Effects of prostaglandin F2α (PGF2α) on cell-death pathways in the bovine corpus luteum (CL)

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Keywords: Apoptosis; Bovine CL; Necroptosis; Prostaglandin F2α; RIPKs.

Fig. 1

Effect of local or systemic PGF_2α analogue (aPGF_2α) administration on the mRNA expression of (a) caspase 3 (CASP3), (b) caspase 8 (CASP8) in the early- and middle- stage corpora lutea (CL), respectively. The gray bars represent the control group, and the black bars represent local or systemic aPGF_2α administered groups. Letters ^a,b indicate statistical differences between all experimental groups in the early and middle- stage CL. Asteriks ^* indicate statistical differences between local/systemic aPGF_2α injected early CL vs local/systemic aPGF_2α injected middle-stage CL

Fig. 2

Effect of local or systemic PGF_2α analogue (aPGF_2α) administration on the protein concentration of (a) caspase 3 (CASP3), (b) caspase 8 (CASP8) in the early- and middle- stage corpora lutea (CL), respectively. The gray bars represent the control group, and the black bars represent local or systemic aPGF_2α administered groups. Letters ^a,b indicate statistical differences between all experimental groups in the early and middle- stage CL. Asteriks ^* indicate statistical differences between local/systemic aPGF_2α injected early CL vs local/systemic aPGF_2α injected middle-stage CL

Fig. 3

Effect of local or systemic PGF_2α analogue (aPGF_2α) administration on the (a) the ratio of BCL2 to BAX mRNA expression and (b) the ratio of BCL2 to BAX protein concentration levels in the early- and middle- stage corpora lutea (CL), respectively. The gray bars represent the control group, and the black bars represent local or systemic aPGF_2α administered groups. Letters ^a,b indicate statistical differences between all experimental groups in the early and middle- stage CL. Asteriks ^* indicate statistical differences between local/systemic aPGF_2α injected early CL vs local/systemic aPGF_2α injected middle-stage CL

Fig. 4

Effect of local or systemic PGF_2α analogue (aPGF_2α) administration on the mRNA expression of: (a) RIPK1, (b) RIPK3 in the early and middle- stage corpora lutea (CL), respectively. The gray bars represent the control group, and the black bars represent local or systemic aPGF_2α administered groups. Letters ^a,b,c indicate statistical differences between all experimental groups in the early and middle- stage CL. Asteriks ^* indicate statistical differences between local/systemic aPGF_2α injected early CL vs local/systemic aPGF_2α injected middle-stage CL

Fig. 5

Effect of local or systemic PGF_2α analogue (aPGF_2α) administration on the protein concentration of: (a) RIPK1, (b) RIPK3 in the early- and middle- stage corpora lutea (CL), respectively. The gray bars represent the control group, and the black bars represent local or systemic aPGF_2α administered groups. Letters ^a,b indicate statistical differences between all experimental groups in the early and middle- stage CL. Asteriks ^* indicate statistical differences between local/systemic aPGF_2α injected early CL vs local/systemic aPGF_2α injected middle-stage CL

Fig. 6

Effect of local or systemic PGF_2α analogue (aPGF_2α) administration on the mRNA expression of: (a) CYLD, (b) MLKL in the early - and middle-stage corpora lutea (CL), respectively. The gray bars represent the control group, and the black bars represent local or systemic aPGF_2α administered groups. Letters ^a,b indicate statistical differences between all experimental groups in the early and middle- stage CL. Asteriks ^* indicate statistical differences between local/systemic aPGF_2α injected early CL vs local/systemic aPGF_2α injected middle-stage CL

Fig. 7

Effect of local or systemic PGF_2α analogue (aPGF_2α) administration on the protein concentration of: (a) CYLD, (b) MLKL in the early- and middle-stage corpora lutea (CL), respectively. The gray bars represent the control group, and the black bars represent local or systemic aPGF_2α administered groups. Letters ^a,b,c indicate statistical differences between all experimental groups in the early and middle- stage CL. Asteriks ^* indicate statistical differences between local/systemic aPGF_2α injected early CL vs local/systemic aPGF_2α injected middle-stage CL

Fig. 8

Representative section of images of localization of: (a, b, c) RIPK1, (d, e, f) RIPK3, (g, h, i) CYLD and (j, k, l) MLKL protein in the bovine early-stage corpora lutea (CL) at 4 h after local or systemic PGF_2α analogue (aPGF_2α) administration. Each small window shows a negative control stained with normal rabbit IgG instead of primary antibody. Positive immunohistochemistry staining was assessed as brown staining. Bar = 20 μm

Fig. 9

Representative section of images of localization of: (a, b, c) RIPK1, (d, e, f) RIPK3, (g, h, i) CYLD and (j, k, l) MLKL protein in the bovine middle-stage corpora lutea (CL) at 4 h after local or systemic PGF_2α analogue (aPGF_2α) administration. Each small window shows a negative control stained with normal rabbit IgG instead of primary antibody. Positive immunohistochemistry staining was assessed as brown staining. Bar = 20 μm

Fig. 10

The arithmetic means of intensities of: (a) RIPK1, (b) RIPK3, (c) CYLD and (d) MLKL in the bovine early- and middle-stage corpora lutea (CL) after local or systemic PGF_2α analogue (aPGF_2α) administration. The gray bars represent the control group, and the black bars represent local or systemic aPGF_2α administered groups. Letters ^a,b,c indicate statistical differences between all experimental groups in the early and middle- stage CL. Asteriks ^* indicate statistical differences between local/systemic aPGF_2α injected early-stage CL vs local/systemic aPGF_2α injected middle-stage CL

Fig. 11

Schematic diagram of the study design. Cows were synchronized via two injections of aPGF_2α administered with an 11-days interval, starting on protocol day − 14. The onset of estrus was considered as day 0 of the estrous cycle. On day 4 or 10 respectively, the cows received an local injection of saline/ aPGF_2α (2.5 mg Dinoprost/0.5 ml) or systemic injection of saline/ aPGF_2α (25 mg Dinoprost/5 ml). At 4 h after treatment the cows were ovariectomized. CLs were collected for gene and protein expression