Age- and species-dependent infiltration of macrophages into the testis of rats and mice exposed to mono-(2-Ethylhexyl) phthalate (MEHP)

Abstract

The mechanism by which noninfectious testicular inflammation results in infertility is poorly understood. Here the infiltration of CD11b+ immunoreactive testicular interstitial cells (neutrophil, macrophages, dendritic cells) in immature (Postnatal Day [PND] 21, 28, and 35) and adult (PND 56) Fischer rats is described at 12, 24, and 48 h after an oral dose of 1 g/kg mono-(2-ethylhexyl) phthalate (MEHP), a well-described Sertoli cell toxicant. Increases of CD11b+ cells are evident 12 h after MEHP exposure in PND 21 and 28 rats. In PND 28 rats, CD11b+ cells remained significantly elevated at 48 h, while in PND 21 rats, it returned to control levels by 24 h. The peak number of CD11b+ cells in PND 35 rat testis is delayed until 24 h, but remains significantly elevated at 48 h. In PND 56 rats, no increase in CD11b+ cells occurs after MEHP exposure. In PND 21, 28, and 35 rats, a significant increase in monocyte chemoattractant protein-1 (MCP-1) by peritubular myoid cells occurs 12 h after MEHP. Interestingly, MEHP treatment of C57BL/6J mice did not incite an infiltration of CD11b+ cells at either PND 21 or 28. The peak level of germ cell apoptosis observed 24 h after MEHP exposure in young rats is not seen in mice at any age or in PND 56 rats. Taken together, these findings implicate MCP-1 released by peritubular myoid cells in provoking the migration of CD11b+ cells into the immature rat testis early after MEHP exposure and point to a role for CD11b+ cells in triggering germ cell apoptosis in an age- and species-dependent manner.

Keywords: interstitial cells; macrophage; myoid cells; reproductive immunology; toxicology.

FIG. 1

Age-dependent MEHP-induced testicular infiltration of CD11b+ cells in rats. Expression of CD11b+ cells in single cell suspension of live testicular interstitial cells after MEHP treatment of PND 21 (A), 28 (B), 35 (C), and 56 (D) rats after 12 h of MEHP exposure. Blue represent MEHP-treated rats (1 g/kg, p.o.) and red is vehicle-treated rats (corn oil, equivalent volume). The fold-change increases in CD11b+ cells after MEHP treatment (1 g/kg, p.o.) at each age and time point are summarized in the table (E). Asterisks (*) indicate significant differences between treatments at specified time points (P < 0.10, Tukey honestly significant difference [HSD] test; PND 21: n = 4, PND 28: n = 6, PND 35: n = 6, PND 56: n = 3 per time point/treatment).

FIG. 2

Dose-dependent MEHP-induced testicular infiltration of CD11b+ cells in rats. Expression of CD11b+ cells in single cell suspension of live testicular interstitial cells after MEHP treatment at 1 g/kg (A), 0.75 g/kg (B), and 0.5 g/kg (C) in PND 28 rats after 12 h of exposure. Blue represent MEHP-treated rats (1 g/kg, p.o.) and red is vehicle-treated rats (corn oil, equivalent volume). The increases in the number of CD11b+ cells after MEHP treatment (1, 0.75, and 0.5 g/kg, p.o.) at PND 28 and time point are summarized in the table (D). Letters indicate significant differences between treatments at specified time points (P < 0.05, Tukey HSD; 1 g/kg: n = 6, 0.75 g/kg: n = 6, 0.5 g/kg: n = 6, control: n = 13 per time point).

FIG. 3

MEHP-induced infiltration of newly arrived CD68+ macrophages into the testis of immature rats and PND 56 rats. Representative photomicrograph of immunostaining for CD68+ macrophages of immature rats—PND 21 (A), 28 (B), and 35 (C)—and mature rats—PND 56 (D)—treated orally for 12 h with MEHP (1 g/kg, n = 3) or vehicle (control, n = 3). Bars = 50 μm; insets magnification ×300.

FIG. 4

MEHP-induced germ cell apoptosis in rats. TUNEL staining demonstrating MEHP-induced germ cells apoptosis in immature rats. Representative photomicrograph of PND 21 (A), 28 (B), 35 (C), and PND 56 (D) rats treated orally for 24 h with MEHP (1 g/kg; lower panel) or vehicle (corn oil; top panel) demonstrate the differences in age sensitivity. Bars = 50 μm; insets magnification ×300. The AI for all the ages and time points are summarized in the table (E). The AI was calculated as the percentage of essentially round seminiferous tubules containing more than three TUNEL-positive germ cells in each cross-section. Asterisks designate significant differences within the age groups (mean ± SEM, P < 0.05, Tukey HSD; n = 3/time point/treatment).

FIG. 5

Migration of macrophages into the testis after MEHP treatment is induced by MCP-1 produced by PTMCs in an age-dependent manner. The level of MCP-1 (ng/ml) within testis of PND 21, 28, and 35 rats gavaged for 12 h with MEHP (1 g/kg) or vehicle (corn oil) was quantified by ELISA. The box plot (A) shows the median (−) and the lowest and highest data points (i.e., the bottom and top of the box, respectively) to convey the level, spread, and symmetry of the distribution. Statistically significant differences (P < 0.05, ANOVA; n = 3/time point/treatment) in treatment (MEHP > control), age-dependent (PND 21 > 28 > 35) total testis concentration, and treatment by age interaction were observed. Representative photomicrograph of MCP-1 immunofluorescence staining of PND 21 (B), 28 (C), 35 (D), and 56 (E) rats treated orally for 12 h demonstrating expression specifically in PTMCs. Bars = 100 μm (B–D) and 50 μm (E); insets magnification ×300.

FIG. 6

Mice lack the MEHP-induced infiltration of CD11b+ cells and associated germ cell-induced apoptosis. PND 21 (A) and 28 (B) C57BL/6J mice were gavaged with MEHP (1 g/kg, n = 5) or vehicle (control, n = 5) for 12, 24, and 48 h. The number of testicular interstitial cells positive for CD11b+ were quantified by flow cytometry (representative histograms; A and B) and showed no differences between treatments at either age (fold-change; C). Representative photomicrograph of TUNEL staining of PND 21 (D) mice treated orally for 24 h with 1 g/kg MEHP (lower panel, n = 6) or vehicle (top panel, n = 6) demonstrate the differences in species sensitivity to severity of germ cell-induced apoptosis. Bars = 50 μm; insets magnification ×300. The AI for PND 21 mice is summarized in the table (E). The AI was calculated as the percentage of essentially round seminiferous tubules containing more than three TUNEL-positive germ cells in each cross-section (mean ± SEM).