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.

Sperm RNA is a sensitive monitoring endpoint for male reproductive toxicants, and a potential biomarker to assess male infertility and sperm quality. However, isolation of sperm RNA is a challenging procedure due to the heterogeneous population of cells present in the ejaculate, the low yield of RNA per spermatozoon, and the absence of 18S and 28S ribosomal RNA subunits. The unique biology of spermatozoa has created some uncertainty in the field about RNA isolation methods, indicating the need for rigorous quality control checks to ensure reproducibility of data generated from sperm RNA. Therefore, we developed a reliable and effective protocol for RNA isolation from rat and human spermatozoa that delivers highly purified and intact RNA, verified using RNA-specific electrophoretic chips and molecular biology approaches such as RT-PCR and Western blot analysis. The sperm RNA isolation technique was optimized using rat spermatozoa and then adapted to human spermatozoa. Three steps in the sperm isolation procedure, epididymal fluid collection, sperm purification, and spermatozoon RNA extraction, were evaluated and assessed. The sperm RNA extraction methodology consists of collection of rat epididymal fluid with repeated needle punctures of the epididymis, somatic cell elimination using detergent-based somatic cell lysis buffer (SCLB) and the use of RNA isolation Kit. Rat sperm heads are more resistant to disruption than human spermatozoa, necessitating the addition of mechanical lysis with microbeads and heat in the rat protocol, whereas the human sperm protocol only required lysis buffer. In conclusion, this methodology results in reliable and consistent isolation of high-quality sperm RNA. Using this technique will aid in translation of data collected from animal models, and reproducibility of clinical assessment of male factor fertility using RNA molecular biomarkers.

The testis is an organ that maintains an immune suppressive environment. We previously revealed that exposure of pre-pubertal rats to an acute dose of a well-described Sertoli cell toxicant, mono-(2-ethylhexyl) phthalate (MEHP), leads to an accumulation of CD11b+ immune cells in the testicular interstitial space that closely correlates with a robust incidence of germ cell (GC) apoptosis. Here, we test the hypothesis that the infiltrating immune cells contribute to GC apoptosis. Postnatal day 28 Fischer rats that received an oral dose of 700 mg/kg MEHP showed a significant infiltration of both CD11bc+/CD68+/CD163- macrophages and neutrophils. The infiltration peaked at 12 h, but had reduced by 48 h. Testicular macrophages from MEHP-treated rats showed significantly upregulated expression of and, and the ratio was reduced compared to controls. However, small increases in anti-inflammatory genes and were also observed. Depletion of circulating monocytes with clodronate liposomes prior to MEHP treatment reduced the macrophage influx into the testis, but did not lower GC apoptosis. Additionally, depletion of neutrophils using an anti-polymorphonuclear cell antibody prevented both macrophage and neutrophil infiltration into the testis, and also did not affect GC apoptosis. Together, these results show that exposure to MEHP leads to a rapid and temporary influx of pro-inflammatory monocytes and neutrophils in the interstitium of the testis. However, with this acute dosing paradigm, these infiltrating leukocytes do not appear to contribute to MEHP-induced testicular GC apoptosis leaving the functional significance of these infiltrating cells in the pathogenesis of MEHP-induced testicular injury unresolved.

Exposure of rodents to the Sertoli cell (SC) toxicant mono-(2-ethylhexyl) phthalate (MEHP) has been reported to trigger an infiltration of macrophages into the testis in an age- and species-dependent manner. Here we challenge the hypothesis that the peripubertal rat-specific infiltration of macrophages after MEHP exposure is due, in part, to an increase in SC-specific inflammatory cytokine expression. To rule out that GC apoptosis itself is responsible for macrophage recruitment, rats were exposed to a direct germ cell toxicant, methoxy acetic acid (MAA), but no infiltration of macrophages was observed. Next, mRNA levels of inflammatory cytokines were evaluated after MEHP exposure. IL-1α, IL-6, and MCP-1 expression was increased in vivo and correlated with macrophage infiltration in a species-specific manner. Additionally, IL-6 and MCP-1 expression was increased in SC-GC co-cultures and ASC-17D SCs. These results indicate that MEHP-injury in pubertal rats specifically stimulates secretion of pro-inflammatory cytokines and alters the immune microenvironment.