Identification and characterization of a novel CD2-positive cell population in the seminiferous tubule of Fischer CDF344 rats

Abstract

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

Graphical Abstract

Graphical Abstract.

Introduction

The ability of an organism to repair and regenerate after a tissue injury is considered a long-standing topic of evolutionary and biomedical interest. The tissue repair or regeneration context depends on the injured tissue type and whether it is quiescent or dividing [1]. For cellular turnover, most tissues harbor stem or progenitor cells; with their innate ability to self-renew and proliferate, they directly compensate for the loss of tissue injury and actively contribute to the post-injury recovery process, thus helping maintain tissue homeostasis. Immune cells are initially recruited to the site of injury and orchestrate the cascade of molecular and cellular events for recovery by closely coordinating with the tissue-resident cells [2, 3]. Moreover, activated immune cells can also promote irreversible tissue damage. The functional switch between the reparative or damaging phenotypes of the immune cells is dictated by the tissue microenvironment at the site of injury [4]. Therefore, during the post-injury phase, it is essential to study the niche where the recruited immune cells localize along with the local tissue-resident cells to understand and elucidate the existing cellular and molecular interactions that instigate the process of tissue repair and recovery.

In the event of tissue damage or injury, inflammation is the first physiological response before the onset of tissue repair, and inflammation resolution occurs during the recovery phase. Initially, inflammatory cells are recruited to the site of injury; among the immune effector cells, macrophages represent the major subset of cells that respond to the inflammatory challenge in the tissue microenvironment. Indeed, the process of recovery or regeneration is achieved through the interaction of macrophages with the tissue-specific stem or progenitor cells. This phenomenon has been demonstrated in organs such as the liver, heart, kidney, nerve, and muscle tissues [5, 6]. Rodent testis contains two distinct macrophage populations: the interstitial macrophage that resides within the testicular interstitial space and the peritubular macrophage (PTMφ) present on the surface of the seminiferous tubules. Peritubular macrophages are primarily localized in regions enriched with undifferentiated and differentiating spermatogonia [7, 8]. Therefore, PTMφs are conceived to be an essential regulator of the spermatogonial stem cell niche. However, the biological function of PTMφs in the process of toxicant-induced testicular injury and post-injury recovery remains elusive. Therefore, it is necessary to establish the reason for their selective localization and cellular interactions with the niche cells.

Published experimental findings from our group have reported the detailed sequence of pathogenic events and the spatiotemporal dynamics of responding immune cells in the testis following exposure to the environmental chemical mono-(2-ethylhexyl) phthalate (MEHP), a well-studied Sertoli cell toxicant [9, 10]. Mono-(2-ethylhexyl) phthalate is a metabolite of di-(2-ethylhexyl) phthalate (DEHP), and more than two decades of work on rats has established MEHP as a potent chemical for modeling testicular toxicity [10, 11]. Studies from our group and others report that the single-dose acute oral exposure of MEHP to prepubertal Wistar and Fischer rats increased germ cell apoptosis due to Sertoli cell dysfunction [12]. Though most studies consider MEHP-induced germ cell apoptosis as a secondary consequence of disrupted Sertoli cell function, only a limited number of studies explore the relevance of MEHP-induced germ cell apoptosis with infiltrating immune cells in the testis. Infiltration of immune cells into the testis occurs concomitantly with a significantly higher rate of germ cell apoptosis [13]. Previous studies showed that acute exposure to MEHP triggers the infiltration of CD11b⁺ leukocytes, such as macrophages, monocytes, and neutrophils, into the testis interstitium. In particular, MEHP exposure significantly increased the number of infiltrating interstitial macrophages and PTMφs in the seminiferous tubule. The testicular transcriptomic profiling in the acute MEHP-exposed injury model revealed that the PTMφs’ numbers were positively correlated with the rodents’ sensitivity to MEHP exposure, dependent on age (profound response seen in pubertal animals versus adults) and species (rats were more sensitive than mice) [13–15]. The underlying mechanistic link for the MEHP-induced infiltration of interstitial macrophages in the testis was found to be associated with the levels of chemokine called monocyte chemoattractant protein-1 (MCP1), which was identified to be secreted primarily by the peritubular myoid cells (PTMCs) [16]. The infiltrating CD11bc⁺/CD68⁺/CD163⁻ neutrophils and interstitial macrophage numbers positively correlated with the extent of germ cell apoptosis in the testis, which appeared to contribute to the loss of germ cells. However, subsequent studies using an anti-polymorphonuclear cell antibody [17] ruled out the role of infiltrating neutrophils contributing to MEHP-induced germ cell apoptosis. Our recently published work reported increases in PTMφs in the seminiferous tubules of peripubertal Fisher CDF344 rats acutely exposed to MEHP. The PTMφ numbers were observed to be sustained through the post-injury recovery phase (2 weeks post-exposure), positively correlating with the number of PLZF⁺ undifferentiated and differentiating spermatogonia in response to MEHP-induced loss of spermatocytes [15]. Thus, it was proposed that PTMφs could influence the kinetics and the recovery of spermatogenesis. The functional importance of PTMφs in the pathophysiology of MEHP-induced testicular injury and their role in the subsequent activation of the recovery mechanism remain unanswered.

While attempting to profile testicular cells that express CD2 protein in whole-mounted seminiferous tubule preparations, we found a distinct subset of cells on the surface of the seminiferous tubule identified to be positive for the CD2 protein. This study presents an in-depth description of novel CD2⁺ cells’ morphology, clonal organization, size, relative spatial localization, and cell division dynamics. In addition, we have distinguished the novel CD2⁺ cells in whole-mount seminiferous tubule preparations with known seminiferous tubule cells and immune cell lineage markers to show that these cells are a unique population of cells in the rat seminiferous tubule. This study reports the cellular interaction between the PTMφs and novel CD2⁺ cells. The dynamics of cellular interaction between the PTMφs and CD2⁺ cells have been explored using the MEHP-induced testicular injury model.

Materials and methods

Animal model and experimental procedure

Animal experimentation protocols for this study were approved (AUP-2023-00060) by the Institutional Animal Care and Use Committee of The University of Texas at Austin, and experiments were performed following the guidelines set by the National Institute of Health. In the current study, peripubertal (3–4 weeks of age) male Fischer CDF344 rats (body weight range 40–60 g) were purchased from Charles River Laboratories. Upon arrival, the animals were maintained in a temperature- and light (12 L:12D cycle)-controlled animal facility with free access to drinking water and standard rat chow. After 72 h of the acclamation period, the rats were randomly assigned into two experimental groups: vehicle control (corn oil, n = 5) or MEHP (n = 5). Animals were exposed on postnatal day (PND) 28. For the current study, peripubertal age and an inbred Fischer CDF344 rat strain are preferred based on the previously published work, which demonstrated the robust infiltration of macrophages in the testis to occur in an age- and species-dependent manner [10]. The administered dose concentration in the study does not reflect an environment-relevant or human-exposure-relevant dose. Rather, the acute-dosing methodology followed in this study helps to stimulate the infiltration of testicular macrophages within the responsive developmental window, thus serving as an effective in vivo model of testicular macrophage manipulation.

Chemical and dosage administration

Mono-(2-ethylhexyl) phthalate was purchased from Wako Chemical Corporation, Osaka, Japan. Animals were exposed to either 700 mg/kg-bw MEHP dissolved in corn oil or an equivalent volume of corn oil via oral gavage. Each experimental group comprised five animals across two cohorts. After gavage, the animals were left undisturbed in their home cage until sacrifice at 48 h (first cohort: acute injury phase) or 2 weeks (second cohort: post-injury recovery phase) post-exposure. During the first 5 h post-exposure, animals were observed for signs of illness. After this period, animals were monitored daily. At the end of the experimentation period, these animals were euthanized by CO₂ gas inhalation followed by thoracotomy. Under aseptic conditions, the testis was removed, one testis was used for paraffin embedding and the other testis was decapsulated, and testicular arteries were discarded and tubules were used for seminiferous tubule preparation and whole-mount immunofluorescence staining.

Paraffin embedding and immunofluorescence staining of testis cross-section

Briefly, for paraffin embedding, the testis was fixed using the Bouin fixative solution (RICCA, Texas). Bouin solution was replaced by ethanol saturated with lithium, further taken for alcohol gradient procedure utilizing an automatic tissue processor (Leica TP 1020). The processed tissue samples were embedded in paraffin (Leica- EG1150C and EG1150H). Embedded tissues were sliced using a microtome (Thermo Scientific, HM355S) to 5 μm thickness and then mounted on glass slides. For the immunofluorescence staining procedure, the tissue sections were processed for dewaxing and rehydration; antigen retrieval was carried out by microwaving in a 10 mM citrate buffer solution of pH = 6.0. Incubation with 3% hydrogen peroxide was performed for 15 min to block endogenous peroxidase activity. The tissue sections were incubated with goat-blocking serum and primary antibody overnight at 4°C, followed the next day by respective secondary antibody incubation at room temperature for an hour in the dark. Lastly, tissue sections were counterstained with DAPI and cover-slipped and used for microscopic observation (Nikon Eclipse Ni microscope and images captured with a Nikon Cool-SNAP digital camera). Primary antibody–omitted negative controls were included to evaluate the background noise.

Seminiferous tubule preparation and immunofluorescence staining

For seminiferous tubule preparation, the tubules were washed four times in ice-cold phosphate-buffered saline (PBS). The tubules were gently separated from the interstitial tissue using forceps and fixed in 4% paraformaldehyde (Electron Microscopy Sciences, 15710-S) overnight at 4°C on an orbital shaker. Finally, the tubules were washed four times in cold PBS and stored in PBS at 4°C until use. This procedure was explicitly adopted to image-isolated seminiferous tubules devoid of interstitial cells.

Isolated tubules were used for the whole-tubule immunofluorescence staining preparation as described by Gillette et al. [15] with primary antibodies (Table 1) and secondary antibodies (Table 2). For super-resolution microscopy (Nikon A1R upgraded with STEDYCON), whole tubule staining preparations were performed using a specialty secondary antibody: star-orange. Hoechst 33342 (Life Technologies—H3570, 1:10 000) was used as a counterstain for nuclei. Stained whole tubules were mounted on super frost slides using Fluoromount-G (Southern Biotech, 0100-01) mounting medium, and the raised coverslip was sealed with clear nail polish.

Table 1.

List of primary antibodies used for immunostaining experiments. Primary antibodies used in dilution range from 1:100 to 1:250

Antibody	Manufacturer	Catalogue number
CD2	Invitrogen	MA1-70018
CD2	Novus Biologicals	NB100-65228DL594
MHC II	Novus Biologicals	NBP3-09015
MHC II	Biolegend	205401
α SMA	Invitrogen	PA5-85070
SOX9	Life Technologies	PA5-81966
DDX4	Abcam	Ab13840
PLZF	Santa Cruz Biotechnology	SC-22839
STRA8	Invitrogen	PA5-115977
CD3	Novus Biologicals	NBP2-29472
TEX14	Proteintech	CL488-67982
Ki67	Invitrogen	MA5-14520
CD45	Novus Biologicals	NBP2-15811

Table 2.

List of secondary antibodies used for immunostaining experiments. Secondary antibodies are used in 1:500 dilution

Antibody	Manufacturer	Catalogue number
Star orange	Abrreior	STORANGE-1001-20UG
Alexa Fluor 594	Invitrogen	A32742
Alexa Fluor 568	Invitrogen	A11036
Alexa Fluor 488	Invitrogen	A11008
Alexa Fluor 488	Invitrogen	A11001

Confocal microscopy, image acquisition, and cell count

Whole-mount seminiferous tubule preparations were imaged on a Nikon A1R confocal microscope. The selection of regions for imaging was performed only under the Hoechst fluorescence filter to overcome the selection bias of PTMφs and CD2⁺ cells in tubules. Tubules distorted or damaged during the tubule isolation and staining processes were excluded from imaging. Images that were used to count PTMφs and PTMφs-CD2⁺ cell interaction were taken at 60× (1024 × 1024 μm) and with a depth of 4.4 μm in 0.4 μm increments from the surface of the tubule close to the objective lens. Whereas the images used to count CD2⁺ cells were captured at 60× (1024 × 1024 μm) with a depth of 2.4 μm in 0.5 μm increments from the surface of the tubule close to the objective lens. All the acquisition settings were maintained consistently throughout the imaging procedure. From our previous work [15], PTMφs were determined to localize up to a z-depth of 4.4 μm from the surface of the tubule, whereas, for the CD2⁺ cells, the z-depth was kept very minimal to distinctly count the CD2⁺ cell and its nuclei, since they are observed to be nestled in between the PTMCs. Three confocal fields (upper, middle, and bottom) were captured for each tubule. A total of 15 individual tubules were captured per animal and used for cell count. The tubule area was measured for computing the density (no. of cells/10⁵ area) of cells positive for the studied cell surface markers (MHC II⁺ for PTMφs and CD2⁺ cells). The violation of non-independence assumptions within statistical tests was circumvented by summing and normalizing the cell count and tubule area of each image for calculating the density of positive cells, also by considering each animal as an individual statistical entity instead of considering each tubule observation as a separate statistical unit. Counting for PTMφs and CD2⁺ cells, PTMφs-CD2⁺ cells’ interaction was made directly on the microscope, considering the advantage of following the positive cells through a series of focal planes. The inclusion criteria for considering CD2⁺ cells as clones of two or clones of three cell categories were based on the number of distinct nuclei in a cell. The captured z-stack images were flattened with ImageJ software (version 1.53 T) using the “Max Intensity” function. Positively stained cells in the flattened images were then used for representation. The three-dimensional (3D) reconstruction and volume rendering of z-stack images were performed with Imaris 9.9 software (Oxford Instruments).

Statistics

The positively stained cell count was compared between the experimental groups; before analysis, the data were assessed for normality using a density plot, measure of central tendency, skewness, and kurtosis and tested statistically using the Shapiro–Wilk test. Since the data were not normally distributed, the Wilcoxon Ranked-Sum test was used for testing statistical significance with P < 0.05. MS Excel was used to make graphical representations and was edited only for style in Inkscape version 1.3.2. R program was used to perform the statistical analyses.

Results

Morphology, clonal organization, and size of CD2⁺ cells

Seminiferous tubules separated from the interstitial tissue obtained from PND 30 Fischer CD344 rats were processed for whole-mount immunofluorescence and utilized for super-resolution and confocal microscopy. A population of CD2⁺ cells was observed on the surface of the seminiferous tubules. Closer examination of CD2⁺ cells using the super-resolution microscopy technique revealed the morphology and the clonal organization of CD2⁺ cells. Primarily, the CD2⁺ cells exhibited an irregular cell shape with non-spherical nuclei. Three distinct cellular states were observed in CD2⁺ cells based on their clonal organization. The CD2⁺ cells were predominantly observed to exist as single cells, followed by clones of two and three cells. Single cells were mainly observed to have a higher nuclear-to-cytoplasmic ratio (Figure 1A-C, from the corn oil group). On rare occasions, cytoplasmic connections were observed between different clones of cells (Figure 1D, from the corn oil group). At times, CD2⁺ cells were also observed to be in a cluster containing different clones (Figure 1E, from the corn oil group). The CD2⁺ cells’ size proportionally increased with an increase in clone size, noticeably the smallest CD2⁺ cells observed to be around 6.8–7 μm (Figure 1F, from the corn oil group). Since CD2⁺ cells’ nuclei morphology was similar to mitotically dividing cells, we have co-localized the expression of the cell proliferation protein marker Ki67 on CD2⁺ cells in whole mount seminiferous tubule preparation. The results show that CD2⁺ cells do not co-localize with the Ki67 protein expression (Figure 1G, from the corn oil group).

Figure 1.

Super-resolution photomicrographs of CD2⁺ cells (green, applies to all the images in the panel) from whole-mount seminiferous tubule preparations from the corn oil-treated peripubertal rats, imaged at 60×—A) CD2⁺ single cells uniquely observed with a higher nuclear-to-cytoplasmic ratio (relatively large nuclei with a thin rim of cytoplasm) and serrated border. (B) CD2⁺ clone of two cells. (C) Clone of three cells. Confocal photomicrographs of the whole-mount seminiferous tubule preparations from the corn oil-treated peripubertal rats. (D) Dotted lines highlighting structures that look like cytoplasmic projections interconnecting the CD2⁺ clone of two cells with a single cell. (E) Cluster of CD2⁺ cells showing markedly different nuclei morphology, size, and cytoplasmic partition. (F) Occasionally observed noticeably small CD2⁺ single cells measuring in size around 6.8–7 μm. (G) CD2⁺ cell nuclei tested negative for Ki67 (cell proliferation marker) protein (red). Scale bars: 15 μm in (A), 5 μm in (B), and 10 μm in (C); 15 μm in (D–G).

CD2⁺ cells test negative for known testicular Sertoli cell, germ cell, and immune cell lineage markers

To determine whether the CD2 protein expression is restricted to our novel identified cells and to establish the location of these cells relative to other known seminiferous tubule cells, we co-stained the isolated seminiferous tubules with CD2 protein and used markers for Sertoli cells-SOX9 (Figure 2A), pan germ cells-DDX4 (Figure 2B), undifferentiated and differentiating spermatogonia-PLZF (Figure 2C), differentiated spermatogonia-STRA8 (Figure 2D), and germ cell intercellular bridges-TEX14 (Supplementary Figure S1). Co-staining analysis shows that CD2 expression does not overlap with other known markers of seminiferous tubule cells. Cross-section staining of the testis with CD2 protein was observed, with negligible staining within the blood vessels but a lack of immunoreactivity inside the tubules and in the interstitium (Supplementary Figure S2). Co-staining of testis cross-section with CD2 along with a pan germ cell marker-DDX4 shows that germ cells do not express CD2 (Supplementary Figure S3).

Figure 2.

Confocal photomicrographs were taken at 60× magnification, and seminiferous tubules collected from the corn oil–treated rats were utilized for staining and microscopy. Co-staining of CD2⁺ cells (green, applies to all the images in the panel) and (A) Sertoli cell marker (SOX9, red). (B) Pan germ cell marker (DDX4, red). (C) Undifferentiated and differentiating spermatogonia (PLZF, red). (D) Differentiated spermatogonia (STRA8, red). (E) Seminiferous tubules collected from the corn oil–treated rats were co-localized for CD2 (green) and CD45 (red) protein, z-depth was 2.6 μm from the surface of the tubule, CD2⁺ cells do not co-localize with the CD45 protein, the arrow points to a distinct CD45⁺ cell within the PTMC layer. (F) Seminiferous tubules collected from the MEHP-treated rats were co-localized for CD2 (green) and CD45 (red), CD2⁺ cells lack co-localization with the CD45 protein, presence of CD45⁺ cell is highlighted with an arrow mark, and z-depth was 2.8 μm. The z-depth varied for the images of (A–D), based on the depth of the desired cell being imaged, scale bar—15 μm applies to all the images in this panel.

High-resolution z-stack captured using the confocal micrograph of the whole mount seminiferous tubule reveals novel CD2⁺ cells present on a focal plane different than the Sertoli cells and the germ cells. Movies created in orthogonal slice mode using Imaris confine the spatial location of CD2⁺ cells to the surface of the seminiferous tubule. In the movie, the appearance of PLZF⁺ (Supplementary video 1) and SOX9⁺ cells (Supplementary video 2) were used as reference points to spatially demarcate the distinct tissue regions of the seminiferous tubule that contain Sertoli and germ cells. Based on the spatial reference points, CD2⁺ cells were observed to localize in the tissue region before the basal and adluminal compartments. We used CD45, a pan-leukocyte marker, to check the immune lineage of CD2⁺ cells. CD2⁺ cells did not stain for CD45; we do observe CD45-positive cells on the PTMC layer in both corn oil and MEHP-treated groups (Figure 2E and F). Further, to determine whether these CD45-positive cells are PTMφs, we double-stained the isolated seminiferous tubules with CD45 and MHC II antibody (a cell marker for PTMφs). The double staining experiment revealed PTMφs to be heterogeneous (MHC II⁺ CD45⁺ and MHC II⁺ CD45⁻) in their expression of CD45 protein. We also observed the presence of CD45⁺ cells other than PTMφs (Supplementary Figure S4). We have double-stained CD2⁺ cells on the testis cross-section and whole mount seminiferous tubule preparation for T cell lineage marker—CD3. Cross-section staining shows the presence of CD3-positive cells in the testicular interstitial space (Supplementary Figure S5A). The novel CD2⁺ cells in the seminiferous tubule stain negative for CD3 (Supplementary Figure S5B).

Novel CD2⁺ cells were spatially interspersed within the PTMC layer and made direct cellular contact with PTMφs

The CD2⁺ cells were in the same focal plane as the PTMC layer. Further detailed observation revealed that the availability of the cellular space between the PTMCs defined the shape of the CD2⁺ cells (Figure 3A-D). Occasionally, PTMφs were present in the same plane or close to the nuclei of CD2⁺ cells (Figure 3E-G). High-resolution confocal imaging followed by surface rendering revealed two distinct types of physical cell-to-cell contact between the PTMφ and CD2⁺ cell (Figure 3H, H′ and I, I′). The movie created with high-resolution confocal z-stacks visualized the coinciding location of CD2⁺ cell (Supplementary video 3) and PTMφ (Supplementary video 4) within the PTMC layer, on the surface of the seminiferous tubule.

Figure 3.

Confocal photomicrographs taken at 60× magnification, seminiferous tubules collected from the corn oil–treated rats utilized for the staining and microscopy, and colored text on the image refers to the immunolabeled entity. (A–D) CD2 cells⁺ are present within the PTMC layer, and their cell shape is irregular; primarily, the CD2⁺ cells’ shape is determined by the cellular space between the PTMCs. Arrow marks highlight the spot of the empty cellular space between the PTMCs where CD2⁺ cells localize. (E–G) Arrow marks highlight the spot of the cellular space between the PTMCs where MHC II⁺ PTMφ localizes. Based on the characteristic nuclear morphology, the asterisk symbol points to the nucleus, which is presumed to be a CD2⁺ cell. (H) Cell-to-cell interaction between an MHC II⁺ PTMφ and CD2⁺ cell, blue—nuclei. (H′) Masked and surface rendered image visualizing the type of cellular interaction where the membrane of the CD2⁺ cell is extended or pinched by the PTMφ, nearly half of the CD2⁺ cell surface was overlapped with PTMφ surface. (I) Another type of cell-to-cell contact. (I′) Surface-rendered image shows a clear cell-to-cell contact between MHC II⁺ PTMφ and CD2⁺ cell, where the membrane of PTMφ overlaps with the boundary of the CD2⁺ cell membrane. Scale bar: 15 μm in (A–G).

The number of PTMφs and their cellular interaction with CD2⁺ cells increase in response to MEHP exposure

For this study, we followed a modified high-magnification imaging procedure from our previously published work, reporting an increase in PTMφs 48 h after exposure to MEHP in peripubertal rats [15]. In the MEHP-exposed animals, the number of PTMφs was significantly (P < 0.007) elevated at 48 h post-exposure; a trend of threefold increase was observed when compared to the vehicle (corn oil)-treated animals (Figure 4A and C). The PTMφ-CD2⁺ cell interaction was observed to be significantly (P < 0.007) higher (approximately 0.8-fold) in MEHP-treated animals when compared to the vehicle group at 48 h post-exposure, the critical time point when a higher number of PTMφs was also observed (Figure 4D and F).

Figure 4.

(A, B) Representative confocal photomicrographs of whole mount seminiferous tubules from rats at 48 h post-exposure, green—PTMφ labeled for MHC II protein, n = 5. (A) Corn oil. (B) MEHP treatment group. (C) Graph presenting the average number of PTMφ per 10⁵ area, MEHP-exposed animals showed a significant increase in MHC II⁺ PTMφs, calculating changes in PTMφs number with n = 5 rats per treatment group. (D) Representative confocal photomicrograph of whole-mount seminiferous tubules from rats 48 h post-exposure from the corn oil–treated animals (red—CD2⁺ cells and green—PTMφ labeled for MHC II protein). (E) A representative confocal photomicrograph from the MEHP treatment. (F) Graph presenting the average ± SEM (standard error of the mean) of PTMφs-CD2⁺ cells (red) interaction per 10⁵ area. The average number of cellular interactions between the PTMφs and CD2⁺ cells increased significantly in the MEHP-exposure group when compared to the corn oil–treated group, (n = 5/group). Scale bar: 15 μm applies to all the images in this panel.

MEHP exposure did not affect the dynamics of CD2⁺ cells’ division

To explore whether the increase in PTMφ-CD2⁺ cell interactions influences the number of CD2⁺ cells during the acute (48 h post-exposure) and post-injury recovery (2 weeks) phases, we analyzed the relative abundance of CD2⁺ clones. No statistically significant (P is not less than 0.05) change in the number of CD2⁺ clones (single, clone of two, and clone of three) was observed between the vehicle and MEHP group. Even the number of CD2⁺ clone cells remained grossly the same at both time points (48 h and 2 weeks post-exposure) within the vehicle and MEHP groups. The major subset of CD2⁺ clone cells was single, followed by clones of two and three cells (Figure 5).

Figure 5.

Representative photomicrographs showing the different CD2⁺ (green) cell clones, images were taken at 60× magnification, scale bar—15 μm. (A) Corn oil group, 48 h post-exposure. (B) MEHP treatment group, 48 h post-exposure. (C) Corn oil group, 2 weeks post-exposure. (D) MEHP treatment group, 2 weeks post-exposure. (E) Graph presenting the average ± SEM of CD2⁺ cell clones per 10⁵ area, MEHP exposure did not affect the average number of the CD2⁺ cells when compared to the respective corn oil–treated data within the categories: single cell (P = 0.30), clone of two (P = 0.54), and clone of three (P = 0.59) at 48 h post-exposure. For the categories: single cell (P = 0.84), a clone of two (P = 0.07), and a clone of three (P = 0.29) at 2 weeks post-exposure. The data were analyzed to be statistically not significant (at P < 0.05), (n = 5/group).

Discussion

Macrophages are recognized to be the predominant leucocyte population in the testis; their contribution to immunological aspects such as immunotolerance providing immune privilege, innate immune response regulation, phagocytosis, and clearance of dying cells or apoptotic particles is well studied in the testis. However, understanding the non-immunological functions associated with testicular development and homeostasis has only evolved recently [18]. A series of studies published by our lab shows that acute MEHP-induced loss of spermatocytes in peripubertal male Fischer rats incite a significant increase in the number of MHC II⁺ PTMφs in the seminiferous tubules, initially starting at 48 h post-exposure acute injury phase, remain sustained through 2 weeks of post-injury recovery, accompanied by an increase in the number of PLZF⁺ undifferentiated and differentiating spermatogonia, highlighting PTMφs’ role in testicular injury recovery and its influence on the spermatogonial kinetics [15]. Further, we also demonstrated that the infiltration of PTMφs into the seminiferous tubule in response to MEHP-induced injury in peripubertal rats mechanistically involves disruption of the blood–testis barrier (BTB) [19, 20]. In brief, we have demonstrated that the exposure of MEHP and cadmium chloride (CdCl₂), a known BTB disruptor in peripubertal male Fischer rats, led to elevated germ cell apoptosis and PTMφ numbers. However, exposure to methoxy acetic acid (MAA), a chemical that causes spermatocyte apoptosis without altering BTB, does not alter the number of PTMφs. Stabilization of BTB using the overexpression of LG3/4/5 peptide, a component of laminin-alpha2 that contributes to BTB integrity, prevented the increase in the number of PTMφs in MEHP-exposed rats [20]. Depletion of the testicular macrophages using clodronate liposomes was shown to reduce the PLZF⁺ undifferentiated and differentiating spermatogonia number, severely compromising the MEHP-induced post-injury recovery [21]. These findings together demonstrate that PTMφ numbers are responsive to BTB disruption in the testis but do not play a role in the mechanisms that trigger germ cell apoptosis in response to the exposure of testicular toxicant but hints at the possible role of PTMφs in the recovery and regulation of the spermatogonial niche.

Previously, we have shown that the chemokine MCP-1, which is a known signaling molecule responsible for the recruitment of macrophages, was found to be significantly increased in the testis of peripubertal rats after MEHP exposure, aligning with the time point that was observed with the increased influx of macrophages into the testis. Immunofluorescent detection revealed that MCP-1 expression was increased in the PTMCs surrounding the seminiferous tubule. Therefore, the events initiating the mobilization of PTMφs and the positive association between the number of PTMφs and the density of undifferentiated and differentiating spermatogonia have been investigated and reported [13, 15, 20]. Nevertheless, the reason for PTMφs to localize at selective regions of seminiferous tubule and their cellular interaction with seminiferous tubule cells remains unknown.

In this study, for the first time, we report the presence of an undocumented cell type expressing CD2 protein in rat seminiferous tubules. Although a study published in 2020 [21] reported CD2 protein as a cell surface marker of spermatogonial stem cells in rats and mice, the authors were not able to detect CD2 protein expression among the germ cells when they attempted to visualize it using testis histology cross-sections. A critical insight from our current study is that the CD2⁺ cell population is interspersed within the PTMC layer surrounding the seminiferous tubules. Therefore, the utilization of tissue cross-sections significantly limits the visualization, especially the cells on the periphery of the seminiferous tubule. Whole mount seminiferous tubule preparation is a useful tool for studying the cells at the periphery of the seminiferous tubule and for examining the type of interaction between the cells, which is not feasible when utilizing testis histology cross-section [22, 23]. In addition, results from our double-staining experiments using the isolated rat seminiferous tubule sample confirm that none of the germ cell subtypes co-express CD2 protein. Moreover, CD2 protein was found to be expressed by novel cells that are present within the PTMC layer. Since the study claiming CD2 protein as a cell surface marker of spermatogonial stem cells could not show the expression of CD2 protein among the germ cells [21], it is not convincing to say that CD2 protein is a surface marker for spermatogonial stem cells. In the present study, double staining of the testis cross-section for the pan germ cell marker DDX4 and CD2 clearly showed that CD2 is not expressed by the germ cells and the interstitial cells. In a spatial context, CD2⁺ cells are present within the PTMC layer of the seminiferous tubule. Therefore, we speculate that CD2⁺ cells could be niche cells whose influence on the spermatogonial kinetics warrants future studies.

In the present study, CD2⁺ cells are found to be localized in the cellular spaces between the PTMCs. Specifically, the available cellular spaces between the PTMCs were observed to define the CD2⁺ cells’ shape directly. CD2⁺ cells are easily distinguished from the αSMA⁺ PTMCs based on their irregular cell shape and nuclear morphology. Also, they are distinct from other known seminiferous tubule cells based on their expression of CD2 protein. Earlier in our published work, we have shown that on many occasions, MHC II⁺ PTMφs localize to the surface of seminiferous tubules. They were observed to be overlying or nestled between the PTMCs, and the nucleus of PTMφ was observed to be under the nucleus of an unknown cell (refer to Figure 1J and K), which had an irregular nuclear morphology and was very distinct from that of PTMCs’ nuclei [15]. In the current study, confocal observation of the seminiferous tubule revealed the existence of cellular interaction between the PTMφs and novel CD2⁺ cells. The details of membrane–membrane interaction between these PTMφs and CD2⁺ cells using high-resolution confocal z-stacks and surface rendering technique surprisingly revealed that in rare instances, the membrane of CD2⁺ cells remained extended or pinched by PTMφs. Also, there was a significant increase in PTMφs interacting with the CD2⁺ cells with cellular contact upon MEHP treatment. Inference from the 3D models qualitatively indicates that the mode of PTMφ- CD2⁺ cell interaction is cellular contact rather than whole-cell engulfment. The functional significance of this cellular interaction is not yet known. However, considering the proposed role of PTMφs in the regulation of spermatogonial stem cell niche reported by Defalco et al. and our previous published findings, and knowing that, in general, cell-to-cell interaction is important for the coordination of cellular activity and signaling, we propose that the cellular interaction between PTMφs - CD2 cells could be an additional layer of complexity associated with the mechanism of recovery of spermatogenesis [8, 15, 20].

This is the first study to visualize and report the existence of a new CD2 protein-expressing cell population within the PTMC layer of rat seminiferous tubule. Super and high-resolution confocal imaging revealed the CD2⁺ cells’ morphological features, topographical/clonal organization, and relative spatial localization with the other seminiferous tubule cells. The CD2⁺ cells were observed to present predominantly in a single, clone of two, and clone of three cells. However, occasionally, we observed clones that are more than three cells; based on the partition of cytoplasm and morphology of the nucleus, we deem that it could be an aggregation or cluster of different clones of cells rather than a varied clonal organization. The presence of hair-like cytoplasmic connections between clones of two cells with a single CD2⁺ cell may indicate clone fragmentation, which could drive the clonal diversity of the CD2⁺ cells. However, it is not clear if the CD2⁺ cells observed on seminiferous tubules are terminally differentiated cells or tissue-resident progenitor cell populations.

Interestingly, the dynamics of the CD2⁺ cells did not change in response to MEHP exposure. Even different post-injury recovery stages did not affect the dynamics of CD2⁺ cell clones. This suggests that the CD2⁺ cells are a dormant population lacking susceptibility to MEHP-induced toxicity. Further supporting this observation, double staining experiments using seminiferous tubule whole mount preparation showed that Ki67 protein, a known marker of cell proliferation and expressed by actively dividing cells, was not detectable in CD2⁺ cells [24, 25]. In a pathological immune microenvironment in non-reproductive tissues, the cellular interaction of macrophages with tissue-resident cells has been reported to influence cell proliferation [26–28]. In our experimental data, though we observed a significant increase in PTMφ-CD2⁺ cell interaction, that did not seem to affect the number of CD2⁺ cells. Further on, analyzing the single-cell transcriptomic profile of the testis or the secretome of the CD2⁺ cells will shed light on the functional outcome of PTMφ-CD2⁺ cell interaction and its possible role in the regulation of spermatogonial stem cell niche.

CD2 is a 55–66 kDa protein, a member of the immunoglobulin superfamily. In general, CD2 protein is mainly expressed as a surface glycoprotein confined to hematopoietic cells, with exceptionally high expression levels reported in T cells and natural killer cells [29]. Particularly in the context of T cell biology, CD2 protein expression was reported to aid in cell adhesion, immune recognition, organization, and signaling at immunological synapses that formed with the antigen-presenting cells [29–31]. It is well known that in rats, T cells and natural killer cells are spatially restricted to the interstitial compartment of the testis [31]. To date, only immune cells reported to be present in proximity to spermatogonial stem cells in the seminiferous tubule are PTMφs [8, 32]. However, because CD2 is reported to be a co-stimulatory protein that the T cells express [33], it is obligatory to rule out CD2⁺ to be an immune cell; we carried out a double immunolabelling experiment to check the expression of a more common leukocyte marker (CD45) on CD2⁺ cells in the whole-mount seminiferous tubule preparations. CD2⁺ cells from the corn oil group stained negative for CD45 expression; although the CD2⁺ cells in the corn oil group were negative for CD45 expression, we sought to check the expression of CD45 on CD2 cells in the MEHP treatment group to find whether the presence of CD2⁺ cells in this group could partly result from the infiltrating immune cells. In the MEHP group, CD2⁺ cells were negative for CD45, implying that CD2 is unlikely to be infiltrating immune cells. Double staining of MHC II and CD45 proteins on whole-mount seminiferous tubule preparations revealed PTMφs to be heterogenous (MHC II⁺ CD45⁺ and MHC II⁺ CD45⁻) in their expression of the CD45 protein. Also, it is interesting to identify the presence of distinct CD45⁺ cells other than PTMφs, which may be another immune cell within the PTMC layer; this warrants future investigation. Lack of expression of the marker CD45 by the CD2⁺ cells indicates a non-immune lineage. To further rule out PTMφ-CD2⁺ cell interaction as a form of immunological synapse between the antigen-presenting cell and a T cell, we carried out a double immunolabelling experiment to validate expression of the T-cell lineage marker CD3 with the CD2 protein using whole-mount seminiferous tubule preparations. The CD2⁺ cells were observed to be negative for CD3.

In previous years, PTMC-related research in rodent testis was focused on the organization of actin cytoskeleton and the functional implication of its role in the maintenance of spermatogonial stem cell niche. However, the explicit organization of the PTMCs and cellular (inter-peritubular) spaces has been understudied [34, 35]. This study used an extensive confocal microscope-based quantification approach to visualize PTMφ-CD2⁺ cell interaction. We determined the clonal dynamics of CD2⁺ cells, which are not feasible with flow-cytometry-based methods. To date, apart from the PTMφs, there is no report regarding any other cell type on the surface of the seminiferous tubule, especially on the cellular spaces between the PTMCs. For the first time, our study not only revealed the presence of distinct cells within the PTMC layer but also reported the dynamics of PTMφ-CD2⁺ cell interaction in response to the MEHP-induced testicular injury.

Author contributions

Conceptualization: C.L. and J.H.R.; Methodology: C.L. and R.T.; Investigation: C.L., R.T., and A.C.; Formal Analysis: C.L.; Writing—original draft: C.L.; Writing—review and editing: C.L. and J.H.R.; Funding Acquisition: J.H.R.

Conflict of interest

The authors have declared that no conflict of interest exists.

Data availability

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