Equine chorionic gonadotropin influence on sheep oocyte in vitro maturation, apoptosis, and follicle-stimulating hormone receptor and luteinizing hormone receptor expression

Abstract

We assessed the effects of equine chorionic gonadotropin (eCG) on oocyte in vitro maturation (IVM), apoptosis, and follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), and gonadotropin-releasing hormone receptor (GnRHR) expression and mRNA levels. Cumulus-oocyte complexes (COCs) were recovered from sheep ovaries and pooled in groups, before being cultured in IVM media containing varying eCG concentrations. Maturation and apoptosis rates were then calculated. Expression of FSHR, LHR, and GnRHR mRNA in oocytes was measured using quantitative reverse transcription polymerase chain reaction. Protein levels were ascertained by western blotting. Matured oocytes displayed and released an intact first polar body. Sheep oocyte maturation rates gradually increased as eCG concentration was raised from 0 to 20 µg/mL. Apoptosis rates of eCG-treated oocytes were lower than those of the control group, and were lowest using 20 µg/mL eCG. FSHR, LHR, and GnRHR mRNA expression increased (P < 0.01, P < 0.05, and P < 0.05, respectively, compared to 0 µg/mL eCG) with eCG concentration, being highest following exposure to 20 µg/mL. FSHR and GnRHR protein levels were significantly higher in oocytes administered 20 µg/mL eCG compared with those matured in the absence of eCG. eCG dose positively correlated with FSHR, LHR, and GnRHR mRNA and protein expression. In conclusion, eCG enhances maturation and decreases apoptosis of oocytes undergoing IVM, and heightens FSHR, LHR, and GnRHR expression. Such increased expression may facilitate oocyte IVM. These findings contribute to our understanding of the mechanisms of underlying hormonal control of sheep oocyte IVM, advancing ovine reproductive methods.

FSHR and LHR Expression and Signaling as Well as Maturation and Apoptosis of Cumulus-Oocyte Complexes Following Treatment with FSH Receptor Binding Inhibitor in Sheep

“…IVM of sheep COCs was performed referring to the early methods [15, 17]. A microwell culture system was used in this experiment.…”

“…Basal maturation media (bMM) were prepared as our previous methods [15]. The bMM was consisted of 9.5 g Medium 199 powders (Sigma, St. Louis, MO, USA), 2.2 g NaHCO 3 , 25 mg sodium pyruvate, 4.8 g 4-(2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES), 50 IU ampicillin sodium, 50 µg streptomycin sulfate, and ultrapure water up to a final volume of 1000 mL.…”

“…Ovaries were collected between May to July of 2016 from 509 immature and noncyclic ewes (6-7 months old) immediately after their slaughter at the local abattoirs [15], and then transported to the laboratory in 30-35°C Dulbecco’s phosphate-buffered saline (DPBS) (Sigma Co. Ltd, Beijing, China) within 3 h after collection. Use of these animals was approved by the Institutional Animal Ethics Committee of Northwest University for Nationalities, and all experiments were conducted according to the conventions of the Committee for the Purpose of Control and Supervision of Experiments on Animals in China.…”

“…The harvested COCs were observed under an inverted fluorescence microscope (Leica, Japan), and those displaying an intact first polar body were identified as matured COCs. Experiments were executed in triplicate [18]. …”

“…Ovine GAPDH gene (GenBank accession number: HM-043737.1) was selected as the reference gene for normalizing expression levels of target genes [15, 18]. The sequences of the primers used in the qPCR were as follows: FSHR, forward 5′-TCTTTGCTTTTGCAGTTGCC-3′ and reverse, 5′-GCACAAGGAGGGACATAACATAG-3′; LHR, forward 5′- CCTGAAGAAGATGCACGATGACGCC-3′ and reverse, 5′- ACCCATTCCCTGTCTGCCAGTCT-3′; GAPDH, forward, 5′- CTTCAACAGCGACACTCACTCT-3′ and reverse, 5′- CCACCACCCTGTTGCTGTA-3′.…”