Role of Wnt Signaling During In-Vitro Bovine Blastocyst Development and Maturation in Synergism with PPARδ Signaling

Abstract

Wnt/β-catenin signaling plays vital role in the regulation of cellular proliferation, migration, stem cells cell renewal and genetic stability. This pathway is crucial during the early developmental process; however, the distinct role of Wnt/β-catenin signaling during pre-implantation period of bovine embryonic development is obscure. Here, we evaluated the critical role of Wnt/β-catenin pathway in the regulation of bovine blastocyst (BL) development and hatching. 6 bromoindurbin-3'oxime (6-Bio) was used to stimulate the Wnt signaling. Treatment with 6-Bio induced the expression of peroxisome proliferator-activated receptor-delta (PPARδ). Interestingly, the PPARδ co-localized with β-catenin and form a complex with TCF/LEF transcription factor. This complex potentiated the expression of several Wnt directed genes, which regulate early embryonic development. Inhibition of PPARδ with selective inhibitor 4-chloro-N-(2-{[5-trifluoromethyl]-2-pyridyl]sulfonyl}ethyl)benzamide (Gsk3787) severely perturbed the BL formation and hatching. The addition of Wnt agonist successfully rescued the BL formation and hatching ability. Importantly, the activation of PPARδ expression by Wnt stimulation enhanced cell proliferation and fatty acid oxidation (FAO) metabolism to improve BL development and hatching. In conclusion, our study provides the evidence that Wnt induced PPARδ expression co-localizes with β-catenin and is a likely candidate of canonical Wnt pathway for the regulation of bovine embryonic development.

Bovine Oviduct Epithelial Cell-Derived Culture Media and Exosomes Improve Mitochondrial Health by Restoring Metabolic Flux during Pre-Implantation Development

“…Immature cumulus oocyte complexes (COCs) were retrieved from follicles (2–8 mm diameter) from the ovaries of Korean native Hanwoo cows, obtained from the local slaughterhouse, and placed in physiological saline (0.9% NaCl) at 37.5 °C. The TL-HEPES medium was used for the collection of oocytes, and afterwards submitted to in vitro maturation (IVM) and in vitro fertilization (IVF) as described previously by [ 37 ]. In a brief, the collected oocytes were cultured for 22–24 h in NUNC 4-well plates (Nunc, Roskilde, Denmark) containing IVM medium, composed of tissue culture media-199 (TCM-199) supplemented with 10% (v/v) fetal bovine serum (FBS), 1 µg/mL estradiol-17β, 10 µg/mL follicle-stimulating hormone, 0.6 mM cysteine, and 0.2 mM sodium pyruvate.…”

“…The effect of in vitro culture condition on cellular proliferation and DNA fragmentation was determined with anti-BrdU labelling and terminal deoxynucleotidyl transferase (TdT) 20-deoxyuridine, 50-triphosphate (dUTP) nick-end labeling (TUNEL) assay by using an In Situ Cell Death Detection Kit (Roche Diagnostics Corp., Indianapolis, IN, USA), as explained previously by [ 37 , 38 ]. Briefly, for cell proliferation assay, day-8 BLs were washed and incubated for 6 h with 100 mM BrdU at 37 °C.…”

Wnt/β-catenin Pathway-Mediated PPARδ Expression during Embryonic Development Differentiation and Disease

“…Several in vivo and in vitro studies showed that the presence of Oct4 and c-Myc expression is crucial during the preimplantation period of embryonic development, maintenance of cell proliferation, and ICM identity [5,23]. Recently, a study on mouse and bovine embryonic development reported that increased Wnt/β-catenin signaling is also manifested by the up-regulated expression of the PPARδ gene in both the ICM and TE cell population [5,6]. The PPARδ expression maintains the higher cell index ratio by sustaining the proliferative signaling in response to β-catenin/Tcf4 signaling [11].…”

“…The PPARδ expression maintains the higher cell index ratio by sustaining the proliferative signaling in response to β-catenin/Tcf4 signaling [11]. In vitro studies of mouse and bovine embryos showed that Wnt3 stimulation simultaneously increases the PPARδ and c-Myc expression in ICM and trophoblast stem cells (TSCs) and supports the progressive proliferation event during early development [5,6]. These pieces of evidence highlight the functional importance of PPARδ expression during embryonic development and the maintenance of ESCs' pluripotent state in coordination with the Wnt/β-catenin pathway.…”

“…Wnt participates in all of these intricate processes either directly or by a crosstalk with other signaling pathways. The peroxisome proliferative activated receptor delta (PPARδ) of the nuclear superfamily receptor, which primarily regulates the lipid metabolism, also plays a crucial role during embryogenesis in conjunction with Wnt/β-catenin signaling [5,6]. The dysregulation of these signaling pathways causes congenital malformations such as cancer, osteoporosis, and different metabolic disorders [4,7].…”