Abstract
PFOS induces Sertoli cell injury using testicular cells isolated from rodent testes, but it remains unknown if PFOS has similar effects in humans. Herein, we maintained human Sertoli cells in a mitotically active state in vitro, thus enabling transfection experiments that altered gene expression to explore the molecular mechanism(s) underlying toxicant-induced cell injury. Human Sertoli cells obtained from men at ages 15, 23, 36 and 40 were cultured in vitro. These differentiated Sertoli cells remained mitotically active when cultured in the presence of 10% FBS (fetal bovine serum), with a replication time of ~1–3 weeks. At ~80% confluency, they were used for studies including toxicant exposure, immunoblotting, immunofluorescence analysis, tight junction (TJ)-permeability assessment, and overexpression of BTB (blood-testis barrier) regulatory genes such as FAK and its phosphomimetic mutants. PFOS was found to induce Sertoli cell injury through disruptive effects on actin microfilaments and microtubule (MT) organization across the cell cytosol. As a consequence, these cytoskeletal networks failed to support cell adhesion at the BTB. Overexpression of a FAK phosphomimetic and constitutively active mutant p-FAK-Y407E in these cells was capable of rescuing the PFOS-induced injury through corrective cellular organization of cytoskeletal elements. Summary: PFOS induces human Sertoli cell injury which can be rescued by overexpressing p-FAK-Y407E mutant.
Highlights
Perfluorooctanesulfonate (PFOS) and its related products (e.g., perfluorooctanoic acid (PFOA)) were first produced by 3M Company in 1949
~90% confluency on human fibronectin-coated bicameral units and cultured in DMEM/F12 medium with other additives and antibiotics in 24-well dishes (Fig. 1A, left panel), they were capable of establishing a tight junction (TJ)-permeability barrier which could be monitored by quantifying TER across the cell epithelium as noted in Fig. 1A
These disruptive effects of PFOS on the TJ barrier were likely mediated by down-regulation and redistribution of the blood-testis barrier (BTB)-associated proteins, such as N-cadherin and its adaptor ß-catenin (Fig. 1B,C; Figure S1)
Summary
Perfluorooctanesulfonate (or perfluorooctanesulfonic acid) (PFOS) and its related products (e.g., perfluorooctanoic acid (PFOA)) were first produced by 3M Company in 1949. Due to the ongoing use of PFOS in consumable products in particular in China, we thought it pertinent to investigate: (i) if PFOS would induce human Sertoli cell injury, and (ii) if p-FAK-Tyr[407], a signaling protein known to promote rat Sertoli cell function by promoting the integrity of the Sertoli cell blood-testis barrier (BTB), would rescue PFOS-induced disruption and Sertoli cell injury in human Sertoli cells cultured in vitro[15,16] This knowledge would provide much needed insight into PFOS-mediated testis injury and inform us further on potential mechanisms of environmental toxicant-induced male reproductive dysfunction
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