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Peripubertal exposure of male rodents to the phthalate metabolite mono-(2-ethylhexyl) phthalate (MEHP) causes testicular inflammation, spermatocyte apoptosis, and disruption of the blood-testis barrier. The MEHP-induced inflammatory response in the testis includes an infiltration of macrophages and neutrophils, although the cause and purpose of this response is unknown. Recently, a population of testicular macrophages known as peritubular macrophages that are phenotypically distinct from those resident in interstitium was described in mice. Peritubular macrophages aggregate near the spermatogonial stem cell niche and are believed to stimulate their differentiation. We hypothesized that if testicular peritubular macrophages do indeed stimulate spermatogonial differentiation, MEHP exposure would result in an increase of peritubular macrophages to stimulate the replacement of lost spermatocytes. Male rats were exposed to 700 mg/kg MEHP or corn oil (vehicle control) via oral gavage at postnatal day 28 and euthanized at 48 h, 1 or 2 weeks later. Seminiferous tubules were stained with immunofluorescent markers for macrophages (major histocompatibility complex class II [MHC-II+]) and undifferentiated spermatogonia (PLZF). Peritubular macrophages were observed in rat testis: MHC-II+ cells on the surface of seminiferous tubules with heterogeneous morphology. Quantification of MHC-II+ cells revealed that, unlike in the mouse, their numbers did not increase through puberty (2-week period). MEHP increased macrophage presence by 6-fold 48 h after exposure and remained elevated by 2-fold 2 weeks after exposure. An increase of differentiating spermatogonia occurred 2 weeks after MEHP exposure. Taken together, our results suggest that peritubular macrophages play a crucial role in the testis response to acute injury and the subsequent recovery of spermatogenesis.

FasL (TNFSF6, CD95L) is hypothesized to trigger testicular germ cell apoptosis that normally occurs during a distinct peripubertal period as well as in response to toxicant-induced Sertoli cell injury. To test this hypothesis, we evaluated the testis of FasL gene-deficient mice (FasL(-/-)) at two distinct developmental ages (postnatal day [PND] 28 and 44) and after toxicant-induced Sertoli cell injury. Testicular cross sections from peripubertal (PND 28) FasL(-/-) mice showed significant increases in the basal germ cell apoptotic index (AI; 20.58 +/- 4.59) as compared to the testis of C57BL/6J wild-type mice (5.16 +/- 0.08) and closely correlated with increased expression of TRAIL protein in the testis of FasL(-/-) mice. A limited, but significant, number of seminiferous tubules in the testis of PND 28 FasL(-/-) mice showed a severe loss of germ cells with only Sertoli cells present. In contrast, no apparent gross histological changes were observed in the testis of adult (PND 44) FasL(-/-) mice. However, PND 44 FasL(-/-) mice did show a 51% reduction in homogenization-resistant elongate spermatids as compared to age-matched C57BL/6J mice. Exposure of PND 28 FasL(-/-) mice to mono-(2-ethylhexyl) phthalate (MEHP), a well-described Sertoli cell toxicant, unexpectedly caused a rapid decrease in the germ cell AI that paralleled increased levels of the CFLAR (c-FLIP) protein, a known inhibitor of death receptor signaling. In contrast, MEHP treatment did not decrease c-FLIP levels in PND 28 C57BL/6J mice. Taken together, these findings indicate that FasL protein expression is required during the peripubertal period for the proper regulation of germ cell apoptosis that occurs normally during this period. The influence of FasL on the cellular regulation of c-FLIP protein levels appears to be a unique mechanism for modulating germ cell apoptosis after toxicant-induced Sertoli cell injury.