Mammalian oocyte maturation depends on the interaction of many factors, and enables oocyte development to reach the metaphase II stage. Among others, steroid and growth factors work together in the regulation of signals associated in the oocyte maturation event. In addition, epidermal growth factor (EGF) has been shown to have positive effects during oocyte in vitro maturation in several species. This study aimed to establish the capacity of equine oocytes to undergo nuclear and cytoplasmic maturation in the presence of EGF and to describe the influence of EGF on steroid secretions (progesterone, oestradiol, testosterone and androstenedione) following in vitro culture.
Ovaries were obtained from horses that had been humanely killed. Cumulus-oocyte complexes (n = 204) were obtained by aspiration and subsequent scraping of equine ovarian follicles, and were matured in TCM 199. They were then cultured in two different treatment groups: control and EGF (50 ng ml-1). Each group was pretreated with either 0, or 10-6 mM tyrphostin A-47, a specific tyrosine kinase inhibitor. At the end of maturation, medium samples were collected for steroid determinations by using EIA. This procedure was as previously validated by Lorenzo et al. (1997). Nuclear maturation was determined as the percentage of oocytes reaching the metaphase II stage at the end of the culture period. Cytoplasmic maturation (measured as cortical granule (CG) migration and distribution) was determined by fluorescein isothiocyanate-labelled Lens culinaris agglutinin with laser confocal microscopy. All statistical comparisons and analysis were done by using ANOVA, using Catmod procedure of SAS.
Results show that treatment with EGF significantly increased (P < 0.05) the incidence of metaphase II stage when compared with the control group (71.6 versus 32.1 % in controls). When oocytes were cultured with EGF, all the matured oocytes showed complete CG migration to the cortex, to form a continuous monolayer under the oolemma but no CG-free domain was observed in oocytes during maturation. The specific tyrosine kinase inhibitor A-47 was effective in suppressing nuclear and cytoplasmic EGF-induced effects on oocytes cultured with EGF. In addition, compared to controls, EGF supplementation in the maturation media was associated with a significant increase of progesterone and androstenedione (P < 0.01), and positive correlations between oestradiol/androstenedione ratios and nuclear maturation were found. These observations together with the increase of nuclear maturation rates, and complete CG migration to the cortex when using EGF in culture media, suggest an important role for EGF in the regulation of equine oocyte maturation.
This work was supported by Programa Del Amo, Universidad Complutense-UCDavis and Club Hípico La Silla, Monterrey, México.