Equine oocyte maturation with epidermal growth factor.
Epidermal growth factor (EGF) has been shown to have a positive effect during oocyte in vitro maturation in several species. This study was performed to establish the capacity of equine oocytes to undergo nuclear maturation in the presence of EGF and to localise its receptor in the equine ovary by immunohistochemical methods. Oocytes were obtained by aspiration and subsequent scraping from equine follicles (15-25 mm diameter) and cultured in 3 different treatment groups for 36 h: control Group (modified TCM 199 with 0.003% BSA), EGF Group (TCM-199 supplemented with 50 ng/ml EGF) and EMS Group (TCM 199 supplemented with 10% v/v oestrous mare serum). Each group was divided further into 3 treatments with tyrphostin A-47, a specific tyrosine kinase inhibitor, at 0, 10(-4) and 10(-6) mmol/l. Maturation was determined as the percentage of oocytes reaching metaphase II stage at the end of the culture period. Immunohistochemical detection of EGF-receptor (EGFR) was performed using a streptoavidin-biotin method. The recovery rate and oocyte retrieval were 84.6% (recovered oocytes/follicles aspirated) and 6.55 (oocytes/mare), respectively. Treatment with EGF significantly (P<0.05) increased the incidence of metaphase II stage compared with the control group (69.4 vs. 26.9% in controls, respectively). The specific-tyrosine kinase inhibitor A-47 was effective in suppressing EGF-effect on EGF-cultured oocytes; no significant differences were observed in EMS-supplemented oocytes when cultured with A-47. EGF-receptor was localised in follicles, with localisation being more prominent in the cumulus than in mural granulosa cells. This finding, together with the increase of oocyte nuclear maturation rate when using EGF in culture media and the inhibition of maturation by tyrphostin A-47, suggests a physiological role for EGF in the regulation of equine oocyte maturation. The results should help successful development of assisted reproductive technology in the horse.
Departamento de Fisiología Animal, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain.
P L
PL
Lorenzo
I K M
IK
Liu
G F
GF
Carneiro
A J
AJ
Conley
A C
AC
Enders
D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000302:isolation & purification* / Q000378:metabolism; D005260:Female; D006736:Horses; D007150:Immunohistochemistry / Q000662:veterinary; D066298:In Vitro Techniques; D009865:Oocytes / Q000187:drug effects / Q000254:growth & development*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D020032:Tyrphostins / Q000494:pharmacology
D004791:Enzyme Inhibitors; D020032:Tyrphostins; C060152:tyrphostin 47; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases