Inhibitors of caspase homologues suppress an apoptotic phenotype in cultured rabbit corpora lutea

Abstract

Apoptosis is a morphologically defined type of cell death initiated by variousstimuli that results in the activation of caspases (cysteine-containingaspartate-specific proteases). In the present study, it was determined thatcaspases are present during, and play a role in, corpus luteum (CL) apoptosisin vitro. Pseudopregnancy was induced in rabbits with100 IU human chorionic gonadotrophin. On Day 11 of pseudopregnancy, CL wereisolated and cultured for 0, 2, 4, 6, and 8 h in the absence of trophicsupport to induce spontaneous apoptosis. Total RNA was extracted and analysedfor caspase-1 expression by Northern blot analysis. The results demonstratedcaspase-1 expression from 4 h. In the second part of the study, CL wereincubated without trophic support for 4 h with increasing concentrations ofthree general caspase inhibitors, sodium aurothiomalate (SAM), iodoacetic acid(IAA) and N-tosyl-L-phenylalanylchloromethylketone(TPCK) , and two specific caspase inhibitors, N-acetyl (Ac)-Tyr-Val-Ala-Asp(YVAD)-chloromethylketone (CMK) (Ac-YVAD-CMK) and Ac-Asp-Glu-Val-Asp(DEVD)-aldehyde (CHO) (Ac-DEVD-CHO). At completion, DNA was isolated andintegrity assessed. Treatment of CL with SAM, IAA or Ac-DEVD-CHO effectivelysuppressed apoptotic DNA fragmentation. The final component of the study wasto examine caspase-3 protein expression. Western blot analysis revealed asignificant increase in caspase-3 expression over the experimentaltime-course. The results of the present study clearly demonstrate atime-dependent link between the caspases, specifically caspase-3 andspontaneous apoptosis in the rabbit CL.Extra keyword: pseudopregnancy.