Effect of timing of development on total cell number and expression profile of HSP-70.1 and GLUT-1 in buffalo (Bubalus bubalis) oocytes and preimplantation embryos produced in vitro.
The present study was designed to compare the expression profile of two developmentally important genes (HSP-70.1 and GLUT-1) and TCN (total cell number) count in fast (group A) and slow (group B) cleaved buffalo embryos to access their in vitro developmental competence. Buffalo COCs (cumulus oocyte complexes) were collected from local abattoir ovaries and subjected to in vitro maturation in: TCM-199 supplemented with 10% FBS (fetal bovine serum), BSA (3 mg/ml), sodium pyruvate (0.25 mM) and 20 ng/ml EGF (epidermal growth factor) at 38.5 degrees C under 5% CO2. In vitro derived embryos were collected at 4-8, 8-16 cell, morula and blastocyst stages at specific time points for gene expression analysis and total cell count. A semiquantitative RT-PCR (reverse transcriptase-PCR) assay was used to determine the HSP-70.1 and GLUT-1 transcripts. Results showed that developmental competence and TCN count in fast (group A)-cleaving embryos was significantly (P<0.05) higher than in the slow group (group B). The gene transcript of HSP-70.1 and GLUT-1 was expressed in oocytes (immature and mature) and throughout the embryonic developmental stages in the fast group (group A), while in the slow (group B) cleaving embryos, the expression of HSP-70.1 was absent in all the embryonic developmental stages, and expression of GLUT-1 was absent after 8-16 cell stage. In conclusion, TCN count and expression profile of HSP-70.1 and GLUT-1 genes in buffalo embryos are different taking into account the cleavage rate. Quality of such embryos for research purposes, TCN and expression profiling of developmentally important genes should be employed to optimize the in vitro culture system to produce superior quality of embryos.
Rajhans
Rajib
R
Reproductive Physiology and ETT Laboratory, Veterinary Physiology and Climatology Division, Indian Veterinary Research Institute, Izatnagar, India.
Kumar
G Sai
GS
Dubey
Pawan K
PK
Sharma
G Taru
GT
D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000502:physiology*; D002020:Buffaloes / Q000196:embryology / Q000235:genetics; D002417:Cattle; D002452:Cell Count; D027342:Excitatory Amino Acid Transporter 2 / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D009865:Oocytes / Q000502:physiology*; D046509:Tissue Culture Techniques
D027342:Excitatory Amino Acid Transporter 2; D018840:HSP70 Heat-Shock Proteins; C081884:heat-shock protein 70.1