title: Analysis of porcine granulosa cell death signaling pathways induced by vinclozolin.
abstract: Recent studies suggest that disturbing androgen-signaling pathways in porcine ovarian follicles may cause granulosa cell (GC) death. For this reason, we investigated which apoptotic pathway is initiated after GC exposure to an environmental antiandrogen, vinclozolin (Vnz), in vitro. Immunocytochemistry, Western blots, and fluorometric assays were used to quantify caspase-3 and -9 expression and activity. To elucidate the specific mechanism of Vnz action and toxicity, GCs were assessed for viability, cytotoxicity, and apoptotic activity using the ApoTox-Glo Triplex Assay. To further determine the mechanism of GC death induced by Vnz, we used the Apoptosis Antibody Array Kit. In response to Vnz stimulus, we found an increased level of caspase-3 protein expression (P ≤ 0.001) and an increase in caspase-3 proteolytic activity (P ≤ 0.001), confirming that Vnz is a potent proapoptotic factor. The strong immunoreaction of caspase-9 after Vnz treatment (P ≤ 0.001) suggests that intrinsic mitochondrial apoptosis pathway was activated during GC death. On the other hand, caspase-8, being a part of the extrinsic receptor pathway, was also activated (P ≤ 0.001). Therefore, it is possible that Vnz induces porcine granulosal apoptosis also through a parallel pathway. Activation of these two pathways was confirmed by the Apoptosis Antibody Array Kit. In conclusion, it is possible that the intrinsic signaling pathway may not act as an initial trigger for GC apoptosis but might contribute to the amplification and propagation of apoptotic cell death in the granulosa layer after treatment with this antiandrogen. Moreover, Vnz disturbs the physiological process of programmed cell death. Consequently, this could explain why atretic follicles are rapidly removed and suggests that normal function of the ovarian follicle may be destroyed.
authors.affiliation: Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Knet
authors.affiliation: Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland.
authors.forename: Kamil
authors.initials: K
authors.lastname: Wartalski
authors.affiliation: Department of Glycoconjugate Biochemistry, Institute of Zoology, Jagiellonian University, Krakow, Poland.
authors.forename: Dorota
authors.initials: D
authors.lastname: Hoja-Lukowicz
authors.affiliation: Department of Experimental Hematology, Institute of Zoology, Jagiellonian University, Krakow, Poland.
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Tabarowski
authors.affiliation: Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland.
authors.forename: Maria
authors.initials: M
authors.lastname: Slomczynska
authors.affiliation: Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland. Electronic address: maja.duda@uj.edu.pl.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Duda
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D005260:Female; D005659:Fungicides, Industrial / Q000633:toxicity*; D006107:Granulosa Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010080:Oxazoles / Q000633:toxicity*; D015398:Signal Transduction / Q000187:drug effects; D013552:Swine / Q000378:metabolism*
chemical_list: D005659:Fungicides, Industrial; D010080:Oxazoles; C025643:vinclozolin
keywords: Apoptosis; Caspase-3; Granulosa cell; Pig; Vinclozolin