Granulocyte-macrophage colony stimulating factor (GM-CSF) enhances cumulus cell expansion in bovine oocytes

Oscar A Peralta,Marcelo H Ratto,Ana Fernandez,Alfredo Ramirez,Pablo Strobel,Ilona Concha,Marco Berland,Danai Bucher

doi:10.1186/1477-7827-11-55

Abstract

BackgroundThe objectives of the study were to characterize the expression of the α- and β-subunits of granulocyte-macrophage colony stimulating factor (GM-CSF) receptor in bovine cumulus cells and oocytes and to determine the effect of exogenous GM-CSF on cumulus cells expansion, oocyte maturation, IGF-2 transcript expression and subsequent competence for embryonic development.MethodsCumulus-oocyte complexes (COC) were obtained by aspirating follicles 3- to 8-mm in diameter with an 18 G needle connected to a vacuum pump at −50 mmHg. Samples of cumulus cells and oocytes were used to detect GM- CSF receptor by immunofluorescence. A dose–response experiment was performed to estimate the effect of GM-CSF on cumulus cell expansion and nuclear/cytoplasmic maturation. Also, the effect of GM-CSF on IGF-2 expression was evaluated in oocytes and cumulus cells after in vitro maturation by Q-PCR. Finally, a batch of COC was randomly assigned to in vitro maturation media consisting of: 1) synthetic oviductal fluid (SOF, n = 212); 2) synthetic oviductal fluid supplemented with 100 ng/ml of GM-CSF (SOF + GM-CSF, n = 224) or 3) tissue culture medium (TCM 199, n = 216) and then subsequently in vitro fertilized and cultured for 9 days.ResultsImmunoreactivity for both α and β GM-CSF receptors was localized in the cytoplasm of both cumulus cells and oocytes. Oocytes in vitro matured either with 10 or 100 ng/ml of GM-CSF presented a higher (P < 0.05) cumulus cells expansion than that of the control group (0 ng/ml of GM-CSF). GM-CSF did not affect the proportion of oocytes in metaphase II, cortical granules dispersion and IGF-2 expression. COC exposed to 100 ng/ml of GM-CSF during maturation did not display significant differences in terms of embryo cleavage rate (50.4% vs. 57.5%), blastocyst development at day 7 (31.9% vs. 28.7%) and at day 9 (17.4% vs. 17.9%) compared to untreated control (SOF alone, P = 0.2).ConclusionsGM-CSF enhanced cumulus cell expansion of in vitro matured bovine COC. However, GM-CSF did not increase oocyte nuclear or cytoplasmic maturation rates, IGF-2 expression or subsequent embryonic development.

Highlights

The objectives of the study were to characterize the expression of the α- and β-subunits of granulocyte-macrophage colony stimulating factor (GM-CSF) receptor in bovine cumulus cells and oocytes and to determine the effect of exogenous GM-CSF on cumulus cells expansion, oocyte maturation, IGF-2 transcript expression and subsequent competence for embryonic development
Data showed that the proportion of oocytes undergoing metaphase II after treatment with 1, 10 or 100 ng/ml of GM-CSF was not significantly different compared to the untreated controls (59.2, 54.2, 59.6 and 54.7%, respectively; Table 1)
We evaluated the effect of GM-CSF during in vitro maturation on the mRNA IGF-2 levels by Q-PCR

Summary

Introduction

The objectives of the study were to characterize the expression of the α- and β-subunits of granulocyte-macrophage colony stimulating factor (GM-CSF) receptor in bovine cumulus cells and oocytes and to determine the effect of exogenous GM-CSF on cumulus cells expansion, oocyte maturation, IGF-2 transcript expression and subsequent competence for embryonic development. The granulocyte-macrophage colony stimulating factor (GM-CSF) is a glycoprotein with several molecularweight species ranging from 18 to 30 kDa [1]. The interaction of GM-CSF with its receptor stimulates multiple signal transduction pathways, including Jak/STAT pathway, Ras/Raf/mitogen-activated protein kinase pathway, phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (PKB) pathway, and protein kinase C (PKC) pathway [5]. GM-CSF promote glucose uptake through PI 3-kinase/ PKB pathway via translocation of glucose transporter 1 (GLUT 1) [4]. GM-CSF induction of the PKB/Akt pathway results in direct cell survival activity and inactivation of proapoptotic factors BAD, caspase 9 and forkhead [6]. Akt promotes cell survival indirectly by regulating a number of processes involved in glucose metabolism [6]

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