106 EFFECTS OF BONE MORPHOGENETIC PROTEIN 4 (BMP4) AND ITS INHIBITOR NOGGIN ON BOVINE IN VITRO EMBRYO DEVELOPMENT

Abstract

Bone morphogenetic protein 4 (BMP4) is a member of the BMP family of conserved morphogenes in charge of many events of differentiation (Chen et al. 2004 Growth Factors 22, 233–241) BMP4 is involved in regulation of pluripotency in humans and mice though the role in bovine early embryo development is still undefined. Noggin is a BMP4 inhibitor (Groppe et al. 2002 Nature 420, 636–642) that does not have a specific receptor but functions by directly binding BMP ligands. The objective of this work was to study the effects of BMP4 and Noggin on early bovine embryo development. Cumulus–oocyte complexes (COC) were aspirated from abattoir ovaries and in vitro matured in TCM containing 10% fetal bovine serum (FBS), 2 mM FSH, 20 mM cysteamine, 1% antibiotic- antimycotic (15240, GIBCO, Grand Island, NY, USA) and 0.1 mM sodium pyruvate. Incubation conditions were a 6.5% CO₂ humidified atmosphere at 39°C. After 22 h, in vitro fertilization was performed. Briefly, frozen–thawed semen was centrifuged twice at 490 × g and resuspended in B.O. solution to a final concentration of 20 × 10⁶ mL^–1 and incubation with COC was performed for 5 h. Presumptive zygotes were randomly cultured in CR2 with 0.3% BSA, free of serum and co-culture (control, n = 217) or supplemented with 100 ng mL^–1 of either BMP4 (n = 218) or Noggin (n = 205). Cleavage and blastocyst rates were evaluated at Days 2 and 9 of culture. Blastocysts cell numbers were analysed by nuclear staining with Hoechst 33342. The expression pattern of the transcription factor Oct-4 was studied by immunocytochemistry and confocal microscope analysis in blastocysts. Chi-square tests were applied for cleavage, blastocyst, and hatching rates. One-way ANOVA was used to compare blastocyst cell number and a proportion test was used for Oct-4 expression. For all, P < 0.05 was considered significant. Cleavage rate was significantly lower in the Noggin group compared to control (51.2% v. 62.3%) whereas the BMP group (61.3%) did not differ from control or Noggin groups. Blastocyst rates for the BMP and Noggin groups were statistically lower than control (9.24% and 11.7% v. 20.6%, respectively). Hatching rate for the control group was significantly higher than both BMP and Noggin groups (4.6% v. 1.4% and 0.49%, respectively). Blastocyst cell number did not differ between groups (130, 117, and 128 for control, BMP4, and Noggin groups, respectively). Oct-4 expressing cells over total cell number was lower in BMP (72%; n = 3) and Noggin (72%; n = 3) groups compared to control (83%; n = 3). In our conditions, BMP inhibition with Noggin or addition of exogenous BMP4 negatively affected developmental rates and altered the proportion of pluripotent (Oct-4 positive) cells. Our results demonstrate the importance of a correct balance within the BMP signalling system for proper bovine in vitro embryo development.