Expression profile of Cyclin B gene in buffalo cumulus oocyte complex
Abstract
The present study was carried out to determine the temporal expression of Cyclin B gene in
follicles of different size using real time PCR. From 1257 ovaries, 740 and 1881 good quality COCs
were recovered by aspiration from large (>6 mm) and medium (2-6 mm) follicle respectively. The
recovered COCs were matured in TCM-199 medium supplemented with 10% FBS, 0.4% BSA and
hormones FSH and LH, along with controls without FBS and BSA, and incubation at zero min, 30 min,
60 min, 90 min, 18 hr, 20 hr, 22 hr, 24 hr and 26 hr in a CO2 incubator with 5% CO2 under humid
conditions at 38.5°C. The maturation was assessed by the expansion of cumulus cells of COCs. From
in vitro matured COCs total RNA was isolated, reverse transcribed and amplified by Cyclin B gene
specific primers. Real time PCR was carried out to study the expression pattern of Cyclin B gene in
follicles of different sizes. The 318 bp amplified product was purified and cloned successfully into
pGET cloning vector using E. coli cell JM107 strain as the host cell. Positive cloneswere sequenced
and the phylogenetic relationship of Cyclin B gene of buffalo was determined with other species.
Result: The recovery rate of COCs per ovary was 2.08. The overall IVM rate of COCs was 81.90% and
50.21% in TCM-199 supplemented with FBS and BSA and control. The expression of Cyclin B was
higher in medium follicles. The expression of Cyclin Bwas highest at zero min in both large and
medium follicles and decreases as the maturation time increases.In the phylogenetic analysis, the
Cyclin B geneof Bubalus bubalis are found more closely related to that of Bos taurusthan to Rattus
norvegicus. Conclusion: Function of Cyclin B is more during early phases of maturation and less
during further stages of maturation.