Pesq. Vet. Bras. 35(7):605-612, julho 2015 DOI: 10.1590/S0100-736X2015000700002

Supplementation of fetal bovine serum alters 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@

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and other known and unknown components that the gro- wing embryo requires (Van Langendonckt et al. 1997), even desenvolvimentoo desenvolvimento pré-implantacional embrionário. O objetivoem 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 suplementação com 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 ao que 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 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 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 affect embryo developmental rates, and remains a in bovine embryos. The source of protein in media is an area of great inte- particularIn cattle, histone where modification much research has hasnot focusedbeen documented on the al- rest.major Fetal field bovine of research. serum (FBS) is the most common protein tered reprogramming of in embryos produced by source in media. Culture media containing FBS generally somatic cell nuclear transfer, (Santos et al. 2003, Morgan produce better blastocyst rates, due to the growth factors

et al. 2005, Wee et al. 2006) fewer studies have examined Pesq. Vet. Bras. 35(7):605-612, julho 2015 607

Supplementation of fetal bovine serum alters histone modification H3R26me2 - Immunocytochemical analysis of histone H3R26 di-methylation ges (Maalouf et al. 2008, Ross et al. 2008) or the effects of The zona pellucida was removed from each embryo by incu- the histone modifications throughout preimplantation sta bating in 0.1% Tyrode’s solution (Sigma) for 30 seconds at 37°C, embryonic stage allow for proper development and suc- then 0.5% (w/v) pronase at 37°c for 10 seconds. Embryos were cessfulculture pregnancyconditions. to Specific ensue epigenetic(Palini et al. patterns 2011). withinBovine each em- then quickly placed in 4% (w/v) paraformaldehyde for 15 to 20 bryos provide another important model for investigating until further processed. the effects of IVP on epigenetic processes. Therefore, the minutes at room temp and stored in PBS with 0.1% Triton-X at 4C, - bovine embryonic development in vitro in the presence or Immunofluorescence analysis was performed as previously- current study examined the profile of H3R26me2 during mindescribed (BSA; Sigma)(Arnold and et al. 0.1% 2008). (v/v) Briefly, Tween-20 after (Sigma)washing in in PBS] PBS, for sam 20 compared to in vivo embryos at the morula stage. ples were exposed to blocking buffer [3% (w/v) bovine serum albu- absence of fetal bovine serum and how H3R26me2 levels 26me2 (07-215; EMD Millipore, Billerica, MA) was diluted 1:200 in MATERIALS AND METHODS minutes at room temperature. Primary antibody for histone H3R - Production of in vitro and in vivo embryos blocking buffer and mixed with samples overnight at 4°C. Samples In vitro fertilized (IVF) embryos were produced from oocytes were then washed in PBS and exposed to an Alexa Fluor 555-con obtained from slaughterhouse. Oocytes were matured in vitro as 33342jugated (10µg/mL; secondary Invitrogen). antibody [diluted Fluorescence 1:200 in was block; visualized (Invitrogen, and - Eugene, Oregon, USA)]. Embryonic DNA was visualized by Hoechst previously described (Bordignon et al. 2003). Briefly, cumulus- abundanceimages captured during using early an development, epifluorescence embryos microscope of different (Olympus/ stages tion,-oocyte St Louis,complexes Missouri, (COC) USA) were supplemented aspirated from with 2 to 10% 7mm (v/v) follicles FBS. (2-cell,IX/FLA/70, 4-cell, Tokyo, 8-cell, Japan) 16-cell, with morula a CCD and camera. blastocyst; To measure n=25-28 protein em- Onlyand washed COCs with in Hepes-buffered several layers TCM199of cumulus (Sigma-Aldrich cells and with Corpora homo- bryos per stage) and treatments (with and without serum) were genous oocyte cytoplasm were selected. Groups of 25 COCs were processed at the same time and mounted on the same microscope cultured in 100µl drops of IVM media [bicarbonate-buffered TCM- slide. In vivo morula embryos were processed with morula stage embryos from both in vitro groups (0% FBS and 2.5% FBS; n=15- 20 per group). Image-capture parameters below saturation were 199 (Gibco BRL, Grand Island, New York, USA) supplement with- -Aldrich),10% FBS, 50µg/ml22µg/ml ofpyruvate LH (Ayerst, (Sigma-Aldrich), London, ON, Canada), and 83.4µg/ml 1µg/ml of for each embryo were recorded under identical conditions. Fluo- amicacinaFSH (Follitropin-V, (Sigma-Aldrich)]. Vetrepharm), Matured 1µg/ml COCs of wereestradiol-17ß fertilized (Sigma in vitro identified for each slide and images for both H3R26me2 and DNA as previously described (Parrish et al. 1986) and cultured in 50µl rescence was quantified using ImageJ 1.36b software (National-

7.2mM KCl, 0.5mM MgCl2 ansInstitutes and standard of Health, errors Bethesda, of the meansMD). For to eachbe calculated. embryo, the staining 0.5mMdrops of glucose modified 0.33mM synthetic pyruvic oviductal acid, 3mM fluid lactic(mSOF;108mM acid, 150µg/ml NaCl, intensity for H3R26me2 was normalized to the DNA, enabling me gentamicin, and 0.01% (w/v), 2.5mM phenol NaHCO3, red) media 1.7mM plus amino CaCl2-H2O, acids Statistical analysis (1.4mM glycine, 0.4mM alanine, 1mM glutamine, 2% (v/v) essen- tial amino acids, 1% (v/v) non-essential amino acids; (Gardner et of SAS. Dependent and independent variables were established

2 and 20% The experimental data were analyzed using the ANOVA model

O2 for 7 days. For the without serum treatment group (0% FBS), test was used to assess differences between stages of develop- 8mg/mlal. 1994) of at fatty39°C acid in humidified free BSA (Sigma)atmosphere was ofadded 5% COto the mSOF ment,according presence to the or experimental absence of serum design. during Duncan’s culture multiple and in range vivo media. Culture media for the serum treatment groups (2.5% FBS) versus in vitro embryos. were supplemented with 2.5% of FBS and 6mg/mL fatty acid free BSA. Embryos at the 2-cell embryos were collected prior to RESULTS culture, while 4-cell, 8-cell, 16-cell, morula and blastocyst stages were collected from each group (0% FBS and 2.5% FBS). All sta- Effect of serum on embryo development ges were collected within the same culture. Embryo collections The presence of 2.5% FBS had no effect on cleavage rates, were replicated 4 times. Embryos were then further processed for - immunocytochemical analysis, as described below velopment to the blastocyst stage was increased when FBS All treatment protocols involving the use of animals were waswhen present compared in the to theculture 0% FBSmedia group (P<0.05). (Table In1). addition, However, em de- approved by the “Comissão de Ética no Uso de Animais” of the bryos cultured in the absence of FBS tended to be delayed in Universidade Estadual Paulista in accordance with regulations of their development, with most reaching the blastocyst stage by day 8, as opposed to day 7 for the serum group. the “Conselho Nacional de Controle de Experimentação Animal following superovulation protocol: on day 0, aspiration of domi- Table 1. Cleavage and development rates of bovine (CONCEA)”. For in vivo (AI) embryos, 8 cows were subjected to the nant follicle (if present) and insertion of a progesterone implant embryos produced in vitro with different concentrations of FBS (0% or 2.5%) everyfor 7 days 12h, (Crestar; in daily decreasing Intervet, Brazil); doses (total on days dose 4-7 of i.m. 200mg); injections on day of Treatment n Cleavage Blastocysts Blastocsyts 6,Follitropin-V 500 g cloprostenol (Bioniche (Sincrocio; Animal Health Ouro Canada, Fino, Brazil); Belleville, on day Canada) 8, 1ug (%) (% total) (% cleavage) Buserelin Acetate (Sincrofort; Ouro Fino); and insemination at 12 0% FBS 993 80.35 ± 2.61 18.83 ± 2.79a 23.38 ± 2.93a μ 2.5% FBS 999 83.58 ± 2.55 29.13 ± 4.77b 34.85 ± 5.43b At 5.5 days post AI, morula stage embryos were collected by a,b Values with different superscripts differ between lines (p<0.05). The and 24h post-Buserelin injection (day 9). - percentages of cleaved embryos were calculated in relation to the total red saline (PBS). Embryos were then further processed for immu- number of oocytes. The percentages of blastocysts were calculated by the nocytochemicalnon-surgical flushing analysis. of the uterus with sterile phosphate buffe total number of oocytes (%) and number of embryos cleaved (% cleaved).

Pesq. Vet. Bras. 35(7):605-612, julho 2015 608 Daniel R. Arnold et al.

across early development in in vitro pro- Fig.1. Immunofluorescence for H3R26me2-

cellsduced (4c), bovine 8 cells embryos. (8c), 16 (16) Immunofluores cells, morula (mol)cence forand H3R26me2 blastocysts (red) (bl) grownin 2 cells with (2c), 0% 4 and with 2.5% fetal bovine serum. Total

DNA is shown in blue.

Immunofluorescene of H3R26me2 during in vitro de- (Fig.2). Both treatment groups had elevated levels at the velopment - (P<0.05) than the 0% group (Fig.2). The 0% FBS groups - had4-cell higher stage, levels with theat the 2.5% 16-cell FBS groupand morula significantly stages, higher when bryosA specific cultured antibody with and to without H3R26me2 fetal bovine was utilized serum (Fig.1).to eva compared to similar stage embryos cultured with 2.5% FBS luate fluctuations during in vitro development of IVP em (Fig.2). Comparing within the 0% FBS treatment, the 16-

Overall levels of H3R26me2 changed across development Pesq. Vet. Bras. 35(7):605-612, julho 2015 609

Supplementation of fetal bovine serum alters histone modification H3R26me2 stage of development of bovine embryos. In addition, fetal bovine serum had an effect both on developmental rates

The presence of 2.5% FBS increased development rates to and H3R26me2 mark at different stages of development. 4-cell stage. When embryos were cultured without FBS, an increasethe blastocyst was witnessed stage and ata higherthe 4-cell level stage, of H3R26me2 as well as inat 16-the cell and morula stage embryos. Interestingly, in vivo morula stage embryos were more similar to the 0% FBS group in - bryos cultured with 2.5% FBS. regardsThe useto H3R26me2of fetal bovine levels, serum both in culturebeing higher media thanhas been em controversial with both positive and negative effects re- ported. Several studies have demonstrated increased blas- - tocyst development when FBS is supplemented, (Lazzari et bryonic development in response to supplementation of 0% Fig.2.and Average 2.5% fetalintensity bovine of serumH3R26me2 (FBS) at to different culture media.stages of*Diffe em- al. 2002, Rizos et al. 2003, Leivas et al. 2011) similar to the rences in levels between 0% (white) and 2.5% (black) FBS. - a,b,cDifferences between embryo stages grown in media sup- sociated with abnormal fetal growth, in subsequent preg- plemented with 0% FBS. †,‡Differences between embryo stages current study. However, the use of FBS has also been as grown in media supplemented with 2.5% FBS. 2-cell embryos were collected prior to culture and set to 1 for comparisons tonancies 10%, (Youngwhile the et currental. 1998, study Lazzari utilized et al. 2.5%.2002, In Farin addition, et al. between embryo stages. Results presented as percentage of FBS2004). has However, an adverse these effect latter on studies embryo supplemented survival after FBS cryo up- *,a,b,c, †,‡P<0.05). preservation, due to increased lipid content within the em- bryos (Rizos et al. 2003). Leivas and co-authors (Leivas et total DNA ( al. 2011) also suggest that embryos from certain breeds of cattle may depend more on the presence of FBS in the cul- ture media. Similar to the current study, they report a 10% increase in blastocyst rates between IVP embryos from Bos indicus cattle cultured with 2% FBS than embryos cultured with BSA alone (Leivas et al. 2011). For this reason, many laboratories in South America, where most cattle are of Bos indicus origin, still utilize FBS during embryo culture. While most research in serum supplementation during in vitro culture have focused on developmental rates and

studies have evaluated the effects on epigenetic processes, gene expression [for review see (Lonergan et al. 2003), few- cations in embryos primarily investigates the asymmetric produced in vitro or in vitro culture supplemented with 0% or distributionsuch as histone between modifications. male and Research female pronucleion histone [(Mason modifi Fig.3.2.5% Average fetal intensitybovine serum. for H3R26me2 Results presented for morula as stage percentage embryos of *,a,b,c P<0.05). -

total DNA ( andet al. vital 2012). these Studies epigenetic addressing marks the are issue during of specificdevelopment. histo 4-cell and morula stage embryos (Fig.2). For the 2.5% FBS Rossne modifications and co-authors are starting(Ross et to al. demonstrate 2008) reported, how dynamicin a bo- cell stage had the highest levels of H3R26me2, followed by ooocytes, declines in mature oocytes and across embryo group, only the 4-cell stage embryos had significantly more developmentvine model, that to thetri-methylation morula stage, of H3K27then increases is highest in inblas GV- H3R26me2Immunofluorescene than all other of H3R26me2 stages (Fig.2). for in vivo and in vi- tocyst embryos. In addition, the enzymes that methylate tro morula stage embryos - To determine which culture condition is more represen- tative of natural embryo development, morula stage em- stage,H3K27 where follow it a declines, similar patternincreasing (Ross again et al.at the2008). blastocyst In hu bryos were collected from superovulated animals. In vivo man embryos, is relatively static to the 8-cell asymmetrically distributed between the ICM and tropho- the in vitro, 0% FBS morula embryos (Fig.3). Protein levels stage (Zhang et al. 2012). In mice blastocysts, H3K27me3 is- morula stage embryos had similar levels of H3R26me2 as mosome in the trophectoderm (Erhardt et al. 2003, Dahl were higher than morulas cultured with 2.5% FBS (Fig.3). blast cells, as well as being enriched on the inactive X chro of H3R26me2 for both in vivo and 0% FBS morula embryos - DISCUSSION pmentet al. 2010). to the Human blastocyst embryos stage also(Zhang have et dynamic al. 2012). changes These - in the permissive histone mark, H3K4me3 during develo- nuclear stage to the 4-cell stage, followed by a decline at The current study demonstrates that the histone modifica authors observed an increase in H3K4me3 from the pro tion H3R26me2 is present and fluctuates depending on the Pesq. Vet. Bras. 35(7):605-612, julho 2015 610 Daniel R. Arnold et al. the 8-cell stage and another increase to the blastocyst stage (Zhang et al. 2012). These authors suggest the changes ob- being performed at this time in vivo produced embryos inhibited this experiment from to 8-cell stage may be associated with embryonic genome also seen at the 16-cell and morula stage. This stage of em- activation,served for both(Zhang H3K4me3 et al. 2012) and H3K27me3 which occurs from during the 4-cell this In the serum-free group, an increase in H3R26me2 was time in human embryos (Dobson et al. 2004). into cells of the inner cell mass and trophectoderm (McLa- - renbryos 1990). also Thesecoincides results with may the suggest first cellular a similar differentiation role for this 26me2, which has not been reported in a bovine model. Di- epigenetic mark as seen in the 4-cell mouse model (Torres- The current study evaluated the histone mark H3R- pression (Di Lorenzo & Bedford 2011). Due to this residue’s such increase was observed. These results suggest that -methylation of H3R26 has been associated with gene ex either-Padilla the et embryosal. 2007). cultured However, in inthe the absence 2.5% ofFBS FBS group, require no - physical proximity on histone H3 to amino acid residue K27, which when methylated is a major repressive mark, methyla factorsalterations present in this in thehistone serum), modification or factors (and within in turnFBS geneinhi- tion of H3R26 may inhibit the Polycomb repressive complex expression, in order to compensate for the lack of essential afrom mouse methylating model. According H3K27 (Di to Lorenzo Torres-Padilla & Bedford et al. 2011). (2007), In address this issue in vivo produced morula stage embryos theaddition, levels H3R26me2 of methylation is critical of arginine for embryo residues development in histone in bit the normal increase in H3R26me2 during this time. To- rula stage embryos produced in vivo were similar to those mouse embryos and may play a role in subsequent cellular observedwere collected in the and 0% FBSevaluated. group andLevels higher of H3R26me2 than those inin moem- H3-specific (H3R26me2, R2me and R17me) are present in bryo supplemented with 2.5% FBS. These results suggest between blastomeres of the 4-cell stage embryos. The blas- differentiation. They reported different levels of H3R26me2 rise to the trophectoderm (Torres-Padilla et al. 2007). In embryos,that the increase and the in presence H3R26me2 of FBS observed inhibits in this the increase0% FBS, inis tomeres with the least amount of H3R26me2 would give more similar to the profile of naturally in vivo developing- - - sultedaddition, in subsequent overexpression development of the histone into the methyltransferase inner cell mass H3R26me2. Future studies are required to determine whi (Torres-Padillathat methylates et H3R26, al. 2007). PRMT4/CARM1, In the current in study, blastomeres differences re ch genes are associated with H3R26me2, and if the presen - ce of serum altered expressionCONCLUSIONS of these genes. embryos,between blastomeresin response were to serum not evaluated. supplementation Our main duringobjec the embryonic preimplantation period in cattle, and its le- culture.tive was Future to determine studies theare plannedprofile of to H3R26me2 assess differences in bovine in vel Thevaries histone during modification development. H3R26me2 is present during These variations between embryo stages coincide with important development milestones of embryo develop- H3R26me2 concentrations between blastomeres, as well as- - 26me2the expression/activation staining intensities ofbetween PRMT4/CARM1. the inner cell However, mass and in rentiation. the trophectodermcurrent study, there of the appears blastocyst. to be a difference in H3R ment;Even embryonic though supplementationgenome activation of and fetal first bovine cellular serum, diffe - during in vitro culture, increased developmental rates to sed at the 4-cell stage, regardless of serum. This result may Concentrations of H3R26me2 were significantly increa when compared to embryos cultured in serum-free media genome activation. Embryonic genome activation (EGA) is orthe produced blastocyst in vivo. stage, the H3R26me2 profile was altered thesuggest moment a possible where role the inembryo priming begins the DNAto produce for embryonic its own These results suggest that when the embryo begins to factors (Telford et al. 1990). Up until EGA, embryonic de- produce its own factors, in the stage of 8-16 cells, unknown factors in fetal bovine serum affect this epigenetic mark, were stored in the mature oocyte. In cattle, EGA occurs which may lead to altered transcriptional regulation that velopment utilizes the maternal mRNA and proteins that could lead to a change in the development and subsequent Kanka and co-authors (Kanka et al. 2009) have demons- embryo loss. tratedin the transcriptionalstage of 8-16 cells activity (Kopecný of a few et genesal. 1989). in 4-cell However, stage This research provides evidence that media compo- bovine embryos. This increase in transcription of selected genes, prior to the global activation at the 8 to 16-cell sta- such effects should be carefully investigated. ge has been referred to as the “minor genome activation” nentsFuture and additivesstudies are can needed influence to epigeneticdetermine processes,how these and va- and has been reported in several species [for review see (Tadros & Lipshitz 2009). Further studies are needed to affect embryonic development, and how to handle these variationsriations on to modification improve the ofin vitrohistones, production like H3R26me2, of embryos. can the 4-cell embryos in the present study are associated with minordetermine activation if the increaseof the embryonic in H3R26me2 genome. levels The observed increase in Acknowledgements.- The authors would like to thank Carlos V. da Rocha,

invaluable technical assistance during the production embryos. Funding with the 2.5% FBS group containing a higher concentra- Roberta Vantini, Natalia Rocha, Beatriz Leão and Priscila Dall`Acqua for H3R26me2 at the 4-cell stage was observed in both groups, Estado de São Paulo, Brazil (FAPESP, grant 2009/50381-5 to F.L. Lopes). D.R.for this Arnold, project C.A.P. was Corrêa, supported L.L.G. by Lorena, the Fundação R.C. Gaspar, de Apoio and F.L.a Pesquisa Lopes had do tion. How these results represent in vivo conditions is of interest. However, the difficulty of collecting 4-cell stage Pesq. Vet. Bras. 35(7):605-612, julho 2015 611

Supplementation of fetal bovine serum alters histone modification H3R26me2 scholarships funded by FAPESP. J.C.T. Penteado holds a scholarship from vitamins, and culturing embryos in groups stimulate development. Biol. Reprod. 50:390-400. (CAPES). the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil of gamete source and culture conditions on the competence of in vitro- Authors contribution: Hansen-produced P.J., Block embryos J., Loureiro for post-transfer B., Bonilla survivalL. & Hendricks in cattle. K.E. Reprod. 2010. Effects Fertil. - Dev. 22:59-66.

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