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Death receptor response in rodent testis after mono-(2-ethylhexyl) phthalate exposure
C John Giammona1, Pragati Sawhney, Yamini Chandrasekaran
1College of Pharmacy, Division of Pharmacology and Toxicology, The University of Texas at Austin, Austin, Texas 78712-1074, USA.
Abstract:
Apoptosis of testicular germ cells is critical for the maintenance of functional spermatogenesis. Previously, we have demonstrated that the Fas (Apo-1, CD95) receptor participates in the regulation of germ cell apoptosis, particularly after toxicant-induced Sertoli cell injury. In this study, we show that germ cells from B6.SMNC3H-Fas(gld,gld) (gld) mice that express a dysfunctional form of FasL still undergo significant apoptosis, albeit at a lower incidence than seen in B6 mice, following mono-(2-ethylhexyl) phthalate (MEHP)-induced Sertoli cell injury. In addition, we show the presence of Fas, TRAIL-R1 (Death Receptor-4, DR4), and TRAIL-R2 (DR5) in the testis of C57BL/6 (B6) and gld mice (4 weeks old), and Sprague-Dawley rats (5 weeks old) and their responsiveness after MEHP treatment. More importantly, Western blot analysis of cellular fractions showed an increase in death receptor localization on the membrane fractions taken from Sprague-Dawley rats. Immunohistochemical analysis indicated localization of Fas and DR5 primarily to the spermatocyte subpopulation of germ cells. Examination of downstream receptor-mediated signals (i.e., cleavage of procaspase-8 and NFkappaB activation) revealed an early increase in NFkappaB-DNA binding and an increase in procaspase-8 processing in mutant gld mice. In summary, germ cell-associated death receptors, as well as downstream signaling elements, appear to be responsive to MEHP-induced Sertoli cell injury. Whether this is directly responsible for the increases in germ cell apoptosis after MEHP exposure is yet to be determined. The observed robust and early increase in Fas in wild-type testis and diminished rates of germ cell apoptosis in mutant testis (gld and lpr(cg)) reiterates the importance of the Fas signaling pathway.
Insights
Germ cell apoptosis is vital for spermatogenesis. This study shows that while Fas signaling is important, other death receptors also respond to toxicant-induced Sertoli cell injury, impacting germ cell death.
Area of Science:
- Reproductive Biology
- Toxicology
- Cell Death Signaling
Background:
- Apoptosis of testicular germ cells is crucial for maintaining spermatogenesis.
- The Fas receptor plays a role in regulating germ cell apoptosis, especially after Sertoli cell injury.
- Toxicants like mono-(2-ethylhexyl) phthalate (MEHP) can induce Sertoli cell injury and subsequent germ cell apoptosis.
Purpose of the Study:
- To investigate the role of Fas and other death receptors in germ cell apoptosis following MEHP-induced Sertoli cell injury.
- To examine the expression and responsiveness of death receptors (Fas, TRAIL-R1, TRAIL-R2) in the testis after MEHP exposure.
- To analyze downstream signaling events, including caspase-8 cleavage and NF-kappaB activation, in response to MEHP treatment.
Main Methods:
- Utilized B6.SMNC3H-Fas(gld,gld) (gld) mice with dysfunctional FasL and wild-type B6 mice, as well as Sprague-Dawley rats.
- Administered mono-(2-ethylhexyl) phthalate (MEHP) to induce Sertoli cell injury.
- Performed Western blot analysis to assess death receptor localization and procaspase-8 processing.
- Conducted immunohistochemical analysis to determine the localization of Fas and DR5.
- Assessed NF-kappaB activation using DNA-binding assays.
Main Results:
- Germ cells in gld mice underwent apoptosis after MEHP exposure, but at a lower rate than in wild-type B6 mice.
- Fas, TRAIL-R1 (DR4), and TRAIL-R2 (DR5) were present in the testes of mice and rats and responded to MEHP treatment.
- Western blot analysis showed increased death receptor localization to membrane fractions in rats after MEHP exposure.
- Immunohistochemistry revealed Fas and DR5 primarily in spermatocytes.
- NF-kappaB-DNA binding and procaspase-8 processing increased early in gld mice after MEHP exposure.
Conclusions:
- Germ cell-associated death receptors and downstream signaling elements are responsive to MEHP-induced Sertoli cell injury.
- The Fas signaling pathway is important for germ cell apoptosis following MEHP exposure, as evidenced by diminished apoptosis in gld mice.
- Further research is needed to determine if these death receptors are directly responsible for increased germ cell apoptosis after MEHP exposure.