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Genome-wide 5-hydroxymethylcytosine modification pattern is a novel epigenetic feature of globozoospermia.

Wang XX, Sun BF, Jiao J, Chong ZC, Chen YS, Wang XL, Zhao Y, Zhou YM, Li D - Oncotarget (2015)

Bottom Line: Discovery of 5-hydroxymethylcytosine (5hmC) in mammalian genomes has excited the field of epigenetics, but information on the genome-wide distribution of 5hmC is limited.Here, utilizing the chemical labeling and biotin-enrichment approach followed by Illumina HiSeq sequencing, we showed that (i) 6664, 9029 and 6318 genes contain 5hmC in normal, abnormal, and globozoospermia sperm, respectively; (ii) some 5hmC-containing genes significantly involves in spermatogenesis, sperm motility and morphology, and gamete generation; (iii) 5hmC is exclusively localized in sperm intron; (iv) approximately 40% imprinted genes have 5hmC modification in sperm genomes, but globozoospermia sperm exhibiting a large portion of imprinted genes lose the 5hmC modification; (v) six imprinted genes showed different 5hmC patterns in abnormal sperm (GDAP1L1, GNAS, KCNK9, LIN28B, RB1, RTL1), and five imprinted genes showed different 5hmC patterns in globozoospermia sperm (KCNK9, LIN28B, RB1, SLC22A18, ZDBF2).These results suggested that differences in genome-wide 5hmC patterns may in part be responsible for the sperm phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University, Shenyang 110004, China.

ABSTRACT
Discovery of 5-hydroxymethylcytosine (5hmC) in mammalian genomes has excited the field of epigenetics, but information on the genome-wide distribution of 5hmC is limited. Globozoospermia is a rare but severe cause of male infertility. To date, the epigenetic mechanism, especially 5hmC profiles involved in globozoospermia progression, remains largely unknown. Here, utilizing the chemical labeling and biotin-enrichment approach followed by Illumina HiSeq sequencing, we showed that (i) 6664, 9029 and 6318 genes contain 5hmC in normal, abnormal, and globozoospermia sperm, respectively; (ii) some 5hmC-containing genes significantly involves in spermatogenesis, sperm motility and morphology, and gamete generation; (iii) 5hmC is exclusively localized in sperm intron; (iv) approximately 40% imprinted genes have 5hmC modification in sperm genomes, but globozoospermia sperm exhibiting a large portion of imprinted genes lose the 5hmC modification; (v) six imprinted genes showed different 5hmC patterns in abnormal sperm (GDAP1L1, GNAS, KCNK9, LIN28B, RB1, RTL1), and five imprinted genes showed different 5hmC patterns in globozoospermia sperm (KCNK9, LIN28B, RB1, SLC22A18, ZDBF2). These results suggested that differences in genome-wide 5hmC patterns may in part be responsible for the sperm phenotype. All of this may improve our understanding of the basic molecular mechanism underlying sperm biology and the etiology of male infertility.

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Sequencing results of 5hmC in normal, abnormal, and globozoospermia sperm genomes(A) Dot blot detection of 5hmC in sperm DNA with positive and negative control. 400 ng, 200 ng, 100 ng and 50 ng genomic DNA purified from abnormal and globozoospermia sperm were blotted on Hybond-N+ nylon-based membrane and immune-blotted using anti-5-hydroxymethylytosine antibody. Synthesized oligonucleotides with and without 5hmC modification were included as positive and negative controls. (B) Mapping results of 5hmC sequencing in normal, abnormal, and globozoospermia sperm genomes. Raw reads were aligned to human UCSC hg19 and peaks calling using MACS (P < 10−5, fold enrichment > 10). (C) 5hmC peak numbers in normal, abnormal, and globozoospermia sperms. 20486, 38282 and 19354 peaks were identified in normal, abnormal, and globozoospermia sperm, respectively. (D) Average value of 5hmC fold enrichment in normal, abnormal, and globozoospermia sperm.
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Figure 2: Sequencing results of 5hmC in normal, abnormal, and globozoospermia sperm genomes(A) Dot blot detection of 5hmC in sperm DNA with positive and negative control. 400 ng, 200 ng, 100 ng and 50 ng genomic DNA purified from abnormal and globozoospermia sperm were blotted on Hybond-N+ nylon-based membrane and immune-blotted using anti-5-hydroxymethylytosine antibody. Synthesized oligonucleotides with and without 5hmC modification were included as positive and negative controls. (B) Mapping results of 5hmC sequencing in normal, abnormal, and globozoospermia sperm genomes. Raw reads were aligned to human UCSC hg19 and peaks calling using MACS (P < 10−5, fold enrichment > 10). (C) 5hmC peak numbers in normal, abnormal, and globozoospermia sperms. 20486, 38282 and 19354 peaks were identified in normal, abnormal, and globozoospermia sperm, respectively. (D) Average value of 5hmC fold enrichment in normal, abnormal, and globozoospermia sperm.

Mentions: To assess the general content of 5hmC, we first evaluated the existence of 5hmC in sperm genome by dot blot assay (Figure 2A). We detected a significant quantity of 5hmC in as little as 100 ng sperm genomic DNA. It is quite interesting to note that the amount of 5hmC is remarkably changed in different tissues in contrast to the stable patterns of 5-methylcytosine (5mC) [8].


Genome-wide 5-hydroxymethylcytosine modification pattern is a novel epigenetic feature of globozoospermia.

Wang XX, Sun BF, Jiao J, Chong ZC, Chen YS, Wang XL, Zhao Y, Zhou YM, Li D - Oncotarget (2015)

Sequencing results of 5hmC in normal, abnormal, and globozoospermia sperm genomes(A) Dot blot detection of 5hmC in sperm DNA with positive and negative control. 400 ng, 200 ng, 100 ng and 50 ng genomic DNA purified from abnormal and globozoospermia sperm were blotted on Hybond-N+ nylon-based membrane and immune-blotted using anti-5-hydroxymethylytosine antibody. Synthesized oligonucleotides with and without 5hmC modification were included as positive and negative controls. (B) Mapping results of 5hmC sequencing in normal, abnormal, and globozoospermia sperm genomes. Raw reads were aligned to human UCSC hg19 and peaks calling using MACS (P < 10−5, fold enrichment > 10). (C) 5hmC peak numbers in normal, abnormal, and globozoospermia sperms. 20486, 38282 and 19354 peaks were identified in normal, abnormal, and globozoospermia sperm, respectively. (D) Average value of 5hmC fold enrichment in normal, abnormal, and globozoospermia sperm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4466632&req=5

Figure 2: Sequencing results of 5hmC in normal, abnormal, and globozoospermia sperm genomes(A) Dot blot detection of 5hmC in sperm DNA with positive and negative control. 400 ng, 200 ng, 100 ng and 50 ng genomic DNA purified from abnormal and globozoospermia sperm were blotted on Hybond-N+ nylon-based membrane and immune-blotted using anti-5-hydroxymethylytosine antibody. Synthesized oligonucleotides with and without 5hmC modification were included as positive and negative controls. (B) Mapping results of 5hmC sequencing in normal, abnormal, and globozoospermia sperm genomes. Raw reads were aligned to human UCSC hg19 and peaks calling using MACS (P < 10−5, fold enrichment > 10). (C) 5hmC peak numbers in normal, abnormal, and globozoospermia sperms. 20486, 38282 and 19354 peaks were identified in normal, abnormal, and globozoospermia sperm, respectively. (D) Average value of 5hmC fold enrichment in normal, abnormal, and globozoospermia sperm.
Mentions: To assess the general content of 5hmC, we first evaluated the existence of 5hmC in sperm genome by dot blot assay (Figure 2A). We detected a significant quantity of 5hmC in as little as 100 ng sperm genomic DNA. It is quite interesting to note that the amount of 5hmC is remarkably changed in different tissues in contrast to the stable patterns of 5-methylcytosine (5mC) [8].

Bottom Line: Discovery of 5-hydroxymethylcytosine (5hmC) in mammalian genomes has excited the field of epigenetics, but information on the genome-wide distribution of 5hmC is limited.Here, utilizing the chemical labeling and biotin-enrichment approach followed by Illumina HiSeq sequencing, we showed that (i) 6664, 9029 and 6318 genes contain 5hmC in normal, abnormal, and globozoospermia sperm, respectively; (ii) some 5hmC-containing genes significantly involves in spermatogenesis, sperm motility and morphology, and gamete generation; (iii) 5hmC is exclusively localized in sperm intron; (iv) approximately 40% imprinted genes have 5hmC modification in sperm genomes, but globozoospermia sperm exhibiting a large portion of imprinted genes lose the 5hmC modification; (v) six imprinted genes showed different 5hmC patterns in abnormal sperm (GDAP1L1, GNAS, KCNK9, LIN28B, RB1, RTL1), and five imprinted genes showed different 5hmC patterns in globozoospermia sperm (KCNK9, LIN28B, RB1, SLC22A18, ZDBF2).These results suggested that differences in genome-wide 5hmC patterns may in part be responsible for the sperm phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University, Shenyang 110004, China.

ABSTRACT
Discovery of 5-hydroxymethylcytosine (5hmC) in mammalian genomes has excited the field of epigenetics, but information on the genome-wide distribution of 5hmC is limited. Globozoospermia is a rare but severe cause of male infertility. To date, the epigenetic mechanism, especially 5hmC profiles involved in globozoospermia progression, remains largely unknown. Here, utilizing the chemical labeling and biotin-enrichment approach followed by Illumina HiSeq sequencing, we showed that (i) 6664, 9029 and 6318 genes contain 5hmC in normal, abnormal, and globozoospermia sperm, respectively; (ii) some 5hmC-containing genes significantly involves in spermatogenesis, sperm motility and morphology, and gamete generation; (iii) 5hmC is exclusively localized in sperm intron; (iv) approximately 40% imprinted genes have 5hmC modification in sperm genomes, but globozoospermia sperm exhibiting a large portion of imprinted genes lose the 5hmC modification; (v) six imprinted genes showed different 5hmC patterns in abnormal sperm (GDAP1L1, GNAS, KCNK9, LIN28B, RB1, RTL1), and five imprinted genes showed different 5hmC patterns in globozoospermia sperm (KCNK9, LIN28B, RB1, SLC22A18, ZDBF2). These results suggested that differences in genome-wide 5hmC patterns may in part be responsible for the sperm phenotype. All of this may improve our understanding of the basic molecular mechanism underlying sperm biology and the etiology of male infertility.

Show MeSH
Related in: MedlinePlus