Introduction
In vitro culture systems for early embryo development have contributed substantially to the success of many assisted reproduction technologies (Vajta et al., Reference Vajta, Rienzi, Cobo and Yovich2010). Bovine in vitro embryo production (IVP) is routinely used to shorten generational intervals and to propagate valuable genetic material among breeding populations. IVP is widely used in commercial cattle breeding systems and 546,628 in vitro produced embryos were transferred in 2013, of which 72.7% were produced in South America, especially from Bos indicus breeds (Perry, Reference Perry2014). However, differences between embryos produced in vivo with respect to those produced in vitro have been reported, which can primarily be associated to abnormal gene expression profiles and aberrant epigenetic marks. These modifications could explain the reported differences in metabolism, cell number, ultrastructure and cryotolerance (see Urrego et al., Reference Urrego, Rodriguez-Osorio and Niemann2014). Thus, studying the expression profile of selected genes and epigenetic marks could result in improved oocyte and embryo selection criteria and a better discrimination between viable and non-viable oocytes and embryos (Wrenzycki et al., Reference Wrenzycki, Herrmann and Niemann2007).
DNA methylation of specific cytosine residues of CpG dinucleotides is a major epigenetic mark catalyzed by DNA methyltransferases (DNMTs) and critically involved in regulation of gene expression during embryonic development and genomic imprinting (Petrussa et al., Reference Petrussa, Van de Velde and De Rycke2014). Development of the preimplantation mammalian embryo is characterized by dynamic changes in DNA methylation that are critically dependent upon gender and cell lineage (Dobbs et al., Reference Dobbs, Rodriguez, Sudano, Ortega and Hansen2013). DNA methylation profiles can be significantly altered by assisted reproductive technologies (ARTs), including in vitro culture (Niemann et al., Reference Niemann, Carnwath, Herrmann, Wieczorek, Lemme, Lucas-Hahn and Olek2010) and several studies found altered expression of DNMT1 (Cirio et al., Reference Cirio, Ratnam, Ding, Reinhart, Navara and Chaillet2008; Golding et al., Reference Golding, Williamson, Stroud, Westhusin and Long2011) and DNMT3A (Sagirkaya et al., Reference Sagirkaya, Misirlioglu, Kaya, First, Parrish and Memili2006; Gómez et al., Reference Gómez, Gutiérrez-Adán, Díez, Bermejo-Alvarez, Muñoz, Rodriguez, Otero, Alvarez-Viejo, Martín, Carrocera and Caamaño2009) in in vitro produced embryos. These enzymes are involved in maintenance and de novo methylation of DNA, and are thus critical for embryo development. Additionally, aberrant transcript profiles of imprinted genes such as IGF2R are particularly critical for inducing developmental disorders (Perecin et al., Reference Perecin, Méo, Yamazaki, Ferreira, Merighe, Meirelles and Garcia2009; Farin et al., Reference Farin, Alexander and Farin2010; Velker et al., Reference Velker, Denomme and Mann2012).
Although, in vitro embryo production is widely used in Bos indicus breeds, little information is known regarding the effect of this biotechnology on the molecular status during preimplantation development. Here, we evaluated the CpG DNA methylation status of bovine testis satellite I (BTS) and Bos taurus alpha satellite I (BTαS) sequences, and concomitantly the relative abundance of selected transcripts, critically involved in DNA methylation (DNMT1 and DNMT3A), growth and development (IGF2R) and pluripotency (POU5F1) in Bos indicus embryos produced in vitro or in vivo.
Materials and methods
In vitro embryo production
In vitro embryo production was performed under conditions similar to commercial in vitro production. Therefore, ready-to-use commercially available media were obtained from Nutricell (Nutrientes Celulares Campinas, Sao Paulo, Brazil). Ovaries were collected from Bos indicus (Brahman) cows at a local slaughterhouse and maintained at 30°C in sterilized saline solution until use. The method used to produce in vitro embryos has been reported recently (Urrego et al., Reference Urrego, Herrera-Puerta, Chavarria, Camargo, Wrenzycki and Rodriguez-Osorio2015). Briefly, Cumulus–oocyte complexes (COCs) were aspirated from 4 to 8 mm follicles using a 18-gauge needle attached to a 10 ml syringe and handled in Tyrode's Albumin Lactate Pyruvate (TALP)-HEPES medium supplemented with 0.4% bovine serum albumin (BSA) (Sigma Chemical, St Louis, MI, USA).
Cumulus–oocyte complexes were classified morphologically according to the oocyte cytoplasm status and the morphology of cumulus cell layers (Khurana & Niemann, Reference Khurana and Niemann2000). Only COCs with a compact cumulus and homogenous (grade I) or slightly heterogeneous (grade II) cytoplasm were used for this study. Groups of 10 COCs were matured in 50 µl drops of maturation medium (Nutricell), supplemented with 10% fetal bovine serum (FBS; Gibco, Life Technologies, Grand Island, NY, USA), covered with mineral oil (Sigma). In vitro maturation was performed for 24 h in a humidified environment of 5% CO2 in air at 38.5 °C.
In vitro fertilization was performed using commercial frozen sperm from a single Brahman bull with proven fertility. Straws were thawed in a water bath at 37°C for 60 s. Spermatozoa were obtained after centrifugation at 700 g for 10 min in a Percoll (Sigma) discontinuous density gradient (45–90%). After maturation, oocytes were washed three times in 100 μl drops of in vitro fertilization (IVF) medium (Nutricell), supplemented with penicillamine, hypotaurine, epinephrine (Nutricell) and heparin (10 μl/ml) and then transferred to 50 μl IVF medium drops under oil. Spermatozoa were added to reach a final concentration of 2 × 106/ml and co-incubated with approximately 10 COCs in vitro matured for 18–21 h at 38.5°C and 5% CO2 in air.
Presumptive zygotes were partially denuded by mechanical pipetting in TALP–HEPES medium and after washing three times in 100 μl medium drops. Zygotes were cultured in groups of 15–20 in 50 µl drops of synthetic oviduct fluid with amino acids (SOFaa) medium (Nutricell), supplemented with 5% FBS. Embryos were cultured in 5% CO2, 5% O2 and a humidified atmosphere at 38.5°C in air. Half of the culture medium was replaced after 72 h post-insemination (hpi) with fresh SOFaa medium, when cleavage rates were determined. Blastocysts rates were assessed at 162 hpi (D7). Grade 1, 2 or 3 blastocysts (Gordon, Reference Gordon2003) were stored at −80°C in pools of five embryos for gene expression and 10 embryos for DNA methylation analysis in 70 μl Trizol® reagent (Ambion, Life Technologies, Carlsbad, CA, USA).
In vivo embryo production
All procedures involving animals were carried out with the approval from the Committee for Ethical Care and Use of Animals of the University of Antioquia (Colombia). Twelve non-pregnant adult Brahman female donors from a commercial herd were selected according to sanitary and reproductive status. Reproductive organs were examined by transrectal palpation and ultrasonography using an Aloka SSD 500 ultrasound system and an 5 MHz linear transducer (Aloka, Inc., Tokyo, Japan). Only cycling animals confirmed by the presence of a functional corpus luteum and with a body condition score of 3 ± 0.5 units were included (scale of 1 to 5, where 1 indicates emaciated and 5 obese).
Cows were synchronized and superstimulated with the following protocol: D0: placement of an intravaginal device containing 1 g progesterone (DIB, Syntex S.A., Buenos Aires, Argentina) and injection of 2.0 mg estradiol benzoate im (Ric-Be, Syntex S.A.); D4 to D7: superstimulation with eight equal doses of 250 IU FSH at 12 h intervals (Pluset®, Calier, Spain); D6: 0.150 mg cloprostenol im (Prolise®, Tecnopec, São Paulo, Brazil); D6.5: removal of the intravaginal device; D8.5 and D9: Two artificial inseminations were performed, with sperm from the same bull used for IVF with a 12 h interval; D15: embryos were non-surgically collected from the uterine horns as previously described (Neto et al., Reference Neto, Sanches, Binelli, Seneda, Perri and Garcia2005). Briefly, to perform uterine flushings, the catheter was gently inserted into the uterus, the balloon was inflated with air and the uterus was flushed three to four times with a total of 1 litre Dulbecco's phosphate-buffered saline (DPBS; Nutricell). For the second flushing, 80–150 ml of DPBS was infused through the catheter and the plunger was closed with a disposable 5 ml syringe. The same experienced operator performed all procedures within this study. Retrieved embryos were evaluated according to developmental stage and quality (Gordon, Reference Gordon2003). Grade 1, 2 or 3 blastocysts were collected, washed and finally stored at −80°C in pools of five and 10 embryos in 70 μl Trizol® reagent (Ambion, Life Technologies) for further molecular analyses as described above for in vitro produced embryos.
RNA extraction and quantitative real-time-polymerase chain reaction (RT-qPCR)
Pools of five blastocysts (produced in vitro and in vivo) were processed for total RNA extraction using Trizol® reagent protocol, according to the manufacturer's instructions. Briefly, 430 μl of Trizol (Ambion, Life Technologies), was added to the tubes containing embryos to adjust the volume up to 500 µl. Immediately thereafter, 10 μg of RNase-free glycogen (Ambion, Life Technologies) and 200 µl of chloroform (Merck, Darmstadt, Germany) were added to the tubes. Samples were vigorously shaken, incubated at room temperature for 2 min, and centrifuged at 12,000 g for 15 min at 4°C. The upper aqueous phase was removed, and 500 µl cold isopropanol (Merck) was added to the pellet. RNA was precipitated overnight at −20°C, followed by centrifugation at 12,000 g, for 10 min at 4°C. After supernatant removal, RNA pellets were washed with 500 µl 75% ethanol (Merck), air-dried, and resuspended in 30 µl RNase-free water.
The recovered total RNA was immediately transcribed into cDNA. Reverse transcription was performed with total RNA using the Superscript™ III first strand synthesis kit (Invitrogen, Life Technologies) with random hexamer primers. The temperatures and times were 25°C for 10 min, 42°C for 50 min, and 85°C for 5 min. Then, 2 IU of E. coli RNase H was added to each tube and the samples were incubated at 37°C for 20 min.
Primers for RT-qPCR were designed using Primer Premier 5 software (Premier Biosoft International, Palo Alto, CA, USA). All primers were designed to span exon–intron boundaries to rule out genomic DNA amplification (Table 1). RNA relative quantification was performed in three biological replicates and three technical replicates and RT-qPCR was performed on a Rotor-Gene™ 6000 Real-Time PCR instrument (Corbett Life Science, Australia). Quantitative assessment was performed by QuantiTec SYBR PCR kit, (Qiagen, Valencia, CA, USA). Reactions were performed in a total volume of 20 µl using cDNA equivalents of 0.2 embryos and gene specific primers. The polymerase chain reaction (PCR) parameters were 95°C for 5 min for denaturation, 50 cycles of 95°C for 15 s at 60°C for 20 s, 72°C for 30 s and for final extension of 72°C for 5 min. After each PCR run, a melting curve analysis was performed for each sample to confirm that a single specific product had been generated. A negative reverse transcription control was performed to check for genomic DNA contamination. Gene expression levels were calculated by efficiently corrected ΔΔCt method, using GAPDH for data normalization. Primer efficiency was calculated using the program LinRegPCR (Ramakers et al., Reference Ramakers, Ruijter, Deprez and Moorman2003) for each reaction. The selected transcripts are related to DNA methylation (DNMT1 and DNMT3A), growth and development (IGF2R, imprinted gene) and reprogramming (POU5F1).
Primer sequences used for gene expression analysis by real-time PCR
a Denotes the endogenous reference gene.
Analysis of CpG methylation
Bos indicus genomic DNA from pools of 10 expanded blastocysts produced in vitro or in vivo was subjected to bisulfite conversion, to study the methylation status of a set of CpG sequences from the BTS and Bos taurus alpha satellite I (BTαS) (Bos taurus genome). The Bos taurus sequences were used due to limited annotation in the Bos indicus genome when these experiments were performed. However, analysis using the Bos indicus databased at the Basic Local Alignment Search Tool (BLAST) from the National Center for Biotechnology Information (NCBI), revealed hits with more than 96% of identity and E-values lower than 3e−61, located in different chromosomes for the studied sequences, indicating conserved homology. For DNA extraction, DNA/RNA from in vitro and in vivo produced blastocysts were isolated simultaneously using Trizol® reagent (Invitrogen) according to the manufacturer's instructions. After complete removal of the aqueous phase, as described above for the RNA isolation protocol, DNA in the interphase was isolated by removing the remaining aqueous phase, and precipitating the DNA with ethanol. For initial homogenization, 150 µl ethanol (Merck) were added, and samples were mixed by inversion. Next, samples were stored at room temperature for 3 min and DNA was precipitated at 2000 g for 5 min at 4°C. The supernatant was removed and DNA pellets were washed twice in a solution containing 0.1 M sodium citrate (Merck) in 10% ethanol (Merck). At each wash, DNA pellets were stored in the washing solution for 30 min at 15 to 30°C (with periodic mixing). After the second wash, DNA was centrifuged at 2000 g for 5 min at 2 to 8°C, and the pellet was resuspended in 750 µl of 75% ethanol (Merck). DNA was incubated for further 15 min at room temperature (with periodic mixing) and centrifuged at 2000 g for 5 min at 4°C. Finally, cleaned DNA samples were dissolved in 50 µL 8 mM NaOH (Merck), frozen and stored at −20°C until further analysis.
Bisulfite conversion was performed using the EZ DNA Methylation-Direct™ Kit (Zymo Research, Freiburg, Germany) according to the manufacturer's instructions as described recently (Bernal et al., Reference Bernal, Heinzmann, Herrmann, Timmermann, Baulain, Großfeld, Diederich, Lucas-Hahn and Niemann2015). Purified extracted DNA was converted using the CT Conversion Reagent provided by the kit, and incubated at 98°C for 8 min followed by 64°C for 3.5 h in a thermal cycler. After DNA conversion, unmethylated cytosines are transformed into uracil, while methylated cytosines remain unchanged. Converted DNA was washed and cleaned using the Zymo-Spin™ IC column and dissolved in 10 μl M-Elution Buffer.
Bovine testis satellite I (BTS) and Bos taurus alpha satellite I (BTαS) sequences were amplified using specific primers (Table 2) (Kang et al., Reference Kang, Lee, Shim, Yeo, Kim, Koo, Lee, Beyhan, First and Han2005; Diederich et al., Reference Diederich, Hansmann, Heinzmann, Barg-Kues, Herrmann, Aldag, Baulain, Reinhard, Kues, Weissgerber, Haaf and Niemann2012). BTS contains 12 highly conserved CpG sites in a 211-bp fragment. For the BTαS sequence, a fragment of 154 bp containing nine CpG sites was analyzed (Kang et al., Reference Kang, Lee, Shim, Yeo, Kim, Koo, Lee, Beyhan, First and Han2005). Satellite sequence-specific PCR fragments were amplified and successful amplification was confirmed by agarose gel electrophoresis. PCR products were cleaned using the Invisorb® Fragment Cleanup System (Stratec Molecular GmbH, Berlin, Germany) according to manufacturer's instructions. PCR products were ligated into the pGEM-T easy vector (Promega Corporation, Madison, USA) and the plasmids were transformed into Escherichia coli XL10-Gold cells (Stratagene, Santa Clara, CA, USA). Clones were screened for successful ligation and transformation; therefore colonies were picked and directly used for PCR amplification of the insert using the universal T7 and SP6 primers (Table 2). Positive clones were submitted to DNA sequencing using the same universal primers on a 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Sequences were analyzed using the BiQ Analyzer program (Bock et al., Reference Bock, Reither, Mikeska, Paulsen, Walter and Lengauer2005). The specific genomic sequence from the bovine genome for each studied satellite was used for comparison and CpG identification on sample sequences. Clone sequences with a conversion rate lower than 90% or with a high number of sequencing errors in the alignment were excluded from the analysis. The methylation profiles for each satellite were evaluated by counting the methylated CpG sites of the total number of analyzed CpG.
Primer sequences used for analysis of satellite sequences
Statistical analysis
Relative expression software tool (REST) was used to compare mRNA abundances in each group. The mathematical model used in REST software is based on PCR efficiencies and the crossing point deviation between samples (Pfaffl, Reference Pfaffl2002). For each group there were three biological and three technical replicates. Methylation profiles were analyzed using the Pearson's chi-squared test from R software (R Development Core Team, 2011). The level of significance for all tests was set at P ≤ 0.05. Data from in vivo and in vitro embryo production are presented descriptively.
Results
In vivo and in vitro production of bovine embryos
To determine the effects of in vitro embryo production on DNA methylation profiles and the expression of genes involved in epigenetic reprogramming during early embryo development in Bos indicus, we produced in vivo and in vitro bovine blastocysts. All donors submitted to multiple ovulations (MO) responded to the stimulation protocol, and two or more corpora lutea (CL) were visualized by ovarian ultrasound examination. In total, 101 embryos with grade 1, 2 or 3 were recovered after uterine flushing for molecular analysis. Cleavage rates were calculated as the proportion of all recovered embryos divided by the number of total obtained ova (88.8 ± 3.6%). The rate of transferable embryos, measured as the proportion of grade 1, 2, and 3 recovered embryos divided by the number of total ova, was 79.4 ± 8.7% (Table 3). After in vitro embryo production, 284 embryos were obtained for this study. Six replicates of the in vitro embryo production, using COCs obtained from slaughterhouse material, were performed. On average, the cleavage rate was 81.1 ± 1.4% at 72 hpi and after 7 days of in vitro embryo culture 39.9 ± 0.5% of the cleaved embryos had developed to the blastocyst stage (blastocysts/cleaved embryos).
Embryo production
a Fertilization rate: proportion of recovered grade 1, 2, 3 or 4 embryos divided by total ova.
b Transferable embryos rate: proportion of recovered grade 1, 2 or 3 embryos divided by total ova.
c Degenerated embryos rate: proportion of grade 4 embryos divided by total ova.
d Six replicates. The blastocyst rate is based on cleaved embryos.
IVM, in vitro matured.
mRNA expression analysis of DNMT1, DNMT3A, IGF2R and POU5F1 genes in bovine blastocysts produced in vitro or in vivo
Expression of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as the internal control. DNA methyltransferase 1 (DNMT1) transcript levels were significantly reduced in in vitro produced blastocysts (P < 0.05) compared to their in vivo derived counterparts. DNA methyltransferase 3A (DNMT3A), IGF2R (Insulin-like growth factor 2 receptor) and POU5F1 (POU class 5 homeobox 1) mRNA abundance were not significantly different between in vivo and in vitro derived blastocysts (Fig. 1).
Transcript levels [(mean ± standard error of the mean (SEM)] for DNMT1, DNMT3A, IGF2R, and POU5F1, analyzed by RT-qPCR in Bos indicus embryos produced in vivo (black columns) and in vitro (grey columns). Each group was analyzed using three biological replicates and three technical replicates. Each biological replicate consisted of a pool of five embryos. a,bDifferent letters in the bars indicate different values (P < 0.05).
Methylation profiles of two satellite DNA sequences
In total, 184 clones, including 1887 CpGs, were evaluated to determine the Bos indicus blastocyst methylation status of the ovine testis satellite I (BTS) and Bos taurus alpha satellite I (BTαS) sequences (Table 4). For in vivo produced embryos, the methylation percentage of BTS was 13.1%, whereas the methylation profile of in vitro produced embryos was significantly higher (18.7%, P < 0.05). The CpG methylation level for the BTαS sequence did not differ significantly between embryos produced in vivo (35.8%) and embryos produced in vitro (32.5%).
Methylation pattern of bovine testis satellite I (BTS) and Bos taurus alpha satellite I (BTαS) sequences.
a,b Rows with different superscript letters per satellite are significantly different (P < 0.05).
c Per cent methylation levels are the proportion of methylated CpG sites relative to the total number.
Discussion
In vitro embryo production has emerged as a useful alternative to conventional embryo transfer and the technology is being used commercially in several countries around the globe. In South America, a significant proportion of cattle embryos has been produced by IVP since 2004, primarily in Bos indicus animals (Camargo et al., Reference Camargo, Freitas, de Sa, de Moraes Ferreira, Serapiao and Viana2010), but little information is known about the effects on embryo quality in this species. Furthermore, numerous studies have indicated that the consequences of exposing embryos to in vitro culture conditions include alterations in gene expression and aberrant DNA methylation (Wrenzycki et al., Reference Wrenzycki, Lucas-Hahn, Herrmann, Lemme, Korsawe and Niemann2002; Niemann et al., Reference Niemann, Carnwath, Herrmann, Wieczorek, Lemme, Lucas-Hahn and Olek2010; Urrego et al., Reference Urrego, Rodriguez-Osorio and Niemann2014). Here, we investigated the effects of in vitro embryo production on satellite DNA CpG methylation status and relative mRNA abundance of four developmentally important genes in expanded Bos indicus blastocysts produced in vivo and in vitro.
In the present study, blastocyst rates using commercial media were consistent with previous reports, in which culture was carried out under similar conditions (Camargo et al., Reference Camargo, Boite, Wohlres-Viana, Mota, Serapiao, Sa, Viana and Nogueira2011; Urrego et al., Reference Urrego, Herrera-Puerta, Chavarria, Camargo, Wrenzycki and Rodriguez-Osorio2015). In vivo blastocyst production was as expected in Bos indicus animals. It is known that Bos indicus cattle differ from their Bos taurus counterparts in several details of reproductive physiology (Baruselli et al., Reference Baruselli, Ferreira, Sales, Gimenes, Sá Filho, Martins, Rodrigues and Bó2011). For instance, Bos indicus have a greater sensitivity to gonadotropins, a shorter duration of estrus, and more often express estrus during the night (Bó et al., Reference Bó, Baruselli and Martínez2003). It has been also reported that Bos indicus cows have a smaller diameter of the dominant follicle and more ovarian follicles recruited per follicular wave than Bos taurus, which in turn may increase embryo production (Sartori et al., Reference Sartori, Bastos, Baruselli, Gimenes, Ereno and Barros2010).
Development of bovine embryos subjected to in vitro culture has been associated with an increased frequency of pre- and postnatal abnormalities that are thought to be the result of changes in epigenetic marks and gene expression profiles (Wrenzycki et al., Reference Wrenzycki, Herrmann, Lucas-Hahn, Korsawe, Lemme and Niemann2005; Rodriguez-Osorio et al., Reference Rodriguez-Osorio, Urrego, Cibelli, Eilertsen and Memili2012; Urrego et al., Reference Urrego, Rodriguez-Osorio and Niemann2014). In the present study, we found that DNMT1 expression was significantly reduced in in vitro produced blastocysts compared with in vivo blastocysts (Fig. 1), whereas no significant differences were found with regard to the relative mRNA abundance for DNMT3A, IGF2R and POU5F1 between in vitro and in vivo derived embryos.
DNMT1 plays a critical role in the maintenance of DNA methylation by restoring the methylation pattern of newly synthesized hemi-methylated DNA strands during replication (Bestor et al., Reference Bestor, Gundersen, Kolsto and Prydz1992; Pradhan et al., Reference Pradhan, Bacolla, Wells and Roberts1999). Previous studies had indicated a pattern of aberrant expression profile for DNMT1. In human embryos, DNMT1 expression was lower in poor quality embryos compared to the reference group (Petrussa et al., Reference Petrussa, Van de Velde and De Rycke2014). Transcript levels of DNMT1 were lower in somatic cell nuclear transfer (SCNT) derived bovine embryos, suggesting that epigenetic programming by DNMT1 is critical for regular bovine preimplantation development (Golding et al., Reference Golding, Williamson, Stroud, Westhusin and Long2011). Likewise, it has been demonstrated that vitrification decreased the mRNA abundance of Dnmt1o in mouse oocytes, probably as an effect of altered epigenetic marks (Zhao et al., Reference Zhao, Ren, Du, Hao, Wang, Qin, Liu and Zhu2013). Therefore, the lower mRNA abundance observed in the in vitro produced blastocysts is probably associated with decreased blastocyst quality.
The DNMT3A protein is a de novo DNA methyltransferase, which acts upon hemi-methylated and non-methylated DNA with equal efficiency during early embryonic development (Okano et al., Reference Okano, Bell, Haber and Li1999). In contrast with DNMT1, transcript levels of DNMT3A were not affected by in vitro culture in the present study. These findings differ from the results reported by Hoffmann et al. (Reference Hoffmann, Niemann, Hadeler, Herrmann and Wrenzycki2006), in which the amount of DNMT3A mRNA was affected by in vitro culture. Various protocols are presently employed to generate bovine IVF embryos, and protocol-specific differences therefore might explain discrepancies in results.
Transcript abundance for IGF2R did not differ in IVP embryos compared with those of the control group. However, loss of IGF2R expression in bovine in vitro produced embryos was associated with excessive fetal and placental growth (Farin et al., Reference Farin, Piedrahita and Farin2006, Reference Farin, Alexander and Farin2010; Farmer et al., Reference Farmer, Farin, Piedrahita, Bischoff and Farin2013). Aberrant expression of IGF2R directly correlated with the large offspring syndrome (LOS) in sheep (Young et al., Reference Young, Fernandes, McEvoy, Butterwith, Gutierrez, Carolan, Broadbent, Robinson, Wilmut and Sinclair2001). In general, SNRPN, H19/IGF2, and IGF2R imprinted genes are extensively hypomethylated in early stage embryos derived from SCNT and to a lesser extent in IVP embryos, indicating that reprogramming of the chromatin and the in vitro culture of oocytes and/or embryos may be associated with epigenetic erasure of imprinted loci (Smith et al., Reference Smith, Therrien, Filion, Bressan and Meirelles2015).
POU5F1 (also called OCT4) is a member of the POU transcription factor family with a germ line-specific expression profile. It is widely used to identify pluripotent cells in different species and plays a critical role in bovine preimplantation development (Kirchhof et al., Reference Kirchhof, Carnwath, Lemme, Anastassiadis, Schöler and Niemann2000; Herrmann et al., Reference Herrmann, Dahl, Lucas-Hahn, Collas and Niemann2013). This study did not find differences in the level of POU5F1 expression. However, it has been shown that the mean transcript level of POU5F1 was significantly higher in KSOMaa cultured blastocysts than in blastocysts produced in SOFaa medium or in vivo, clearly indicating sensitivity of OCT-4 expression to culture environment (Purpera et al., Reference Purpera, Giraldo, Ballard, Hylan, Godke and Bondioli2009).
Epigenetic reprogramming of the mammalian genome after fertilization creates the methylation patterns needed for normal development by activation and silencing of specific genes (Reik et al., Reference Reik, Dean and Walter2001; Haaf, Reference Haaf2006). The global methylation of the bovine genome declines to a nadir at the 6–8-cell stage and increases thereafter (Dobbs et al., Reference Dobbs, Rodriguez, Sudano, Ortega and Hansen2013), rendering early embryos specifically vulnerable to ART-related epigenetic defects (El Hajj & Haaf, Reference El Hajj and Haaf2013). Genome-wide abnormalities in DNA methylation patterns or cytosine methylation levels after IVP have been observed in bovine embryos (Hou et al., Reference Hou, Liu, Lei, Cui, An and Chen2007; Suzuki et al., Reference Suzuki, Therrien, Filion, Lefebvre, Goff and Smith2009; Niemann et al., Reference Niemann, Carnwath, Herrmann, Wieczorek, Lemme, Lucas-Hahn and Olek2010). Our results revealed significant hypermethylation for BTS in IVP embryos compared with their in vivo derived counterparts, while no significant difference was observed for BTαS between the two groups of embryos.
Satellite DNA is found in centromeric and pericentromeric regions and is highly conserved in mammals (Enukashvily and Ponomartsev, Reference Enukashvily and Ponomartsev2013). Considered as ‘junk DNA’ for many years, it is known that satellite DNAs have significant impact on genomic functions including chromosome organization and segregation. Furthermore, their transcripts have functional roles on formation and maintenance of heterochromatin structure (Plohl et al., Reference Plohl, Meštrović and Mravinac2012), and have been related to stress, embryogenesis, mitosis, senescence and carcinogenesis (Enukashvily & Ponomartsev, Reference Enukashvily and Ponomartsev2013). Some DNA satellites sequences have been successfully analyzed for monitoring epigenetic changes in early embryos related to the origin of oocytes and embryos (Suzuki et al., Reference Suzuki, Therrien, Filion, Lefebvre, Goff and Smith2009; Sawai et al., Reference Sawai, Takahashi, Fujii, Moriyasu, Hirayama, Minamihashi, Hashizume and Onoe2011; Bernal et al., Reference Bernal, Heinzmann, Herrmann, Timmermann, Baulain, Großfeld, Diederich, Lucas-Hahn and Niemann2015). The hypermethylation observed in the BTS satellite sequence in the in vitro generated embryos compared with their in vivo produced counterparts, could indicate perturbation of demethylation of specific sequences. The decreased DNMT1 expression detected in the in vitro produced embryos could be the result of a compensation mechanism to control global DNA hypermethylation. However, future studies are necessary to confirm these observations.
Similar to our results, increased DNA methylation levels of BTS have been reported previously for blastocysts produced in vitro (Bernal et al., Reference Bernal, Heinzmann, Herrmann, Timmermann, Baulain, Großfeld, Diederich, Lucas-Hahn and Niemann2015). Satellite DNA hypermethylation has been observed in aborted cloned fetuses (Zhang et al., Reference Zhang, Wang, Han, Duan, Lv and Li2014) and abnormal placenta (Perrin et al., Reference Perrin, Ballestar, Fraga, Frappart, Esteller, Guerin and Dante2007). Furthermore, highly methylated DNA may be associated with gene silencing. The bisulfite sequencing approach in the present study does not allow discrimination between 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC), and the identification of non-CpG methylation sites, but it provides a general overview of CpG DNA methylation status, indicating that in vitro embryo production in Bos indicus cattle affects embryo quality at the molecular level as reported previously in other cattle species (Hou et al., Reference Hou, Liu, Lei, Cui, An and Chen2007). When interpreting these findings, one has to take into account that in vivo derived embryos used for these experiments, were collected after superovulatory treatment of the donors. Although superovulation can affect embryonic gene expression (Mundim et al., Reference Mundim, Ramos, Sartori, Dode, Melo, Gomes, Rumpf and Franco2009; Diederich et al., Reference Diederich, Hansmann, Heinzmann, Barg-Kues, Herrmann, Aldag, Baulain, Reinhard, Kues, Weissgerber, Haaf and Niemann2012; Urrego et al., Reference Urrego, Rodriguez-Osorio and Niemann2014), it is the only way to produce sufficient in vivo derived embryos for research purposes.
In conclusion, our study reports changes in gene expression profiles and aberrant methylation patterns of satellite DNA in Bos indicus blastocysts produced in vitro (Fig. 2). The present results are consistent with previous findings, in which bovine embryos respond to alterations in their environment by modifying DNA methylation and transcription, thus confirming the impact of ARTs on epigenetic marks during embryo development in different species. Further research in Bos indicus cattle is needed to clarify the effects of ARTs on in vitro production to improve the quantitative and qualitative efficiency of the process.
Influence of IVP on epigenetic profiles and gene expression in Bos indicus embryos. Relative transcript abundance for genes was compared in embryos produced in vivo (control) vs. embryos produced in vitro. In vitro produced embryos had significantly lower amounts of DNMT1 (P < 0.05), while DNA methylation was significantly higher (P < 0.05) in the bovine testis satellite I (BTS) sequence in embryos produced in vitro compared with that of in vivo produced embryos.
Acknowledgments
The authors thank Administrative Department of Science, Technology and Innovation (COLCIENCIAS) for providing a scholarship for the first author. COLCIENCIAS COD 122852128473, CES University and the Sustainability Strategy 2013–2014 from CODI (Universidad de Antioquia), supported this work. We thank Jose Tamayo for assistance in figure design.
Conflicts of interests
None of the authors has any conflict of interest to declare.
