
TY  - JOUR
A1  - Palasz, A. T. 
A1  - Beltrán Breña, P. 
A1  - De la Fuente, J. 
A1  - Gutiérrez-Adán, A. 
T1  - 183 THE EFFECT OF BOVINE EMBRYO CULTURE WITHOUT ANY PROTEIN SOURCES UNTIL DAY 4 ON TRANSCRIPTION OF HYALURONAN SYNTHASES AND RECEPTORS AND mtDNA CONTENT
JF  - Reproduction, Fertility and Development
JO  - Reprod. Fertil. Dev.
Y1  - 2007
VL  - 20
IS  - 1
SP  - 171
EP  - 171
KW  - 
UR  - https://doi.org/10.1071/RDV20N1AB183 
N2  - 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&#8211;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 &#181;L drops in the following groups; Group 1, control, SOF + 5 mg mL&#8211;1 BSA, and Group 2, SOF only. The number of zygotes &#8804;8 cells was recorded 96 h pi, and 20 &#181;L of fresh media were added as follows: G-1, (positive control) SOF + BSA, no change; G-1a, SOF + BSA + 1 mg mL&#8211;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. 
ER  -

