12-Oxoeicosatetraenoic acid, a candidate signal for placenta separation, activates matrix metalloproteinase and induces apoptosis in bovine trophoblast cells

Abstract

Objective: 12-oxo-5Z,8Z,10E,14Z-eicosatetraenoic acid (12-KETE), a metabolite of arachidonic acid, is a strong candidate signal for placenta separation following calf discharge at delivery. In the present study, the effects of 12-KETE on bovine trophoblast cells were investigated to determine its function in the placentome at delivery.

Methods: Bovine trophoblast cells derived from blastocysts were used. They were cocultured with or without fibroblasts derived from bovine placentome and/or bovine uterine epithelial cells. 12-KETE was added to the culture medium.

Results: Bovine trophoblast cells contained binucleate cells and strongly expressed caudal type homeobox 2 (CDX-2) genes. Addition of 12-KETE to the trophoblast cell colony without feeder cells or that on a fibroblast monolayer induced rapid exfoliation of the colony. After 12-KETE addition, trophoblast cells emitted strong fluorescence caused by the degradation of dye-quenched collagen, indicating that 12-KETE activated matrix metalloproteinase of the trophoblast cells. Exfoliated cell colonies were stained with YOPRO-1, but not propidium iodide (PI). Moreover, DNA fragmentation and Bcl-2 associated X protein (Bax) gene (apoptosis stimulator) upregulation were observed in exfoliated cells, indicating that 12-KETE induced trophoblast cell apoptosis. These results were consistent with previous in vivo observations; however, even a lower concentration of 12-KETE activated trophoblast protease. Meanwhile, fibroblasts derived from the bovine placentome converted arachidonic acid to 12-KETE.

Conclusion: These observations indicate that 12-KETE may serve as a signal for placenta separation at delivery.

Keywords: 12-Oxoeicosatetraenoic Acid; Apoptosis; Bovine; Matrix Metalloproteinase; Trophoblast.

Figure 1

Preparation of trophoblast cells derived from bovine blastocyst. (A) Regressed blastocyst (day 5 of culture), (B) TRO in a culture of FIB derived from the placentome (day 30 of culture), (C) TRO in a culture of uterine epithelial cells (day 5 of culture), (D) TRO in a mixed culture of uterine EPI and FIB derived from the placentome (day 5 of culture), (E) Formation of vesicles (day 25 of culture), (F) Formation of baby vesicles (white arrow) on the surface of trophoblast vesicle in EPI culture (day 13 after transplantation). (G) Formation of the trophoblast colony in EPI culture (day 33 of culture), (H) Trophoblasts colony without feeder cells (day 22 after transplantation), (white bars in A–H: 200 μm). (I) Floating vesicles (white bar: 100 μm). (J) Hoechst staining + phase-contrast image. White arrow shows a binucleate cell, (white bar: 50 μm). (K) qPCR results of CDX2 gene expression (TRO: n = 5, EPI: n = 3, FIB: n = 3). TRO, trophoblast colony; FIB, fibroblasts; EPI, epithelial cells; CDX2, caudal type homeobox 2. ^a,b Different lowercase letters indicate statistically significant differences among cell types (p<0.001). (L1, L2) Immunostaining (white bar: 100 μm). L1, cytokeratin; L2, vimentin.

Figure 2

Induction of exfoliation of the trophoblast colony without (A–C) or with (D–F) feeder cells (fibroblasts derived from the placentome) by 12-KETE addition. (A, D) Control, (B) +40 μM of 12-KETE (2.5 h after addition), (E) +40 μM of 12-KETE (5 h after addition), (C) +4 μM of 12-KETE (8 h after addition), (F) +10 μM of 12-KETE (38 h after addition), (white bars: 200 μm). Replications (n≥10) of experiments showed the same results. 12-KETE, 12-oxo-5Z,8Z,10E,14Z-eicosatetraenoic acid.

Figure 3

Induction of MMP activation of the trophoblast colony without (n = 3) (A, B) or with (n = 3) (C, D) feeder cells (fibroblasts derived from the placentome) by 12-KETE. Trophoblast colonies were preincubated with dye-quenched collagen for 2 h before 12-KETE addition. (A) No addition of 12-KETE, (B) At 2.5 h after 40 μM of 12-KETE addition, (white bars in A, B: 100 μm), (C) No addition of 12-KETE, (D) At 24 h after 40 μM of 12-KETE addition, (white bars in C, D: 50 μm). Replications (n = 5) of experiments showed the same results. MMP, matrix metalloproteinases; 12-KETE, 12-oxo-5Z,8Z,10E,14Z-eicosatetraenoic acid.

Figure 4

Induction of apoptosis of trophoblast colony in the presence or absence of feeder cells (fibroblasts derived from the placentome) by 12-KETE. Double staining of trophoblast colony without (A, B) (n = 3) or with (C, D) (n = 3) feeder cells by YOPRO-1 (A, C) or PI (B, D), 4 h (A, B) or 5 h (C, D) after 12-KETE treatment (40 μM), (white bars in A, B: 50 μm, in C, D: 25 μm). Detection of DNA fragmentation in trophoblast colony in the absence (E, F) (n = 1) or presence (G, H) (n = 3) of feeder cells. (E) At 19 h after 40 μM of 12-KETE, (F) No addition of 12-KETE, (white bars: 50 μm), (G) At 24 h after 40 μM of 12-KETE, (H) No addition of 12-KETE, (white bars: 50 μm). (J1, J2) qPCR results of Bax and Bcl-2 gene expression. * p<0.05. Replications (n = 3) of experiments showed the same results. 12-KETE, 12-oxo-5Z,8Z,10E,14Z-eicosatetraenoic acid; qPCR, quantitative polymerase chain reaction; Bax, Bcl-2 associated X protein; Bcl-2, B-cell/CLL lymphoma 2.

Figure 5

Protease inhibitor-induced suppression of cell exfoliation at a low concentration of 12-KETE. (A) DNA fragmentation (24 h after addition of 10 μM 12-KETE) in the presence of feeder cells (n = 3), (B) YOPRO-1 staining (3 h after addition of 10 μM 12-KETE) in the presence of feeder cells (n = 3), (C) MMP activation (18 h after addition of 10 μM 12-KETE) in the presence of feeder cells (n = 3), (white bars in A, B, C: 25 μm). Results in the absence of feeder cells were the same as those in their presence (data not shown). (D) At 23.5 h after addition of 10 μM 12-KETE in the presence of feeder cells, (E) At 23.5 h after addition of 10 μM 12-KETE plus 0.05 mg/mL Pefabloc in the presence of feeder cells. Replications (n = 3) of experiments showed the same results. (F) At 8 h after addition of 10 μM 12-KETE in the absence of feeder cells, (G) At 8 h after addition of 10 μM 12-KETE plus 0.2 mg/mL Pefabloc in the absence of feeder cells, (white bars in D-G: 200 μm). Replications (n = 4) of experiments showed the same results. 12-KETE, 12-oxo-5Z,8Z,10E,14Z-eicosatetraenoic acid; MMP, matrix metalloproteinases.

Figure 6

HPLC analysis of free Ara in the cultured uterine epithelial cells with or without hydrocortisone (0.1, 0.3 μM) treatments (n = 3). ^a,b Different lowercase letters indicate statistically significant differences among treatments (p<0.05). HPLC, high performance liquid chromatography.

Figure 7

HPLC chromatograph of the fibroblast culture medium. (A) Cultured medium without Ara, (B) Cultured medium containing 75 μM of Ara. (C) Cultured medium spiked with a 12-KETE standard (1.53 ng). Black arrows show 12-KETE peaks. Replications (n = 3) of experiments showed the same results. HPLC, high performance liquid chromatography; Ara, eicosatetraenoic acid; 12-KETE, 12-oxo-5Z,8Z,10E,14Z-eicosatetraenoic acid.