Developmental and molecular biological studies of in vitro produced cat embryos in different culture systems

Abstract

EXP. 1 aimed to investigate the effect of roscovitine (ROS) on the developmental competence of cat oocytes matured in vitro. Groups of COCs were cultured in 0, 12.5, 25, 50, 100, and 200 µM ROS for 24 h and were either fixed to assess the stages of nuclear maturation or additionally matured in vitro for 24 h before fixation. Cumulus cells from the COCs treated with ROS were examined for late apoptosis. The developmental competence of cat oocytes after ROS treatment and in vitro fertilization was determined. ROS reversibly arrested cat oocytes at an immature stage in a dose-dependent manner. ROS at 12.5 and 25 µM demonstrated less efficiency to arrest the oocytes compared with other doses. However, higher doses of ROS induced cumulus cell apoptosis and resulted in a high number of degenerated oocytes after in vitro maturation (P<0.05). ROS at 12.5 and 25 µM, which gave rise to the highest rate of mature stage (MII) (P<0.05), were therefore used to evaluate their effect on embryo development. Pretreatment with 12.5 and 25 µM ROS prior to in vitro maturation decreased the developmental competence of cat oocytes compared with non–ROS-treated controls (P<0.05). In conclusion, ROS reversibly maintained cat oocytes at the germinal vesicle stage without detrimental effect on nuclear maturation. However, it negatively affected cumulus cell viability and developmental competence. EXP. 2 aimed to define the effects of culture media and culture volume in cat embryos. Groups of 8 to 10 embryos were cultured in SOF, modified Tyrode’s solution and MK-1 medium in different volumes (20-, 50- and 100-µl drops). SOF supplemented with different concentrations of glucose (1.5, 3.0 and 6.0 mM) was used to examine the effect of glucose in culture medium on embryo development. Quantitative polymerase chain reaction was used to determine the relative transcripts of BAX, BCL-2 and GLUT-1 genes in blastocysts derived from various concentrations of glucose. SOF and MK-1 supported feline embryo development better than modified Tyrode’s solution (P<0.05). Embryos cultured in 20-µl droplets showed decreased development in all three media (P<0.05). Increasing the glucose concentration in SOF to 6.0 mM adversely affected embryo development and tended to increase the BCL-2 transcript in blastocysts (P<0.05). In conclusion, type of culture medium, culture volume and glucose concentration affected the development of domestic cat embryos. Decreased culture volume (20 µl) and high glucose concentration (6 mM) negatively affected embryo development. The increase of anti-apoptotic BCL-2 expression found in blastocysts cultured in 6.0 mM glucose may prevent an increase of apoptosis. In the present study, it was clearly demonstrated that differential gene expression occurred in embryos with similar morphology. EXP. 3 aimed to determine the effects of embryo density and number on feline embryo development. Embryos were randomly cultured in group (n=10 and 5) or singly in different medium volume (12.5, 25, 50, 100 and 200 µl). They were examined for their developmental competence and fragmentation of blastocyst cell nuclei using DNA labeling. Only expanded blastocysts acquired from different density and numbers were collected to examine their mRNA transcripts of BAX, BCL-2 and HSP70 genes using quantitative polymerase chain reaction. Embryos cultured in groups tended to develope better than those cultured singly. For group cultured embryos (n=10), embryos acquired from low culture density (1:5, 1:10 and 1:20) could develop better than those acquired from high density (1:1.25 and 1:2.5) (P<0.05). Moreover, fragmentation of the blastocyst cell nuclei tended to increase in high culture density. On the other hand, there was no significant difference of developmental competence among embryos cultured singly in varied densities. Blastocysts derived from high culture density (1:1.25) also significantly up-regulated BAX and HSP70 transcripts comparing with those of low culture densities (1:5 and 1:20) (P<0.05). However, there was no significant difference in relative transcripts among varied embryo numbers (n=10, 5, 1) cultured in fixed 200 µl culture medium. In conclusion, high density negatively affected the developmental competence and up-regulated pro-apoptotic (BAX) and stress response (HSP70) transcripts in embryos. This highlighted the mechanisms used to protect the embryo against suboptimal culture condition.