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Effect of cryopreservation on the synthesis of DNA by the inner cell mass of in vitro-produced bovine blastocysts
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Theriogenology EFFECT OF CRYOPRESERVATION ON THE SYNTHESIS INNER CELL MASS OF IN VITRO-PRODUCED BOVINE
OF DNA BY THE BLASTOCYSTS
M. Takagi, I. Sakonju and T. Suzuki United Graduate School of Veterinary Science, Yamaguchi University, 753 Yamaguchi, Japan. The objective of this study was to examine the rate of DNA synthesis in inner cell mass (ICM) cells of frozen-thawed IVF derived embryos using 3 types of cryoprotectants (CP). Excellent quality blastocysts and expanded blastocysts on Day 7 of culture cryopreserved by standard slow freezing procedures in were either 1.6 M propylene glycol (PG), 1.8 M ethylene glycol (EG) or 1.4 M glycerol and 0.25 M sucrose procedures (GL) by earlier (Theriogenology described 39:325,1993). Thawed and diluted embryos were co-cultured with cumulus cells for 24 h in culture media (TCM-199) supplemented with 3 mg/ml bromodeoxyuridine (Brau) _ No siqnificant differences were found in the rate of development of embryos to blastocysts following cryopreservation in the three cryoprotectants (82%. 73% and 79% for embryos frozen, respectively, in EG, PG and GL). Only the ICM of embryos developing beyond the blastocyst stage after 24 h of culture were evaluated. ICMs were isolated by immunosurgery described previously (Cryobiology 31:398,1994) as and DNA synthesis was evaluated by immunocytochemical staining using a anti-BrdU antibody. monoclonal Briefly, ICMs were placed in hypotonic citrate, fixed and spread on a glass slide. Slides reconstituted incubated in nuclease/anti-5-bromo-2'were deoxyuridine monoclonal antibody for 1 h at room temperature, washed and then incubated with peroxidase anti-mouse IgG2a for were washed with saline and then incubated in 30 min. Slides 3,3'-Diaminobenzidine tetrahydrochloride solution for 20-25 min. The number of BrdU-immunoreactive ICM cells were counted. The ICM cells then number of was determined by counter total staining with 3% methyl green. Controls consisted of unfrozen in vitro-produced blastocysts, blastocysts exposed to Brdu but not monoclonal stained with anti-BrdU antibody and (Br(+ 1) blastocysts that were not cultured in Brau (Br(- )). Results are shown in Table 1. Table 1. Mean of post-thawed ICM cell number Total SP (8) Note. SP:
total cell number and percentage of S-phase cells IVF bovine embryos frozen using 3 types of CP. Cryoprotectant Unfrozen EG Br (-1) Br(p ) (;;4;) ($2;) (;;lZ) (;;2;) (;;2;) (;;2G) 51.3a S-phase
32:9b cell.
4019ab
35:Ob
l.Oc
2.3~
The rates of DNA synthesis in ICM cells of frozen-thawed embryos with each CP were lower than that of unfrozen embryos. Cryoprotectants, especially in the cases of EG and GL were significantly lower than unfrozen embryos (PcO.05, ANOVA, Table rates of ICM 1) - Our results suggest that the cell proliferation cells of frozen-thawed bovine embryos during 24h culture tend to be lower than that of unfrozen embryos irrespective of the CP used.
Theriogenology EFFECT OF CRYOPRESERVATION ON THE SYNTHESIS INNER CELL MASS OF IN VITRO-PRODUCED BOVINE
OF DNA BY THE BLASTOCYSTS
M. Takagi, I. Sakonju and T. Suzuki United Graduate School of Veterinary Science, Yamaguchi University, 753 Yamaguchi, Japan. The objective of this study was to examine the rate of DNA synthesis in inner cell mass (ICM) cells of frozen-thawed IVF derived embryos using 3 types of cryoprotectants (CP). Excellent quality blastocysts and expanded blastocysts on Day 7 of culture cryopreserved by standard slow freezing procedures in were either 1.6 M propylene glycol (PG), 1.8 M ethylene glycol (EG) or 1.4 M glycerol and 0.25 M sucrose procedures (GL) by earlier (Theriogenology described 39:325,1993). Thawed and diluted embryos were co-cultured with cumulus cells for 24 h in culture media (TCM-199) supplemented with 3 mg/ml bromodeoxyuridine (Brau) _ No siqnificant differences were found in the rate of development of embryos to blastocysts following cryopreservation in the three cryoprotectants (82%. 73% and 79% for embryos frozen, respectively, in EG, PG and GL). Only the ICM of embryos developing beyond the blastocyst stage after 24 h of culture were evaluated. ICMs were isolated by immunosurgery described previously (Cryobiology 31:398,1994) as and DNA synthesis was evaluated by immunocytochemical staining using a anti-BrdU antibody. monoclonal Briefly, ICMs were placed in hypotonic citrate, fixed and spread on a glass slide. Slides reconstituted incubated in nuclease/anti-5-bromo-2'were deoxyuridine monoclonal antibody for 1 h at room temperature, washed and then incubated with peroxidase anti-mouse IgG2a for were washed with saline and then incubated in 30 min. Slides 3,3'-Diaminobenzidine tetrahydrochloride solution for 20-25 min. The number of BrdU-immunoreactive ICM cells were counted. The ICM cells then number of was determined by counter total staining with 3% methyl green. Controls consisted of unfrozen in vitro-produced blastocysts, blastocysts exposed to Brdu but not monoclonal stained with anti-BrdU antibody and (Br(+ 1) blastocysts that were not cultured in Brau (Br(- )). Results are shown in Table 1. Table 1. Mean of post-thawed ICM cell number Total SP (8) Note. SP:
total cell number and percentage of S-phase cells IVF bovine embryos frozen using 3 types of CP. Cryoprotectant Unfrozen EG Br (-1) Br(p ) (;;4;) ($2;) (;;lZ) (;;2;) (;;2;) (;;2G) 51.3a S-phase
32:9b cell.
4019ab
35:Ob
l.Oc
2.3~
The rates of DNA synthesis in ICM cells of frozen-thawed embryos with each CP were lower than that of unfrozen embryos. Cryoprotectants, especially in the cases of EG and GL were significantly lower than unfrozen embryos (PcO.05, ANOVA, Table rates of ICM 1) - Our results suggest that the cell proliferation cells of frozen-thawed bovine embryos during 24h culture tend to be lower than that of unfrozen embryos irrespective of the CP used.