Pesq. Vet. Bras. 35(7):605-612, julho 2015 DOI: 10.1590/S0100-736X2015000700002 Supplementation of fetal bovine serum alters histone modification H3R26me2 during preimplantation development of in vitro produced bovine embryos1 Daniel R. Arnold2, Carolina A.P. Corrêa3, Laura L.G. Lorena4, Roberta C. Gaspar3, Guilherme F. Rossi3, Aderson M. Ifran3, João C.T. Penteado4, Gisele Mingoti4 and Flavia L. Lopes4* ABSTRACT.- Arnold D.R., Corrêa C.A.P., Lorena L.L.G., Gaspar R.C., Rossi G.F., Ifran A.M., Pen- teado J.C.T., Mingoti G. & Lopes F.L. 2015. Supplementation of fetal bovine serum alters histone modification H3R26me2 during preimplantation development of in vitro produced bovine embryos. Pesquisa Veterinária Brasileira 35(7):605-612. Departamen- to de Apoio, Produção e Saúde Animal, Faculdade de Medicina Veterinária de Araçatuba, Universidade Estadual Paulista, Rua Clovis Pestana 793, Araçatuba, SP 16050-680, Brazil. E-mail: In vitro production (IVP) of bovine embryos is not only of great economic importance to the [email protected] industry, but is also an important model for studying embryo development. - -implantation development of IVP bovine embryos cultured with or without serum sup- plementationThe aim of this and study how was these to in evaluate vitro treatments the histone compared modification, to in vivoH3R26me2 embryos during at the premo- rula stage. After in vitro maturation and fertilization, bovine embryos were cultured with either 0 or 2.5% fetal bovine serum (FBS). Development was evaluated and embryos were collected and fixed at different stages during development (2-, 4-, 8-, 16-cell, morula and Embryosblastocyst). cultured Fixed embryoswith 2.5% were FBS then developed used for to immunofluorescence blastocysts at a greater utilizing rate thanan antibody 0%FBS groupsfor H3R26me2. (34.85+ 5.43%Images vs. of 23.38stained+ embryos were analyzed as a percentage of total DNA. was at the 4-cell (P<0.05), 16-cell2.93%; (P<0.05) P<0.05). and morula Levels (P<0.05)of H3R26me2 stages. changed In the 2.5%FBSfor both groups over development. In the 0%FBS group, the greatest amount of H3R26me2 staining Morula stage in vivo - group, only 4-cell stage embryos were significantly higher than all other stages (P<0.01). embryos had similar levels as the 0%FBS group, and both were signifi culturecantly higher conditions than thegreatly 2.5%FBS alter this group. regulation. These results suggest that the histone modification H3R26me2 is regulated during development of pre-implantation bovine embryos, and that serum. INDEX TERMS: Bovine, embryo development, H3R26me2, in vitro produced, fetal bovine RESUMO.- [Suplementação com soro fetal bovino altera in vitro.] A produção in vitro (PIV) de embriões de bovinos a modificação de histona H3R26me2 durante o período não é apenas de grande importância econômica para a pe- pré-implantacional em embriões bovinos produzidos cuária, mas é também um importante modelo para estudar 1 Received on September 11, 2014. Accepted for publication on July 23, 2015. 2 ticabal,4 Departamento SP 14884-900, de Apoio, Brazil. Produção E-mails: e [email protected]úde Animal, Faculdade de Medi- cinacom, Veterinária [email protected], de Araçatuba, Unesp, [email protected] Rua Clovis Pestana 793, Araçatuba, usp.br In Vitro Brasil S/A, Rodovia SP-340 Km 166, Cx. Postal 238, Mogi Mirim, - SP3 13800-970,Faculdade de Brazil. Ciências E-mails: Agrárias [email protected], e Veterinárias, Universidade carolinaalves@ Estadual Paulista (Unesp), Via de Acesso Prof. Paulo Donato Castellane s/n, Jabo- fmva.unesp.brSP 16050-680, Brazil. E-mails: [email protected], jctpenteado@hot mail.com, [email protected]; *Corresponding author: flavialopes@ 605 606 Daniel R. Arnold et al. and other known and unknown components that the gro- wing embryo requires (Van Langendonckt et al. 1997), even desenvolvimentoo desenvolvimento pré-implantacional embrionário. O objetivo em embriões deste bovinos estudo though early embryo development may be delayed (Gard- produzidosfoi avaliar a inmodificação vitro, cultivados de histona, com H3R26me2ou sem suplementa durante o- ner 1998, Thompson 2000). In addition, the use of FBS ção de soro fetal bovino (SFB), bem como comparar essa in IVP systems appears to be more important for certain in vitro e breeds of cattle. Leivas and co-authors (Leivas et al. 2011) in vivo in vitro e fertilização, embriões reported improved blastocyst rates for IVP Bos indicus em- forammodificação cultivados específica com suplementação entre mórulas produzidasde 0 ou 2,5% SFB. O bryos cultured with 2% FBS than embryos cultured with desenvolvimento. Após a maturação embrionário foi avaliado e embriões fo- - for embryos produced from Bos taurus oocytes (Leivas et al. 2011).BSA alone. These No results differences suggest in blastocyst another difference rates were in identified sub-spe- ram coletados e fixados em diferentes fases durante o de- - senvolvimento (2, 4, 8 e 16 células, mórula e blastocisto). embriõesOs embriões corados fixados foram foram analisadas avaliados porbaseadas imunofluorescên na porcen- andcies. theHowever, risk of the contamination. drawbacks to In using addition, serum the include presence varia of cia utilizando um anticorpo para H3R26me2. Imagens de bility in composition between batches, its undefined nature - to cryopreservation, when compared to in vivo embryos [for tocistotagem domaior DNA que total. o grupo Embriões que não cultivados recebeu suplementaçãocom 2,5% SFB reviewserum duringsee Lonergan culture etalters al. 2006). gene expressionSimilar results and aretolerance found tiveram uma taxa de desenvolvimento ao estágio de blas - pression may be due to effects on epigenetic regulation. com SFB (34.85±5,43% vs 23.38±,93%; P<0,05). Níveis de- in miceEpigenetic (Khosla mechanisms et al., 2001). appear These todifferences be the most in gene sensiti ex- 26me2H3R26me2 foi mais variaram intensa para nos ambosestágios os de grupos 4 células ao (P<0,05),longo do ve factors affected by environmental changes like culture desenvolvimento. No grupo 0% SFB, a marcação para H3R apenas os embriões de 4 células tiveram marcação signi- 16 células (P<0,05) e mórula (P<0.05). No grupo 2.5% SFB, epigeneticconditions mechanism(Niemann & that Wrenzycki is critical 2000, to embryo Duranthon develop et al.- in vivo - ment2008, ofKohda several et al. species 2012). such Histone as mice modification (Corry et is al. one 2009), such 26me2ficativamente semelhantes maior aoque grupo todas 0% as SFB, outras e ambos fases foram(P<0,01). sig- cattle (Santos et al. 2003, Enright et al. 2005, Wee et al. Mórulas produzidas apresentaram níveis de H3R- 2006, Maalouf et al. 2008, Ross et al. 2008), pig (Park et - énificativamente regulada durante maiores o desenvolvimento que o grupo 2.5% pré-implantacional SFB. Estes re man (Zhang et al. 2012). In mice, Sarmento et al. (2004), desultados embriões sugerem bovinos, que e a que modificação as condições de histona de cultura H3R26me2 alteram describesal. 2009, Gao two et types al. 2010), of histone sheep patterns (Hou et during al. 2008) preimplan and hu- de maneira importante esta regulação. tation development, a consistent, stable mark during deve- lopment and a dynamic, reversible mark. The highly dyna- mic marks are often associated with critical events, such as in vitro, soro fetal bovino. TERMOS DE INDEXAÇÃO: Bovino, desenvolvimento embrionário, cellular differentiation from totipotent cells into the inner H3R26me2, produção INTRODUCTION cellthe transitionmass and totrophectoderm embryonic genome [for review activation see Mason or the et first al. In vitro production (IVP) of embryos has revolutionized the way infertility is addressed. Since the birth of Louise Brown in 1978 (Steptoe & Edwards 1978), fertility clinics with2012). the Two trophectoderm examples are (Erhardt trimethylation et al. 2003). of lysines Epigenetic 9 and worldwide have adopted this procedure. Besides humans, 27 of histone H3 which are enriched in the ICM compared another industry where IVP has become widely accepted development. is the cattle industry. In 2011, 453,471 bovine IVP em- modificationsMethylation are of relatedarginine to residues important in histonestages of proteins embryonic are bryos were produced, with 343,927 being used for embryo also critical during preimplantation. Like lysine methyla- transfer (Stroud 2012). While production of in vitro em- tion, arginine methylation has been associated with both bryos is common, the rate of development is still very low transcriptional activation and inhibition (Di Lorenzo & Be- dford 2011). In murine embryos, arginine methylation has 2010). These low results are primarily due to the inability been linked with determining cell fate. Torres-Padilla et al. towhen replicate compared the in to vivo embryos environment produced in ina laboratoryvivo (Hansen setting. et al. blastomeres of 4-cell stage embryos. They also describe implantation are vital for proper embryonic development (2007) reported differences in H3R26me2 levels between Conditions for which embryos are exposed prior to would subsequently give rise to the trophoblast cells, whe- 2010; Besenfelder et al. 2012). This is most evident in IVP reasthat the remainingblastomere blastomeres with the least will amount develop ofinto H3R26me2 the inner embryos,and successful where establishment changes to culture of pregnancy conditions (Hansen can drama et al.- cell mass (Torres-Padilla et al. 2007). Regulation of this tically