9-cis retinoic acid improves developmental competence and embryo quality during in vitro maturation of bovine oocytes through the inhibition of oocyte tumor necrosis factor-α gene expression1

Abstract

Retinoic acid (RA; all-trans RA and 9-cis RA) enhances embryo developmental competence and quality through multiple mechanisms affecting the oocyte and preimplantation embryo. Folliculogenesis and oocyte maturation are influenced by tumor necrosis factor-α (TNF-α) via inhibition of aromatase activity and estradiol secretion in granulosa cells. Retinoic acid inhibits TNF-α production in various cell lines. The aim of the present study was to determine whether oocyte TNF-α concentrations regulate developmental competence and embryo quality and if the beneficial effects of 9-cis RA are mediated through attenuation of oocyte TNF-α production. Bovine cumulus oocyte complexes collected from abattoir ovaries were matured in maturation medium in the absence (control) or presence of 5 nM 9-cis RA (RA), 100 ng/mL of recombinant bovine TNF-α (TNF), or 5 nM 9-cis RA + 100 ng/mL of recombinant bovine TNF-α (RA+TNF). Oocytes were subsequently collected for gene expression analysis or subjected to in vitro fertilization and culture. Apoptosis and gene expression were analyzed in d-8 blastocysts. Results indicated that 9-cis RA downregulated (P < 0.01) both basal and TNF-α-induced TNF-α mRNA in oocytes (1.0-fold in control, 0.4-fold in RA, 2.1-fold in TNF, and 0.7-fold in RA+TNF). The 9-cis RA increased (P < 0.001) blastocyst development rates (37.1 ± 6.9 vs. 23.6 ± 8.0%) and total cell number (138.4 ± 19.2 vs. 120.2 ± 24.5) and reduced (P < 0.001) the percentage of apoptotic cells (3.3 ± 2.0 vs. 5.6 ± 2.3%) compared with controls. Expression of caspase 3 (0.4- vs. 1.0-fold) and TNF-α (0.4- vs. 1.0-fold) mRNA was downregulated (P < 0.05) in RA-treated blastocysts compared with controls. Moreover, 9-cis RA rescued (P < 0.001) development rates (24.5 ± 11.1 vs. 15.6 ± 9.0%), increased total cell number (124.6 ± 36.5 vs. 106.9 ± 31.1), and reduced apoptosis (5.8 ± 2.0 vs. 8.1 ± 3.1%) in blastocysts exposed to TNF-α (TNF group). Caspase 3 (0.8-fold in RA+TNF vs. 2.2-fold in TNF) and TNF-α (0.3-fold in RA+TNF vs. 2.8-fold in TNF) mRNA expression was attenuated (P < 0.05) in TNF-α-treated blastocysts. In conclusion, the present study suggests that 9-cis RA exerts its beneficial roles on oocyte developmental competence and embryo quality by attenuating oocyte TNF-α mRNA expression.

Coagulansin-A has beneficial effects on the development of bovine embryos in vitro via HSP70 induction

“…Ovaries were washed in fresh Dulbecco's PBS (D-PBS) and cumulus-oocyte complexes (COCs) were recovered as described by Deb et al [13]. In brief, COCs were recovered from follicles with a diameter of 2-8 mm using an 18-gauge needle attached to a vacuum pump.…”

“…In vitro matured COCs were fertilized with frozen-thawed bovine sperm as described by Deb et al [13]. Semen was thawed at 39…”

“…Briefly, fixed embryos (n = 68) were washed twice with 0.3 % (w/v) polyvinylpyrrolidone (PVP) prepared in 1 M PBS (PVP-PBS) before being permeabilized [0.5 % (v/v) Triton X-100 and 0.1 % (w/v) sodium citrate] for 30 min at room temperature [13]. After permeabilization, embryos were washed twice with PVP-PBS and incubated in the dark with fluorescently conjugated terminal deoxynucleotide transferase dUTP for 1 h at 37…”

“…Oocytes were cultured in maturation medium as described by Deb et al [13]. In brief, COCs were washed three times in maturation medium (TCM-199) supplemented with 10 % (v/v) FBS, 1 mg/ml oestradiol-17β, 10 mg/ml follicle-stimulating hormone, 0.6 mM cysteine and 0.2 mM sodium pyruvate, and then transferred to a 4-well plate containing 700 ml of IVM media for 22-24 h at 38.5…”

Efficient Production of Bovine Tetraploid Embryos by Electrofusion of In Vitro Produced Two-Cell Stage Embryos

“…Bovine embryos were produced in vitro as previously described [25]. In brief, ovaries were obtained from Korean native Hanwoo cows at a local abattoir, and transported within 2 h to the laboratory in physiological saline (0.9% [w/v] NaCl) held at 35-37°C.…”