183 THE EFFECT OF BOVINE EMBRYO CULTURE WITHOUT ANY PROTEIN SOURCES UNTIL DAY 4 ON TRANSCRIPTION OF HYALURONAN SYNTHASES AND RECEPTORS AND mtDNA CONTENT

Abstract

We previously showed (Palasz et al. 2005 Reprod. Fertil. Dev. 17, 219 abst) that addition of hyaluronan (HA) at Day 4 of culture improved bovine embryo quality and development. At the same time it was reported that cells cultured on solid surfaces without surface-active compound produce larger amounts of HA than those grown in suspension (Kraemer and Barnhart 1978 Exp. Cell Res. 114, 153–157). The objective of the present study was to quantify mRNA transcripts of the HA synthase 3 isoforms (Has1, 2, and 3), the HA-receptors CD44 and RHAMM, and mitochondrial DNA(mtDNA) copy numbers in bovine embryos cultured until Day 4 (96 h post-insemination (pi)) without protein supplement. A total of 1966 oocytes (11 replicates) were fertilized, and presumptive zygotes were initially cultured in 30 µL drops in the following groups; Group 1, control, SOF + 5 mg mL^–1 BSA, and Group 2, SOF only. The number of zygotes ≤8 cells was recorded 96 h pi, and 20 µL of fresh media were added as follows: G-1, (positive control) SOF + BSA, no change; G-1a, SOF + BSA + 1 mg mL^–1 HA; G-2, SOF + BSA; G-2a, SOF + BSA + HA; and G-2b, (negative control) SOF only. Embryos were cultured under paraffin oil at 39°C and 5% CO₂ in humidified air. Cleavage rates were recorded on Day 2 and the number of the blastocysts was registered on Days 7, 8, and 9. Five Day 7 blastocysts from each replicate from each treatment were frozen for evaluation of gene expression patterns and mtDNA copy analysis. Real-time reverse transcriptase-polymerase chain reaction was used for individual mRNA expressions. Embryo development among groups was compared by chi-square analysis and data on mRNA expression by one-way repeated-measures ANOVA. The cleavage rates and numbers of embryos that developed to d8 cells at Day 4 were no different between 2 groups: G-1, 77.8 and 46.7%; G-2, 77.2 and 46.2%, respectively. However, more (P < 0.05) blastocysts developed at Days 7, 8, and 9 in G-1, G-1a, and G-2, 26.7, 22.7, and 27.7%, respectively, than in G-2a, 17.3%, and G-2b, 9.7%. Of the 3 Has isoforms, only Has2 gene expressionwas increased (P < 0.05) in both BSA only-supplemented G-2 and G-2a, but not in remaining 3 groups. Expression of CD44 receptor gene was lower (P < 0.05) in G-2b (SOF only), but not different among the remaining 4 groups. However, expression of RHAMM receptors was highly dependent on HA addition to media containing BSA and were the highest in G-1a and lowest (P < 0.05) in G-2b (SOF only group) as compared to both BSA only-supplemented groups that were not different. Mitochondria number was higher (P < 0.05) in HA + BSA than in the BSA only group and lowest in the remaining 3 groups. We conclude that embryo culture without proteins until 96 h pi did not affect development and the addition of HA and/or BSA after 96 h pi increased expression of RHAMM and Has2 but not CD44, Has1, and Has3 genes. Addition of both HA and BSA at Day 4 increased mtDNA copy number. The consequences of those findings must be elucidated further.