Real – Time PCR Based Expression Study of Cyclin B Gene in Buffalo Cumulus Oocyte Complex

Received: Revised: Accepted: 2013–11–2


INTRODUCTION
In vitro maturation (IVM) and in vitro fertilization (IVF) procedures performed on oocytes obtained from slaughterhouse derived ovaries have recently provided a practical means for producing large number of bovine zygotes at low cost for research and commercial purpose (Hansen, 2006).The results for IVF in buffalo are very poor as compared to cattle because majority of IVF studies in buffalo use the protocols which are established for cattle (Yousaf and Chohan, 2003).The overall success rate of embryo production following IVF in buffalo is reported to be not more than 10-15 percent which is very low when compared to that of cattle (Chauhan et al., 1998;Nandi et al., 2001;Raghu et al., 2002).Nandi et al. (2002) have reported approximately 80 percent of buffalo oocytes fail to reach the blastocyst stage and most of blastocysts are not capable of further development.The poor success rate of in vitro fertilization and lower developmental competence of in vitro produced embryos present a principal barrier for the successful implementation of other assisted reproductive techniques like animal cloning, animal transgenesis, stem cell culture and nuclear transfer etc. in buffaloes.The maturation of oocytes under in vitro condition has significant influence on the number of embryos that develop to the blastocyst stage (Thompson et al., 1995;Jones, 2000;Rizos et al., 2002).The oocyte undergoes a series of modifications during maturation and these are necessary to acquire developmental competence.Therefore more advanced research for the development of suitable in vitro maturation (IVM) system is important.
Cyclins control the transition between the phases of the cell cycle as regulatory subunits of the cyclin-dependent kinases (CDKs) (Donjerkovic and Scott, 2000).The amount of Cyclin B (which binds to Cdk1) and the activity of the Cyclin B-Cdk complex rise through the cell cycle until mitosis (Ito, 2000), where they fall abruptly due to degradation of Cyclin B (Cdk1 is constitutively present).Cyclin B protein is degraded by ubiquitin pathway (Hershko, 1999).The complex of Cdk and Cyclin B is called maturation promoting factor or mitosis promoting factor (MPF; Maller, 1990).Activated Cyclin B-Cdk complex promotes several of the events of early mitosis.The active complex phosphorylates and activates 13S condensin, which helps to condense chromosomes (Kimura et al., 1998).
Maturation-inducing hormone (MIH) induces oocytes to synthesize Cyclin B (Hirai et al., 1992;Katsu et al., 1999).The rate of Cyclin B synthesis controls the length of the first meiotic M phase (Polanski et al., 1998).Human Cyclin B protein microinjected into cycloheximide-treated bovine oocytes triggered meiotic resumption.Thus, Cyclin B has a

Cumulus Oocyte Complex
ISSN: 2307-8316 (Online); ISSN: 2309-3331 (Print) critical role in meiotic resumption in bovine oocytes (Lévesque and Sirard, 1996).The rates of Cyclin B synthesis and degradation determine the timing of the major events taking place during meiotic maturation of the mouse oocyte (Ledan et al., 2001).GVBD in mouse oocytes is sensitive to Cyclin B abundance and the changes in distribution of Cyclin B contribute to progression through Meiosis I stage (Marangos and Caroll, 2004).Cyclin B and cdc2 (cell division control) were expressed at the mRNA level in GV oocytes but cdc2 was the only pre-MPF protein detected at that stage, thus meiosis resumption seems to be regulated by the translation of Cyclin B mRNA.Cyclin B and CDC2 mRNA were high at 10 h of IVM and decreased gradually until 26 h, where most of oocytes reached the meiosis-II stage (Khalil et al., 2010).
Thus for improving reproductive performance of buffaloes, better understanding of expression level of different genes, which have a role in meiotic resumption, is important.The present study has been conducted to determine the expression pattern of Cyclin B gene in buffalo COCs from different size follicles at different time interval during in vitro maturation.

Collection of Ovaries
Buffalo ovaries were collected from MCD slaughter house, Ghazipur, New Delhi.All ovaries were carried to the quality control laboratory of slaughter house within 15 minutes of slaughter in normal saline solution (NSS) (at 37°C) supplemented with antibiotics (Penicillin 100 IU mL and streptomycin sulphate 100 μg/mL).All the visible ovarian follicles were graded as medium follicles (2-6 mm) and large follicles (more than 6 mm) based on their diameter and oocytes were aspirated in the aspiration media containing TCM-199, Glucose, Sodium Pyruvate, Sodium Bicarbonate and antibiotics, using 18G needle.The aspirated contents were transferred to 60 mm petridish and searched under stereozoom microscope (Kyowa, Japan).

In Vitro Maturation of Oocytes
COCs were washed serially thrice in 35 mm petridish containing 100 µL drops of oocyte washing medium containing TCM-199, supplemented with 10% FBS and finally transferred into 100µL of washing drops prepared from maturation drops supplemented with hormones PMSG (10 IU/mL) and hCG (10 IU/mL).The COCs from graded follicles were matured in maturation media drops, TCM-199 supplemented with 10 percent FBS containing antibiotics (streptomycin 100 µg/mL, Penicillin 100 IU/mL) and hormones PMSG (10 IU/mL) and hCG (10 IU/mL).The maturation drops were covered with warm (35-37ºC) light weight mineral oil and kept for different time period (Zero minute, 30 min, 60 min, 90 min, 18 hr, 20 hr, 22 hr, 24 hr and 26 hr) in CO 2 incubator at 38.5ºC under 5% CO 2 pressure and with a relative humidity of 90 to 95%.Maturation of oocytes was evaluated by the cumulus cell expansion under stereo zoom microscope (Figure 1).

Real Time PCR
For the analysis of temporal expression profile of gene, real time PCR was carried out using real time PCR Applied Biosystems 7500.For the real time PCR reaction, SYBER Green dye i.e.Quantitect SYBER Green PCR kit ® (Qiagen) was used and all the instructions were followed as per the supplier.The reaction for the target gene, Cyclin B gene, and the endogenous control, GAPDH gene was carried out in triplicate along with NTC (Non-template Control) as a negative control for each sample.The reaction mixture used to carry out the real time PCR reaction for Cyclin B and GAPDH gene a total reaction volume of 25 µL, was as: 2X Quantitect SYBER Green PCR Mix (Qiagen, 12.5 µL), primers (forward and reverse 0.3 M each), NFW (variable), 25mM MgCl 2 (Fermentas, 2 µL), and template (2 µL).The cyclic conditions used for amplification of Cyclin B and GAPDH transcript cDNA were according to the instructions of the supplier.The set of primers used for the real time PCR are as shown in Table 1.

Relative Quantification by Comparative C T Method (∆∆C T Method)
The average C T (Threshold cycle) value obtained for the Cyclin B (target) gene was normalized to GAPDH (endogenous control).The data obtained was subjected to comparative C T method (Livak and Schmittgen, 2001) for the analysis of the expression levels of target (Cyclin B) gene and an endogenous control.The sample at 26 hour of incubation was selected as calibrator.

Cloning and Sequencing of PCR Products
In the present study the 318 bp PCR products of Cyclin B gene (Figure 2) of buffalo was used for cloning in CloneJET TM PCR Cloning Kit using pJET 1.2 blunt end cloning vector.The product was sequenced at Department of Animal Biotechnology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences.

Nucleotide Sequence Analysis
The sequence obtained was analyzed by using BLASTn.The phylogenetic tree was constructed using Phylogeny.fr(http://www.phylogeny.fr/)server to show relatedness among the species with respect to Cyclin B cDNA.

Expression Profile of Cyclin B Gene
The differential expression level of Cyclin B gene transcripts in in vitro matured oocytes was studied by relative quantification method.The level of target mRNA in different gorups was determined by comparative CT method (∆∆CT method).The level of Cyclin B mRNA was more in medium follicles than large follicles at any point of time.The expression level of Cyclin B mRNA decreases as the maturation time increases.The relative abundance of Cyclin B in COCs aspirated from graded follicles was higher just after aspiration (i.e.zero minute) as compared to maturation times of 24 and 26 hr.After 22 hrs the level of expression of Cyclin B remained low and almost constant (Figure 3 and Table 2.1, 2.2).The expression level of Cyclin B was highest at zero minutes in both large and medium sized follicles.
The phylogenetic tree was constructed using Phylogeny.frserver to show relatedness between the species with respect to Cyclin B fragment (Figure 4).In phylogenetic tree, Cyclin B of Bubalus bubalis were found more closely related to Bos Taurus than to Sus scrofa.From the phylogenetic tree it was observed that the Rattus norvegicus Cyclin B gene diverged much earlier in the evolution.

Sequence of Cyclin B (Partial mRNA)
318 bp product length was successfully sequenced using BigDye terminator chemistry and used as Query Sequence for constructing phylogenetic tree.In present study the expression of mRNA for Cyclin B gene in in vitro matured COCs was studied by real time PCR.Our study revealed that the expression of Cyclin B gene was higher in the oocytes collected from medium sized follicles than large size follicle Majority of the oocytes completed maturation between 22 and 26 hr.These results were in agreement with those reported by Gasparrini et al. (2008), who found that the majority of buffalo oocytes accomplish nuclear maturation between 21 and 24 hr after the start of IVM.In another study, Nandi et al. (2002) found that cumulus expansion and extrusion of first polar body in buffalo oocytes commence at 6-17 hr post-maturation to reach the maximum levels at 22-24 hr.However, the results of the present study were in disagreement with the study of Neglia et al. (2001), in which the majority of buffalo oocytes reached the MII stage between 15 and 19 hr after the start of IVM and an increased incidence of degenerated oocytes was observed at later times.Large variations in the timing of the oocyte maturation process in vitro have also been reported in cattle (Ward et al., 2002;Park et al., 2005).The different oocyte maturation time-scale recorded among different buffalo studies may be accounted by different conditions of IVM and particularly by oocyte quality, which in this species, is also likely to be affected by seasonal factors.
The cytoplasmic maturation of the oocytes is a key parameter, which determines the success rate of in vitro production of embryos (Blondin and Sirard, 1995).Gaining knowledge about the variation/chronology of gene expression during oocyte maturation is crucial for optimization of IVF and other artificial reproduction technologies.Studies involving expression pattern of developmentally important genes in oocytes and embryos have not been widely investigated (Gaudette et al., 1993;Lechniak, 2002).Based on the Cyclin B mRNA transcripts in buffalo oocytes in our study, mRNA for genes that encode components of MPF were detected immediately after oocytes collection, at zero minute of IVM.Wu et al. (1997) reported that activity of MPF is low in GV-stage bovine oocytes and increases around GVBD to peak at the MI stage.In the current study, Cyclin B mRNA was high at zero minute of IVM and decreased gradually until 26 h, where most of oocytes reached the MII stage.This result correlates the resumption of meiosis with the abundance of MPF component mRNAs.The depletion of Cyclin B mRNAs could be associated with mRNA degradation or translation into MPF-component proteins.
This study is also in agreement with the results of Robert et al. (2000) who reported that bovine GV-stage oocytes, recovered from ovaries immediately after slaughter, possess a stockpile of maternal Cyclin B mRNA.Interestingly, it seems that Cyclin protein can accumulate before oocyte maturation, when the ovary transportation time is long (Levesque and Sirard, 1996).Cyclin B mRNA probably is in a translationally inactive state (masked) in immature GV-stage bovine oocytes.Therefore, initiation of Cyclin B translation could be one of the early events leading to oocyte meiotic resumption.These studies clearly indicate that the role of Cyclin B is crucial during the early stages of folliculogenesis and its function and expression diminishes towards the later stages.Thus, the functions of Cyclin B is more conspicuous in the early phases of maturation and less during later stages of maturation of oocyte.

CONCLUSIONS
The mRNA expression of Cyclin B gene in buffalo cumulus oocyte complex was higher in medium follicles followed by large follicles.Cyclin B gene expression is found in all the IVM COCs irrespective of size of follicular source.The relative abundance of Cyclin B in COCs aspirated from graded follicles was higher just after collection i.e. at zero minute compared to those at 24 and 26 hrs of maturation.This expression pattern of Cyclin B reaffirms its role in germinal vesicle breakdown (GVBD) and early folliculogenesis and thereby proves that Cyclin B is an important factor regulating in vitro maturation of COCs.

Table 1 :
Sequence of primers used in real time PCR experiments

Table 2 .
1:∆∆CTrelative quantitation of Cyclin B transcript in medium follicle Table 2.2: ∆∆CT relative quantitation of Cyclin B transcript in large follicle

Table 3 :
Maximum homology search using BLASTn