Prostaglandin F 2α (PGF 2α) may differentially affect viability of luteal cells by inducing either proliferation or cell death (via apoptosis or necroptosis). The diverse effects of PGF 2α may depend on its local vs. systemic actions. In our study, we determined changes in expression of genes related to: (i) apoptosis: caspase (CASP) 3, CASP8, BCL2 associated X (BAX), B-cell lymphoma 2 (BCL2) and (ii) necroptosis: receptor-interacting protein kinase (RIPK) 1, RIPK3, cylindromatosis (CYLD), and mixed lineage kinase domain-like (MLKL) in the early and mid-stage corpus luteum (CL) that accompany local (intra-CL) vs. systemic (i.m.) analogue of PGF 2α (aPGF 2α) actions. Cows at day 4 (n = 24) or day 10 (n = 24) of the estrous cycle were treated by injections as follows: (1) systemic saline, (2) systemic aPGF 2α (25 mg; Dinoprost), (3) local saline, (4) local aPGF 2α (2.5 mg; Dinoprost). After 4 h, CLs were collected by ovariectomy. Expression levels of mRNA and protein were investigated by RT-q PCR, Western blotting and immunohistochemistry, respectively.
We found that local and systemic administration of aPGF 2α in the early-stage CL resulted in decreased expression of CASP3 ( P < 0.01), but CASP8 mRNA expression was up-regulated ( P < 0.05). However, the expression of CASP3 was up-regulated after local aPGF 2α treatment in the middle-stage CL, whereas systemic aPGF 2α administration increased both CASP3 and CASP8 expression ( P < 0.01). Moreover, we observed that both local and systemic aPGF 2α injections increased RIPK1, RIPK3 and MLKL expression in the middle-stage CL ( P < 0.05) while CYLD expression was markedly higher after i.m. aPGF 2α injections ( P < 0.001). Moreover, we investigated the localization of necroptotic factors (RIPK1, RIPK3, CYLD and MLKL) in bovine CL tissue after local and systemic aPGF 2α injections in the bovine CL.
Our results demonstrated for the first time that genes related to cell death pathways exhibit stage-specific responses to PGF 2α administration depending on its local or systemic actions. Locally-acting PGF 2α plays a luteoprotective role by inhibiting apoptosis and necroptosis in the early CL. Necroptosis is a potent mechanism responsible for structural CL regression during PGF 2α-induced luteolysis in cattle.