Introduction
The oocyte maturation conditions used influence long-term embryo development significantly. The cumulus cells that surround oocytes play an important role in regulating oocyte maturation. Gonadotropins added to the maturation medium induce cytoplasmic maturation in oocytes (Sirard et al., Reference Sirard, Parrish, Ware, Leibfried-Rutledge and First1988). Follicle stimulating hormone (FSH) induced the production of estrogen, stimulates follicular development to the preovulatory stage and luteinizing hormone (LH) surge induced secondary factors such as progesterone, prostaglandin (PGE2) and epidermal growth factor (EGF) that mediate cumulus expansion and oocyte maturation (Shimada et al., Reference Shimada, Hernandez-Gonzalez, Gonzalez-Robayna and Richards2006).
The quality of oocyte maturation was assessed morphologically with the scoring of expansion of cumulus–oocyte complexes (COCs), PB and spindles, so far. The quality of matured oocytes predicted on the basis of morphology is still controversial. Therefore, some critical factors from the cumulus cells that are related to oocyte quality are the subject of considerable research. The present study was conducted to investigate the effect of FSH and LH on bubaline oocytes cumulus expansion in culture. Because of their important roles in cell signalling and follicular growth FSH receptor (r), LHr and Cx43 mRNAs may be important markers that could be used to predict oocyte competence and their expression were analysed at differing qualities at 0 h, 6 h, 12 h, 18 h and 24 h time points during in vitro maturation (IVM) in all the treatment groups.
Material and methods
All reagents were purchased from Sigma Aldrich, USA unless otherwise noted.
Collection of oocytes and cumulus expansion assessment
Buffalo oocytes were isolated from abattoir ovaries by follicular aspiration using an 18-gauge needle. Oocytes were washed in TCM-199 with 10% fetal bovine serum (FBS), gentamycin sulfate (10 μg/ml). Pools of 20–25 COCs and denuded oocytes were matured separately in TCM-199 with 10% FBS and sodium pyruvate (2.5 mM) + FSH and LH treatment groups. Group 1: control (without FSH and LH), Group 2: 1000 ng/ml FSH, Group 3: 1000 ng/ml LH and Group 4: 1000 ng/ml FSH + 1000 ng/ml LH in 4-well plates at 38.5 °C in 5% CO2 in air. Cumulus expansion was scored according to the 0–4 scale and the cumulus expansion index (CEI) was calculated as described by Fagbohun & Downs (Reference Fagbohun and Downs1990).
Reverse transcription
Ten freshly matured COCs and denuded oocytes from each treatment groups were treated using cells to cDNA II kit (Ambion). Preparation of lysate and reverse transcription was performed according to manufacturer's protocol.
Gene expression analysis
PCR primers were designed using the software Beacon Designer 7.0 (Premier Biosoft International) (Table 1). Gene expression was analysed with 2× SYBR Green master mix (Invitrogen) in 25 μl reaction on all the samples in triplicates on real-time PCR (Mx3000p Stratagene). PCR conditions were 95 °C for 10 min, then 50 cycles consisting of denaturation at 95 °C for 10 s, annealing at 56 °C for 10 s and extension at 72 °C for 15 s. Subsequent to the PCR, a dissociation curve analysis programme was run to confirm a single specific peak and to detect primer/dimer formation using the programme of 0 s at 95 °C, 10 s at 56 °C then for 0 s at 95 °C with acquisition on the step mode. The comparative CT method was used for relative quantification of target gene expression levels (Bustin, Reference Bustin2000). Quantification was normalized to the internal control GAPDH gene.
Table 1 Details of primers used for real-time PCR experiments.
Statistical analysis
SPSS 7.5 (USA) programme was used for statistical analysis. Average and standard deviation was calculated for cumulus expansion and polar body formation rate at p ≤ 0.05 level of significance. After testing for normality and equal variance, one-way ANOVA followed by post hoc multiple pair wise comparisons using Duncan's multiple range tests for variable were employed to determine the difference in the gene expression pattern in matured oocytes.
Results and Discussion
Gonadotropins FSH and LH hormones were supplemented in maturation medium to improve the maturation efficiency. Cumulus cells and oocytes are functionally and physically connected, establishing a sophisticated network of mutual interaction, which ultimately confers to the oocyte development competence. According to Modina et al. (Reference Modina, Beretta, Lodde, Lauria and Luciano2004) cumulus cells are very important for the maturation of the oocytes however, this is species specific.
In the present studies, bubaline oocytes cumulus expansion was assessed in four different treatments of FSH and LH hormones. Cumulus expansion percentage, polar body formation percentage (Table 2) and degree of cumulus expansion (Table 3) in maturation medium with 1000 ng/ml FSH and 1000 ng/ml LH were significantly greater than medium without hormonal supplement, or 1000 ng/ml FSH and 1000 ng/ml LH alone. Results indicate that FSH and LH act synergistically to enhance maturation rate.
Table 2 Effect of FSH and LH on developmental rate.
aPolar body formation rate was scored in oocytes with cumulus expansion only. b1000 ng/ml.
Table 3 Effects of FSH and LH supplements on cumulus cell expansion.
a1000 ng/ml.
FSHr mRNA expression was directly affected by time of maturation and oocyte quality. FSHr expression was higher in COCs in comparison to denuded oocytes at all time points (Fig. 1A). FSHr mRNA abundance decreased in both COCs as well as denuded oocytes after 6h of maturation. COCs had more FSHr mRNA levels than denuded oocytes but the differences were insignificant. This finding is consistent with the down-regulation of FSHr mRNA in bovine COCs (Calder et al., Reference Calder, Caveney, Smith and Watson2003) and suggests that gene expression in the surrounding cumulus cells is essential for the meiotic resumption of oocytes.
Figure 1 Relative expression level of FSHr (A), LHr (B) and Cx43 (C) mRNA in matured COCs (black bar) and denuded oocytes (grey bar) at 6, 12, 18 and 24 h of maturation. Significant differences at different time points are indicated by different letters (COCs: A–D = p ≤ 0.05; Denuded oocytes: a–d = p ≤ 0.05), whereas values with the same symbol (*) indicate significant differences (p ≤ 0.05) between oocytes at the same time point.
Likewise FSHr there was an effect of oocyte quality and maturation time on LHr expression level too. LHr expression was significantly higher in COCs than denuded oocyte at each time point (Fig. 1B). Relative abundance of LHr tended to increase from 6 h to 24 h maturation. According to Eppig et al. (Reference Eppig, Wigglesworth, Pendola and Hirao1997) advancing nuclear maturation in the form of cumulus expansion of oocytes may allow increased LHr mRNA expression. It is observed that LHR mRNA abundance is positively correlated with maturation rate.
Cx43 mRNA abundance was significantly higher in COCs compared with denuded oocytes (Fig. 1C) which indicates that cumulus cells have a unique role in the expression of gap junction protein. Differential expression of Cx43 mRNA among COCs and denuded maturing oocytes may be a useful marker of oocyte maturation competence. Cx43 mRNA abundance decreased in COCs and denuded oocytes from 6h to 24h of maturation agreed with the result observed in the outer cumulus layers of COCs (Shimada et al., Reference Shimada, Maeda and Terada2001).
From our studies we concluded that 1000 ng/ml FSH + 1000 ng/ml LH in maturation medium exhibited better maturation rate and indicate that in vitro maturation medium have the greatest effect on oocyte quality and further chemical modification in oocyte culture medium in the form of hormonal supplement can improve the oocyte maturation rate. Relative abundance of FSHr, LHr and Cx43 mRNAs are dependent on time of oocyte maturation and oocyte quality and these genes can be used as a marker gene to check the developmental competence of matured oocytes.
