Abstract
BackgroundProstaglandin F2α (PGF2α) may differentially affect viability of luteal cells by inducing either proliferation or cell death (via apoptosis or necroptosis). The diverse effects of PGF2α 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 PGF2α (aPGF2α) 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 aPGF2α (25 mg; Dinoprost), (3) local saline, (4) local aPGF2α (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.ResultsWe found that local and systemic administration of aPGF2α 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 aPGF2α treatment in the middle-stage CL, whereas systemic aPGF2α administration increased both CASP3 and CASP8 expression (P < 0.01). Moreover, we observed that both local and systemic aPGF2α injections increased RIPK1, RIPK3 and MLKL expression in the middle-stage CL (P < 0.05) while CYLD expression was markedly higher after i.m. aPGF2α 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 aPGF2α injections in the bovine CL.ConclusionOur results demonstrated for the first time that genes related to cell death pathways exhibit stage-specific responses to PGF2α administration depending on its local or systemic actions. Locally-acting PGF2α plays a luteoprotective role by inhibiting apoptosis and necroptosis in the early CL. Necroptosis is a potent mechanism responsible for structural CL regression during PGF2α-induced luteolysis in cattle.
Highlights
Prostaglandin F2α (PGF2α) may differentially affect viability of luteal cells by inducing either proliferation or cell death
The aim of the present study was to examine the differences in expression of genes related to: (i) apoptosis (CASP3, caspase 8 (CASP8), B-cell lymphoma 2 (BCL2) associated X (BAX), BCL2) and (ii) necroptosis (RIPK1, receptorinteracting protein kinase 3 (RIPK3), CYLD, mixed lineage kinase domain-like (MLKL)) in response to intra-corpus luteum (CL) or i.m. analogue of PGF2α (aPGF2α) injections in the early- vs. middle-stage bovine CL
Local and systemic administration of aPGF2α resulted in decreased messenger RNA (mRNA) expression (P = 0.0073, P = 0.0003, respectively; Fig. 1A) and protein concentration of Caspase 3 (CASP3) (P = 0.0021, P = 0.0038, respectively; Fig. 2A) in the early-stage CL, while both aPGF2α treatments increased CASP3 mRNA expression (P < 0.0001; Fig. 1A) and protein concentration (P < 0.0001; Fig. 2A) in the middle-stage CL
Summary
Prostaglandin F2α (PGF2α) may differentially affect viability of luteal cells by inducing either proliferation or cell death (via apoptosis or necroptosis). 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 PGF2α (aPGF2α) actions. Hojo et al [10, 11] proposed that necroptosis (CASP – independent cell death pathway) is an alternative luteolytic mechanism responsible for death of luteal steroidogenic cells (LSC) and luteal endothelial cells (LEC) and for their elimination from the bovine CL during luteolysis This process is characterized by disrupted cellular membranes with leakage of their intracellular contents and tissue damage [12, 13]. The activation of RIPK3 and RIPK3 substrate-mixed lineage kinase domain-like (MLKL) by its phosphorylation [17] are key steps during the execution of necroptosis [18]
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