Influence of equine growth hormone, insulin-like growth factor-I and its interaction with gonadotropins on in vitro maturation and cytoskeleton morphology in equine oocytes

Abstract

In horses, successful in vitro fertilization procedures are limited by our inability to consistently mature equine oocytes by in vitro methods. Growth hormone (GH) is an important regulator of female reproduction in mammals, playing an important role in ovarian function, follicular growth and steroidogenesis. The objectives of this research were to investigate: the effects of equine growth hormone (eGH) and insulin-like growth factor-I (IGF-I) on the in vitro maturation (IVM) of equine oocytes, and the effects of eGH in addition to estradiol (E2), gonadotropins (FSH and LH) and fetal calf serum (FCS) on IVM. We also evaluated the cytoskeleton organization of equine oocytes after IVM with eGH. Equine oocytes were aspirated from follicles <30 mm in diameter and matured for 30 h at 38.5°C in air with 5% CO2. In experiment 1, selected cumulus–oocyte complexes (COCs) were randomly allocated as follows: (a) control (no additives); (b) 400 ng/ml eGH; (c) 200 ng/ml IGF-I; (d) eGH + IGF-I; and (e) eGH + IGF-I + 200 ng/ml anti-IGF-I. In addition to these treatment groups, we also added 1 μg/ml E2, 5 IU/ml FSH, 10 IU/ml LH and 10% FCS in vitro (experiment 2). Oocytes were stained with markers for microtubules (anti-α-tubulin antibody), microfilaments (AlexaFluor 488 Phalloidin) and chromatin (TO-PRO3-iodide) and assessed via confocal microscopy. No difference was observed when eGH and IGF-I was added into our IVM system. However, following incubation with eGH alone (40%) and eGH, E2, gonadotropins and FCS (36.6%) oocytes were classified as mature v. 17.6% of oocytes in the control group (P < 0.05). Matured equine oocytes showed that a thin network of filaments concentrated within the oocyte cortex and microtubules at the metaphase spindle showed a symmetrical barrel-shaped structure, with chromosomes aligned along its midline. We conclude that the use of E2, gonadotropins and FCS in the presence of eGH increases the number of oocytes reaching oocyte competence.