Introduction
The success of an in vitro embryo production (IVEP) programme depends on the developmental competence of oocytes. Oocyte developmental competence itself is regulated by several factors such as origin of oocytes (Lonergan et al., Reference Lonergan, Monaghan, Rizos, Boland and Gordon1994), follicular health (Hagemann, Reference Hagemann1999), culture milieu (Lee et al., Reference Lee, Bettegowda, Wee, Ireland and Smith2009) and communication between oocytes and their surrounding cumulus cells (CCs) (Krisher, Reference Krisher2004). This bidirectional communication in cumulus–oocyte complexes (COCs) is mediated through gap junctions (Conti et al., Reference Conti, Hsieh, Park and Su2006; Gilchrist et al., Reference Gilchrist, Lane and Thompson2008) and is crucial for the promotion of cell growth (Li et al., Reference Li, Norman, Armstrong and Gilchrist2000), prevention of cell death (Hussein et al., Reference Hussein, Froiland, Amato, Thompson and Gilchrist2005), suppression of luteinization (Sutton-McDowall et al., Reference Sutton-McDowall, Mottershead, Gardner, Gilchrist and Thompson2012) and maintenance of CC metabolism (Gilchrist et al., Reference Gilchrist, Lane and Thompson2008; Sutton-McDowall et al., Reference Sutton-Mcdowall, Purdey, Hannah, Brown, Mottershead, Cetica, Dalvit, Goldys, Gilchrist, Gardner and Thompson2015). An understanding of the role of oocyte secretary factors (OSFs) that regulate oocyte developmental competence has helped to improve IVEP protocols.
OSFs are amongst the important mediators of bidirectional communication, which regulates gene expression in CCs that are associated with oocyte maturation and subsequent embryo development. Major OSFs that regulate oocyte developmental competence are growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) (Galloway et al., Reference Galloway, McNatty, Cambridge, Laitinen, Juengel, Jokiranta, McLaren, Luiro, Dodds, Montgomery, Beattie, Davis and Ritvos2000; Hanrahan et al., Reference Hanrahan, Gregan, Mulsant, Mullen, Davis, Powell and Galloway2004). The function and potential of both these proteins differ markedly between species (Crawford & McNatty, Reference Crawford and McNatty2012). In mono-ovulatory animals, the ratio of GDF9 and BMP15 is nearly equivalent, suggesting that both factors are important for follicular development. However, in the oocytes of multi-ovulatory animals, GDF9 expression is higher than BMP15 expression, suggesting a minor role for BMP15 (Crawford & McNatty, Reference Crawford and McNatty2012). In humans, rare mutations of GDF9 and BMP15 are closely linked with polycystic ovarian syndrome (Wang et al., Reference Wang, Zhou, Wang, Liu, Ni, Zhang, Zhao, Ma and Chen2010) and ovulation failure (Dixit et al., Reference Dixit, Rao, Padmalatha, Kanakavalli, Deenadayal, Gupta, Chakravarty and Singh2005). Hussein et al. (Reference Hussein, Thompson and Gilchrist2006) reported the positive effect of OSFs on oocyte developmental competence. They found enhanced bovine oocyte developmental competence after the addition of 175 ng/ml GDF9 and 10% v/v BMP15. As buffalo and bovine are related species, similar doses were chosen for this study. Rossi et al. (Reference Rossi, Portela, Passos, Cunha, Silva, Costa, Saraiva, Donato, Peixoto, Van Den Hurk and Silva2015) evaluated the effect of different concentrations of BMP4, as well as the interaction between BMP4 and FSH on growth, ultrastructural integrity, and mRNA expression for GDF-9, BMP15, maternal antigen that the embryo requires (MATER) and nucleoplasmin-2 in bovine secondary follicles cultured in vitro. To date, limited information has been available regarding the role of GDF9 and BMP15 during the maturation process in buffalo oocytes and no reliable molecular markers of oocyte developmental competence have been identified.
Therefore, the present study was designed with the hypothesis that OSFs (GDF9 or BMP15 alone, or in combination) may enhance oocyte developmental competence by upregulating the expression of oocyte quality predictor genes in CCs. Large follicle (LF)-derived and small follicle (SF)-derived oocytes were cultured independently to analyze the effect of OSF supplementation on oocytes from differently sized follicles. We investigated the role of DOs as a source of endogenous OSFs in COC co-culture during in vitro maturation (IVM) and compared the control with the exogenous recombinant OSF-supplemented oocytes. We also evaluated the effect of OSFs on expression of epidermal growth factor receptor (EGFR), hyaluronan synthase 2 (HAS2) and cluster differentiation 44 (CD44) genes as potential oocyte competence markers.
Material and Methods
Chemicals
All chemicals used in this study were procured from Sigma Chemicals Co. (St. Louis, MO, USA), except when specifically mentioned in the text. All these chemicals were culture grade and endotoxin free. Plastic dishes and tubes were procured from Nunc (Thermo Scientific, Denmark). All chemicals for reverse transcription were purchased from Thermo Scientific, Denmark.
Oocyte recovery and in vitro maturation
Oocyte recovery
Buffalo oocyte collection and in vitro maturation was performed as per the established laboratory protocol (Bhardwaj et al., Reference Bhardwaj, Ansari, Parmar, Chandra and Sharma2016b). In brief, buffalo ovaries were collected from the local abattoir and transported to the laboratory in pre-warmed saline (35–37°C). COCs were aspirated separately from the LFs (≥6 mm) and SFs (<6 mm) using a 18G needle fitted with a 5-ml syringe. Culture-grade oocytes (COCs with more than three layers of compact cumulus and evenly granular ooplasm) were selected under a stereozoom microscope for further experimental study. Different experiments were performed in five replicates.
Generation of DOs
DOs were generated by removing CCs from COCs by washing them with 0.01% hyaluronidase for 1–2 min. Any remaining leftover CCs were removed by pipetting followed by four to six times serial washing with TCM-199 medium (M2154, Sigma).
In vitro maturation
Culture-grade oocytes were washed four to five times in maturation medium containing TCM-199 (HEPES modified) supplemented with 5 μg/ml LH, 0.5 μg/ml FSH, 1 μg/ml estradiol-17β, 0.25 mM sodium pyruvate, 0.68 mM l-glutamine, 10 μg/ml gentamicin, 3 mg/ml bovine serum albumin (BSA) and 10% FBS. This medium was used as a basal medium for in vitro maturation. COCs of both follicle sizes were matured in vitro in basal medium supplemented either with GDF-9 (175 ng/ml; human recombinant; SRP4872-20UG, Sigma) or BMP15 (100 ng/ml; human recombinant; 5096-BM-005, Sigma) alone or in combination. To determine the role of DOs as a source of endogenous OSFs, COCs were co-cultured with DOs at a ratio of 2:1. COCs harvested from the LFs and SFs that were cultured in basal medium were used as the control. In total, about 10–15 COCs were kept for maturation in 50 μl droplets of maturation medium overlaid with mineral oil for 24 h at 38.5°C and 5% CO2 in air with maximum relative humidity. Cumulus expansion was graded on a scale of +1 to +5.
In vitro fertilization and embryo production
Buffalo embryos were produced in vitro as per the established laboratory protocol (Bhardwaj et al., Reference Bhardwaj, Ansari, Parmar, Chandra and Sharma2016b). In brief, in vitro matured COCs of all groups were fertilized with frozen–thawed buffalo bull semen using FerTALP medium supplemented with 0.2 mM sodium pyruvate, 6 mg/ml BSA, 20 µg/ml heparin and 50 µg/ml gentamycin. After 18 h co-incubation of sperms and oocytes, presumptive zygotes were washed in modified synthetic oviductal fluid (mSOF) containing BSA (3 mg/ml fraction V), 10% FBS, sodium pyruvate (0.25 mM), l-glutamine (0.68 mM) with essential and non-essential amino acids. Presumptive zygotes were then cultured (10–15 zygotes/50 µl droplet) in mSOF at 38.5°C, 5% CO2 and maximum relative humidity. Medium was changed on alternate days until the blastocyst stage or day 8 post insemination.
RNA isolation and reverse transcription
For the isolation of RNA, CCs of all the groups were separated from the oocytes (n = 20 for each sample) after 24 h of in vitro maturation by vortexing for 1 min. All the denuded oocytes were removed under a stereozoom microscope and CCs were suspended in phosphate-buffered saline (PBS)-PVP and then were transferred with a pipette in an autoclaved Eppendorf tube (0.5 ml). Cells were centrifuged at 1500 rpm for 10 min, followed by washing in PBS. The pellet formed was used for RNA isolation. Total RNA was isolated from CCs using TRIzol® reagent (Ambion cat. no. 15596-026). In total, 1 µg of total RNA was reverse transcribed using a single-stranded cDNA synthesis kit (Thermo Scientific, cat. no. AB1453/B) as per the manufacturer's instructions. The quality of cDNA was assessed by amplification reaction for housekeeping genes (GAPDH and β-actin). The PCR product was analyzed using 1.8% agarose gel electrophoresis.
Quantitative real-time PCR
Quantitative real-time PCR (qPCR) was carried out in duplicate for relative quantification of mRNA expression of competence markers (EGFR, HAS2, and CD44) and reference genes (GAPDH and β-actin).The primer sequences and amplicon sizes are summarized in Table 1. qPCR was performed using the DyNAmo Flash SYBR green qPCR kit (Thermo Scientific, cat#415L) and Stratagene Mx3000P (Agilent Technology, Santa Clara, CA USA) as per the manufacturer's instructions. In brief, a master mix contained 10 µl of SYBR green, 0.5 µl of forward and reverse primers each (20 pM), 8 µl nuclease free water and 1 µl of cDNA. The reaction was performed for 40 cycles with the following cycling conditions: denaturation at 95°C for 30 s, annealing at 60°C for 15 s and extension at 72°C for 30 s. A non-template control was run to check for any contamination and to minimize errors. The geometric mean of Ct values of the β-actin and GAPDH were used as the Ct of reference genes for the calculation of relative gene expression by the Pfaffl method (Pfaffl, Reference Pfaffl2001). For each group, three samples were taken and used for statistical analysis. LF-derived oocytes from the control group were used as the endogenous control for relative expression analysis.
Gene-specific oligonucleotide primers pairs with annealing temperature, amplicon sizes and references
Statistical analysis
For the analysis of cleavage and blastocysts data, all replicated proportional development data were arc-sin transformed prior to analysis. Statistical analysis within groups was carried out by independent t-tests and one-way analysis of variance (ANOVA) to make comparison between the groups using SAS 9.2 software (SAS Institute Inc., Cary, NC, USA) using Tukey's test and differences with a P-value < 0.05 were considered as statistically significant. All data were presented as mean ± standard error (SE). qPCR data were analyzed statistically by independent t-test for within group analysis followed by one-way ANOVA for comparison between the groups using SAS 9.2 software (SAS Institute Inc., Cary, NC, USA) and Graph Pad In Stat Software (Peter Russell, Royal Veterinary College) and a P-value < 0.05 was considered to be statistically significant.
Results
Effect of OSFs on in vitro embryo production
Cumulus expansion was improved in the GDF9–BMP15 combination group followed by the DO, GDF9, BMP15 and control groups and was better in the LF groups than in the SF groups for all respective treatments (Fig. 1 and Table 2). The LF-derived oocytes depicted significantly (P < 0.05) higher cleavage and blastocyst rates as compared with the SF-derived oocyte for the control groups compared with other groups; no significant (P > 0.05) differences were noticed among other groups. Cleavage and blastocyst rates were significantly (P < 0.05) higher in the GDF9–BMP15 combination group and the DO group than in the other groups, irrespective of follicle sizes (Table 2). No significant difference (P > 0.05) was observed between the GDF9–BMP15 combination group and the DO group. Similarly, there were no significant differences (P > 0.05) between control, GDF9 and BMP15 groups.
Cumulus expansion in different experimental groups supplemented with OSFs: cumulus expansion was observed at 24 h of maturation. Expansion of cumulus cells is an index of measurement for the developmental competence of oocytes.
Effect of OSFs on LF-derived and SF-derived oocytes in different experimental groups
A,B,a,b Values (mean ± SE) along column represented by different superscript letters differ significantly (P < 0.05).
Values (mean ± SE) within group represented by asterisk mark differ significantly (P < 0.05).
Five replicates were taken for each group.
Cumulus expansion was graded on a scale of +1 to +5.
Cleavage rate: proportion of total oocytes cultured resulting in two-cell embryos.
Blastocyst rate: proportion of cleaved oocytes resulting in blastocyst development.
Genes expression profile of oocyte competence markers
All the oocyte competence markers, EGFR, HAS2, and CD44, were expressed in CCs of in vitro matured oocytes, irrespective of follicle sizes. Although, mRNA expression analysis revealed no specific pattern when compared between LF-derived and SF-derived oocytes, overall EGFR, HAS2, and CD44 expression was higher in LF oocytes than SF oocytes (Fig. 2). The relative expression of EGFR was higher in CCs from LF-derived oocytes compared with the SF-derived oocytes in all the treated groups. Supplementation of GDF9 in culture medium significantly (P < 0.05) upregulated EGFR expression as compared with BMP15 supplementation; while, the combination of GDF9–BMP15 exhibited significantly (P < 0.05) higher expression as compared with the control. A significant (P < 0.05) upregulation in EGFR expression in oocytes matured in the presence of DOs as compared with the other groups was noted. Similarly, HAS2 was upregulated significantly (P < 0.05) by GDF9 supplementation, but was further upregulated by BMP15 supplementation and further upregulated significantly (P < 0.05) in DOs, as well in the GDF9–BMP15 combination group (Fig. 2). CD44 expression was relatively not influenced (P > 0.05) by supplementation of GDF9 and BMP15 independently, irrespective of the follicle size. However, it was upregulated significantly (P < 0.05) in the GDF9–BMP15 combination group in comparison with all other groups. A non-significant (P > 0.05) increase in CD44 expression was registered in SF-derived oocytes.
Relative gene expression of oocyte competence markers (EGFR, HAS-2 and CD44). Data are presented as mean ± standard error (SE) and groups with different superscripts differ significantly (P < 0.05). Capital and small letters represents LF-derived and SF-derived oocytes respectively. Subgroups with different superscripts vary significantly. EGFR: epidermal growth factors receptor, HAS2: hyaluronan synthase, CD44: cluster of differentiation 44.
Discussion
The present study investigated the effect of OSF supplementation during IVM on buffalo oocyte developmental competence and the effect of OSFs on the expression profile of competence markers in CCs. Fatehi et al. (Reference Fatehi, van den Hurk, Colenbrander, Daemen, van Tol, Monteiro, Roelen and Bevers2005) reported that BMP2, BMP4, and the types I and II receptor systems play an important role in development and functioning of follicles rather than in final oocyte maturation and cumulus expansion. During the study, the effect of OSF supplementation during IVM on blastocyst rate varied among the groups. Co-culture of COCs (derived from LFs and SFs) with denuded oocytes during IVM significantly improved the cleavage and blastocyst rates. This result indicated that the denuded oocytes secrete some mitogenic factors that stimulated further cytoplasmic maturation of oocytes. Improved developmental competence has been documented following DO–COC co-culture in mouse (Sudiman et al., Reference Sudiman, Ritter, Feil, Wang, Chan, Mottershead, Robertson, Thompson and Gilchrist2014), pig (Gomez et al., Reference Gomez, Kang, Koo, Kim, Kwon, Park, Atikuzzaman, Hong, Jang and Lee2012), cattle (Hussein et al., Reference Hussein, Sutton-McDowall, Gilchrist and Thompson2011; Yeo et al., Reference Yeo, Gilchrist, Thompson and Lane2008) and goat (Romaguera et al., Reference Romaguera, Morato, Jimenez-Macedo, Catala, Roura, Paramio, Palomo, Mogas and Izquierdo2010; Dey et al., Reference Dey, Deb, Ha, Lee, Bang, Lee and Kong2012).
No significant difference was noticed for the cleavage and blastocyst rates following supplementation of GDF9 and BMP15 independently in LF-derived and SF-derived oocytes in comparison with their respective controls. This finding is in agreement with the study conducted by Sudiman et al. (Reference Sudiman, Ritter, Feil, Wang, Chan, Mottershead, Robertson, Thompson and Gilchrist2014) in cattle, in which no significant difference was observed on supplementation of GDF9 and BMP15 independently in comparison with the control. However, in contrast with these results, increase in blastocyst rate has been reported by other groups on administration of OSFs independently in cattle (Hussein et al., Reference Hussein, Thompson and Gilchrist2006) and mice (Sudiman et al., Reference Sudiman, Ritter, Feil, Wang, Chan, Mottershead, Robertson, Thompson and Gilchrist2014). Cleavage and blastocyst rates were significantly improved in the GDF9 and BMP15 combination and DO groups in comparison with other groups, although there was no significant difference between the GDF9 and BMP15 combination and DO groups. GDF9 and BMP15, therefore, act synergistically and thus improve developmental competence. This simple process, of addition of OSFs, helped overcome the deficiency in endogenous OSFs during IVM. It is possible that BMP15 and GDF9, in combination, activate SMAD1/5/8 and SMAD2/3 pathways, respectively, that are involved in the regulation of oocyte developmental competence. Activation of these pathways is important for oocyte development in mono-ovulatory large animals (Hanrahan et al., Reference Hanrahan, Gregan, Mulsant, Mullen, Davis, Powell and Galloway2004). BMP15 supplementation during IVM is likely to promote the uniform distribution of active mitochondria, thereby improving functional competence (Sutton-McDowall et al., Reference Sutton-McDowall, Mottershead, Gardner, Gilchrist and Thompson2012). Hussein et al. (Reference Hussein, Sutton-McDowall, Gilchrist and Thompson2011) depicted qualitative temporal changes in oocyte paracrine factor production during maturation and Li et al. (Reference Li, Li, Ou, Zhang, Ren, Wei, Zhang and Yang2014) found that GDF9 and BMP15 mRNA expression levels were closely associated with oocyte maturation, fertilization, embryo quality, and pregnancy outcome. OSFs exert synergistic beneficial effects on nuclear and cytoplasmic maturation, rapid utilization of energy and management of oxidative stress (Romaguera et al., Reference Romaguera, Morato, Jimenez-Macedo, Catala, Roura, Paramio, Palomo, Mogas and Izquierdo2010). In contrast with our report, Hussein et al. (Reference Hussein, Thompson and Gilchrist2006) observed no increase in blastocyst yield when supplementing the combination of GDF9 and BMP15 in cattle COCs. In this study, cleavage and blastocysts rates were significantly higher in LF-derived than SF-derived oocytes in control groups and by supplementation of GDF9 or BMP15 or DOs. There was no significant difference between LF-derived and SF-derived oocytes, this result indicated that such supplementation is more effective in SF-derived oocytes than in LF-derived oocytes. This result is in corollary with the fact that large follicle-derived oocytes are more competent than small follicle-derived oocytes (Lonergan et al., Reference Lonergan, Monaghan, Rizos, Boland and Gordon1994).
Competent oocytes express higher levels of CCs transcripts, such as HAS2, GREM1, EGFR and TNFAIP6, which are responsible for cumulus expansion (Bhardwaj et al., Reference Bhardwaj, Ansari, Pandey, Parmar, Chandra, Kumar and Sharma2016a). The effect of OSF supplementation during in vitro maturation on relative gene expression of competence markers in CCs was also studied. A significant increase in gene expression was observed in both the DO co-cultured and in the GDF9–BMP15 combination group in LF oocytes.
CD44 and HAS2 expression in the BMP15 supplemented group was significantly higher than in the control and was related to hyaluronic acid production in CCs (Schoenfelder & Einspanier, Reference Schoenfelder and Einspanier2003) as HAS2 mRNA expression in CCs correlates with hyaluronic acid (HA) production. HAS2 was differentially expressed in human CCs, as the quality of human oocytes varies with HAS2 expression (Cillo et al., Reference Cillo, Brevini, Antonini, Paffoni, Ragni and Gandolfi2007). Hyaluronic acid was found to delay death and prevent fragmentation of porcine oocytes (Tunjung et al., Reference Tunjung, Yokoo, Hoshino, Miyake, Kadowaki and Sato2009). In SF-derived oocytes, the DO group and the GDF9–BMP15 combination group had a significantly higher transcript level of oocyte competence markers, whereas no significant differences were noticed in the other two groups compared with the control. CD44 expression was higher in the DO-supplemented group than in the GDF9 and control groups, with no significance for the BMP15-supplemented experimental group. CD44 is a cell adhesion receptor with HA as its main ligand. It is mainly involved in cell adhesion, migration, inflammatory response and apoptosis inhibition (Lesley et al., Reference Lesley, Gal, Mahoney, Cordell, Rugg, Hyman, Day and Mikecz2004; Eshkar et al., Reference Eshkar, Ronen, Levartovsky, Elkayam, Caspi, Aamar, Amital, Rubinow, Golan, Naor and Zick2007). CD44 has only been reported in mature oocytes, while it was absent in humans (Campbell et al., Reference Campbell, Swann, Aplin and Seif1995), cattle (Furnus et al., Reference Furnus, Valcarcel, Dulout and Errecalde2003), pigs (Toyokawa et al., Reference Toyokawa, Harayama and Miyake2005) and mice (Wheatley et al., Reference Wheatley, Isacke and Crossley1993). These findings suggest the positive effect of BMP15 on the expression of CD44 and HAS2 transcripts which in turn further improved oocyte developmental competence. CD44 is absent in immature oocytes, as matrix formation during this stage would affect nutrient exchange and paracrine signaling (Yokoo et al., Reference Yokoo, Kimura and Sato2010).
A significant difference was observed in EGFR expression of both LF-derived and SF-derived oocytes, thus EGFR can be considered as an important determinant of oocyte competence. EGFR expression was significantly higher in the DO group than in the control and GDF9 supplemented groups, with no significant difference compared with the GDF9–BMP15 combination group. OSFs facilitate cross-talk between CCs and oocytes via upregulation of EGFR, HAS2, and CD44 expression, therefore the developmental competence of oocytes is increased.
It can be concluded from this study that LF-derived oocytes have higher developmental competence than SF-derived oocytes. Supplementation of OSFs modulates the developmental competence of buffalo oocytes, irrespective of follicular size, by increasing the relative abundance of cumulus-enabling factors and thereby increasing cleavage and the quality of blastocyst production. Furthermore, EGFR, HAS2, and CD44 expression could be used as markers in CCs for the selection of competent buffalo oocytes for in vitro embryo production.
Acknowledgements
The authors acknowledge the Director of the ICAR-IVRI for providing funds and facilities for this research work.
