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

Jitender Yadav, Vishal Sharma, Aman Kumar, Richa Khirbat, Trilok Nanda^*,

*Corresponding author: [email protected]

ARTICLE CITATION: Yadav J, Sharma V, Kumar A, Khirbat R, Nanda T (2014). Real–Time PCR based expression study of cyclin B gene in buffalo cumulus oocyte complex. Adv. Anim. Vet. Sci. 2 (3): 142 – 147.
Received: 2013–11–20, Revised: 2013–12–16, Accepted: 2013–12–16
The electronic version of this article is the complete one and can be found online at ( http://dx.doi.org/10.14737/journal.aavs/2014/2.3.142.147) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited

ABSTRACT

The success rate of in vitro fertilization (IVF) in buffalo is very low, though a research of more than a decade has been carried out. One of the reasons could be the lack of standardised protocol for in vitro maturation of oocytes. The present study was conducted to determine the temporal expression of Cyclin B gene in follicles of different size. A good quality cumulus oocyte complexes (COCs) were recovered from large (>6 mm) and medium (2–6 mm) follicles of buffalo ovaries by aspiration. The recovered COCs were matured in Tissue Culture Medium–199 medium supplemented with 10% foetal bovine serum (FBS), 0.4% bovine serum albumin (BSA) and hormones pregnant mare’s serum gonadotropin (10 IU/mL) and human chorionic gonadotropins (10 IU/mL) and incubated for 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. Total RNA was isolated from COCs, reverse transcribed and real time PCR was done to study the expression pattern of Cyclin B gene in follicles of different sizes. Real time PCR assay revealed higher expression of Cyclin B in medium size follicles. However temporal expression of Cyclin B was highest at zero min in both large and medium follicles and decreases as the maturation time increases. These results suggest that the role of Cyclin B is more during early phases of maturation and less during later stages of maturation.

INTRODUCTION

In vitro maturation (IVM) and in vitro fertilization (IVF) procedures performed on oocytes obtained from slaughter–house 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 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.

MATERIALS AND METHODS

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)

Reverse Transcription (cDNA Synthesis); RNA Extraction and Preparation of cDNA
Total RNA was isolated from in vitro matured COCs by using TRIzol® LS reagent (Life technologies) as per the manufacturer’s instruction. Total RNA was quantified using Qubit® 2.0 fluorometer (Invitrogen). To ascertain minimum variation among different groups, a pool of 66 COCs was used for RNA isolation. Reverse transcription was carried out with total reaction volume of 20 µL. Briefly, NFW (8.00 µL), 5X RT buffer (Fermentas) (5 µL), 10mM dNTPs (Fermentas) (0.5 µL), total RNA (4 µL), M–MuLV–RT 200 IU/µL (Promega) (1 µL), DMSO (1 µL), decamer primers (Ambion) (0.5 µL). The RT–PCR cyclic conditions were as: annealing at 25oC for 10 min, reverse transcription at 42oC for 1 hour, and denaturation at 90oC for 5 min in thermal cycler (Eppendorf thermocycler). The cDNA was stored at –20oC till further use.

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 MgCl2 (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.

Phylogenetic Analysis of Nucleotide Sequences of Cyclin B
The identity analysis of the buffalo Cyclin B sequence with the other species GenBank sequence was done using BLASTn. The NCBI Genbank accession numbers of the sequences of Cyclin B of the various species considered were XR_083650.1 (Bos taurus), NM_031966.3 (Homo sapiens), XM_845305.2 (Canis lupus familiaris), XM_001491280.2 (Equus caballus), NM_001170768.1 (Sus scrofa), NM_001261149.1 (Macaca mulatta) and X60768.1 (Rattus norvegicus). The Cyclin B was found 94% identical to Bos taurus, 92% to Homo sapiens, 92% to Canis lupus familiaris, 92% to Equus caballus, 92% to Sus scrofa, 92% to Macaca mulatta and 88% to Rattus norvegicus (Table 3). The phylogenetic tree was constructed using Phylogeny.fr server 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.

318 bp product length was successfully sequenced using BigDye terminator chemistry and used as Query Sequence for constructing phylogenetic tree.

DISCUSSION

Cell proliferation is an elementary cellular function and is tightly controlled by physiological signals as well as internal molecular mechanisms. In eukaryotic cells, the maturation promotion factor (MPF) is the master regulator of cell proliferation. MPF is a heterodimer of Cyclin B and cyclin dependent kinase. In this study, we report the partial sequence of buffalo Cyclin B cDNA. The identity analysis had shown that the Cyclin B gene of Bubalus bubalis is more closely related to Bos Taurus and lesser to Rattus norvegicus. Thus Rattus norvegicus Cyclin B gene has diverged much earlier in the evolution. But overall the sequence of Cyclin B has remained conserved throughout the evolution. 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 B 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.

ACKNOWLEDGEMENTS

We would like to acknowledge the help and support received from Dr. M.L. Sharma (VPH Specialist, MCD) and Dr. Senthil Kumar (Manager, Allana, Delhi) for their help for the providing the sample for our research. We would also like to acknowledge Department of Biotechnology, DBT, for the financial assistance.

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