Dose-dependent effects of luteinizing hormone and follicle stimulating hormone on in vitro maturation, apoptosis, secretion function and expression of follicle stimulating hormone receptor and luteinizing hormone receptor of sheep oocytes

Abstract

TThe present study compared the effects of follicle stimulating hormone (FSH) and luteinizing hormone (LH) on in vitro maturation (IVM), apoptosis, and secretion function in sheep oocytes, as well as gene expressions of the receptors (FSHR, LHR, and GnRHR) in cumulus-oocyte complexes (COCs). The COCs were recovered from sheep ovaries and pooled in groups. The COCs were cultured for 24 hours in IVM medium supplemented with various concentrations of LH (5–30 μg/mL) and FSH (5–30 IU/mL). They were allocated to LH-1 (5 µg/mL), LH-2 (10 µg/mL), LH-3 (20 µg/mL), and LH-4 (30 µg/mL) groups, and FSH-1 (5 IU/mL), FSH-2 (10 IU/mL), FSH-3 (20 IU/mL), and FSH-4 (30IU/mL) groups. The apoptosis of COCs was assessed by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). The maturation rates of oocytes improved gradually as LH and FSH concentration increased from 0 to 10 μg/mL(IU/mL), reaching a peak value of 44.1% of LH-2 and 48.5% of FSH-2 group. Oocyte apoptosis rates of LH-2 and FSH-2 groups were the lowest among LH- and FSH-treated groups, respectively. The germinal vesicle breakdown (GVBD) rate of the FSH-2 group was higher than that of the control group (CG) and FSH-4 groups. The GVBD rate of LH-2 group also increased in comparison with the CG group. FSH concentration of the FSH-2 group was greater than that of CG. Expression levels of FSHR, LHR, and GnRHR mRNAs of FSH-2, LH-3, and LH-3 group, respectively, were higher than CG. Levels of FSHR proteins in FSH-2 and FSH-3 groups were greater than CG. Levels of GnRHR proteins were increased with a maximum increment of FSH-4. The FSH and LH supplemented into the IVM medium could promote the maturation rate, reduce the apoptosis rate of sheep oocytes, and increase FSH concentrations in IVM medium fluid. Additionally, FSH and LH enhanced expression levels of FSHR, LHR, and GnRHR mRNAs of sheep COCs.

Keywords: Apoptosis, cumulus-oocyte complexes, germinal vesicle breakdown, protein expression, receptor