Distribution of 5-methylcytosine and 5-hydroxymethylcytosine in bovine fetal tissue of the placenta

5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) are modified cytosines found in mammals that are involved in the regulation of gene expression. The aim of this study was to characterize the global patterns of 5-mC and 5-hmC of the fetal placenta of Nellore cattle as well as blood and sperm as controls. 5-mC and 5-hmC levels were determined using MethylFlash Methylated/Hydroxymethylated DNA Quantification Kit, respectively. Placenta tissues showed lower levels of 5-mC and 5-hmC compared to sperm. The male cotyledon showed higher levels of 5-hmC than the female. For the first time, the levels of 5-mC and 5-hmC in Bos taurus indicus were characterized, which may contribute to our understanding of the mechanisms of epigenetic regulation in the placenta. The presence of 5-hmC in somatic tissues suggest that 5-hmC has its own biological function and it is not only a byproduct from the oxidation of 5-mC. These results may be of interest in ARTs, especially in cloning in the diagnosis/prognosis of aberrant placentation and the viability of pregnancies.

INDEX TERMS:
Methylation; hydroxymethylation; fetal tissue; placenta; epigenetics; cattle; physiology

Authorship

Anelise S. Mendonça

Laboratório de Reprodução Animal, Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica, Avenida W5 Norte (final), Asa Norte, Brasília, DF 70770-917, Brazil. E-mails: thiagobraga86@gmail.com, eomelo@gmail.com, margot.dode@embrapa.brEmbrapa Recursos Genéticos e BiotecnologiaBrazilBrasília, DF, BrazilLaboratório de Reprodução Animal, Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica, Avenida W5 Norte (final), Asa Norte, Brasília, DF 70770-917, Brazil. E-mails: thiagobraga86@gmail.com, eomelo@gmail.com, margot.dode@embrapa.br

Faculdade de Medicina Veterinária, Universidade Federal de Uberlândia (UFU), Rua Ceará s/n, Umuarama, Uberlândia, MG 38400-902, Brazil. E-mail: anelise.mendonca@yahoo.com.brUniversidade Federal de UberlândiaBrazilUberlândia, MG, BrazilFaculdade de Medicina Veterinária, Universidade Federal de Uberlândia (UFU), Rua Ceará s/n, Umuarama, Uberlândia, MG 38400-902, Brazil. E-mail: anelise.mendonca@yahoo.com.br

Thiago F. Braga

Eduardo O. Melo

Margot A.N. Dode

Maurício M. Franco ^**Corresponding author: mauricio.franco@embrapa.br

Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia (UFU), Avenida Pará 1720, Umuarama, Uberlândia, MG 38400-902.Universidade Federal de UberlândiaBrazilUberlândia, MG, BrazilInstituto de Genética e Bioquímica, Universidade Federal de Uberlândia (UFU), Avenida Pará 1720, Umuarama, Uberlândia, MG 38400-902.

*Corresponding author: mauricio.franco@embrapa.br

Conflict of interest statement.- The authors have no competing interests.

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables

Figures (4)
Tables (3)

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Fig.1.
Levels of 5-hmC and 5-mC in the blood, placenta tissues and sperm of Bos taurus indicus cattle. 5-hydroxymethylcytosine (5-hmC), 5-methylcytosine (5-mC) and 5-hmC + 5-mC are represented as bars in each of the analyzed tissues. Data are shown as the mean ± SEM. Lower case (5-hmC), capital letters (5-mC) and Greek letters (5-hmC + 5-mC) were used to show the differences among tissues. Different letters represent different levels of methylated/hydroxymethylated cytosine among tissues (p<0.05).

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Fig.2.
Levels of 5-hmC and 5-mC in blood, extraembryonic tissues and sperm of Bos taurus indicus cattle. 5-Hydroxymethylcytosine (5-hmC), 5-methylcytosine (5-mC) and 5-hmC + 5-mC are represented as bars in each of the analyzed tissues. Data are shown as the mean ± SEM. Lower case (5-hmC), capital letters (5-mC) and Greek letters (5-hmC + 5-mC) were used to show the differences among tissues. Different letters represent different levels of methylated/hydroxymethylated cytosine among tissues (p<0.05).

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Fig.3.
5-mC/5-hmC ratios in blood, allantochorion, cotyledon, funiculus umbilicalis and sperm of Bos taurus indicus cattle. Data are shown as the mean ± SEM. Bars indicate the ratio of 5-methylcytosine/5-hydroxymethylcytosine (5-mC/5-hmC) for the different tissues.

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Fig.4.
Levels of 5-hmC, 5-mC and 5-hmC + 5-mC in blood and embryonic tissues by gender. Data of 5-hydroxymehyltcytosine (5-hmC), 5-methycytosine (5-mC) and 5-hmC + 5-mC are shown as the mean ± SEM for each gender. Different letters represent different levels of methylated and hydroxymethylated cytosine between genders (p<0.05).

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Table 1.
5-hydroxymethylcytosine (5-hmC) and 5-methylcytosine (5-mC) levels (mean ± SEM) in blood, allantochorion, cotyledon, funiculus umbilicalis and sperm of Bos taurus indicus cattle

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Table 2.
5-hydroxymethylcytosine (5-hmC) and 5-methylcytosine (5-mC) levels (mean ± SEM) in blood, placenta, funiculus umbilicalis and sperm of Bos taurus indicus cattle

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Table 3.
5-hydroxymethylcytosine (5-hmC) and 5-methylcytosine (5-mC) levels (mean ± SEM) in blood, allantochorion, cotyledon and funiculus umbilicalis of Bos taurus indicus cattle according to gender

Fig.1. Levels of 5-hmC and 5-mC in the blood, placenta tissues and sperm of Bos taurus indicus cattle. 5-hydroxymethylcytosine (5-hmC), 5-methylcytosine (5-mC) and 5-hmC + 5-mC are represented as bars in each of the analyzed tissues. Data are shown as the mean ± SEM. Lower case (5-hmC), capital letters (5-mC) and Greek letters (5-hmC + 5-mC) were used to show the differences among tissues. Different letters represent different levels of methylated/hydroxymethylated cytosine among tissues (p<0.05).

Fig.2. Levels of 5-hmC and 5-mC in blood, extraembryonic tissues and sperm of Bos taurus indicus cattle. 5-Hydroxymethylcytosine (5-hmC), 5-methylcytosine (5-mC) and 5-hmC + 5-mC are represented as bars in each of the analyzed tissues. Data are shown as the mean ± SEM. Lower case (5-hmC), capital letters (5-mC) and Greek letters (5-hmC + 5-mC) were used to show the differences among tissues. Different letters represent different levels of methylated/hydroxymethylated cytosine among tissues (p<0.05).

Fig.3. 5-mC/5-hmC ratios in blood, allantochorion, cotyledon, funiculus umbilicalis and sperm of Bos taurus indicus cattle. Data are shown as the mean ± SEM. Bars indicate the ratio of 5-methylcytosine/5-hydroxymethylcytosine (5-mC/5-hmC) for the different tissues.

Fig.4. Levels of 5-hmC, 5-mC and 5-hmC + 5-mC in blood and embryonic tissues by gender. Data of 5-hydroxymehyltcytosine (5-hmC), 5-methycytosine (5-mC) and 5-hmC + 5-mC are shown as the mean ± SEM for each gender. Different letters represent different levels of methylated and hydroxymethylated cytosine between genders (p<0.05).

Table 1. 5-hydroxymethylcytosine (5-hmC) and 5-methylcytosine (5-mC) levels (mean ± SEM) in blood, allantochorion, cotyledon, funiculus umbilicalis and sperm of Bos taurus indicus cattle

Sample	5-hmC (%)	5-mC (%)	5-hmC + 5-mC (%)
Blood (n=19)	3.47 ± 0.44	12.68 ± 1.55	16.15 ± 1.76
Allantochorion (n=12)	2.86 ± 0.10	17.26 ± 4.35	20.11 ± 3.01
Cotyledon (n=11)	2.43 ± 0.64	12.94 ± 1.65	15.36 ± 2.06
Funiculus umbilicalis (n=18)	6.69 ± 1.17	22.64 ± 2.99	29.65 ± 3.19
Sperm (n=5)	14.35 ± 1.25	34.87 ± 2.31	49.22 ± 3.98
p value	< 0.001	< 0.001	< 0.001

Table 2. 5-hydroxymethylcytosine (5-hmC) and 5-methylcytosine (5-mC) levels (mean ± SEM) in blood, placenta, funiculus umbilicalis and sperm of Bos taurus indicus cattle

Sample	5-hmC (%)	5-mC (%)	5-hmC + 5-mC (%)
Blood (n=19)	3.47 ± 0.44	12.68 ± 1.55	16.15 ± 1.76
Placenta (n=11)	2.63 ± 0.82	15.19 ± 3.46	17.82 ± 2.37
Funiculus umbilicalis (n=18)	6.69 ± 1.17	22.64 ± 2.99	29.65 ± 3.19
Sperm (n=5)	14.35 ± 1.25	34.87 ± 2.31	49.22 ± 3.98
p value	< 0.001	< 0.001	< 0.001

Table 3. 5-hydroxymethylcytosine (5-hmC) and 5-methylcytosine (5-mC) levels (mean ± SEM) in blood, allantochorion, cotyledon and funiculus umbilicalis of Bos taurus indicus cattle according to gender

Sample	Male			Female
Sample	5-hmC (%)	5-mC (%)	5-hmC + 5-mC (%)	5-hmC (%)	5-mC (%)	5-hmC + 5-mC (%)
Blood (n=19)	2.67 ± 0.40	12.77 ± 2.29	15.44 ± 2.30	4.37 ± 0.73	12.57 ± 2.20	16.94 ± 2.82
Allantochorion (n=12)	2.99 ± 1.54	10.71 ± 2.99	13.21 ± 2.13	2.72 ± 1.45	23.80 ± 7.56	26.06 ± 7.57
Cotyledon (n=11)	4.15 ± 0.74	12.99 ± 1.86	17.15 ± 2.52	0.98 ± 0.46	12.89 ± 2.78	13.87 ± 3.00
Funiculus umbilicalis (n=18)	7.91 ± 2.10	24.13 ± 4.31	31.25 ± 4.47	5.32 ± 0.61	20.78 ± 4.25	26.10 ± 4.55

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Distribution of 5-methylcytosine and 5-hydroxymethylcytosine in bovine fetal tissue of the placenta

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[1] *Corresponding author: mauricio.franco@embrapa.br