Introduction
The in vitro production (IVP) of embryos has been largely successful in cattle and might improve their genetic background by shortening significantly the generation gap, when compared with artificial insemination and embryo transfer (ET). However, oocyte quality (Lonergan et al., Reference Lonergan, Monaghan, Rizos, Boland and Gordon1994; Otoi et al., Reference Otoi, Yamamoto, Koyama, Tachikawa and Suzuki1997) and in vitro maturation (IVM) conditions (Warzych et al., Reference Warzych, Peippo, Szydlowski and Lechniak2007) may determine the proportion of embryos that are suitable for ET.
It is well known that oocyte competence is acquired during oocyte growth, but is only concluded during the final period of maturation in vivo (Ferreira et al., Reference Ferreira, Vireque, Adona, Meirelles, Ferriani and Navarro2009). Some ultrastructural cytoplasmic changes in the oocyte occur after 18 h and extrusion of the polar body occurs 19 h after the luteinizing hormone (LH) surge (Ferreira et al., Reference Ferreira, Vireque, Adona, Meirelles, Ferriani and Navarro2009). However, the maturation process in vitro is influenced by culture environment; alpha minimum essential medium (α-MEM) defined medium (DCM) has been used to mimic the physiological in vivo conditions as it maintains oestrogen concentrations, aromatase expression in follicular cell walls (Vasconcelos et al., Reference Vasconcelos, Salles, Oliveira e Silva, Gulart, Souza, Torres, Bocca and Rosa e Silva2013) and blockage of oocyte nuclear maturation (Oliveira e Silva et al., Reference Oliveira e Silva, Vasconcelos, Caetano, Gulart, Camargo, Báo and Rosa e Silva2011).
Several growth factors [e.g. epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), and insulin] are known to be involved in mammalian oocyte growth and maturation within ovarian follicles (van den Hurk & Zhao, Reference Van den Hurk and Zhao2005). These factors have been used widely for IVM of bovine cumulus–oocyte complexes (COCs) to improve the results achieved by in vitro procedures (Sagirkaya et al., Reference Sagirkaya, Misirlioglu, Kaya, First, Parrish and Memili2007; Vireque et al., Reference Vireque, Camargo, Serapião, Rosa E Silva, Watanabe, Ferreira, Navarro, Martins and Ferriani2009). The combination insulin–transferrin–selenium (ITS) has also been used to improve the conditions in defined IVM systems (Quirk et al., Reference Quirk, Harman and Cowan2000). The combination of IGF1, insulin, androstenedione, transferrin, and sodium selenite, for maturation of bovine oocytes in serum-free and albumin-free minimum essential medium (DCM) in the absence of follicle-simulating hormone (FSH) could achieve similar results to those of the non-defined medium supplemented with serum in terms of blastocysts production and expression of HSP70-1 and BAX genes (Vireque et al., Reference Vireque, Camargo, Serapião, Rosa E Silva, Watanabe, Ferreira, Navarro, Martins and Ferriani2009).
The role of FSH in the maturation of bovine COC has also been well described in the literature and it acts to enhance cAMP intracellular levels and activate proteins that modulate gene expression (Rodriguez & Farin, Reference Rodriguez and Farin2004). Insulin interacts with FSH in the steroidogenic pathway in granulosa cells (Adashi et al., Reference Adashi, Resnick, Hernandez, Svoboda and Van Wyk1988; Minegishi et al., Reference Minegishi, Hirakawa, Kishi, Abe, Abe, Mizutani and Miyamoto2000; Spicer et al., Reference Spicer, Chamberlain and Maciel2002).
Insulin induces the cellular uptake of glucose and presents mitogenic and anti-apoptotic activity (Augustin et al., Reference Augustin, Pocar, Wrenzycki, Niemann and Fischer2003). Insulin receptor mRNA expression is increased in the pre-ovulatory dominant follicles in granulosa cells (Shimizu et al., Reference Shimizu, Murayama, Sudo, Kawashima, Tetsuka and Miyamoto2008). Insulin also seems to be involved in modulating the response of granulosa cells to gonadotrophins (Willis & Franks, Reference Willis and Franks1995) and has positive effects on the developmental potential of bovine oocytes (Ocaña-Quero et al., Reference Ocaña-Quero, Pinedo-Merlin, Ortega- Mariscal and Moreno-Millan1998) during the IVM process. However, excess insulin may have detrimental effects (Chaves et al., Reference Chaves, Duarte, Rodrigues, Celestino, Silva, Lopes, Almeida, Donato, Peixoto, Moura, Lobo, Locatelli, Mermillod, Campello and Figueiredo2012; Grazul-Bilska et al., Reference Grazul-Bilska, Borowczyk, Bilski, Reynolds, Redmer, Caton and Vonnahme2012; Rhee et al., Reference Rhee, Choi, Lee, Park, Kim, Yi, Oh, Cha, Chang and Lee2013).
Insulin can influence the expression of several genes (O’Brien et al., Reference O’Brien, Streeper, Ayala, Stadelmaier and Hornbuckle2001), but the direct effect of insulin on the levels of transcripts associated with metabolic profile, cellular competence and stress in oocytes is not yet clear. The response to different energy substrates and antioxidant profile of oocytes in vitro might provide an insight into their metabolic requirements. Glucose transport type 1 (GLUT1) mRNAs have been detected in bovine oocytes. It is well known that developmental competence of oocytes decreases with low or high glucose levels, diminishing cytoplasmic maturation and impairing nuclear maturation (Sutton-McDowall et al., Reference Sutton-McDowall, Gilchrist and Thompson2010), therefore glucose uptake is imperative to the metabolic state and viability of the cell. Peroxiredoxin 1 (PRDX1) demonstrated an antioxidative action, is present in the oocyte cytoplasm and the transcript levels decrease after oocyte maturation (Neumann et al., Reference Neumann, Krause, Carman, Das, Dubey, Abraham, Bronson, Fujiwara, Orkin and Van Etten2003; Thélie et al., Reference Thélie, Papillier, Pennetier, Perreau, Traverso, Uzbekova, Mermillod, Joly, Humblot and Dalbiès-Tran2007; Pereira et al., Reference Pereira, Machado, Costa, Serapião, Viana and Camargo2010).
Heat shock proteins (HSP) promote cell protection against heat damage, preventing protein denaturation (Kregel, Reference Kregel2002), blocking apoptosis, and its transcription rate is increased by cellular stress (Kiang & Tsokos, Reference Kiang and Tsokos1998).
Growth differentiation factor 9 (GDF9) is an oocyte-secreted factor, a member of TGF-β family of growth factors, with critical importance in the regulation of normal cumulus cell function, and thus COCs competence (Fair et al., Reference Fair, Carter, Park, Evans and Lonergan2007; Gilchrist & Thompson, Reference Gilchrist and Thompson2007). In addition, GDF9 acts in follicular cells and along with other members of the TGF-β family is crucial for follicle development (Knight & Glister, Reference Knight and Glister2006).
Based on oocyte meiotic arrest (Oliveira e Silva et al., Reference Oliveira e Silva, Vasconcelos, Caetano, Gulart, Camargo, Báo and Rosa e Silva2011) and embryo development (Vireque et al., Reference Vireque, Camargo, Serapião, Rosa E Silva, Watanabe, Ferreira, Navarro, Martins and Ferriani2009) promoted by the use of DCM, the aim of the present study was to evaluate the dose–response effect of insulin in DCM, without IGF1 and in the presence of FSH, by analysing oocyte nuclear maturation, the relative levels of transcripts of genes associated with metabolism, competence and cellular stress, and subsequent embryo development in vitro.
Materials and methods
All reagents used in the study were from Sigma Chemical Co. unless otherwise stated.
Oocyte recovery and selection
Cattle ovaries were obtained at a local slaughterhouse and shipped to the laboratory in warm saline solution (supplemented with 0.1 g/1 of streptomycin). COCs were obtained by aspiration of 3–8 mm follicles using a 21-gauge needle in a 10-ml syringe manipulated with Dulbecco's phosphate-buffered saline (D-PBS; Gibco, Grand Island, NY, USA) supplemented with 0.4% bovine serum albumin (BSA). Only oocytes showing homogeneous cytoplasm and with more than three layers of granulosa cells were used.
Oocyte culture medium composition
The composition of the defined culture medium (DCM) is reported in a patent filled by the Fundação Universidade de Brasília and was used with modifications to evaluate the effect of the different doses of insulin. The DCM was composed of α-MEM medium (Invitrogen-Gibco/BRL) supplemented with polyvinyl alcohol (PVA), 10 ng/ml FSH, androstenedione, non-essential amino acids (Invitrogen-Gibco/BRL), transferrin, sodium selenium, sodium bicarbonate (Invitrogen-Gibco/BRL), HEPES, and antibiotics (penicillin and streptomycin).
Experimental design
Four maturation groups were established, one insulin free (DCM control) and three with COCs matured in DCM supplemented with bovine insulin (Sigma, USA): 1 ng/ml (DCM1); 10 ng/ml insulin (DCM10); and 100 ng/ml (DCM100).
In vitro maturation and fertilization
The in vitro maturation and fertilization procedures were adapted from a previous study (Camargo et al., Reference Camargo, Viana, Ramos, Serapião, de Sa, Ferreira, Guimarães and do Vale Filho2007). Briefly, COCs were matured in vitro in DCM for 22–24 h in a humidified atmosphere with 5% CO2 in air and at 38.5°C. For in vitro fertilization in vitro matured COCs were separated into groups of 25–30, washed and transferred to 100-μl drops of fertilization medium under mineral oil. Motile spermatozoa were obtained after centrifugation in a Percoll discontinuous density gradient (45–90%) and added to the fertilization drop at a final concentration of 1 × 106 ml−1. IVF was performed in Fert–TALP medium (Gordon, Reference Gordon1994), supplemented with penicillamine, hypotaurine, epinephrine, and heparin for 22 h under same conditions used during maturation.
In vitro embryo culture
After fertilization, oocytes were partially stripped by mechanical pipetting in CR2aa medium (previously equilibrated in 5% CO2 in air and in a humidified atmosphere at 38.5°C) until one or two layers of cumulus cells remained. Groups of 15–20 presumptive zygotes with their respective cumulus cells were subsequently cultured in 50 μl of CR2aa medium supplemented with 10% fetal calf serum (FCS) and covered with mineral oil. Embryo culture was performed in 5% CO2 in air in a humidified atmosphere at 38.5°C. The rates of cleavage and blastocyst generation were assessed at 72 h post-insemination (hpi), and 192 hpi (day 8), respectively.
Oocyte nuclear maturation evaluation
At the end of the IVM period, 236 COCs were denuded and fixed in 3:1 ethanol:acetic acid solution for 48 h and subsequently transferred to glass slides in small drops. Vaseline and paraffin were used to maintain the coverslip in contact with the oocytes. Slides were stained with 1% lacmoid for the oocyte nuclear maturation analysis under a phase contrast microscope at ×400 or ×1000 magnification (Sirard and Coenen, Reference Sirard and Coenen1993). Oocytes were classified in stages as immature (germinal vesicle and germinal vesicle breakdown), intermediary (metaphase I to telophase I), and mature (metaphase II).
Total RNA extraction and reverse transcription
After IVM, three pools of 10 oocytes from each group had their cumulus cells removed by repeated pipetting in PBS medium supplemented with PVA and then were frozen in liquid nitrogen. These samples were stored for 2 weeks at –80°C before RNA extraction. Total RNA was extracted using the RNeasy Micro Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions, and treated with DNase I (27 units for 15 min at room temperature for every sample). The RNA samples were reverse transcribed using the SuperScript III First-Strand Synthesis Supermix (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's instructions. Reactions were performed using oligo(dT)20 primers, dNTP mix, Superscript™ III RT, RNaseOUT™, MgCl2, RT buffer in a final volume of 20 μl. The samples were first incubated at 65°C for 5 min and then for 50°C for 50 min. The reaction was terminated at 85°C for 5 min and then chilled on ice. After that, RNase H was added to the samples and these were incubated at 37°C for 20 min. The RNA and cDNA quantification and purity for each sample was performed using 1 μl of sample in spectrophotometer ND-100 (NanoDrop, Wilmington, DE, USA).
Relative quantification by real-time polymerase chain reaction (PCR)
Relative quantification was performed in triplicate using real-time PCR (ABI Prism 7300 Sequence Detection Systems, Foster City, CA, USA). Reactions were prepared using a mixture of SYBR Green PCR Master Mix (Applied Biosystems), primers, nuclease-free water, and cDNA. The amounts of primers and cDNA used in the reactions were previously standardized to achieve greater primer efficiency. The following cDNA amounts per reaction were used: 600 ng for the peroxiredoxin 1 (PRDX1) and heat shock protein 70.1 (HSP70-1) genes, 400 ng for the glucose transporter type 1 (GLUT1), and 200 ng for the ACTB and differentiation factor 9 (GDF9) genes.
The cycling conditions were 95°C for 10 min, 45 cycles at 95°C for 15 s, the gene-specific primer annealing temperature for 30 s (Table 1), and 60°C for 30 s. After each PCR run, a melting curve analysis was performed to confirm that a single specific product was generated. No-template controls (NTC), comprised of the PCR reaction mix without nucleic acid, were also run with each primer to confirm the absence of contaminations. Primer efficiency was calculated using LinRegPCR software (Ramakers et al. Reference Ramakers, Ruijter, Deprez and Moorman2003) for each reaction. The primer efficiency for target genes was 1.85 ± 0.03, 1.74 ± 0.03, 1.85 ± 0.03 and 1.85 ± 0.08 for GDF9, GLUT1, PRDX1 and HSP70-1 genes, respectively. Expression of ACTB and GAPDH genes were analysed in order to identify a better endogenous reference gene (endogenous control) for this experiment. ACTB showed lower coefficient of variation of threshold cycles (1.5%) and higher primer efficiency (1.95 ± 0.06) among replicates than GAPDH (1.7% and 1.93 ± 2.2) gene and it was then chosen as reference gene. Relative abundance (RA) analyses were performed using the Relative expression software tool (REST) (Pfaffl et al., Reference Pfaffl, Horgan and Dempfle2002) and based on primer efficiency.
Sequences of target bovine gene primers and melting temperatures
F, forward primer; R, reverse primer.
Statistical analysis
The differences in oocyte nuclear maturation, cleavage and blastocyst rates after seven replicates were assessed by the chi-squared (×2) test. The relative gene expression analyses were performed using the REST (Pfaffl et al., Reference Pfaffl, Horgan and Dempfle2002) using a pairwise fixed reallocation randomization test. Differences with P < 0.05 were considered to be significant.
Results
Dose–response of insulin effect on nuclear maturation of COCs in vitro
The nuclear maturation rates of 236 oocytes are presented in Fig. 1. The presence of insulin in the defined medium did not change the nuclear maturation rates (immature, intermediary, or mature) in all groups analysed after 24 h of IVM (P > 0.05) (Fig. 1).
Nuclear maturation status of bovine COCs (n = 236) matured for 24 h in DCM supplemented with different doses of insulin. Results are expressed in percentage (%).aIndicates that there is no difference within each nuclear maturation stage among the four groups of insulin (P < 0.05), determined by chi-squared test. DCM control, DCM without insulin supplementation. DCM1, DCM control supplemented with 1 ng/ml of bovine insulin. DCM10, DCM control supplemented with 10 ng/ml of bovine insulin. DCM100, DCM control supplemented with 100 ng/ml of bovine insulin.
Dose–response effect of insulin in relative quantification of transcripts in oocytes
The relative transcript levels of GDF9, GLUT1, PRDX1, and HSP70-1 in oocyte were assessed on COCs matured in DCM control, DCM1, DCM10 or DCM100. These relative amounts of mRNAs were compared with the calibrator, the insulin-free group (DCM control).
The insulin concentrations used in DCM did not change the relative levels of the GLUT1 transcript, while DCM1, DCM10, and DCM100 decreased the levels of the GDF9 and HSP70-1 transcripts (Figs. 2 and 3). The relative levels of PRDX1 transcripts only decreased in the DCM10, and DCM100 groups (Fig. 3).
Relative abundance of transcripts related to maturation (GDF9) and glucose transporter 1 (GLUT1) in bovine oocytes matured in DCM supplemented with different doses of insulin. *Indicates significant difference (P < 0.05) in comparison with DCM 0 ng/ml insulin versus DCM with 1, 10 or 100 ng/ml. The results are shown as mean and standard error using the medium as DCM–calibrator (score 1). DCM control, DCM without insulin supplementation; DCM1, DCM control supplemented with 1 ng/ml of bovine insulin; DCM10, DCM control supplemented with 10 ng/ml of bovine insulin; DCM100, DCM control supplemented with 100 ng/ml of bovine insulin.
Relative abundance of transcripts related to cell stress (PRDX1 and HSP70-1) in bovine oocytes matured in DCM supplemented with different doses of insulin. *Indicates difference (P < 0.05) in comparison with DCM 0 ng/ml insulin versus DCM with 1, 10 or 100 ng/ml. The results are shown as mean and standard error using the medium as DCM–calibrator (score 1). DCM control, DCM without insulin supplementation; DCM 1, DCM control supplemented with bovine insulin at 1 ng/ml; DCM 10, DCM control supplemented with bovine insulin at 10 ng/ml; DCM 100, DCM control supplemented with bovine insulin at 100 ng/ml.
Dose–response effect of insulin in IVM on embryo production rate in vitro
No significant differences for oocyte maturation were observed in embryo cleavage rates among DCM control, DCM1, DCM10, and DCM100 groups (P > 0.05) (Table 2). A significant higher blastocyst formation rate was observed in the DCM100 group in comparison with the DCM control group (P < 0.05) (Table 2). No difference was observed in the blastocyst generation rates among DCM control, DCM1, and DCM10 groups (P > 0.05) (Table 2).
Effect of insulin in defined culture medium (DCM) of COCs on cleavage and blastocyst production
a ,b Different letters in the same column differ statistically by chi-squared test (P < 0.05).
No., total number of oocytes/group.
DCM control, DCM without insulin supplementation; DCM1, DCM control supplemented with 1 ng/ml of bovine insulin; DCM10, DCM control supplemented with 10 ng/ml of bovine insulin; DCM100, DCM control supplemented with 100 ng/ml of bovine insulin.
Discussion
This experiment evaluated the developmental competence of bovine COCs matured in α-MEM medium supplemented with different concentrations of insulin and with a fixed concentration of FSH. Our data show that use of α-MEM serum-free medium produced suitable bovine COC maturation and embryos. Insulin supplementation increased the in vitro production of embryos and was associated with changes in the levels of GDF9, PRDX1, and HSP70-1 transcripts.
In the present study, three insulin concentrations and a fixed concentrations of FSH, plus transferrin and sodium selenium were also used, to detect any direct effect of insulin. Several studies have suggested that insulin alone (Lee et al., Reference Lee, Kang, Lee and Hwang2005), or in combination with transferrin and selenium in IVM systems, improves the developmental competence of COCs in prepubertal bovine (Córdova et al., Reference Córdova, Morató, de Frutos, Bermejo-Álvarez, Paramio, Gutiérrez-Adán and Mogas2011), porcine (Jeong et al., Reference Jeong, Hossein, Bhandari, Kim, Kim, Park, Lee, Park, Jeong, Lee, Kim and Hwang2008), and buffalo (Raghu et al., Reference Raghu, Nandi and Reddy2002). Similarly, our results demonstrated that the nuclear maturation stage was not affected by the presence of different doses of insulin, however nuclear maturation was significantly lower compared with commercial medium supplemented with higher doses of FSH and serum (data not shown). Cytoplasmatic maturation progression and low rates of nuclear maturation have been described previously as a consequence of DCM culture conditions (Oliveira e Silva et al., Reference Oliveira e Silva, Vasconcelos, Caetano, Gulart, Camargo, Báo and Rosa e Silva2011) and due to the presence of low FSH concentrations in culture (de Souza et al, Reference de Souza, Tuany, Nobre, Sousa and Rosa a Silva2013).
Insulin promotes the proliferation and steroidogenic activity induced by FSH in granulosa cells (Spicer et al., Reference Spicer, Chamberlain and Maciel2002). Insulin and IGF1 increase the activity of granulosa cells in culture in part by increasing the number of FSH receptors (rFSH) and levels of rFSH mRNA (Adashi et al., Reference Adashi, Resnick, Hernandez, Svoboda and Van Wyk1988; Minegishi et al., Reference Minegishi, Hirakawa, Kishi, Abe, Abe, Mizutani and Miyamoto2000). The role of FSH in the maturation of bovine COCs is well known. FSH acts on cumulus cells by increasing the levels of cAMP that is transmitted to oocytes via gap junctions. This temporary increase in the cAMP levels in the oocyte activates protein kinase II (PKAII), which in turn stimulates the transcription of genes necessary for maturation (Rodriguez & Farin, Reference Rodriguez and Farin2004). Therefore, it is possible that the cumulus cells also exhibit higher levels of rFSH after insulin stimulation and influence oocyte maturation, however more experiments need to be done to elucidate these physiological interactions in vitro.
Transcripts related to metabolic profile, cellular competence and stress were analysed in order to investigate oocyte competence after culture in different doses of insulin, bearing in mind that the maturation process per se can lead to RNA degradation compared with the immature oocyte (Thélie et al., Reference Thélie, Papillier, Pennetier, Perreau, Traverso, Uzbekova, Mermillod, Joly, Humblot and Dalbiès-Tran2007).
Insulin reduced the transcript levels of GDF9, which is an oocyte-specific member of the TGF-β family that is involved in proliferation, differentiation, and regulation of cumulus cells (Eppig, Reference Eppig2001; Gilchrist & Thompson, Reference Gilchrist and Thompson2007). This oocyte factor seems to be a cumulus expansion-inductor factor that enables granulosa cells to respond to FSH by producing hyaluronic acid (Eppig, Reference Eppig2001). It has been shown previously that in vivo matured oocytes have lower abundance of GDF9 transcripts than their in vitro matured counterparts (Lonergan et al., Reference Lonergan, Gutiérrez-Adán, Rizos, Pintado, Fuente and Boland2003), despite their higher developmental competence (Humblot et al., Reference Humblot, Holm, Lonergan, Wrenzycki, Lequarré, Joly, Herrmann, Lopes, Rizos, Niemann and Callesen2005). As the transcription activity in oocytes during meiotic maturation is low (Bettegowda & Smith, Reference Bettegowda and Smith2007) and transcripts may be degraded during maturation (Thélie et al., Reference Thélie, Papillier, Pennetier, Perreau, Traverso, Uzbekova, Mermillod, Joly, Humblot and Dalbiès-Tran2007), the decrease of GDF9 transcripts in oocytes with high competence may be due to their translation, favoring further development, coupled to RNA degradation.
Insulin in the defined medium did not alter the abundance of GLUT1 transcripts. Analysis of GLUT1 transcripts in oocytes and embryos has been used to better understand the metabolic process in these structures (Lequarre et al., Reference Lequarre, Grisart, Moreau, Schuurbies, Massip and Dessy1997, Lopes et al., Reference Lopes, Wrenzycki, Ramsing, Herrmann, Niemann, Lovendahl, Greve and Callesen2007). The levels of GLUT1 mRNA in bovine oocytes reduces by half during maturation, perhaps because of the natural degradation of mRNAs during this period (Lequarre et al., Reference Lequarre, Grisart, Moreau, Schuurbies, Massip and Dessy1997). The RA of GLUT1 in mature oocytes does not change with the source of protein used, being oestrous cow serum or PVA (Wrenzycki et al., Reference Wrenzycki, Herrmann, Carnwath and Niemann1999). In addition, studies have indicated that oocytes make use of substrates of glucose metabolism from cumulus cells (Sutton et al., Reference Sutton, Gilchrist and Thompson2003), and glucose metabolism is low in the oocyte during maturation (Rieger & Loskutoff Reference Rieger and Loskutoff1994; Sutton-McDowall et al., Reference Sutton-McDowall, Gilchrist and Thompson2010). These findings indicate that GLUT1 transcript levels in oocytes are largely reduced during maturation and slightly influenced by the maturation medium, and suggest that the positive effect of insulin during oocytes maturation on further embryo development is not associated with an increase in glucose uptake via GLUT1 transporters.
Insulin may play a role in oxidative stress through regulation of antioxidant enzymes (Wang et al., Reference Wang, Tao and Hai2012), but no information is available about the effect of insulin on expression of peroxiredoxins (PRDX) and HSP genes in oocytes. The PRDX1 and HSP70-1 genes have demonstrated antioxidant proprieties in several cells and are present in bovine oocytes (Christians et al., Reference Christians, Zhou, Renard and Benjamin2003, Leyens et al., Reference Leyens, Knoops and Donnay2004, Pereira et al., Reference Pereira, Machado, Costa, Serapião, Viana and Camargo2010). In this study, the addition of 10 or 100 ng/ml of insulin decreased the abundance of PRDX1 and HSP70-1 transcripts. In addition, just 1 ng/ml of insulin induced the downregulation of the HSP70-1 gene. Pereira and collaborators (Pereira et al., Reference Pereira, Machado, Costa, Serapião, Viana and Camargo2010) have observed that, at the end of IVM, oocytes showed relatively higher levels of PRDX1 and HSP70-1 transcripts in medium in which the source of the macromolecule was PVA instead of serum. Our results showed that the addition of 100 ng/ml of insulin in the serum-free defined maturation medium reduced the amount of transcripts associated with protection against oxidative stress and increased the production of embryos. These findings may suggest that insulin may stimulate the translation of transcripts associated with cellular stress during oocyte maturation, increasing protection against stress and, consequently, generating oocytes with low amounts of antioxidant transcripts but with high competence.
The present results provide evidence that the addition of 100 ng/ml of insulin in the DCM increased the bovine blastocyst rate compared with the other groups and independent of FSH action. In addition, the cleavage and blastocyst generation rates were similar to those reported by Vireque and collaborators (Vireque et al., Reference Vireque, Camargo, Serapião, Rosa E Silva, Watanabe, Ferreira, Navarro, Martins and Ferriani2009). In present study, insulin supplemented at 100 ng/ml increased the bovine blastocyst rate when compared with the insulin-free medium, whereas no significant differences was found for lower insulin concentrations. A previous study on antral follicles cultured in the presence of insulin has reported cytoplasmic changes associated with oocyte maturation. Many of these changes occurred before nuclear maturation (Fouladi-Nashta & Campbell, Reference Fouladi-Nashta and Campbell2006). Therefore, we speculate that insulin at appropriated concentrations might contribute to cytoplasmic maturation, making oocytes that reached metaphase II more competent for the production of embryos, as also observed by Oliveira e Silva and collaborators (Oliveira e Silva et al., Reference Oliveira e Silva, Vasconcelos, Caetano, Gulart, Camargo, Báo and Rosa e Silva2011).
The present study indicates the possibility of using insulin in a chemically defined medium, as a factor that is able to reduce the cellular stress response promoted by in vitro conditions. It highlights DCM as an option for serum-free oocyte maturation. However, the results of transcript expression related to oocyte development, metabolism and cellular stress, need to be confirmed at the protein level and additional studies are being conducted by our research group in this regard. In conclusion, our findings indicate that bovine COCs matured in serum-free DCM presented better blastocyst rates when 100 ng/ml insulin is added to the IVM. Furthermore, the use of insulin during maturation influences the levels of GDF9, HSP 70.1, and PRDX1 transcripts in bovine oocytes.
Acknowledgements
This study was supported financially by FAP-DF/CNPq (PRONEX 193.000.577/2009). The authors thank Embrapa Dairy Cattle, Fapemig and FAP-DF/CNPq for financial support for molecular analysis. We also thank CAPES for providing a scholarship for the first author.
