The presence of macrophages and inflammatory responses in an in vitro testicular co-culture model of male reproductive development enhance relevance to in vivo conditions

Abstract

Our 3-dimensional testis co-culture system (3D-TCS) represents a promising model of male reproductive toxicity which captures sensitive processes of male reproductive development and contains the main testes cell types (germ, Leydig and Sertoli cells). Macrophages are another cell type important for testicular function and help to modulate immuno-endocrine processes during testes development. Chemicals such as phthalate esters (PE’s) affect macrophage function and testosterone production in the testes in vivo. The aim of this study was to determine whether macrophages were present in the 3D-TCS and investigate responses in our model that may be related to immuno-endocrine functions. We observed consistent expression of the resident macrophage marker ED2 as well as increases in inflammatory cytokines produced by macrophages and testes cells (IL-6, TNF-α and KC/GRO) after exposure to toxic PE’s. Pathway analysis of gene expression changes after exposure to PE’s showed that IL-6 and TNF-α signaling pathways were enriched after treatment with reproductively toxic, but not non-reproductively toxic phthalates. These results indicate that macrophages and inflammatory processes are captured in the 3D-TCS and that these processes are impacted by exposure to reproductive toxicants. These processes represent a major mode of action for in vivo testis toxicity for a variety of compounds and our novel in vitro model is able to capture toxicant perturbation of immune function.

MEHP-induced rat testicular inflammation does not exacerbate germ cell apoptosis

“…CXCL1 protein was indeed found to be upregulated in the testes of MEHP-treated rats. These findings are in agreement with other reports that phthalates (DBP and DEHP) can upregulate Cxcl1 and Cxcl10 gene expression in the testis in vivo (Lahousse et al , 2006; Johnson et al , 2007), and in an in vitro testicular co-culture model (Harris et al , 2016). How expression of CXCL1 is induced remains to be investigated, but TNFα and Il-1β are likely candidates, as they have been reported to induce CXCL1 expression in peritubular myoid cells (PTMCs) and SCs, respectively (Aubry et al , 2000), and we have previously shown both cytokines to be increased upon MEHP exposure (Stermer et al , 2017).…”

Effect of prenatal DINCH plasticizer exposure on rat offspring testicular function and metabolism

“…Immunohistochemical analyses of the inflammatory marker CD68 revealed an increased presence of macrophages in the interstitial areas of PND60 animals, suggesting a possible activation of the inflammatory pathway, which has been proposed to lead to fibrosis 27 . This result correlated well with our previous research on DEHP and inflammation in adipose tissue 48 and with other reports that describe proinflammatory effects of phthalates in the testes 49 – 51 . Interestingly at PND200 the effect was lost, suggesting that age-related changes in the testes affect susceptibility to DINCH.…”

Elevated CCL2 causes Leydig cell malfunction in metabolic syndrome

“…Testicular interstitium contains several cell types, including Leydig cells, macrophages, and endothelial cells, capable of producing proinflammatory cytokines such as IL-1β, IL-6, and TNF-α to modulate testis functions (25)(26)(27)(28)(29). CCL2 is a chemokine expressed constitutively in testis by Leydig cells and peritubular cells (30,31).…”