The use of purified rat Leydig cells complements the H295R screen to detect chemical-induced alterations in testosterone production†

Abstract

Exposure to endocrine disrupting chemicals has been associated with compromised testosterone production leading to abnormal male reproductive development and altered spermatogenesis. In vitro high-throughput screening (HTS) assays are needed to evaluate risk to testosterone production, yet the main steroidogenesis assay currently utilized is a human adrenocortical carcinoma cell line, H295R, which does not synthesize gonadal steroids at the same level as the gonads, thus limiting assay sensitivity. Here, we propose a complementary assay using a highly purified rat Leydig cell assay to evaluate the potential for chemical-induced alterations in testosterone production by the testis. We evaluated a subset of chemicals that failed to decrease testosterone production in the HTS H295R assay. The chemicals examined fit into one of two categories based on changes in substrates upstream of testosterone in the adrenal steroidogenic pathway (17α-hydroxyprogesterone and 11-deoxycorticosterone) that we predicted should have elicited a decrease in testosterone production. We found that 85% of 20 test chemicals examined inhibited Leydig cell testosterone production in our assay. Importantly, we adopted a 96-well format to increase throughput and efficiency of the Leydig cell assay. We identified a selection criterion based on the AC50 values for 17α-hydroxyprogesterone and 11-deoxycorticosterone generated from the HTS H295R assay that will help prioritize chemicals for further testing in the Leydig cell screen. We hypothesize that the greater dynamic range of testosterone production and sensitivity of the Leydig cell assay permits the detection of small, yet significant, chemical-induced changes not detected by the HTS H295R assay.

Evaluating Chemicals for Thyroid Disruption: Opportunities and Challenges with in Vitro Testing and Adverse Outcome Pathway Approaches

“…2017; U.S. EPA 2014), as well as steroidogenesis pathways (Botteri Principato et al. 2018; Haggard et al.…”

SARS-CoV-2 Enters Human Leydig Cells and Affects Testosterone Production In Vitro

“…As human testis samples are difficult to obtain and Leydig cells from commercial sources neither express the human Leydig cell marker 3β-HSD nor produce T in response to stimulation, we employed hLLCs, the human Leydig-like cell model we previously reported [ 12 ], to explore the human Leydig cell tropism of SARS-CoV-2. Due to the overlap in gene and protein expression profiles of steroidogenesis between H295-R cells (human adrenal cortical cells) and Leydig cells [ 20 ], we utilized H295-R cells as a suitable reference for examining whether various steroid-producing cells possess similar mechanisms for SARS-CoV-2 entry. ACE2, the receptor for SARS-CoV-2 and a commonly found receptor in multiple tissues, was previously shown to be one of the main culprits enabling SARS-CoV-2 to infect multiple organs.…”

Isolation of Leydig cells from adult rat testes by magnetic‐activated cell sorting protocol based on prolactin receptor expression

“…[3][4][5] Due to the critical roles played in both reproduction and general well-being of the males, LCs have been studied intensively over years in both in vivo and in vitro settings, including studies in the toxicological field. 6 In vitro studies of LCs require the cells be isolated and cultured in the best possible condition as it could be, in order to examine the cell functions at optimal physiological condition with minimal interfering from other testicular cells. Various methods/procedures have been developed over years for the isolation of LCs from rodent testes.…”

“…Defects in LCs, either in the numbers or their functions, could result in low T with complicated symptoms including changes in body composition, increased fatigue, sexual dysfunction, depressed mood, decreased cognitive function and reduced immune response 3–5 . Due to the critical roles played in both reproduction and general well‐being of the males, LCs have been studied intensively over years in both in vivo and in vitro settings, including studies in the toxicological field 6 . In vitro studies of LCs require the cells be isolated and cultured in the best possible condition as it could be, in order to examine the cell functions at optimal physiological condition with minimal interfering from other testicular cells.…”