injury in different ages and species of animals remains limited.

Sensitivity to phthalates is known to vary between different ages and species of rodents. Specifically, young rodents are more sensitive to phthalate’s toxic effects on the testis than adult rodents. We have previously shown that MEHP exposure in peripubertal rats elicits a robust infiltration of macrophages and neutrophils compared with adult rats (Murphy et al., 2014; Voss et al., 2018), this increased sensitivity might be attributed to the fact that the immature developing physiological systems of the young animals (Blacher et al., 2022; Gardner, 1980), makes them more vulnerable to the toxicity of MEHP but capable of repair and regeneration. In addition to age, we have also observed species differences in susceptibility to MEHP, with rats being more sensitive than mice. The extent of germ cell apoptosis in peripubertal C57BJ/6 male mice was less impacted as compared with the peripubertal rats, and mice had no significant infiltration of CD11b+ monocytes after MEHP administration (Murphy et al., 2014). Previous investigations into PTMφ responses to MEHP were limited to the peripubertal Fischer CDF344 rats, considered a “sensitive” age and species group. The PTMφ response to MEHP in adult rats and peripubertal mice, which are well-known age and species rodents that are less sensitive to the effects of MEHP and have not yet been subjected to comprehensive examination. Understanding the responses of PTMφs in these less responsive ages and species could provide clues as to whether MEHP in and of itself is inducing the response of the PTMφs or if they are increasing in numbers in response to the disruptive effects of MEHP.

The current study investigates whether the extent of MEHP injury correlates with the number of PTMφs in the testis. To address this objective, we have quantified the PTMφ number in adult (post-natal day/PND 75) male Fischer CDF344 rats (less sensitive age group) and peripubertal PND 26 C57BJ/6 mice representing the less sensitive species group compared with peripubertal rats. Experimental results indicate that the basal levels of PTMφs in less sensitive rodent animals are higher in number as compared with the MEHP-sensitive peripubertal Fischer CDF 344 rats, and no significant increase in PTMφ numbers was observed following MEHP exposure. Correspondingly, 3′ Tag sequencing data suggest that upon MEHP administration, peripubertal rats have the most differential changes in their transcriptomic profiles compared with adult rats and peripubertal mice.

Materials and methods

 

 

Animals and housing

Male Fischer CDF344 rats were purchased from Charles River (Wilmington, Massachusetts) and male C57BJ/6 mice were purchased from Jackson Labs (Bar Harbor, Maine). Animals were maintained in a controlled temperature (22°C ± 0.5°C) and lighting (12L:12D) environment and allowed to acclimate for 3 days before experimental procedures. Standard lab chow (Purina Mills Lab Diet No. 5LL2, St Louis, Missouri) and tap water were supplied ad libitum. All animal procedures were performed per the guidelines and approval of The University of Texas at Austin’s Institutional Animal Care and Use Committee.

Chemicals and antibodies

MEHP (97.3% purity) was obtained from Wako Chemicals (Richmond, Virginia). Hematoxylin was purchased from VWR North American (Missouri, Texas). Eosin Yellowish solution was received from Fischer Diagnostics (Hampton, New Hampshire). Primary antibodies were purchased from the following sources: Caspase 3 (Cat No. 9664L), Cell Signaling (Danvers, Massachusetts); PLZF (Cat No. sc-28319) and F4/80 (Cat No. sc-226642), Santa Cruz Biotechnology (Dallas, Texas); MHCII (Cat No. 205401), BioLegend (San Diego, California); Notch1 (Cat No. SAB5700255), Millipore Sigma (Burlington, Massachusetts); CD68 (Cat No. MA5-13324) and Ctnna1 (Cat No. 13-9700), Thermo Fisher (Waltham, Massachusetts). Secondary antibodies including Alexa Fluor—488 goat anti-mouse (Cat No. A11001), —488 goat anti-rabbit (Cat No. A11008), —488 goat anti-rat (Cat No. A11006), —568 goat anti-rabbit (Cat No. A11036), and —594 goat anti mice (Cat No. A11032) were all purchased from Invitrogen (Waltham, Massachusetts).

Animal treatment and tissue collection

Exact age PND (post-natal day) 26- and PND 75-male Fischer F344 rats and PND 26 C57BJ/6 mice were treated by oral gavage with a single oral dose of MEHP (700 mg/kg in corn oil) or an equivalent volume of vehicle (corn oil, 2 ml/kg, p.o.). Rats and mice were randomly assigned to 2 experimental groups: the control group (n = 7) and the treatment group (n = 7). 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). Following gavage, animals were observed regularly for general health until sacrifice 48 h after treatment. Animals were euthanized by CO2 asphyxiation followed by cervical dislocation. The testes were rapidly removed aseptically and weighed. One testis was randomly selected for seminiferous tubule staining and was immediately placed in ice phosphate-buffered saline (PBS, Gibco, Cat No. 10010-023) on ice. The other testis 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. Animals were euthanized in another independent cohort experiment following the above-mentioned exposure paradigm their testes were snap-frozen in liquid nitrogen and stored at −80°C until further utilized for RNA extraction and sequencing experiments.

Seminiferous tubule staining and imaging

Imaging of PTMφs and spermatogonia in isolated seminiferous tubules was performed according to our previously published methods (Gillette et al., 2021). In brief, the testis was decapsulated by puncturing and tearing the tunica, and the tubules were placed into ice PBS contained in a glass Petri dish. The tubules were gently teased apart, and then the interstitial tissue and blood vessels were removed with blunt-end 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 in an orbital shaker, washed an additional 4 times in ice-cold PBS and then stained with antibodies MHCII or F4/80 (for the PTMφs) and PLZF (for undifferentiated spermatogonia) for immunofluorescence microscopy. Stained seminiferous tubules were imaged on a Zeiss laser scanning confocal microscope (Zeiss, LSM 710). Images that were used to count MHCII+ macrophages and PLZF+ spermatogonial were captured at 20× (425.10 × 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 1.49T) using the “Max Intensity” method. Positive cells were then counted with ImageJ. Cell counts were normalized to tubule area and are represented as cell counts per 105 pixel area. Cell counts were calculated based on 10 individual tubules within each animal by summing the cell count and tubule area of each image and calculating the density of positive cells. This methodology considers each animal as the individual statistical unit rather than each tubule observation as the individual statistical unit and avoids violations of nonindependence assumptions within statistical tests. All the seminiferous tubule staining figures were further checked for the quantification results utilizing an artificial intelligence (AI)-based non-biased quantification technique called NIS-Elements NIS.ai General Analysis 3 (GA3) developed from NIKON.

Immunofluorescent staining of testis cross sections

Paraffin-embedded tissue blocks are sliced to 5 micrometer width using Microtome HM355S (Thermo Scientific) and mounted on standard glass slides. Sections were dewaxed and rehydrated; antigen retrieval was performed by microwaving in a 10-mM citrate buffer solution (pH = 6.0). Endogenous peroxidase activity was blocked by incubation with a solution of 3% hydrogen peroxide for 15 min. The tissue sections were incubated in goat-blocking serum and incubated with primary antibody for overnight at 4°C chamber, and followed the next day by incubating with respective secondary antibody for an hour in the dark at room temperature. Finally, sections were counterstained with DAPI and cover-slipped for microscopic observation. For Caspase 3 staining, the apoptotic index (AI) was calculated as the percentage of essentially round seminiferous tubules containing more than 3 Caspase 3-positive GCs in each cross section, as described in previous study (Voss et al., 2018). An isotype-matched rabbit IgG antibody was used to evaluate the nonspecific binding of the primary antibody. We also performed a negative control in which the primary antibody was omitted. The negative control did not show any immunofluorescence that corresponded with Caspase-3 expression. For each rat, at least 100 seminiferous tubules were analyzed.

RNA extraction and Tag sequencing

Testes that were stored at −80°C were used to isolate total RNA using AllPrep DNA/RNA/miRNA Universal Kit (QIANGEN, Germany) following the manufacturer’s recommendations. Total RNA was quantified, and after confirmation of its concentration and integrity using Nanodrop spectrophotometer (Thermo Scientific, Waltham, Massachusetts) and BioAnalyzer 2100 (Agilent, USA) respectively, RNA was submitted to the Genomic Sequencing and Analysis Facility at the University of Texas at Austin for library preparation and 3′ Tag sequencing experiments.

Tag sequencing analysis

The raw data utilized in this study were acquired from the Genomic Sequencing and Analysis Facility at the University of Texas at Austin. The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE229734. Raw data underwent a quality control assessment utilizing Fastqc. The sequences were further processed by trimming adaptors and removal of low-quality sequences. Reads that were shorter than 50 bp were discarded. The reference transcript sequences for mice were obtained from Gencode, whereas the rat reference transcript sequences were obtained from Ensembl. The remaining reads were aligned to the respective reference genomes (Rnor_6.0 for rats and mm10 for mice) using the Bowtie2 tool. The gene expression was quantified utilizing Samtools. The differential expression of genes was analyzed through the application of DESeq2, and Gene Ontology (GO) enrichment analysis was performed using the GO-MWU package, which was developed by Dr Mikhail Matz of the University of Texas at Austin and can be downloaded via https://github.com/z0on/GO_MWU. GO enrichment was performed to identify ages- or species-specific differences and similarities. Additionally, the benchwork data obtained above were compared with the gene expression profiles obtained from DESeq2 to exhibit consistent expression patterns in both the experimental and bioinformatics data that were age- and species-specific. The visualization of the sequencing results was performed utilizing R 4.2.0 and Python, Heatmaps and GO charts were created to illustrate the similarities and differences between different age or species animals.

Statistics

Results from experiments are presented as individual data points and means ± SEM. Each replicate represents a biological replicate and the number of replicates is indicated in each figure legend. The data were subjected to Student’s t test or parametric 1-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). p-values of less than .05 were considered statistically significant.

Results

 

MEHP-sensitive PND26 rats showed acute spermatocyte apoptosis with MEHP treatment

The current investigation evaluated the extent of testicular spermatocyte apoptosis resulting from MEHP exposure in PND 26 and PND 75 rodents by assessing the extent of caspase 3 immunofluorescence staining. A pronounced increase in the number of apoptotic germ cells was observed in the peripubertal PND 26 Fischer CDF344 rats following a single exposure to MEHP. As depicted in Figure 1, the AI increased significantly from 15 to 28. However, the AI did not increase significantly when adult PND 75 Fischer CDF344 rats were exposed to MEHP.

Figure 1.

Figure 1.

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Spermatocytes apoptosis. Representative figures of peripubertal (A) and adult (B) Fischer CDF344 rats’ sections were stained for active caspase-3 immunofluorescence in N = 7 rats per treatment group. Positively stained apoptotic cells are suggested by white arrows. The apoptotic index (AI) was calculated and is shown on the corresponding panels on the right side of the figure.

Adult rodents have a higher basal level of peritubular macrophages that were not further increased in response to MEHP treatment

We hypothesized that if adult rats and peripubertal mice are less sensitive toward MEHP toxicity than peripubertal rats, then MEHP treatment would not significantly change the numbers of PTMφs in the testes. As shown in Figure 2, a 6-fold increase in PTMφ numbers occurs in PND 26 Fischer rats 48 h after MEHP exposure. However, adult Fischer rats had a greater basal level of PTMφ than PND 26 rats, averaging 1.8 PTMφs per 105 pixel area. Interestingly, compared with peripubertal rats, the numbers of PTMφs did not significantly increase after MEHP exposure. Peripubertal C57BJ/6 mice had a basal level of PTMφ that is 5 times higher than that of peripubertal rats under basal conditions, with an average number of 13 PTMφs per 105 pixel area. Contrary to peripubertal CDF344 rats, no discernible increase in PTMφs in peripubertal C57BJ/6 mice was observed following MEHP exposure.

Figure 2.

Figure 2.

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Peritubular macrophages (PTMφs) responses and spermatogonia level changes. Representative whole seminiferous tubule staining results of PND 25 peripubertal rats (A), PND 75 adult rats (B), and PND 25 peripubertal cC57BJ/6 mice (C). Control (left, CO) versus MEHP (right), PTMφs are stained by MHCII (rats) or F4/80 (mice), green color, positively stained PTMφs are suggested by white arrows, and differentiating spermatogonia is stained by PLZF, red color. Quantification results of PTMφ and PLZF+ cell level changes with N = 7 rats (30 area per rat) per treatment group are shown on the corresponding panels on the right side of the figure.

MEHP treatment did not significantly change PLZF+ spermatogonial levels in less sensitive rodents

As shown in Figure 2, MEHP administration did not significantly enhance the numbers of PLZF+ undifferentiated and differentiating germ cells in adult Fischer CDF344 rats. On the other hand, in peripubertal rats exposed to MEHP, there was a correlation between the increases in PLZF+ undifferentiated and differentiating spermatogonia and the increase in PTMφs, suggesting that PTMφs may play a role in replacing lost spermatocytes. Peripubertal C57BJ/6 mice do not exhibit a substantial increase in PLZF+ spermatogonial cells in response to MEHP exposure, in contrast to peripubertal Fischer rats. This observation is consistent with earlier findings that the observed changes in PTMφ and PLZF+ levels is positively correlated.

MEHP-sensitive peripubertal rats had notable testicular transcriptomic profile changes with MEHP treatment

To validate whether the variability in the PTMφ response is reflected in the testicular transcriptomic profiles, 3′ Tag sequencing was utilized to explore and compare the transcriptomic profile changes between peripubertal Fischer CDF344 rats, adult Fischer CDF344 rats, and peripubertal C57BJ/6 mice with or without MEHP treatment. Principal component analysis (PCA) plots were generated using 3′ Tag sequencing data from different testis samples, Figure 3A represents rats and Figure 3B represents mice, respectively. The plots showed a clear separation between each data point, indicating distinct gene expression patterns in each animal. Peripubertal Fischer CDF344 rats and adult Fischer CDF344 rats with or without MEHP treatment clearly formed distinct and separate clusters, whereas samples from peripubertal C57BJ/6 mice with or without MEHP treatment are randomly distributed. The PCA plot of rats also revealed 2 peripubertal MEHP-treated rats fell into the cluster of the peripubertal control rat group, suggesting they are potential non-respondents.

Figure 3.

Figure 3.

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Principal component analysis (PCA). PCA results of Fischer CDF344 rats (A) and C57BJ/6 mice (B). In (A), arCO represents PND75 adult Fischer CDF344 rat in control group (green), armehp represents PND 75 adult Fischer rat in MHEP-treated group (yellow), pCO represents PND 25 peripubertal Fischer rats in control group(purple), and pMEHP represents PND 25 peripubertal Fischer rats in MEHP-treated group (red). In (B), co represents PND 25 peripubertal C57BJ/6 mice in control group (green) and MEHP represents PND 25 peripubertal C57BJ/6 mice in MEHP-treated group (yellow). Each dot represents an individual animal.

Differential expression analysis was then performed on the tag seq data to identify genes that were differentially expressed between the 2 conditions (control vs MEHP-treated). Figure 4 displays the representative top 20 genes whose expression in treated groups has changed most noticeably. MEHP treatment dramatically altered the gene expressions in peripubertal Fischer CDF344 rats. In sum, the expression of more than 13 000 genes corresponding to the MEHP-treated group was differentially expressed from that of the control group in peripubertal Fischer CDF344 rats (processed data GEO Series accession number GSE 229734). However, although a similar gene expression change trend was also seen in adult rats, it was not to the same extent as in peripubertal Fischer CDF344 rats. Lastly, peripubertal C57BJ/6 mice did not exhibit the same degree of differential gene expression as Fischer CDF344 rats that had either received MEHP exposure or not.

Figure 4.

Figure 4.

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Differential gene expression analysis. Heatmaps of the top 20 significantly changed genes of Fischer CDF344 rats (A) and C57BJ/6 mice (B). Warm color indicates high expression level, cold color indicates low expression level. In the heatmap of rats, arCO represents PND 75 adult rat in control group (green), armehp represents PND 75 adult rat in MEHP-treated group (yellow), pCO represents PND 25 peripubertal rats in control group (purple), and pMEHP represents PND 25 peripubertal rats in MEHP-treated group (red).

The alterations in gene expression were further classified into functional categories, or “GO,” to gain an understanding of the various biological processes involved. Figure 5 provides a summary of the top 15 GO terms of all the groups associated with the most differentially expressed genes. In peripubertal Fischer CDF344, rats were primarily categorized as part of the immune system gene set and those involved in the response to organic cyclic compounds, regulation of anatomical structure morphogenesis, and regulation of locomotion. In adult Fischer CDF344 rats, GO enrichment was more heavily concentrated in the immune system-related gene set, though some of the other categories were still present to a certain extent. Similarly, peripubertal C57BJ/6 mice exposed to MEHP show GO enrichment changes to a substantially lesser level. Only a small number of altered genes clustered in signaling receptor binding activity or DNA binding categories were observed. To be noted, the log10 fold change in peripubertal Fischer CDF344 rats of top 15 GO terms is between 20 and 30, in adult Fischer CDF344 rats is in the range of 10–20, however, in peripubertal C57BJ/6 mice they are all below 10, indicating that the transcriptomic profiles of peripubertal Fischer CDF344 rats had the most intense changes with MEHP treatment.

Figure 5.

Figure 5.

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Gene Ontology (GO) analysis. GO results of PND 25 peripubertal Fischer CDF344 rats (A), PND 75 adult Fischer CDF344 rats (B), and PND 25 peripubertal C57BJ/6 mice (C). Adjusted p-value which ≤e−04 is utilized to set the threshold.

Discussion

The objective of this study was to ascertain if MEHP-induced increases in the numbers of PTMφs in the testis of rodents correspond to the extent of germ cell apoptosis observed after MEHP exposure. We have previously described that the testis-specific subtype of macrophages, the PTMφ, is recruited to the testis of peripubertal rats after exposure to MEHP (Gillette et al., 2021). The mechanisms and signaling pathways that account for the recruitment of PTMφs into the testis is not known. Nevertheless, it is well-recognized that peripubertal-aged rats and mice show significant increases in the incidence of spermatocyte apoptosis in the testis upon phthalates treatment, whereas those of adults in either species do not (Murphy et al., 2014). We compared the responses of PTMφs between these germ cell apoptosis “sensitive” versus “less sensitive” ages and strains to discern if the observed increase in PTMφs depends on MEHP-induced germ cell apoptosis. In addition, we performed a comparative transcriptomic analysis to understand better the potential signaling mechanisms that may be involved in both the recruitment of the PTMφs and their possible function in the testis after toxicant-induced injury.

The study utilized adult male Fischer CDF344 rats to examine the PTMφ response to MEHP in an age well recognized to be less sensitive to MEHP-induced germ cell apoptosis. Caspase-3 was utilized as a marker for apoptotic germ cells due to its established role in regulating programmed cell death (Degterev et al., 2003; Elmore, 2007). As expected, a significant increase in spermatocyte apoptosis was observed in peripubertal-aged Fischer CDF344 rats, but not in adults, confirming the age-dependent susceptibility to MEHP-induced testicular germ cell apoptosis (Figure 1). Additionally, our previous study examined the responses of PTMφs through whole seminiferous tubule staining and found that the number of PTMφs increased significantly along with undifferentiated and differentiating PLZF+ spermatogonia in peripubertal Fischer CDF344 rats following MEHP exposure, suggesting their potential involvement in the testicular response to acute injury and spermatogenesis recovery (Gillette et al., 2021). Notably, in the current study, adult Fischer CDF344 rats exhibited a higher baseline level of PTMφs compared with peripubertal rats. However, their numbers were not significantly altered following MEHP exposure. It is plausible that, in addition to its role in aiding testicular injury recovery, the PTMφs may also harbor a protective function against the toxicity induced by MEHP. Emerging research suggests that macrophages may have a protective role among different toxicant and disease models (Cao et al., 2013; Liao et al., 2012; Polfliet et al., 2001). Specifically, Lee et al. found that macrophages displayed a nontraditional activated phenotype during renal ischemia reperfusion repair phase (Lee et al., 2011). Moreover, in the central nervous system, researchers found that meningeal and perivascular macrophages play a protective role during bacterial meningitis (Polfliet et al., 2001). In the present study, adult rats exhibited a higher baseline level of PTMφs. Because this age of rodents is also less sensitive to the effects of MEHP-induced germ cell apoptosis, it is plausible to hypothesize that PTMφs could contribute to safeguarding the adult rat testes from the effects of MEHP. The potential correlation between the higher basal numbers in adult rats and in both peripubertal and adult mice could underlie the mechanism for the diminished sensitivity of adult rats compared with peripubertal rats. Further research is clearly needed to test this intriguing novel function of PTMφs.

To explore the transcriptomic alterations associated with PTMφ responses and MEHP exposure, 3′-Tag sequencing was employed. Earlier studies had reported significant changes in testicular gene expression upon MEHP exposure, but PTMφs were not specifically investigated (Lahousse et al., 2006). In contrast, this study focused on elucidating the relationship between PTMφ responses and testicular toxicity caused by MEHP exposure. The PCA plot revealed 4 distinct clusters, which represented the effect of MEHP treatment and different age groups of rats. Moreover, the results of the heatmap and GO term analysis demonstrated significant disparities in the transcriptomic profiles of Fischer CDF344 rats exposed to MEHP compared with the control group, particularly in peripubertal rats. These alterations included the upregulation of apoptotic genes (Apaf1, Bbc3, Egr3) and downregulation of anti-apoptotic genes (Birc2, Cflar), as well as changes in genes associated with cell proliferation (Rbm3) and stem cell self-renewal (Ly6h) (Holmes and Stanford, 2007).

Those gene level changes are aligned with our benchwork data presented above that there is a significant increase in the germ cell apoptosis in the peripubertal male Fischer CDF344 rats upon MEHP exposure. Additionally, the expression pattern of tight-junction genes (Ctnnb1, Itgb1, Sorbs1) suggested the potential involvement of the blood-testis barrier (BTB) in PTMφ response, consistent with previous observations of BTB disruption by MEHP exposure in peripubertal Fischer CDF344 rats (Tiwary and Richburg, 2023). In prior studies, we also demonstrated that exposure to MEHP in peripubertal Fischer CDF344 rats resulted in a significant influx of immune cells when compared with adult rats (Murphy et al., 2014; Voss et al., 2018). In line with those findings, the current study observed downregulation of several normal macrophage marker genes (C1qa, C1qb, CX3CR1) and upregulation of the monocyte lineage gene marker CD68, as well as upregulation of M2 macrophage-associated genes (Arg1, Mmp9, Mafb). These gene expression alterations were also observed in adult rats, albeit to a lesser extent. These results suggest that the efficiency of PTMφ immune response declines with age in Fischer CDF344 rats. In order to gain an improved understanding of the complex genetic etiology of PTMφs in the testis and compare the global transcriptomic changes between adult and peripubertal rats a GO analysis was performed. It is important to identify if the differentially expressed genes are members of similar biological pathways or whether they are important to the testicular injury response process. As shown in Figure 5, in the peripubertal rats the top 5 are GO: regulation of locomotion, regulation of cellular component movement, regulation of immune system process, negative regulation of multicellular organismal process, and positive regulation multicellular organismal process. And the top 5 GO term in adult rats are with an emerging theme of immune responses: immune system process, regulation of immune system process, defense response, immune response, and response to biotic stimulus. It is well-recognized that MEHP administration would cause immune responses and therefore the immune-related gene changes are well-understood (Dalgaard et al., 2001; Stermer et al., 2017; Voss et al., 2018). Interestingly, 4 out of 5 top GO terms in peripubertal rats are about cell movement and cell-cell interactions. Those GO terms should be noted and they are possibly relevant to the increase of PTMφs and their unique spermatogonia stem cell niche localization. Additionally, there are other GO terms such as positive regulation of signal transduction and regulation of response to external stimulus reported, which indicate the complex signaling processes and also partially explained the sensitivity in peripubertal rats exposed to MEHP.

PTMφs responses were also investigated in a less-sensitive species via utilizing male peripubertal C57BJ/6 mice. Although both belonging to the Rodentia order, rats and mice exhibit significant differences in their biology, physiology, and metabolism (Bahamonde et al., 2018; Hok et al., 2016). One notable distinction lies in their metabolic pathways and responses to xenobiotics. Although both species metabolize xenobiotics, mice are often to have a metabolism that more closely resembles human metabolic processes. Rats tend to metabolize xenobiotics at a faster rate than mice, primarily due to the differences in enzymes and pathways involved in xenobiotic metabolism, particularly the cytochrome P450 enzymes (Hammer et al., 2021; Lewis et al., 1998). Prior studies in our group suggest that peripubertal mice have increases in spermatocyte apoptosis upon MEHP treatment, however, it is not as robust as that observed in peripubertal Fischer CDF344 rats (Murphy et al., 2014). Although DeFalco et al. previously studied PTMφs in mice under basal conditions (DeFalco et al., 2015), the responses of PTMφs in a mouse toxicant model remain untested. The current study provides novel insights by demonstrating, for the first time, that in mice, PTMφ response is not sensitive to MEHP treatment. Peripubertal male C57BJ/6 mice exhibited the highest baseline level of PTMφs, but no significant loss of spermatocytes or an increase in PTMφ numbers was observed following MEHP exposure. Analysis of the RNA sequencing data for peripubertal C57BJ/6 mice shows that most of the observed gene expression changes were immune-related genes (Serpina3n, Cd74, Itih2, C2, and Tapbp) (Daveau et al., 1998; Fingerle-Rowson et al., 2009; Sergi et al., 2018; Siebenkäs et al., 2017), although there were no significant alterations in the overall transcriptome or morphological profile of mice following MEHP exposure. These findings suggest that peripubertal C57BJ/6 mice do not undergo similar significant transcriptomic changes in response to MEHP exposure as those observed in rats.

Although this study successfully revealed the differences between MEHP “sensitive” and “insensitive” ages of rats and mice, with a specific focus on PTMφs, there are several directions for future research that could enhance our understanding of the functional participation of PTMφs after MEHP exposure. Are PTMφs participating in resolving the inflammation that could be due to the BTB? Or do they have a function in helping to recover spermatogenesis after MEHP-induced injury? Our previous study indicated that the increase in the numbers of PTMφs was associated with increases in the numbers of spermatogonia and that the PTMφs may be making close contact with the basal membrane of Sertoli cells (Gillette et al., 2021) suggesting that these cells may be playing a more active role in stimulating the recovery of the lost spermatocytes. Therefore, beyond measuring the changes of PTMφs numbers, it would be beneficial to explore other factors that might influence their performance, such as alterations in their morphology and immunoreactive profiles. Secondly, it was observed that there exists a varying basal level of PTMφs among different ages or species of rodent animals, with the apoptosis of spermatocytes being correlated with the basal level of PTMφs. This may be attributed to PTMφs dual role as both stimulators and protectors, rather than PTMφs solely contributing to testis recovery following spermatogenesis injury. For instance, the upregulation of anti-apoptosis genes (Birc2, Cflar) and tight-junction genes (Cdh2, Itgb1) in adult Fischer CDF344 rats suggests that PTMφs may offer some degree of protection to the testis. This hypothesis warrants further investigation to elucidate the underlying mechanisms. Lastly, although the study identified significant differential expression of gene sets in the whole testes of rodents with or without MEHP exposure, future research could employ sequencing techniques that specifically target the transcriptomic profiles of PTMφs. Ongoing research in our group involving the isolation of PTMφs and subsequent single-cell RNA sequencing may provide further insights into this macrophage population more closely.

In conclusion, this study significantly contributes to our understanding of the correlation between MEHP-induced testicular germ cell apoptosis and the presence of PTMφs. The findings reveal age- and species-dependent differences in PTMφ responses, transcriptomic profiles associated with immune responses, and spermatogenesis. Because the changes in testicular PTMφs responses correlate with the extent of MEHP-induced spermatocytes apoptosis, but not due to the MEHP exposure itself, this strongly indicates that the PTMφs are reacting to the germ cell injury. Additionally, the various baseline levels of PTMφs and differentiated extent of MEHP-induced spermatocyte apoptosis between sensitive and less sensitive rodents suggest the potential involvement of PTMφs in the toxicant protection process. In the current study, the focus on the PTMφs holds immense promise for advancing our understanding of phthalate toxicity and its impact on reproductive health. Firstly, these macrophages actively participate in immune regulation, producing cytokines and other signaling molecules which can influence neighboring cells. Phthalates exposure can alter the immune responses orchestrated by PTMφs and potentially involved with the disruption of the BTB. Disruption in those processes have direct implications for male fertility, as they can impair sperm development, maturation, and overall reproductive function. Moreover, understanding how PTMφs responds to phthalates can provide invaluable insights into the initial as well as the repair stages of xenobiotic metabolism and elimination, shedding light on the body’s defense and recovery mechanisms against environment toxins. Therefore, by investigating the specific interactions between phthalates and PTMφs, the current study aims to unravel the intricate responses by which MEHP affect the male reproductive system at a cellular and molecular level. The knowledge gained from this study can not only provide fundamental insights into the toxicological functions of PTMφs, but also contribute to the development of targeted inventions and therapeutic strategies to mitigate the adverse effects of PTMφs on male fertility. By elucidating the interplay between PTMφs, phthalates, and the male reproductive system, our study has the potential to inform public health policies, enhance reproductive health assessments and pave the way for innovative approaches to protect individuals from the detrimental consequences of phthalates exposure.

Declaration of conflicting interests

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

Contributor Information

Xin Fang, Interdisciplinary Life Sciences Graduate Program, The University of Texas at Austin, Austin, Texas 78712, USA; Division of Pharmacology and Toxicology, College of Pharmacy, Center for Molecular Carcinogenesis and Toxicology, The University of Texas at Austin, Austin, Texas 78712, USA.

Richa Tiwary, Division of Pharmacology and Toxicology, College of Pharmacy, Center for Molecular Carcinogenesis and Toxicology, The University of Texas at Austin, Austin, Texas 78712, USA.

Vivian P Nguyen, Division of Pharmacology and Toxicology, College of Pharmacy, Center for Molecular Carcinogenesis and Toxicology, The University of Texas at Austin, Austin, Texas 78712, USA.

John H Richburg, Division of Pharmacology and Toxicology, College of Pharmacy, Center for Molecular Carcinogenesis and Toxicology, The University of Texas at Austin, Austin, Texas 78712, USA.

Funding

NIH (R01ES016591).

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