Introduction
It is well known that pre-antral follicles (primordial, primary and secondary) represent 90% of the follicular population, but that less than 1% of this vast population will ovulate, while most suffer atresia (Bonnet et al., Reference Bonnet, Dalbiès-Tran and Sirard2008). Several techniques for isolating and culturing these follicles have been developed in an attempt to avoid this huge oocyte loss in the ovary. Several research groups have cultured secondary follicles (of approximately 0.2 mm) up to antral stages of development (bovine: Gutierrez et al., Reference Gutierrez, Ralph, Telfer, Wilmut and Webb2000; ovine: Cecconi et al., Reference Cecconi, Barboni, Coccia and Mattiol1999; caprine: Magalhães et al., Reference Magalhães, Duarte, Araújo, Brito, Soares, Lima, Lopes, Campello, Rodrigues and Figueiredo2010) and, recently, after maturation and after in vitro fertilization of oocytes from secondary follicles grown in vitro, production of embryos have been described in caprine (Magalhães et al., Reference Magalhães, Duarte, Araújo, Brito, Soares, Lima, Lopes, Campello, Rodrigues and Figueiredo2010; Saraiva et al., Reference Saraiva, Rossetto, Brito, Celestino, Silva, Faustino, Almeida, Bruno, Magalhães, Matos, Campello and Figueiredo2010) and ovine (Arunakumari et al., Reference Arunakumari, Shanmugasundaram and Rao2010) species. However, the number of embryos produced in vitro is still very small, because the majority of the cultured follicles is not viable for this purpose. Thus, to optimize embryo production after fertilization of oocytes collected from in vitro grown pre-antral follicles, it is very important to study the expression of factors belonging to the IGF system in in vitro grown follicles and in differently sized in vivo follicles. It is known that the IGF system is involved in the growth and differentiation of ovarian follicles (reviewed by Silva et al., Reference Silva, Figueiredo and Van den Hurk2009).
The IGF system includes two ligands, IGF1 (IGF-I) and IGF2 (IGF-II), two receptors (IGF1R and IGF2R) and a family of high-affinity IGF binding proteins (IGFBP-1, IGFBP-2. IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6). The type 1 receptor mediates most of the actions of both IGF1 and IGF2. The affinity of the receptor for IGF1 is slightly higher than for IGF2, and much higher than for insulin (Le Roith et al., Reference Le Roith, Bondy, Yakar, Jun-Li and Butler2001). The type 2 receptor binds IGF1 and IGF2 with very low affinity, but does not bind insulin. IGFBPs regulate the availability of IGFs to their target cells by either inhibiting or potentiating their action. IGFBPs can sequester extracellular IGFs and hence reduce the bioavailability of IGF (Giudice, Reference Giudice1992; Silva et al., Reference Silva, Figueiredo and Van den Hurk2009). In ovine and bovine ovaries, IGFBP-2 and IGFBP-5 are synthesized locally in the follicle wall by granulosa cells, and IGFBP-4 and IGFBP-6 in theca cells (Armstrong et al., Reference Armstrong, Baxter, Gutierrez, Hogg, Glazyrin, Campbell, Bramley and Webb1998). In follicular fluid, the levels of IGFBP-2, IGFBP-4 and IGFBP-5 decrease during growth and development of bovine dominant follicles (Spicer & Echternkamp, Reference Spicer and Echternkamp1995; Stewart et al., Reference Stewart, Spicer, Hamilton, Keefer, Dawson, Morgan and Echternkamp1996). However, the mRNA levels for these IGF-system components in bovine follicles at early stages of development have not yet been reported.
The aim of the present study is to investigate: (1) the variation in expression levels of IGF1 and IGF2, their type 1 and type 2 receptors and their six binding proteins (IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6) in bovine follicles with a diameter of 0.2, 0.5 and 1.0 mm; and (2) to compare the levels of mRNA for these IGF-system members in in vivo and in vitro grown follicles.
Material and methods
Ovaries and follicle isolation
Ovaries from adult cows (n = 20) demonstrating cyclicity, shown by the presence of the dominant follicle or corpus luteum, were collected at a local abattoir immediately after slaughter. After collection, the ovaries were washed once in ethanol 70% for about 10 s, and then twice in 0.9% saline solution and transported to the laboratory at 4°C within 1 h. Next, the ovaries were carefully dissected and placed immediately in warmed α-MEM culture medium. Briefly, ovarian cortical slices (1–2 mm thick) were cut from the ovarian surface and follicles were visualized under a stereomicroscope (SMZ645; Nikon, Tokyo, Japan), manually isolated using 26-gauge needles attached to a syringe and washed twice in α-MEM. After isolation, follicles were transferred to 100-μl droplets that contained fresh medium at room temperature for evaluation. For this study, follicles of 0.21 ± 0.055, 0.52 ± 0.053 and 0.96 ± 0.057 in diameter with an intact basal membrane and a centrally located oocyte, surrounded by compact layers of granulosa cells, were selected and distributed randomly into three groups of 10 follicles each and stored at –80°C until RNA extraction.
In vitro culture of secondary follicles
For in vitro culture, healthy follicles of 0.21 ± 0.50 mm (n = 24) were isolated from five ovaries and randomly transferred to 100-μl droplets that contained fresh medium under mineral oil, to evaluate the follicular quality. Then, healthy pre-antral follicles with a visible oocyte, surrounded by granulosa cells, an intact basement membrane and no antral cavity were selected and individually cultured in 100-μl droplets of culture medium cell culture plates with 24 wells (TTP, Switzerland). Fresh medium was prepared and immediately incubated for 1 h at 39°C prior to use. The culture medium was designated modified α-MEM and consisted of α-MEM (pH 7.2–7.4) supplemented with 1.25 mg/ml bovine serum albumin (BSA), insulin 6.25 μg/ml, transferrin 6.25 μg/ml and selenium 6.25 ng/ml (ITS), 200 mM glutamine, 50 μg/ml of ascorbic and 100 ng/ml activin-A (R&D Systems, Minneapolis, USA). A previous study has demonstrated that activin-A has a beneficial effect on growth of bovine secondary follicles (McLaughlin et al., Reference McLaughlin, Bromfield, Albertini and Telfer2010). Incubation of follicles was conducted at 39°C for 12 days, a period in which the follicles reached a mean diameter of 0.5 mm. Pre-antral follicles were distributed randomly in modified α-MEM, which was supplemented with 100 ng/ml of ovine follicle-stimulating hormone (FSH, Sigma, USA; from day 0 [D 0] to day 6 [D 6]) and 500 ng/ml FSH (from day 6 [D 6] to day 12 [D 12]). Every 2 days, 50 μl of the culture medium was added. Before and after culture, three groups of eight cultured follicles were collected and stored at –80°C until RNA extraction.
RNA extraction and cDNA synthesis
Isolation of total RNA was performed using the Trizol® Plus purification kit (Invitrogen, São Paulo, Brazil). DNA digestion was performed using RNase-free DNase (340 Kunitz units/ml) for 15 min at room temperature. After extraction, RNA concentration was estimated by reading absorbance at 260 nm and was checked for purity at 280 nm in a spectrophotometer (Amersham, Biosciences Cambridge, UK). For each sample, RNA concentrations were adjusted to 44 ng/ml and used to synthesize cDNA. Before the reverse transcription reaction, samples of RNA were incubated for 5 min at 70°C and then cooled in ice. The reverse transcription was performed in a total volume of 20 μl composed of 10 μl of sample RNA, 4 μl reverse transcriptase buffer (Invitrogen, São Paulo, Brazil), 8 units RNasin, 150 units of reverse transcriptase Superscript III, 0036 U random primers, 10 mM DTT and 0.5 mM of each dNTP (Invitrogen, São Paulo, Brazil). The mixture was incubated at 42°C for 1 h, subsequently at 80°C for 5 min, and finally stored at –20°C. The negative control was prepared under the same conditions, but without the addition of reverse transcriptase.
Polymerase chain reaction (PCR) amplification and determination of gene stability
Quantification of mRNA in ovarian follicles has been performed successfully using real-time PCR (Kawashima et al., Reference Kawashima, Okazaki, Noma, Nishibori, Yamashita and Shimada2008; Chen et al., Reference Chen, Yu, Wang, Xu and Yang2009), but it is very important to use housekeeping genes that show only minimal changes in expression levels between samples. These genes are used to normalize mRNA levels of genes of interest. To identify the most stable housekeeping gene to use in bovine follicles, quantification of mRNA for GAPDH, β-tubulin, β-actin, PGK, 18S rRNA, ubiquitin and RPL-19 was performed with the use of SYBRGreen. Each reaction in real time (20 μl) contained 10 μl of SYBR Green Master Mix® (Applied Biosystems, Warrington, UK), 7.3 μl of ultrapure water and 1 μl of cDNA. Primers were optimized to determine the minimum primer concentration that gave the maximum result, and therefore a final concentration of 0.85 μM, which demonstrated high efficiency for each primer, was used. Real-time PCR was performed in a thermocycler (Master Cycler, Eppendorf, Germany). The primers chosen to carry out amplification of different housekeeping genes, as shown in Table 1, were designed using PrimerQuest™ software (http://www.idtdna.com). cDNA reactions for PCR amplification consisted of initial denaturation and polymerase activation for 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, 30 s at 58°C and 30 s at 60°C. The final extension period was 20 min at 72°C. After amplification, the melting curve was analyzed to evaluate the presence of specific PCR products.
Primer pairs used in real-time PCR for quantification of housekeeping genes in fresh bovine follicles
Concentration of each primer = 0.85 μM.
Gene stability was evaluated using geNorm software (Vandesompele et al., Reference Vandesonpele, De Preter, Pattyn, Poppe, Van Rou, De Paepe and Speleman2002). Briefly, for this calculation, the expression ratio of two perfect reference genes would be identical in all samples in all experimental conditions or cell types. Variation in the expression ratios between different samples reflects expression instability of one or both of the genes. Therefore, increasing variation in this ratio corresponds to decreasing expression stability. geNorm software can be used to calculate the gene expression stability measure (M), which is the mean pair-wise variation for a gene compared with all other tested control genes. Genes with higher M-values have greater variation in expression. The stepwise exclusion of the gene with the highest M-value allows the ranking of the tested genes according to their expression stability.
Quantification of mRNA for IGFs, IGF receptors and binding proteins
The relative mRNA quantities for IGFs, IGF receptors and IGFBPs, respectively, were determined for in vivo grown follicles of approximately 0.2, 0.5 and 1.0 mm. In addition, mRNA from these IGF members was quantified in follicles with a diameter of 0.2 mm (at day 0 of culture) and in 12-day cultured follicles, which reached a diameter of approximately 0.5 mm. The primers chosen to carry out amplification of different IGF-system components (IGF1 and IGF2, IGF1R and IGF2R, and IGFBP-1, IGFBP-2. IGFBP-3, IGFBP-4, IGFBP-5, and IGFBP-6) are shown in Table 2. The ΔΔCT method, that transforms CT values into normalized relative expression by relating the CT value of the target gene of a sample to a calibrator (sample with lowest CT) and to the CT value of reference genes, was used to normalize mRNA expression data (Livak & Schmittgen, Reference Livak and Schmittgen2001). The two most stable reference genes were used to determine a normalization factor, that is basically the geometric mean of the transcription levels of these reference genes (Vandesompele et al., Reference Vandesonpele, De Preter, Pattyn, Poppe, Van Rou, De Paepe and Speleman2002).
Primer pairs used in real-time PCR for quantification of growth factor mRNAs in fresh bovine follicles
Concentration of each primer = 0.85 μM.
Statistical analysis
In the selected differently sized in vivo grown bovine follicles (i.e. those with a diameter of approximately 0.2, 0.5 or 1.0 mm), levels of mRNA for the various IGF-system components under study were analyzed using the non-parametric Kruskal–Wallis test (P < 0.05). The levels of mRNA for the various IGF-system components in uncultured follicles that were approximately 0.2 mm or approximately 0.5 mm in diameter, as well as in 12-day cultured follicles (approximately 0.5 mm in diameter) were compared using a t-test.
Results
Stability of housekeeping genes in bovine follicles
The analysis of starting cDNA determined the gene expression stability in bovine follicles of 0.2 mm, 0.5 mm or 1.0 mm in diameter, and resulted in gene expression stability values (M) for each gene. Therefore, stepwise exclusion of unstable genes and the subsequent recalculation of average M-values resulted in a ranking of the genes based on their M-values, with the two most stable genes leading the ranking. After stepwise elimination of the least stable gene (18S rRNA), GAPDH and ubiquitin were the genes with the highest stability in bovine follicles of 0.2 mm, 0.5 mm or 1.0 mm in diameter (Fig. 1A, B) and, thus, were used to normalize expression of the IGF system in these follicles.
Stability of housekeeping genes in fresh bovine follicles of 0.2 mm, 0.5 mm, or 1 mm in diameter before (A) and after (B) elimination of the least stable housekeeping gene (18S RNA).
Levels of mRNA for IGF-system components in fresh isolated follicles
Real-time PCR analyses demonstrated that mRNA levels for IGF1 and IGF2 (Fig. 2) did not differ between the three follicular size classes (P > 0.05). The mRNA expression level for IGF1R was highest in 0.5 mm follicles (Fig. 2), but only significantly different from 1 mm follicles (P < 0.05). mRNA expression levels for IGF2R were also highest in 0.5 mm follicles (Fig. 2), but differed only from 0.2 mm follicles (P < 0.05). The levels of expression for IGFBP-1 (Fig. 2) were significantly higher in 0.2 mm follicles than in the two larger follicle size classes (P < 0.05). IGFBP-2 was significantly more expressed in 0.5 mm follicles compared with 1.0 mm follicles (P < 0.05), while this expression did not significantly differ from that in 0.2 mm follicles (Fig. 2). Transcripts for IGFBP-3 and IGFBP-4 (Fig. 2) were most abundant in 1.0 mm follicles, the expression of IGFBP-3 was significantly different from that in 0.2 mm follicles (P < 0.05), and that of IGFBP-4 was significantly different from that in 0.5 mm follicles (P < 0.05). The levels of mRNAs expression for IGFBP-5 and IGFBP-6 (Fig. 2) did not differ among follicular size classes (P > 0.05).
Expression levels of mRNA (mean ± SEM) for IGF1 (A), IGF2 (B), IGF1R (C), IGF2R (D), IGFBP-1 (E), IGFBP-2 (F), IGFBP-3 (G), IGFBP-4 (H), IGFBP-5 (I) and IGFBP-6 (J) in bovine follicles with a diameter of approximately 0.2, 0.5 and 1,0 mm. a,bDifferent superscripts indicate significantly (P < 0.05) different means among follicle categories.
Levels of mRNA for IGF-system components in cultured follicles
After culture for 12 days, follicles reached a diameter of approximately 0.5 mm (0.48 ± 0.51 mm). In these follicles, significant reduction was detected in mRNA expression levels for IGF1, IGF1R, IGFBP-3, IGFBP-5 and IGFBP-6 (Fig. 3), when compared with the corresponding levels in non-cultured (day 0) follicles with a diameter of approximately 0.2 mm (P < 0.05). In these non-cultured follicles, the levels of mRNA for IGF2, IGF2R, IGFBP-1, IGFBP-2 and IGFBP-4 (Fig. 3) were not significantly different from the corresponding levels in 12-day cultured follicles (P > 0.05). Results from real-time PCR analysis were also comparable with levels of expression of mRNA from in vivo grown follicles (of approximately 0.5 mm) in relation to approximately 0.5 mm in vitro grown follicles. mRNA expression levels for IGFBP-1, IGFBP-2, IGFBP3 and IGFBP-6 (Fig. 4) were significantly lower in follicles (approximately 0.5 mm) cultured for 12 days when compared with 0.5 mm follicles that had been grown in vivo. Significant changes to the IGF system in each category of follicle analyzed (0.2 mm, 0.5 mm or 1.0 mm in diameter) can be observed in Fig. 5.
Expression levels of mRNA (mean ± SEM) for IGF1 (A), IGF2 (B), IGF1R (C), IGF2R (D), IGFBP-1 (E), IGFBP-2 (F), IGFBP-3 (G), IGFBP-4 (H), IGFBP-5 (I), IGFBP-6 (J) in bovine follicles of approximately 0.2 mm at the start of In vitro culture (D0) and in 12-day cultured follicles with a diameter of approximately 0.5 mm. *Significant difference between day 0 and day 12 (P < 0.05).
Expression levels of mRNA (mean ± SEM) for IGF1 (A), IGF2 (B), IGF1R (C), IGF2R (D), IGFBP-1 (E), IGFBP-2 (F), IGFBP-3 (G), IGFBP-4 (H), IGFBP-5 (I), IGFBP -6 (J) in fresh bovine follicles of approximately 0.5 mm (D0) and in 12-day cultured follicles of approximately 0.5 mm. *Significant difference between in vivo and in vitro grown follicles (P < 0.05).
Schematic drawing of expression levels for IGF-system components in the different follicular categories analyzed.
Discussion
This study is the first to demonstrate and compare mRNA expression for the IGF system in bovine pre-antral and early antral follicles. IGF1 and IGF2 mRNA levels did not differ between bovine follicles of approximately 0.2, 0.5 or 1.0 mm diameter, Previously, using in situ hybridization, Perks et al. (Reference Perks, Peters and Wathes1999) were unable to demonstrate gene expression for IGF1 in bovine follicles. These data emphasize the importance of using other techniques, such as real-time PCR, to detect even small amounts of mRNA. However, these authors did detect intense IGF2 expression in small antral follicles (2.0–5.0 mm) and demonstrated that this expression decreased with antral follicular growth. This finding was confirmed by Llewellyn et al. (Reference Llewellyn, Fitzpatrick, Kenny, Murphy, Scaramuzzi and Wathes2007), who found higher thecal IGF2 mRNA expression in bovine follicles with diameters of 1.0–2.5 mm compared with those of 2.5–5.0 mm. In sheep, IGF2 mRNA was demonstrated in theca cells of early healthy atretic follicles, with increased expression in small antral follicles (Hastie & Haresign, Reference Hastie and Haresign2006).
The levels of mRNA for IGF1R and IGF2R in 0.5 mm bovine follicles were higher than those observed in 1.0 or 0.2 mm follicles, respectively. Increased follicular vascularity and permeability of blood vessels are strongly associated with the growth of an antral follicle (Van den Hurk & Zhao, Reference Van den Hurk and Zhao2005). This increased vascularity may explain the reduced levels of mRNA for IGF type-I receptor in 1.0 mm follicles, as stronger follicular vascularization promotes the increase of IGF1 levels exogenously produced by the liver (Vergani et al., Reference Vergani, Bartke and Mayerhofer1997), and consequently can down-regulate the expression of its receptor. Armstrong et al. (Reference Armstrong, Baxter, Hogg and Woad2002) showed that IGF1R is expressed in granulosa cells and oocytes of bovine pre-antral follicles, while Schams et al. (Reference Schams, Berisha, Kosmann and Amselgruber2002) reported that during final growth of bovine antral follicles, IGF1R mRNA is expressed in the granulosa and internal theca. IGF2R is found in both granulosa cells and in theca cells (Spicer et al., Reference Spicer, Voge and Allen2004). Increase in the number of theca cells in early antral follicles (Parrott & Skinner, Reference Parrott and Skinner2000) may explain the currently observed increased levels of this IGF receptor in 0.5 mm follicles.
During follicular in vivo growth from 0.2 to 1.0 mm, mRNA levels for IGFBP-1 declined in the bovine follicles under study. In a previous study, follicular IGFBP-1 mRNA expression was undetectable in cattle, regardless of the follicle size or follicle health (Llewellyn et al., Reference Llewellyn, Fitzpatrick, Kenny, Murphy, Scaramuzzi and Wathes2007), probably because of the use of a different detection technique. In sheep, IGFBP-1 mRNA was expressed in both healthy and atretic follicles (Muñoz-Gutiérrez et al., Reference Muñoz-Gutiérrez, Findlay, Adam, Wax, Campbell, Kendall, Khalid, Forsberg and Scaramuzzi2005; Hastie & Haresign, Reference Hastie and Haresign2006). Concerning the expression levels of IGFBP-2, a peak of mRNA expression was observed in 0.5 mm follicles, but its level was reduced in 1.0 mm follicles. A decrease in IGFBP-2 was previously reported in antral follicles during their growth and development to a dominant stage (cow: Spicer & Echternkamp, Reference Spicer and Echternkamp1995; Stewart et al., Reference Stewart, Spicer, Hamilton, Keefer, Dawson, Morgan and Echternkamp1996; sheep: Monget et al., Reference Monget, Fabre, Mulsant, Lecerf, Elsen, Mazerbourg, Pisselet and Monniaux2002). In monkeys, IGFBP-2 mRNA is expressed in granulosa cells of both pre-antral and antral follicles (Vendola et al., Reference Vendola, Zhou, Wang, Famuyiwa, Bievre and Bondy1999).
In our study, expression of IGFBP-3 mRNA increased during bovine follicular growth and reached its highest level in 1.0 mm follicles. Little information is known about IGFBP-3 expression in ovarian follicles. Armstrong et al. (Reference Armstrong, Baxter, Hogg and Woad2002) showed that IGFBP-3 is expressed in granulosa cells and oocytes of bovine pre-antral follicles while, in monkeys, IGFBP-3 mRNA is localized in oocytes of both pre-antral and antral follicles (Arraztoa et al., Reference Arraztoa, Monget, Bondy and Zhou2002). Antral follicle growth is accompanied by a slight increase in IGFBP-3 protein in the follicular fluid content of sow (Grimes et al., Reference Grimes, Guthrie and Hammond1994) and ewe (Monget et al., Reference Monget, Monniaux, Pisselet and Durand1993).
IGFBP-4 mRNA expression was significantly higher in 1.0 mm follicles than in 0.5 mm follicles. In other species, antral follicular growth is accompanied by an increase in IGFBP-4 proteolytic degradation (ewe: Besnard et al., Reference Besnard, Pisselet, Monniaux, Locatelli, Benne, Gasser, Hatey and Monget1996; sow: Besnard et al., Reference Besnard, Pisselet, Monniaux and Monget1997). In ruminants, levels of IGFBP-4 mRNA were found to decrease dramatically in follicles during terminal follicular growth (Monget et al., Reference Monget, Fabre, Mulsant, Lecerf, Elsen, Mazerbourg, Pisselet and Monniaux2002). In the ewe, sow, cow and mare, intrafollicular concentrations of IGFBP-4 protein decreased strongly during the development of follicles from 1–2 mm to pre-ovulatory sizes (Monget et al., Reference Monget, Monniaux, Pisselet and Durand1993).
In fresh bovine follicles, the levels of mRNA for IGFBP-5 and IGFBP-6 did not differ between the three categories analyzed. In ovine thecal cells from healthy follicles, IGFBP-5 expression slightly decreased during follicular growth (Wandji et al., Reference Wandji, Gadsby, Simmen, Barber and Hammond2000). In contrast, IGFBP-5 mRNA expression was found to be enhanced in granulosa cells from bovine and ovine atretic follicles (Monget et al., Reference Monget, Fabre, Mulsant, Lecerf, Elsen, Mazerbourg, Pisselet and Monniaux2002). Information on follicular IGFBP-6 is scarce. This protein was detected in theca cells (Schams et al., Reference Schams, Berisha, Kosmann, Einspanier and Amselgruber1999; Llewellyn et al., Reference Llewellyn, Fitzpatrick, Kenny, Murphy, Scaramuzzi and Wathes2007) in parallel with the expression of IGF2 (Baxter, Reference Baxter2000). During growth of small antral follicles to the pre-ovulatory stage, there was a progressive reduction in IGFBP levels, mainly due to changes in the process of their synthesis and degradation (Monget et al., Reference Monget, Fabre, Mulsant, Lecerf, Elsen, Mazerbourg, Pisselet and Monniaux2002). Proteases degrade the IGFBPs in the follicles (Beg & Ginther, Reference Beg and Ginther2006) and this degradation could explain the increased levels of mRNA for IGFBPs as an attempt to balance the changed levels of IGFBPs in the follicles.
Bovine follicles that had reached a diameter of approximately 0.5 mm during in vitro culture showed a reduction in IGF1, IGF1R, IGFBP-3, IGFBP-5 and IGFBP-6 mRNA levels when compared with non-cultured approximately 0.2 mm follicles. Reduction of IGF1 and IGF1R expression can negatively affect both proliferation and differentiation of bovine granulosa cells (Silva et al., Reference Silva, Figueiredo and Van den Hurk2009). Compared with fresh follicles of approximately 0.5 mm in size, when cultured for 12 days these follicles showed a significant reduction in IGFBP-1, IGFBP-2, IGFBP-3 and IGFBP-6 mRNA levels. Reduction of IGFBP expression could be a way to increase IGF1 bioavailability, as its level was reduced in in vitro grown follicles. It is important to consider that, in vivo, several hormones and growth factors interact to promote optimal follicle development, and our results show that culture systems need to be improved to increase the rate of oocyte maturation. The reduction in expression of IGF1 and its receptors in cultured follicles could be an important cause of the previously described (caprine: Saraiva et al., Reference Saraiva, Rossetto, Brito, Celestino, Silva, Faustino, Almeida, Bruno, Magalhães, Matos, Campello and Figueiredo2010; Magalhães et al., Reference Magalhães, Duarte, Araújo, Brito, Soares, Lima, Lopes, Campello, Rodrigues and Figueiredo2010; ovine: Arunakumari et al., Reference Arunakumari, Shanmugasundaram and Rao2010) low efficiency of embryo production from oocytes collected from in vitro grown pre-antral follicles.
In conclusion, IGFs, their receptors and IGFBPs showed variable expression in the follicular size classes analyzed. After 12 days of culture of 0.2-mm sized pre-antral follicles, the levels of mRNA for IGF1, IGF1R, IGFBP-3, IGFBP-5 and IGFBP-6 were reduced in follicles that reached a diameter of 0.5 mm. When compared with 0.5 mm fresh follicles, cultured follicles of the same size show reduction in their expression of IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-6. This variation in expression in the IGF system in in vitro grown follicles can contribute to an understanding of the molecular events that are responsible for reduced rates of maturation in oocytes collected from in vitro grown pre-antral follicles. In addition, data on mRNA expression for the IGF system can be very useful for the improvement of the culture system. The development of large numbers of good quality oocytes that can be used successfully for in vitro maturation and fertilization procedures, that result in high embryo rates, needs to be achieved.
Acknowledgements
This study was supported by CNPq (Grant No 477025/2009–9), and we thank all members of the animal reproduction research group of the Biotechnology Nucleus of Sobral.
