Introduction
Folic acid (FA) is a synthetic and stable compound that is converted into folate, an essential component in cell division where it is required as a coenzyme for nucleotide and amino acid biosynthesis. During early embryo development, folate is required to prevent neural tube defects (NTD). As FA is not produced endogenously, a diet supplementation is needed (Scaglione and Panzavolta, Reference Scaglione and Panzavolta2014). Several years ago FA started to be added to basic dietary components such as wheat and corn flour (Berry et al., Reference Berry, Bailey, Mulinare, Bower and Dary2010). However, these concentrations were not sufficient to prevent NTD and a daily dose of 0.4 mg is recommended for women who want to conceive; the dose, as well as the period of intake, may vary between countries (Gomes et al., Reference Gomes, Lopes and Pinto2016; Krupenko, Reference Krupenko, De Caterina, Martinez and Kohlmeier2019; Hussain and Sharma, Reference Hussain and Sharma2020).
Due to its role as a ‘carry one-carbon’ unit and in the transformation of homocysteine to methionine, FA is an important source of methyl groups during DNA methylation, an epigenetic marker that controls gene expression throughout development (Wagner, Reference Wagner2001). DNA methylation is the main mechanism behind genomic imprinting, an epigenetic process with monoallelic gene expression in a parent-of-origin effect, essential for mammalian development and placentation. The biallelic expression of imprinted genes or loss of imprinting (LOI) leads to growth and developmental disorders, such as large offspring syndrome (LOS) in bovines and others ruminants (Suzuki et al., Reference Suzuki, Therrien, Filion, Lefebvre, Goff, Perecin, Meirelled and Smith2011; Chen et al., Reference Chen, Robbins, Wells and Rivera2013; Urrego et al., Reference Urrego, Rodriguez-Osorio and Niemann2014).
At chromosome 29 in bovines, there are two important imprinting control regions (ICRs) that are differentially methylated regions (DMRs) in the maternal and paternal genomes. The H19 DMR is located upstream of the H19 promoter and contains a CTCF-binding domain. CTCF is an insulator protein and, in the unmethylated maternal allele, blocks enhancer activity on the IGF2 promoter, which is silenced during H19 expression. In the paternal allele, methylation induces changes in the H19 promoter, which is silenced, and enhancer-blocking activity is prevented, allowing paternal IGF2 activation by the enhancer (Bell and Felsenfeld, Reference Bell and Felsenfeld2000; Lewis and Reik, Reference Lewis and Reik2006). The second region, the KvDMR1, is located in the intronic region of the KCNQ1 gene that overlaps with the promoter region of KCNQ1OT1 (or LIT1). This region is methylated on the maternal allele, therefore the KCNQ1OT1 gene is silenced, whereas the KCNQ1 gene is maternally expressed. The opposite process occurs in the paternal allele, where KCNQ1OT1 is expressed (Fig. 1). KvDMR1 also controls the maternally expressed CDKN1C gene (Mitsuya et al., Reference Mitsuya, Meguro, Lee, Katoh, Schulz, Kugoh, Yoshida, Niikawa, Feinberg and Oshimura1999; Horike et al., Reference Horike, Mitsuya, Meguro, Kotobuki, Kashiwagi, Notsu, Schulz, Shirayoshi and Oshimura2000; O’Neill, Reference O’Neill2005).
Schematic representation of H19 DMR and KvDMR1 imprinting control regions (ICRs) at bovine chromosome 29. The H19 DMR controls H19 and IGF2, which are maternally and paternally expressed, respectively. This is an enhancer competition model with an insulator CTCF blocking activity. The maternal unmethylated allele allows binding of the CTCF protein that prevents IGF2 activation by the enhancer, whereas H19 is expressed. Paternal methylation blocks insulator activity, allowing IGF2 activation by the enhancer and silencing of the H19 gene. KvDMR1 is located in the promoter region of the KCNQ1OT1 gene that overlaps with an intronic region of the KCNQ1 gene. Methylation in the maternal allele silences KCNQ1OT1, whereas KCNQ1 and CDKN1C neighbouring genes are expressed. In the paternal allele, KvDMR1 is unmethylated and allows KCNQ1OT1 expression and the KCNQ1 and CDKN1C are silenced.
Establishment of maternal imprinting and other epigenetic markers during oogenesis is essential for oocyte competence, fertilization and embryo development (Reik et al., Reference Reik, Dean and Walter2001; Macdonald and Mann, Reference MacDonald and Mann2014; DeAngelis et al., Reference DeAngelis, Martini and Owen2018). Oocyte contributions towards organelles, mRNAs and nutrients are fundamental to the maintenance of early embryo development prior to zygotic genome activation (ZGA) and microenvironment modifications may affect epigenetic reprogramming in the oocyte (Meirelles et al., Reference Meirelles, Caetano, Watanabe, Ripamonte, Carambula, Merighe and Garcia2004; Urrego et al., Reference Urrego, Rodriguez-Osorio and Niemann2014; Siqueira et al., Reference Siqueira, Silva, Panetto and Viana2020).
Assisted reproductive technologies (ARTs), as in in vitro maturation of oocytes during in vitro production (IVP) of embryos, is related to aberrant expression of imprinted genes, directly affecting embryo production, pregnancy rate and adulthood health (Kussano et al., Reference Kussano, Leme, Guimarães, Franco and Dode2016; DeAngelis et al., Reference DeAngelis, Martini and Owen2018; Harper et al., Reference Harper, Aittomaki, Borry, Cornel, Werts, Dondorp, Geraedts, Gianaroli, Ketterson, Liebaers, Lundin, Mertes, Morris, Pennings, Sermon, Spits, Soini, van Montfoort, Veiga, Vermeesch, Viville and Macek2018). Even so, those technologies are important in the treatment of infertility, as well as in large-scale production of embryos of domestic animals and storage for lineage maintenance of genetically superior animals (Sirard, Reference Sirard2017; Viana, Reference Viana2018).
It has been reported that FA may be beneficial for oocyte maturation and posterior embryo production, as early embryonic development depends on endogenous folate stored during oocyte maturation (O’Neill, Reference O’Neill1998; Kwong et al., Reference Kwong, Adamiak, Gwynn, Singh and Sinclair2010; Kooistra et al., Reference Kooistra, Trasler and Baltz2013; Sato et al., Reference Sato, Sakurai, Monji, Kuwayama and Iwata2013; Liu et al., Reference Liu, Zhi, Shen, Li, Yao and Yang2016). FA supplementation during oocyte growth may influence embryo epigenetic reprograming and, as consequence, alter the expression of important imprinted genes (Mann and Watson, Reference Mann and Watson2013), therefore we evaluated the influence of different FA concentrations on in vitro maturation of bovine oocytes during embryo production and on expression of IGF2 and KCNQ1OT1 genes.
Materials and methods
In vitro production of embryo
In vitro maturation (IVM) of oocytes
Bovine ovaries were collected from slaughtered animals. Follicles sized between 2–8 mm were aspirated using an 18-gauge needle connected to a 10 ml syringe. Oocytes were selected according to Leibfried and First (Reference Leibfried and First1979) and, in total, 1087 grade I (GI) cumulus–oocyte complexes (COCS) and 1072 grade III (GIII) COCs were selected. Oocytes were matured in the absence or presence of 10 μM, 30 μM or 100 μM FA (8758-5G, Thermo Fischer Scientific, USA). The groups (GI and GIII) without FA were placed in microdrops of IVM medium [TCM199 with Earle’s salts, glutamine, NaHCO3 pyruvate (22 μg/ml), 10% of FBS, FSH (0.5 mg/ml), LH (50 μg/ml), amikacin (83 μg/ml), and estradiol (1.0 μg/ml)], over 22–24 h at 38.8°C, high humidity and 5% CO2. Oocytes treated with FA were matured in IVM medium supplemented with 10 μM, 30 μM or 100 μM FA under the same conditions of temperature, humidity and CO2 in air (20 oocytes/100 μl microdrops).
In vitro fertilization (IVF)
Semen samples from a single bull and from same batch were used in all experiments. The semen sample was centrifuged (342 g) through a Percoll (Sigma-Aldrich, USA) gradient (45:90) for 30 min. After centrifugation, the pellet was analyzed for sperm concentration and motility; each microdrop of the IVF medium contained approximately 2 × 106 sperm/ml. After IVM, oocytes were washed twice with IVF medium (TALP) supplemented with heparin (10 mg/ml), pyruvate (22 μg/ml), BSA FAF (fatty acid free) (6 μg/ml), PHE solution (2 μM of penicillamine, 1 μM of hypotaurine, and 0.25 μM of epinephrine) and amikacin (83 μg/ml) and placed with sperm in IVF microdrops (20 oocytes/100 µl). The gametes were incubated for 18 h at 38.8°C, in high humidity and 5% CO2 in air.
Embryos in vitro culture (IVC)
After the fertilization period, presumptive zygotes (1-cell embryo; day 0 – D0) were transferred to CR2 culture medium supplemented with 5% FBS, 0.03 g BSA V, 0.03 g BSA FAF V, 100 μl alanine, 100 μl glycine and amikacin (83 μg/ml). Embryos were cultured at 38.8°C, in high humidity and 5% CO2 in air for 7 days for preimplantation development. On the seventh day of development (D7), embryos were classified according to the International Embryo Transfer Society guidelines as initial blastocyst, blastocyst, expanded blastocyst or hatched blastocyst; embryo production was evaluated based on viable blastocysts on D7 in relation to zygotes that initiated culture on D0. To analyze gene expression, pools of 12 expanded blastocysts from each treatment were washed in phosphate-buffered saline and stored at −80°C until RNA extraction. Three experimental replicates were evaluated for each treatment.
IGF2 and KCNQ1OT1 gene expression
RNA extraction was performed using the RNeasy® MicroKit (Qiagen, USA) following the manufacturer’s protocol. cDNA synthesis was performed using the SuperScript® III First-strand Synthesis System for RT-PCR (Thermo Fischer Scientific, USA) and oligo(dT) and random hexamers primers (70/30 ratio), following the manufacturer’s recommendations. IGF2 and KCNQ1OT1 gene expression was evaluated using quantitative PCR (qPCR) and the SYBR® Green PCR Master Mix (Thermo Fischer Scientific, USA) with the StepOne Real-Time PCR System (Applied Biosystems, USA). Amplification conditions were: 95°C for 10 min, followed by 45 cycles of 95°C for 15 s and 59ºC for 1 min. Sequences for IGF2, KCNQ1OT1 and the reference gene GAPDH were obtained from the National Center for Biotechnology Information (ncbi.nlm.nih.gov). Primers were designed using PREMIER Biosoft software. Primers for the reference gene ACTB have been described previously by Rios et al. (Reference Rios, Lemos, Fernandes, Andrea, Gomes, Lôbo, Mazucato and Ramos2007). Primer sequences are listed in Table S1. The comparative quantification cycle (Cq) was used to evaluate gene expression based on the Pfaffl method (Reference Pfaffl2001).
Statistical analysis
Statistical analysis was performed using the R platform; data distribution was evaluated using the Shapiro–Wilk test. To evaluate embryo production from GI and GIII oocytes and to compare the influence of FA treatment (10, 30 and 100 μM) the Mann–Whitney or Student’s t-test was used, depending on data distribution. FA influence on IGF2 and KCNQ1OT1 gene expression was compared using analysis of variance followed by Tukey post-hoc test. All results were compared considering a level of significance of 5%.
Results
Embryo production
When comparing the embryo production from GI and GIII oocytes without FA addition, we observed a higher embryo production rate from GI oocytes (51.85% ± 6.7) compared with GIII oocytes (36.10% ± 4.8) (P = 0.00003) (Table 1).
In vitro production of bovine embryos
GI, Grade I oocytes; GIII, Grade III oocytes; COCs, cumulus–oocytes complexes; D0, zygotes; D7, embryos at blastocyst stage; SD, standard deviation.
*, P < 0.05.
Addition of FA to IVM medium altered the production rate depending on concentration and oocyte quality (Table 2). Addition of the lowest FA dose (10 μM) during GI and GIII oocyte maturation did not alter the embryo production rate (P = 0.181 and P = 0.075, respectively). When FA supplementation was increased to 30 μM during GIII oocyte maturation, an increase in embryo production was observed (P = 0.012). Conversely, 30 μM of FA in GI oocytes seemed not to affect embryo production (P = 0.600). Treatment with the highest FA concentration (100 μM) influenced GI oocyte maturation, reducing embryo production (P = 0.043), but did not influence embryo production from GIII oocytes (P = 0.362).
In vitro production of bovine embryos in GI and GIII oocytes matured in the absence or presence of folic acid
COCs, cumulus–oocytes complexes; NA, not added; D0, zygotes; D7, embryos at blastocyst stage; SD, standard deviation.
a Statistical analysis between NA group and treatments. *, P < 0.05.
IGF2 and KCNQ1OT1 gene expression
Expression of IGF2 and KCNQ1OT1 genes was analyzed on pools of expanded blastocysts. IGF2 expression was detected in embryos derived from GI oocytes without FA supplementation and in the lowest FA concentrations (10 and 30 μM). Addition of FA to the IVM medium seemed not to affect IGF2 expression, irrespective of the concentration (10 μM, P = 0.680 and 30 μM P = 0.956) when compared with the control group (without FA supplementation). In addition, when 100 μM of FA was added, IGF2 expression was not detected. Finally, for embryos from GIII oocytes, IGF2 expression was detected only in the control group without FA supplementation.
Although IGF2 expression was detected in GI oocytes, expression levels were interestingly low compared with expression of the KCNQ1OT1 gene, which was observed in all groups. For expanded blastocysts from GI oocytes, KCNQ1OT1 expression was not altered after FA supplementation (10 μM, P = 0.998; 30 μM, P = 0.998; 100 μM, P = 0.458). However, for embryos from GIII oocytes, the lowest dose of FA (10μM) reduced KCNQ1OT1 expression (P = 0.05), while moderate (30 μM, P = 0.878) and high FA doses (100μM, P = 0.817) did not alter gene expression compared with the control group without FA. In addition, increased expression of KCNQ1OT1 in GIII oocytes with 30 μM FA (P = 0.038) was observed when compared with 10 μM (Fig. 2).
Relative expression of IGF2 and KCNQ1OT1 genes on expanded blastocysts from GI and GIII oocytes matured in the absence or presence of FA (10 μM, 30 μM and 100 μM).
Note: different letters indicate a statistical difference (P < 0.05). FA, folic acid.
Discussion
Addition of FA during IVM of bovine oocytes influenced embryo production and gene expression depending on oocyte quality and FA concentration. Moderate FA concentrations (30 μM) improved embryo production from GIII oocytes whereas, in GI oocytes, the highest concentration (100 μM) reduced embryo production rate and IGF2 gene expression. Conversely, the lowest concentration of FA (10 μM) decreased KCNQ1OT1 expression in embryos derived from GIII oocytes.
FA is the synthetic form of folate, considered to be more stable and appropriate to be processed, cooked and stored (Institute of Medicine, 1998). Its daily ingestion is recommended during the periconceptional period and also during pregnancy to avoid, mainly, neural-tube defects (Smithells et al., Reference Smithells, Sheppard and Schorah1976; Czeizel and Dudás, Reference Czeizel and Dudás1992; Tamura and Picciano, Reference Tamura and Picciano2006; Hoek et al., Reference Hoek, Koster, Schoenmarkers, Willemsen, Koning, Steegers and Steegers-Theunissen2019). Nevertheless, before incorporation into the folate pool, FA needs to be reduced by dihydrofolate reductase (DHFR) enzyme into smaller molecules such as tetrahydrofolate (THF). THF is converted to 5,10-methylenetetrahydrofolate (5,10-MTHF) and catalyzed by methylenetetrahydrofolate reductase (MTHFR) to form 5-methylenetetrahydrofolate (5-MTHF). 5-MTHF participates in the one-carbon cycle, donating methyl groups to many chemical reactions (Yamada et al., Reference Yamada, Strahler, Andrews and Matthews2005; Troen et al., Reference Troen, Mitchell, Sorensen, Wener, Johnsin, Wood, Selhub, McTiernan Yasui, Oral, Potter and Ulrich2006; Enciso et al., Reference Enciso, Sarasa, Xanthopoulou, Bristow, Bowles, Fragouli, Delhanty and Wells2016; Cornet et al., Reference Cornet, Clement, Clement and Ménézo2019). As a consequence, only cells that contain DHFR and MTHFR enzymes are able to take up and metabolize to folate the FA present in its microenvironment. Kwong and colleagues (Reference Kwong, Adamiak, Gwynn, Singh and Sinclair2010) demonstrated that these enzymes are present in bovine oocytes and preimplantation embryos, suggesting that they are able to perform this biochemical conversion.
Folate products are essential for early embryo development due to their participation in homocysteine methylation and thymidine synthesis, being important components of DNA replication during cell division (O’Neill, Reference O’Neill1998; Benkhalifa et al., Reference Benkhalifa, Montjean, Cohen-Bacrie and Ménézo2010; Froese et al., Reference Froese, Kopec, Rembeza, Bezerra, Oberholzer, Suormala, Lutz, Chalk, Borkowska, Baumgartner and Yue2018). O’Neill (Reference O’Neill1998) also suggested that FA captured by oocytes during the maturation period could be used by embryos to assist metabolism during development up to ZGA. In animal models, a positive relationship between FA and oocyte maturation has been shown previously after addition to the maturation medium, and was related to the uniform distribution of granulosa cells and formation of polar body in developing follicles (Huang et al., Reference Huang, Gao, Hou and Wu2013).
We observed that, without FA supplementation, morphological differences between oocytes before IVM seem to be enough to influence production rate, as embryo production from GI oocytes was higher compared with that from GIII oocytes. Morphological differences observed in bovine oocytes before IVM regarding the number of cumulus cells layers in the COCs and the ooplasm appearance (homogeneous or heterogeneous) separated the oocytes into three groups – GI, GII and GIII. These morphological characteristics may imply the ability to complete maturation and could determine the fertilization success rate, as those classified as GI were considered to be of better quality than the GIII group (Boni et al., Reference Boni, Cuomo and Tosti2002; Saini et al., Reference Saini, Singh, Shah, Singh, Kaushik, Manik, Singla, Palta and Chauhan2015; Walker and Biase, Reference Walker and Biase2020).
Addition of FA during the IVM improved embryo production depending on oocyte morphology. The lowest dose of FA (10 μM) during oocyte maturation did not affect embryo production rate on the seventh day, regardless of oocyte quality (GI or GIII). Similar results were found by Sato et al. (Reference Sato, Sakurai, Monji, Kuwayama and Iwata2013) when 10 μM FA was added during porcine oocyte maturation. Nevertheless, moderate FA concentrations (30 μM) increased the production rate of embryos in low quality oocytes (GIII group). Although it is still unclear how FA acts during oocyte maturation and early embryo development (Crider et al., Reference Crider, Yang, Berry and Bailey2012), bovine oocytes seem to produce both DHFR and 5-MTHFR (Kwong et al., Reference Kwong, Adamiak, Gwynn, Singh and Sinclair2010) enzymes, responsible for FA metabolization and that positively correlates with embryo production. Furthermore, adequate FA doses may assist oocyte maturation and competence, especially for low quality oocytes, directly affecting fertilization and early embryo development (Boxmeer et al., Reference Boxmeer, Macklon, Lindemans, Beckers, Eijkemans, Laven, Steegers and Steegers-Theunissen2009; Gaskins et al., Reference Gaskins, Afeiche, Wright, Toth, Williams, Gilman, Hauser and Chavarro2015; Schaefer and Nock, Reference Schaefer and Nock2019). This finding is important when considering reproductive treatments in humans, as oocyte quality may be impaired depending on age and infertility.
Addition of the highest FA dose (100 μM) during IVM of GI oocytes seemed to be detrimental and reduced embryo production. In the murine model, high folate intake before and during pregnancy may be harmful, influencing the DNA methylation pattern, causing delay to embryo development and also impairing nervous system development, depending on the time of exposition (Pickell et al., Reference Pickell, Brown, Li, Wang, Deng, Wu, Selhub, Luo, Jerome-Majewska and Rozen2011; Mikael et al., Reference Mikael, Deng, Paul, Selhub and Rozen2013; Rahimi et al., Reference Rahimi, Martel, Karahan, Angle, Behan, Chan, MacFarlane and Trasler2019). FA incorporation by cells has a limit and conversion of FA into its active folate form is restricted by the action of DHFR. Excess levels of FA induce the formation of unmetabolized FA (UMFA) (Troen et al., Reference Troen, Mitchell, Sorensen, Wener, Johnsin, Wood, Selhub, McTiernan Yasui, Oral, Potter and Ulrich2006; Banerjee and Ray, Reference Banerjee and Ray2016; Pizano and Willimson, Reference Pizano, Willimson, Noland, Drisko and Wagner2020) during pregnancy and may influence fetal growth, leading to the development of insulin resistance in childhood (Krishnaveni et al., Reference Krishnaveni, Veena, Karat, Yajnik and Fall2014; Patel and Sobczyńska-Malefora, Reference Patel and Sobczyńska-Malefora2017).
Addition of FA during oocyte maturation and its influence on embryo production as observed in those findings reinforced the hypothesis that some FA used by embryos during early development may be from gametes, especially from oocytes (O’Neill, Reference O’Neill1998). During the maturation period, oocytes stock several essential components and mRNAs to assist early embryo development, mainly before the ZGA, to possibly prevent epigenetic disturbances in the offspring (Meirelles et al., Reference Meirelles, Caetano, Watanabe, Ripamonte, Carambula, Merighe and Garcia2004; Assou et al., Reference Assou, Boumela, Haouzi, Anahory, Dechaud, Vos and Hamamah2010). ARTs are frequently associated with epigenetics changes, such as loss of DNA methylation in the offspring, which may alter the expression of important genes related to embryo growth and development, leading to many diseases, as imprinting disorders (Urrego et al., Reference Urrego, Rodriguez-Osorio and Niemann2014; Salilew-Wondim et al., Reference Salilew-Wondim, Saeed-Zidane, Joelker, Gebremedhn, Poirier, Pandey, Tholen, Neuhoff, Held, Besenfelder, Havlicek, Rings, Fournier, Gagné, Sirard, Robert, Gad, Schellander and Tesfaye2018; Hattori et al., Reference Hattori, Hiura, Kitamura, Miyauchi, Kobayashi, Takahashi, Okae, Kyono, Kagami, Ogata and Arima2019; Siqueira et al., Reference Siqueira, Silva, Panetto and Viana2020). Addition of FA during ART may be useful, mainly because FA acts as a methyl donor and acceptor, assisting nucleotide production during cell division and in the establishment and/or maintenance of epigenetic markers during gametic and embryonic reprogramming (Kim et al., Reference Kim, Seo, Eum, Lim, Kim, Yoon and Lee2009; Huang et al., Reference Huang, Gao, Hou and Wu2013; Friso et al., Reference Friso, Santis, Pizzolo and Patel2020).
Understanding the influence of oocyte IVM on gene expression levels during early embryonic development is an important step to improve ARTs and increase the success rates in IVP (Moore, Reference Moore2001; Goossens et al., Reference Goossens, Soom, Poucke, Vandaele, Vandesompele, Zeveren and Peelman2007; Murakami et al., Reference Murakami, Oshimura and Kugoh2007). However, there is a lack of studies that describe possible influences of FA on imprinted genes. It is important to highlight that, in mammals, imprinted genes are about 1% of the genome and have important roles in embryo and fetus development (Busso et al., Reference Busso, Santander, Salas-Pérez, Santos, De Caterina, Martinez and Kohlmeier2020), as well as in placentation (Miranda-Furtado et al., Reference Miranda-Furtado, Salomão, Verruma, Leite, Rios, Bialecka, Moustakas, Mei, Paz, Duarte, Lopes and Ramos2019). Abnormal expression of imprinted genes in the H19 DMR and KvDMR1 (Fig. 1) may lead to the development of LOS in ruminants, an overgrowth syndrome that has several phenotypic similarities to the Beckwith–Wiedemann syndrome (BWS) in humans (Chen et al., Reference Chen, Robbins, Wells and Rivera2013, Reference Chen, Hagen, Elsik, Ji, Morris, Moon and Rivera2015; Hattori et al., Reference Hattori, Hiura, Kitamura, Miyauchi, Kobayashi, Takahashi, Okae, Kyono, Kagami, Ogata and Arima2019).
In addition to a similar phenotype, LOS and BWS also have similar epigenetic changes. Modifications in the DNA methylation pattern and IGF2 and KCNQ1OT1 gene expression have been reported in both ruminant and human syndromes (DeBaun et al., Reference DeBaun, Niemitz and Feinberg2003; Murrell et al., Reference Murrell, Heeson, Cooper, Douglas, Apostolidou, Moore, Maher and Reik2004; Chen et al., Reference Chen, Robbins, Wells and Rivera2013; Hattori et al., Reference Hattori, Hiura, Kitamura, Miyauchi, Kobayashi, Takahashi, Okae, Kyono, Kagami, Ogata and Arima2019). The paternally expressed IGF2 and KCNQ1OT1 show different biological effects during development, whereas IGF2 is one of the most important growth factors, the KCNQ1OT1 gene transcribes a regulatory lncRNA that controls the expression of surrounding genes, as CDKN1C, beyond its function as a tumour suppressor (Mitsuya et al., Reference Mitsuya, Meguro, Lee, Katoh, Schulz, Kugoh, Yoshida, Niikawa, Feinberg and Oshimura1999; Smith et al., Reference Smith, Garfield and Ward2006; Stampone et al., Reference Stampone, Caldarelli, Zullo, Bencivenga, Mancini, Ragione and Borriello2018; Hattori et al., Reference Hattori, Hiura, Kitamura, Miyauchi, Kobayashi, Takahashi, Okae, Kyono, Kagami, Ogata and Arima2019).
IGF2 expression was detected in embryos from GI oocytes without and at the lowest and moderate concentrations (10 and 30 μM) of FA. The addition of 10 and 30 μM of FA did not affect IGF2 expression in GI oocytes, however the highest tested dose (100 μM) negatively affected IGF2 expression, which was not detected. In addition, in embryos from GIII oocytes, IGF2 expression was detected only in those cultured without FA supplementation. Although the appropriate dose of FA to be used in IVM has not been well established, there is evidence of its influence during oocyte maturation and embryo early development (Kim et al., Reference Kim, Seo, Eum, Lim, Kim, Yoon and Lee2009; Sato et al., Reference Sato, Sakurai, Monji, Kuwayama and Iwata2013) and concentration seems to affect IGF2 gene expression. It is possible that the stock of FA provided to the embryo could influence the DNA methylation pattern of the H19 DMR and, consequently, the IGF2 expression. Steegers-Theunissen and colleagues (Reference Steegers-Theunissen, Obermann-Borst, Kremer, Lindemans, Siebel, Steegers, Slagboom and Heijmans2009) associated FA periconceptional ingestion by the mother with increased DNA methylation in the H19 DMR and consequently alteration in IGF2 expression in babies immediately after birth.
IGF2 was expressed at low levels on expanded blastocysts and its expression did not seem to be related to oocyte quality. These findings corroborates those of Jiang and colleagues (Reference Jiang, Dong, Zheng, Marjani, Donovan, Chen and Tian2015) that demonstrated low levels of IGF2 transcripts in oocytes and during the preimplantation period. The IGF2 gene product is an essential growth factor that influences body weight in cattle (Goodall and Schmutz, Reference Goodall and Schmutz2007), but biallelic expression of IGF2 is related to overgrowth syndromes and cancer development in humans (Brioude et al., Reference Brioude, Lacoste, Netchine, Vazquez, Auber, Audry, Gauthier-Villars, Brugieres, Gicquel, Le Bouc and Rossignol2013; Carli et al., Reference Carli, Riberi, Ferrero and Mussa2020). Overexpression of IGF2 and the increased presence of some types of miRNAs are also related to LOS and BWS (Li et al., Reference Li, Hagen, Ji, Bakhtiarizadeh, Frederic, Traxler, Kalish and Rivera2019).
Unlike IGF2, expression of the KCNQ1OT1 gene was detected in all groups. FA supplementation on embryos from GI oocytes did not seem to affect KCNQ1OT1 expression. Interesting, in embryos from GIII oocytes, the lowest dose of FA (10 μM) reduced KCNQ1OT1 gene expression but, when 30 μM of FA was added, its expression was increased. KCNQ1OT1 is a paternally expressed gene whose expression can be detected at the first stages of early embryo development (Mohammad, et al., Reference Mohammad, Mondal and Kanduri2009). Its lncRNA is classified as a guide and acts in the cell nucleus in chromatin remodelling and the expression of surrounding genes, being able to silence them (Lewis et al., Reference Lewis, Green, Dawson, Redrup, Huynh, Lee, Hemberger and Reik2006; Mancini-DiNardo et al., Reference Mancini-DiNardo, Steele, Levorse, Ingram and Tilghman2006; Pandey et al., Reference Pandey, Mondal, Mohammad, Enroth, Redrup, Komorowski, Nagano, Mancini-DiNardo and Kanduri2008; Bouckenheimer et al., Reference Bouckenheimer, Assou, Hou, Philippe, Sansac, Lavabre-Bertand, Commes, Lemaítre, Boureux and Vos2016).
In general, lncRNAs are found at high concentrations during oocyte growth and embryo preimplantation period, however, in some of them, expression is stage specific. After genome activation, lncRNAs represent a great proportion of embryo transcripts, suggesting a fundamental role during developmental dynamics (Bouckenheimer et al., Reference Bouckenheimer, Assou, Hou, Philippe, Sansac, Lavabre-Bertand, Commes, Lemaítre, Boureux and Vos2016). In overgrowth syndromes, LOI in KvDMR1 and biallelic expression of KCNQ1OT1 are reported (DeBaun et al., Reference DeBaun, Niemitz, McNeil, Brandenburg, Lee and Feinberg2002, Reference DeBaun, Niemitz and Feinberg2003; Chen et al., Reference Chen, Robbins, Wells and Rivera2013; Li et al., Reference Li, Hagen, Ji, Bakhtiarizadeh, Frederic, Traxler, Kalish and Rivera2019) in several tissues from IVP-derived fetuses, which were detected at a lower frequency in in vivo-derived embryos (Chen et al., Reference Chen, Robbins, Wells and Rivera2013, Reference Chen, Hagen, Elsik, Ji, Morris, Moon and Rivera2015).
In humans and animal models, ARTs might alter DNA methylation levels and expression of important genes for embryo development (DeBaun et al., Reference DeBaun, Niemitz and Feinberg2003; Gomes et al., Reference Gomes, Huber, Ferriani, Amaral Neto and Ramos2009; Katari et al., Reference Katari, Turan, Bibikova, Erinle, Chalian, Foster, Gaughan, Coutifaris and Sapienza2009; Weksberg et al., Reference Weksberg, Shuman and Beckwith2010; Chen et al., Reference Chen, Robbins, Wells and Rivera2013; Hattori et al., Reference Hattori, Hiura, Kitamura, Miyauchi, Kobayashi, Takahashi, Okae, Kyono, Kagami, Ogata and Arima2019). Despite the important results regarding embryo production and gene expression, the absence of DNA methylation analysis of the H19 DMR and KvDMR1 may be considered a limitation of this study, as FA is an important methyl donor. The correlation between DNA methylation and gene expression of those regions may bring important contributions to our findings. However, to our knowledge, this report is one of the first to evaluate the effects of FA supplementation during bovine oocyte maturation and its consequences for embryo production and IGF2 and KCNQ1OT1 expression.
Epigenetic changes observed in in vitro-derived embryos seem to be mostly due to exposure to ART procedures, such as gamete and embryo manipulation, and their environmental conditions rather than to parent subfertility, as the same changes have been observed in bovines, ovines (Urrego et al., Reference Urrego, Rodriguez-Osorio and Niemann2014; Smith et al., Reference Smith, Therrien, Filion, Bressan and Meirelles2015; Hansen, Reference Hansen2020) and murines (Market-Velker et al., Reference Market-Velker, Zhang, Magri, Bonvissuto and Mann2010). Animal models are of key relevance to understanding the mechanisms and pathways affected by ARTs, and to minimizing them, reducing developmental errors in the fetus, and increasing pregnancy rates and postnatal health. The addition of FA during IVM of oocytes influenced bovine embryo production and IGF2 and KCNQ1OT1 expression, depending on the oocyte quality and the concentration used. Further studies might provide new insights regarding the ideal FA concentration capable of improving embryo development. Nevertheless, FA supplementation during oocyte maturation seems to be a promising strategy to improve embryo production, especially in low quality oocytes, an important finding when considering reproductive treatments in human. These are the first steps to understanding the effects of FA supplementation on oocyte growth and embryo development, as well as their related molecular mechanisms and pathways.
Acknowledgements
The authors would like to thank Daniel Pascoalino Pinheiro for English review.
Financial support
This work was supported by Coordenação de Aperfeiçoamento de Pessoal do Nível Superior (CAPES), Associação Nacional de Criadores e Pesquisadores (ANCP) and Fundação de Apoio ao Ensino, Pesquisa e Assistência (FAEPA).
Declaration of interest
There is no conflict of interest
Ethical standards
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Supplementary material
To view supplementary material for this article, please visit https://doi.org/10.1017/S0967199421000022
