Introduction
The in vitro production (IVP) of preimplantation embryos is a useful tool to increase the reproductive efficiency of valuable animals, allowing the increase of genetic gain, reduction of generation intervals, and serving as a platform for generation of cloned and transgenic goats (Keefer et al., Reference Keefer, Baldassarre, Keyston, Wang, Bhatia, Bilodeau, Zhou, Leduc, Downey, Lazaris and Karatzas2001; Peippo et al., Reference Peippo, Kurkilahti and Bredbacka2001; Baldassarre & Karatzas, Reference Baldassarre and Karatzas2004). Despite its potential, IVP applications in goats are limited by their low efficiency (Baldassarre & Karatzas, Reference Baldassarre and Karatzas2004; Cognié et al., Reference Cognié, Poulin, Locatelli and Mermillod2004; Chiamenti et al., Reference Chiamenti, Filho, Moura, Paula-Lopes, Neves, Neto, Gonçalves, Lima and Oliveira2013; Souza-Fabjan et al., Reference Souza-Fabjan, Panneau, Duffard, Locatelli, de Figueiredo, Freitas and Mermillod2014).
Several approaches have been pursued in order to increase the quantity and quality of goat embryos, including variations on IVP conditions (Chiamenti et al., Reference Chiamenti, Filho, Moura, Paula-Lopes, Neves, Neto, Gonçalves, Lima and Oliveira2013; Souza-Fabjan et al., Reference Souza-Fabjan, Panneau, Duffard, Locatelli, de Figueiredo, Freitas and Mermillod2014). The addition of hormones, vitamins and growth factors in maturation and/or culture media increases blastocyst development (Bormann et al., Reference Bormann, Ongeri and Krisher2003; Chiamenti et al., Reference Chiamenti, Filho, Moura, Paula-Lopes, Neves, Neto, Gonçalves, Lima and Oliveira2013; Zhang et al., Reference Zhang, Wu, Liu, Qiu, Liu, Zhang and Quan2013; Conceição et al., Reference Conceição, Moura, Ferreira-Silva, Deus, Silva, Cantanhede, Chaves, Lima and Oliveira2015). Moreover, the understanding of mechanism by which supplements enhance embryonic development may allow the establishment of more efficient IVP protocols using supplementation regimens with additive properties.
Vitamin A and its metabolites are collectively named retinoids, which have a profound effect upon cellular survival, proliferation, differentiation and embryonic morphogenesis (Clagett-Dame & DeLuca, Reference Clagett-Dame and DeLuca2002; Noy, Reference Noy2010; Rhinn & Dollé, Reference Rhinn and Dollé2012). Retinoids induce cellular differentiation in vitro due to gene expression modulation of homeobox genes, growth factors and their receptors (Mohan et al., Reference Mohan, Malayer, Geisert and Morgan2001). The addition of retinoids during oocyte maturation and embryo culture enhanced bovine and goat embryo production (Duque et al., Reference Duque, Gómez, Hidalgo, Facal, Fernández and Díez2002, Lima et al., Reference Lima, Oliveira, Goncalves, Montagner, Reichenbach, Weppert, Cavalcanti Neto, Pina and Santos2004; Chiamenti et al., Reference Chiamenti, Aguiar Filho, Freitas Neto, Chaves, Paula-Lopes, Lima, Gonçalves, Cavalcanti Neto and Oliveira2010, Reference Chiamenti, Filho, Moura, Paula-Lopes, Neves, Neto, Gonçalves, Lima and Oliveira2013; Conceição et al., Reference Conceição, Moura, Ferreira-Silva, Deus, Silva, Cantanhede, Chaves, Lima and Oliveira2015).
The ability of growth factors to control preimplantation development has received great attention due to their capacity to increase embryo quality under defined IVP conditions (Sirisathien et al., Reference Sirisathien, Hernandez-Fonseca and Brackett2003). The insulin-like growth factor I (IGF-I) acts as a mitogenic factor increasing bovine and goat embryonic development (Lima et al., Reference Lima, Oliveira, Goncalves, Montagner, Reichenbach, Weppert, Cavalcanti Neto, Pina and Santos2004, Reference Lima, Oliveira, Santos, Reichenbach, Weppert, Paula-Lopes, Cavalcanti Neto and Goncalves2006; Kooijman, Reference Kooijman2006; Chiamenti et al., Reference Chiamenti, Filho, Moura, Paula-Lopes, Neves, Neto, Gonçalves, Lima and Oliveira2013). Moreover, IGF-I diminishes apoptosis during preimplantation development in several species (Spanos et al., Reference Spanos, Becker, Winston and Hardy2000; Makarevich & Markkula, Reference Makarevich and Markkula2002). Retinoids solely (Chiamenti et al., Reference Chiamenti, Filho, Moura, Paula-Lopes, Neves, Neto, Gonçalves, Lima and Oliveira2013; Conceição et al., Reference Conceição, Moura, Ferreira-Silva, Deus, Silva, Cantanhede, Chaves, Lima and Oliveira2015) and combinations of retinoids and IGF-I supplementation during goat oocyte maturation and/or embryo culture enhanced blastocyst yields (Chiamenti et al., Reference Chiamenti, Aguiar Filho, Freitas Neto, Chaves, Paula-Lopes, Lima, Gonçalves, Cavalcanti Neto and Oliveira2010, Reference Chiamenti, Filho, Moura, Paula-Lopes, Neves, Neto, Gonçalves, Lima and Oliveira2013; Conceição et al., Reference Conceição, Moura, Ferreira-Silva, Deus, Silva, Cantanhede, Chaves, Lima and Oliveira2015), suggesting non-overlapping activities under the concentrations tested. However, the effect of retinoids and IGF-I supplementation during embryo culture on apoptosis incidence in goat embryos remained unknown.
The present report was aimed to evaluate if addition of retinoids during oocyte maturation and retinoids and/or IGF-I during embryo culture improves in vitro development of goat embryos and if it affects the incidence of apoptosis.
Materials and methods
Ovary collection and oocyte recovery
Goat ovaries were obtained at local slaughterhouses (Pernambuco State, Brazil), and transported to our laboratory at 30ºC in 0.9% NaCl solution containing 30 μg mL–1 of gentamycin sulfate. Cumulus–oocyte complexes (COC; n = 4,320) were recovered from 2–6 mm ovarian follicles by an 18G needle coupled to a 5 ml syringe and follicular content was deposited in a tube containing washing medium [8.0 mg sodium bicarbonate, 45.0 mg glucose, 5.6 mg sodium pyruvate, 11.9 mg HEPES, 2.5 mg gentamycin sulfate, 20.0 mg polyvinyl alcohol (PVA)] in 50 mL TALP.
In order to recover COCs, follicular fluid remained settling down for 15 min at 39ºC. After discarding the supernatant, COCs were selected based on morphological criteria (Chiamenti et al., Reference Chiamenti, Filho, Moura, Paula-Lopes, Neves, Neto, Gonçalves, Lima and Oliveira2013), oocytes were washed five times in washing medium and pools of 25 oocytes were randomly distributed to one of the experimental groups [Control, Retinyl Acetate (RAc), 9-cis-Retinoic Acid (RA)] in 100 μl of maturation medium covered by sterile paraffin oil.
Oocyte maturation
Maturation medium consisted of TCM-199 supplemented with 50 μg mL–1 sodium pyruvate, 2.6 mg mL–1 sodium bicarbonate, 50 μg mL–1 of gentamycin sulfate, 5.0 μg mL–1 FSH/LH (Pluset®) and 1 mg mL–1 PVA. RAc oocytes (n = 1334) were treated with maturation medium enriched with 0.3 μM retinyl acetate and RA oocytes (n = 1,358) were treated with maturation medium containing 0.5 μM 9-cis-retinoic acid. Control oocytes (n = 1380) were cultured in maturation medium only. Furthermore, oocytes were incubated at 39ºC with 5% of CO2 in air with saturated humidity for 24 h. After in vitro maturation (IVM) and evaluation of cumulus expansion, oocytes were randomly selected and destined for in vitro fertilization (IVF).
In vitro fertilization
Briefly, IVF was performed as described earlier (Chiamenti et al., Reference Chiamenti, Filho, Moura, Paula-Lopes, Neves, Neto, Gonçalves, Lima and Oliveira2013). In brief, 0.1 ml of freshly collected semen was resuspended in 1.5 ml of Modified Dulbecco's Medium (MDM), containing 0.125 g glucose, 0.1552 g sodium bicarbonate, 0.0069 g sodium pyruvate, 0.05 g PVA, 0.05 g caffeine and 0.025 g penicillamine (Keskintepe et al., Reference Keskintepe, Simplicio and Brackett1998). The tube was placed at a 45º angle to select viable sperm cells by swim-up. After 45 min, 0.8 mL was aspirated and centrifuged at 350 g for 10 min. After discarding the supernatant, 200 μl of MDM containing 10 μg ml–1 heparin was added to 200 μl of the pellet obtained after centrifugation.
Before IVF, oocytes were assessed for cumulus expansion and COC that did not expand were discarded. Pools of 25 oocytes were transferred to 150 μl MDM drops containing a final sperm suspension of 2.0 × 106 spermatozoa ml–1 under sterile paraffin oil. Gametes were co-incubated for 18 h at 39ºC with 5% of CO2 in air with saturated humidity.
Embryo culture
Presumptive zygotes were mechanically denuded and transferred to 100-μl drops containing Potassium Simplex Optimized Medium (KSOM) and co-cultured on a monolayer of oviduct cells (Chiamenti et al., Reference Chiamenti, Aguiar Filho, Freitas Neto, Chaves, Paula-Lopes, Lima, Gonçalves, Cavalcanti Neto and Oliveira2010, Reference Chiamenti, Filho, Moura, Paula-Lopes, Neves, Neto, Gonçalves, Lima and Oliveira2013). After IVF, presumptive zygotes were placed in fresh KSOM supplemented with 0.3 μM RAc, 0.5 μM RA, 50 ng ml–1 IGF-I, RAc + IGF-I and RA + IGF-I (Table 1). After 48 h of embryo culture, unfertilized oocytes were removed and 30% of embryo culture medium was changed. Embryos were further cultured until day 8 at 39ºC in air with 5% of CO2 in air with saturated humidity. Embryonic development was recorded at day 3 (D3) and day 8 (D8). Blastocysts at D8 were used for caspase and terminal deoxynucleotidyl transferase enzyme (TUNEL) analysis. The experiment was replicated 10 times.
Table 1 Goat embryo production after supplementation with retinoids and insulin-like growth factor I during oocyte maturation and embryo culture
a,b; a,d; b,c Different superscript letters within columns denote significant differences by Tukey's test (P < 0.05). Least squares mean (± SEM).
IGF-I: 50 ng ml–1 insulin-like growth factor I; RA: 0.5 μM 9-cis-retinoic acid; RAc: 0.3 μM retinal acetate.
Caspase activity assay
Blastocysts were washed three times in 100 µl PBS drops supplemented with 1 mg ml–1 polyvinylpyrrolidone (PBS-PVP) and incubated in drops of 25 µl PBS-PVP containing 5 μM de PhiPhiLux G1D2 for 40 min at 39°C protected from light. Moreover, oocytes were washed in PBS-PVP and caspase enzymatic activity was determined by fluorescence microscopy.
TUNEL assay
The TUNEL assay was performed as previously described, with minor modifications (Paula-Lopes and Hansen, Reference Paula-Lopes and Hansen2002a,Reference Paula-Lopes and Hansenb; Roth and Hansen, Reference Roth and Hansen2004). Blastocysts was denuded and fixed in 100 µl of 4% paraformaldehyde for 1 h at room temperature. Blastocysts were washed three times in 100 µl de PBS-PVP and incubated in 100 µl of permeabilization buffer (0.5% Triton X-100 containing 0.1% sodium citrate) for 1 h and stored at 4°C. To perform the TUNEL assay, samples were washed three times in 100 µl PBS-PVP drops and incubated in 15 μl of terminal deoxynucleotidyl transferase enzyme (TUNEL) solution for 1 h at 37°C. Moreover, oocytes were washed in PBS-PVP and incubated with a DNA-specific stain 4′,6-diamidino-2-phenylindole (DAPI) for 15 min, further washed in 100 μl of PBS-PVP, and transferred to slides with coverslips and evaluated by fluorescence microscopy.
Statistical analysis
Analysis of variance was performed by minimum squares method by PROC GLM (for fixed variables) and PROC MIXED (for fixed and random variables) on SAS STAT package (SAS Institute, Cary, NC, USA). The data were previously analyzed for variance analysis (variable homogeneity and residual normality). Dependent and independent variables were established based on experimental design. Statistical models considered main effects and all possible interactions. Differences with 5% probability were considered significant.
Results
Goat blastocysts were initially produced under several supplementation regimens of RAc, RA and IGF-I during oocyte maturation and embryo culture (Table 1). The effect of different treatments of RAc, RA and IGF-I did not affect cleavage rates (Table 1), except for RAc/IGF-I and RAc/RAc + IGF-I groups, which showed lower cleavage rates. In contrast, blastocyst production was achieved with efficiencies for all groups treated solely with retinoids (Table 1). Moreover, addition of IGF-I during embryo culture increased blastocyst yields irrespectively of retinoid addition (Table 1).
In order to address if the increase in IVP embryos by retinoids and IGF-I is accompanied by reduction in the incidence of apoptosis, blastocysts were evaluated for two hallmarks of apoptosis, namely DNA fragmentation and caspase activation. Blastocysts from all experimental groups displayed similar frequencies of DNA damage when evaluated for DNA fragmentation using the TUNEL assay (Table 2). The incidence of caspase-3 activity was also similar within blastocyst from all experimental groups (Table 3).
Table 2 Incidence of DNA fragmentation in goat blastocysts after supplementation with retinoids and insulin-like growth factor I during oocyte maturation and embryo culture
a,b; a,d; b,c Different superscript letters within columns denote significant differences by Tukey's test (P < 0.05). Least squares mean (± SEM).
IGF-I: 50 ng mL–1 insulin-like growth factor I; RA: 0.5 μM 9-cis-retinoic acid; RAc: 0.3 μM retinal acetate.
DNA fragmentation evaluated by the terminal deoxynucleotidyl transferase enzyme (TUNEL) assay.
Table 3 Incidence of caspase-3 in goat blastocysts after supplementation with retinoids and insulin-like growth factor I during oocyte maturation and embryo culture
a,b; a,d; b,c Different superscript letter (ab, ad, bc) within columns denote significant differences by Tukey's test (P < 0.05). Least squares mean (±SEM).
IGF-I: 50 ng mL–1 insulin-like growth factor I; RA: 0.5 μM 9-cis-retinoic acid; RAc: 0.3 μM retinal acetate.
DNA fragmentation evaluated by the terminal deoxynucleotidyl transferase enzyme (TUNEL) assay.
Discussion
Embryo IVP applications in small ruminants are currently restricted to particular contexts, such as the production of cloned and transgenic animals (Baldassarre & Karatzas, Reference Baldassarre and Karatzas2004). The main limitation of goat IVP embryos is their lower quality when compared with their in vivo counterparts (Baldassarre & Karatzas, Reference Baldassarre and Karatzas2004; Cognié et al., Reference Cognié, Poulin, Locatelli and Mermillod2004), resulting in diminished pregnancy rates and poor cryosurvival. The lower quality of IVP embryos is mostly due to the IVP conditions, since several studies show that more closely mimicking in vivo conditions improves embryonic development and embryo quality (Walker et al., Reference Walker, Hill, Kleemann and Nancarrow1996; Wan et al., Reference Wan, Hao, Zhou, Wu, Yang, Cui, Liu and Zeng2009).
Retinoids and IGF-I are compounds found in the female reproductive tract and enhance embryonic development in vitro in several species (Duque et al., Reference Duque, Gómez, Hidalgo, Facal, Fernández and Díez2002, Lima et al., Reference Lima, Oliveira, Goncalves, Montagner, Reichenbach, Weppert, Cavalcanti Neto, Pina and Santos2004; Chiamenti et al., Reference Chiamenti, Aguiar Filho, Freitas Neto, Chaves, Paula-Lopes, Lima, Gonçalves, Cavalcanti Neto and Oliveira2010, Reference Chiamenti, Filho, Moura, Paula-Lopes, Neves, Neto, Gonçalves, Lima and Oliveira2013). Since embryotrophic properties of retinoids during embryo culture require co-culture conditions in goats (Chiamenti et al., Reference Chiamenti, Aguiar Filho, Freitas Neto, Chaves, Paula-Lopes, Lima, Gonçalves, Cavalcanti Neto and Oliveira2010), the supplementations of retinoids and IGF-I were performed using a monolayer of oviduct cells. Moreover, the sole addition of RA, RAc, IGF-I to embryo culture system increased goat embryonic development as described for several species (Spanos et al., Reference Spanos, Becker, Winston and Hardy2000; Makarevich & Markkula, Reference Makarevich and Markkula2002; Chiamenti et al., Reference Chiamenti, Filho, Moura, Paula-Lopes, Neves, Neto, Gonçalves, Lima and Oliveira2013). However, as described here, retinoids and IGF-I synergically increase blastocyst development as previously reported (Chiamenti et al., Reference Chiamenti, Filho, Moura, Paula-Lopes, Neves, Neto, Gonçalves, Lima and Oliveira2013). The ability to replicate these results demonstrates the strong effect of retinoid and IGF-I treatments despite undefined embryo culture conditions (e.g. co-culture with oviduct cells). Despite beneficial paracrine activity of somatic cells, co-culture conditions may result in efficiency variability due to the stochastic nature of interactions between somatic cells, embryos and culture media.
The effect of retinoids and IGF-I supplementation on embryo quality remained to be investigated. In order to evaluate the effect of retinoids and IGF-I on blastocyst quality, goat embryos were scored for two apoptosis markers (DNA fragmentation and caspase-3 activity). Apoptosis is a development or context-dependent process characterized by selective cell death. During preimplantation development, apoptosis is first detected during embryonic genome activation (Hansen & Fear, Reference Hansen and Fear2011) and represents a physiological process during preimplantation development (Paula-Lopes & Hansen, Reference Paula-Lopes and Hansen2002a,Reference Paula-Lopes and Hansenb; Hansen & Fear, Reference Hansen and Fear2011). More importantly, incidence of apoptosis has been described as a marker of embryo quality (Watson et al., Reference Watson, De Sousa, Caveney, Barcroft, Natale, Urquhart and Westhusin2000; Makarevich & Markkula, Reference Makarevich and Markkula2002). Despite the positive effect of retinoids and IGF-I supplementation, blastocyst quality based upon DNA fragmentation and caspase-3 activity did not differ between groups. Further research may help dissect the interaction between embryos and somatic cells in order to address this question.
It is relevant to consider the beneficial role of IGF-I on embryonic development, especially when used with co-culture systems. Morula yields grew from 33 to 45% after IGF-I addition to embryo culture and blastocyst rate from 11 to 17%, (Prelle et al., Reference Prelle, Stojkovic, Boxhammer, Motlik, Ewald, Arnold and Wolf2001). Caspase activity in blastocysts was not influenced by IGF-I addition to culture medium. This result is in accordance with Watson et al. (Reference Watson, De Sousa, Caveney, Barcroft, Natale, Urquhart and Westhusin2000), where IGF-I increased blastocyst yield, but apoptosis incidence remained unchanged. The demonstration that RAc and RA had similar effects upon embryonic development and apoptosis, suggested no retinoid metabolite specific effects.
In conclusion, retinoids and IGF-I increase blastocyst production through mechanism(s) that are independent of apoptosis inhibition or alternatively, these factors diminish apoptosis by similar pathways.
Acknowledgements
This study was financially supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Ciência e Tecnologia de Pernambuco (FACEPE). Marcelo T. Moura is a CAPES postdoctoral fellow.
