Citation
- Authors: Wang X. et al.
- Year: 2022
- Journal: Theriogenology 1973 58-67
- Applications: in vitro / DNA, shRNA plasmid / jetOPTIMUS
- Cell types:
- Name: Human primary luminal cells
- Name: Trophoblast stem cells
Method
LECs and STCs were cocultured at 1:1 in a Matrigel-coated 6-well plate (#2–3032; CHI Scientific, China) until reaching 60%–80% confluency. The cocultures were transfected with pEGFP-C1 vectors expressing enJSRV-Env or short hairpin RNAs (shRNAs) against enJSRV-Env using the jetOPTIMUS® transfection reagent (Polyplus, France) following the manufacturer's instruction. The empty vectors and vectors expressing negative control shRNAs were used as negative controls. The transfection conditions were optimized by observing the green fluorescence under the microscope at 48 h after transfection. Cells were stained with Giemsa. The number of multinucleated cells was counted under an inverted microscope at magnification 10×.
Abstract
Background: Endogenous Jaagsiekte sheep retrovirus envelope protein (enJSRV-Env) plays an important role in trophoblast cell fusion in sheep. However, the underlying mechanism remains unclear.
Methods: Primary endometrial luminal epithelial cells (LECs) were isolated from the sheep uterus and cocultured with sheep trophoblast cells (STCs). Giemsa staining was conducted to count multinucleated cells in the coculture system. Gain- and loss-of-function assays were performed to explore the role of enJSRV-Env in trophoblast cell fusion in the coculture system. Co-immunoprecipitation and mass spectrometry were carried out to identify the interacting partner of enJSRV-Env in the cocultures. Western blot analysis were conducted to determine the activation of protein kinase A (PKA)/mitogen-activated extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase 1/2 (ERK1/2) signaling.
Results: Primary LECs were identified by the expression of epithelial marker cytokeratin 18. Overexpression of enJSRV-Env promoted the formation of multinucleated cells in the coculture system. enJSRV-Env activated and physically interacted with PKA, along with the activation of MEK/ERK1/2 signaling. PKA inhibition completely reversed enJSRV-Env-induced MEK/ERK1/2 activation, and ERK1/2 inhibition abolished enJSRV-Env-induced formation of multinucleated cells in the coculture system.
Conclusion: enJSRV-Env promotes trophoblast cell fusion in the sheep placenta by activating PKA/MEK/ERK1/2 signaling. This finding reveals a novel mechanism underlying the contribution of enJSRV-Env to trophoblast cell fusion during placental morphogenesis.