Mono-(2-ethylhexyl)phthalate (MEHP)-induced disruption on the crosstalk between sertoli cells and germ cells
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Mono-(2-ethylhexyl) phthalate (MEHP), the toxic metabolite of di-(2-ethylhexyl) phthalate (DEHP), can cause testicular injury in animal models. This thesis examines the molecular mechanisms which lead to the loss of germ cells following MEHP exposure in the testis through germ cell apoptosis and germ cell detachment. MEHP-induced Sertoli cell injury in peri-pubertal rodents results in the stimulation of germ cell apoptosis through the FasL/Fas signaling pathway. In the seminiferous tubules, Sertoli cells express FasL while germ cells produce Fas. In this thesis, I report that following MEHP exposure, Sertoli cells transcriptionally up-regulate the expression of the FasL gene through activation of the transcription factors NFκB and Sp-1. MEHP also induces an increase in the production of soluble TNF-α in germ cells, which mediates NF-κB activation and causes robust increases in Sertoli cell FasL, indicating the participation of germ cells in triggering their own cell death in response to Sertoli cell injury. Metalloproteinases (MPs) and tissue inhibitors of metalloproteinases (TIMPs) are essential for processing the TNF-α precursor to its soluble form which is required for its ability to bind to TNFR-1. TIMP-2 is predominately expressed in primary rat Sertoli cells, and its protein level decreases time-dependently after MEHP exposure, which contributes to MMP-2 activation in the seminiferous tubule. The addition of SB-3CT, a specific gelatinase inhibitor, decreases the activity of MMP-2 and significantly reduces MEHP-enhanced soluble TNF-α production as well as MEHP-induced testicular germ cell apoptosis. Therefore, the decrease in Sertoli cell TIMP-2 expression may be the initial injury following MEHP exposure, which further stimulates the process of germ cell apoptosis by activating MMP-2. Local cell-cell communication through junctional complexes is crucial in the regulation of mammalian spermatogenesis and in the development of the male phenotype, so the disturbance of junctional complexes within the seminiferous epithelium may contribute to MEHP-induced abnormal spermatogenesis. MEHP exposure decreases occludin expression, possibly contributing to the opening of tight junctions between adjacent Sertoli cells. MEHP exposure also suppresses the expression of laminin-γ3 and integrin-β1 in apical ectoplasmic specializations in a time-dependent manner. MMP-2 activation enhances the disconnection of germ cells from Sertoli cells, and specific MMP-2 inhibitors (TIMP-2 and SB-3CT) significantly suppress MEHP-induced germ cell sloughing by altering the expression of junction proteins in vitro and in vivo, suggesting that MEHP-activated MMP-2 plays an important role in regulating the physical contact between Sertoli cells and germ cells. Interestingly, the addition of the caspase inhibitor (z-VAD-FMK) does not inhibit MEHP-induced germ cell detachment, indicating that MEHP-induced germ cell sloughing is independent of germ cell apoptosis. Taken together, the observations in this thesis indicate a distinct role of TIMP-2 and MMP-2 in response to toxicant-induced Sertoli cell injury, providing further insights into the mechanism by which Sertoli cells control the sensitivity of germ cells to undergo apoptosis during the peri-pubertal period.