title: Inhibition of ovarian KIT phosphorylation by the ovotoxicant 4-vinylcyclohexene diepoxide in rats.
abstract: In vitro exposure of Postnatal Day 4 (PND4) rat ovaries to the occupational chemical 4-vinylcyclohexene diepoxide (VCD) destroys specifically primordial and primary follicles via acceleration of atresia. Because oocyte-expressed c-kit (KIT) plays a critical role in follicle survival and activation, a direct interaction of VCD with KIT as its mechanism of ovotoxicity was investigated. PND4 rat ovaries were cultured with and without VCD (30 μM) for 2 days. When assessed by Western analysis or mobility shift detection, phosphorylated KIT (pKIT) was decreased (P < 0.05) by VCD exposure, while total KIT protein was unaffected. Anti-mouse KIT2 (ACK2) antibody binds KIT and blocks its signaling pathways, whereas anti-mouse KIT 4 (ACK4) antibody binds KIT but does not block its activity. PND4 rat ovaries were incubated for 2 days with and without VCD with and without ACK2 (80 μg/ml) or ACK4 (80 μg/ml). ACK2 decreased pKIT; however, ACK4 had no effect. Conversely, ACK2 did not affect a VCD-induced decrease in pKIT, whereas ACK4 further reduced it. Because ACK2 and ACK4 (known to directly bind KIT) affect VCD responses, these results support the fact that VCD interacts directly with KIT. The effect of these antibodies on VCD-induced follicle loss was measured after 8 days of incubation. ACK2 further reduced (P < 0.05) VCD-induced follicle loss, whereas ACK4 did not affect it. These findings demonstrate that VCD induces ovotoxicity by direct inhibition of KIT autophosphorylation of the oocyte. The data also further support the vital function of KIT and its signaling pathway in primordial follicle survival and activation, as well as its role in VCD-induced ovotoxicity.
authors.affiliation: Department of Physiology, University of Arizona, Tucson, Arizona, USA.
authors.forename: Connie J
authors.initials: CJ
authors.lastname: Mark-Kappeler
authors.forename: Nivedita
authors.initials: N
authors.lastname: Sen
authors.forename: Ashley
authors.initials: A
authors.lastname: Lukefahr
authors.forename: Laurel
authors.initials: L
authors.lastname: McKee
authors.forename: I Glenn
authors.initials: IG
authors.lastname: Sipes
authors.forename: John
authors.initials: J
authors.lastname: Konhilas
authors.forename: Patricia B
authors.initials: PB
authors.lastname: Hoyer
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D019138:Antibodies, Blocking / Q000378:metabolism; D000937:Antigen-Antibody Reactions / Q000187:drug effects; D053138:Cyclohexenes / Q000037:antagonists & inhibitors / Q000633:toxicity*; D004785:Environmental Pollutants / Q000037:antagonists & inhibitors / Q000633:toxicity*; D005260:Female; D005496:Follicular Atresia / Q000187:drug effects; D008024:Ligands; D058990:Molecular Targeted Therapy; D008970:Molecular Weight; D009924:Organ Culture Techniques; D006080:Ovarian Follicle / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism; D010053:Ovary / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000037:antagonists & inhibitors / Q000633:toxicity*; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D019009:Proto-Oncogene Proteins c-kit / Q000819:agonists / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D014753:Vinyl Compounds / Q000037:antagonists & inhibitors / Q000633:toxicity*
chemical_list: D019138:Antibodies, Blocking; D053138:Cyclohexenes; D004785:Environmental Pollutants; D008024:Ligands; D047428:Protein Kinase Inhibitors; D014753:Vinyl Compounds; C012606:4-vinyl-1-cyclohexene dioxide; D019009:Proto-Oncogene Proteins c-kit