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Male fertility status is associated with DNA methylation signatures in sperm and transcriptomic profiles of bovine preimplantation embryos

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ABSTRACT

Background: Infertility in dairy cattle is a concern where reduced fertilization rates and high embryonic loss are contributing factors. Studies of the paternal contribution to reproductive performance are limited. However, recent discoveries have shown that, in addition to DNA, sperm delivers transcription factors and epigenetic components that are required for fertilization and proper embryonic development. Hence, characterization of the paternal contribution at the time of fertilization is warranted. We hypothesized that sire fertility is associated with differences in DNA methylation patterns in sperm and that the embryonic transcriptomic profiles are influenced by the fertility status of the bull. Embryos were generated in vitro by fertilization with either a high or low fertility Holstein bull. Blastocysts derived from each high and low fertility bulls were evaluated for morphology, development, and transcriptomic analysis using RNA-Sequencing. Additionally, DNA methylation signatures of sperm from high and low fertility sires were characterized by performing whole-genome DNA methylation binding domain sequencing.

Results: Embryo morphology and developmental capacity did not differ between embryos generated from either a high or low fertility bull. However, RNA-Sequencing revealed 98 genes to be differentially expressed at a false discovery rate < 1%. A total of 65 genes were upregulated in high fertility bull derived embryos, and 33 genes were upregulated in low fertility derived embryos. Expression of the genes CYCS, EEA1, SLC16A7, MEPCE, and TFB2M was validated in three new pairs of biological replicates of embryos. The role of the differentially expressed gene TFB2M in embryonic development was further assessed through expression knockdown at the zygotic stage, which resulted in decreased development to the blastocyst stage. Assessment of the epigenetic signature of spermatozoa between high and low fertility bulls revealed 76 differentially methylated regions.

Conclusions: Despite similar morphology and development to the blastocyst stage, preimplantation embryos derived from high and low fertility bulls displayed significant transcriptomic differences. The relationship between the paternal contribution and the embryonic transcriptome is unclear, although differences in methylated regions were identified which could influence the reprogramming of the early embryo. Further characterization of paternal factors delivered to the oocyte could lead to the identification of biomarkers for better selection of sires to improve reproductive efficiency.

Electronic supplementary material: The online version of this article (doi:10.1186/s12864-017-3673-y) contains supplementary material, which is available to authorized users.

No MeSH data available.


Relative expression of TFB2M in control compared to gapmer supplemented blastocysts. Expression is relative to control blastocysts. Error bars represent the standard error of the mean fold change in expression across n = 3 and n = 2 IVF replicates for 1 μM and 5 μM gapmer supplemented blastocyst, respectively
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Fig2: Relative expression of TFB2M in control compared to gapmer supplemented blastocysts. Expression is relative to control blastocysts. Error bars represent the standard error of the mean fold change in expression across n = 3 and n = 2 IVF replicates for 1 μM and 5 μM gapmer supplemented blastocyst, respectively

Mentions: To further assess the roles of differentially expressed genes in embryonic development, the TFB2M gene was selected as a proof-of-principle for functional analysis because it was a highly expressed gene in embryos derived from high fertility sires and expression was validated by qRT-PCR analysis. The gene was silenced at the zygotic stage using antisense oligonucleotide gapmer technology. The gapmer oligonucleotide is comprised of modified locked nucleic acids (LNA) which flank DNA monomers specific to a target mRNA of interest [27, 28]. Gene silencing is mediated when the gapmer DNA monomers bind to the target mRNA and upon the formation of the DNA:RNA heteroduplex, RNase H will cleave the RNA target strand [27, 28]. Cell culture experiments have demonstrated effective uptake of gapmers from culture media in the absence of transfection agents and efficient repression of gene expression within cells [29, 38]. Following supplementation of 1 μM TFB2M-specific gapmer to the culture media of zygotes, the blastocyst rate of supplemented embryos was significantly reduced by 10.92% (P < 0.001), which was about 70% of the control embryos (Table 4). Similarly, blastocyst rate was reduced by 9.58% with 5 μM gapmer (P < 0.05) in comparison to control non-supplemented zygotes (Table 4). Further examination of the mRNA expression revealed a significant reduction in gene expression using 1 μM TFB2M gapmer supplemented blastocysts in comparison to controls (P < 0.05; Fig. 2). The relative expression of the 5 μM TFB2M gapmer was also greatly reduced and tended towards significance, however, expression was variable across the 2 IVF replicates (P = 0.11; Fig. 2).Table 4


Male fertility status is associated with DNA methylation signatures in sperm and transcriptomic profiles of bovine preimplantation embryos
Relative expression of TFB2M in control compared to gapmer supplemented blastocysts. Expression is relative to control blastocysts. Error bars represent the standard error of the mean fold change in expression across n = 3 and n = 2 IVF replicates for 1 μM and 5 μM gapmer supplemented blastocyst, respectively
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC5382486&req=5

Fig2: Relative expression of TFB2M in control compared to gapmer supplemented blastocysts. Expression is relative to control blastocysts. Error bars represent the standard error of the mean fold change in expression across n = 3 and n = 2 IVF replicates for 1 μM and 5 μM gapmer supplemented blastocyst, respectively
Mentions: To further assess the roles of differentially expressed genes in embryonic development, the TFB2M gene was selected as a proof-of-principle for functional analysis because it was a highly expressed gene in embryos derived from high fertility sires and expression was validated by qRT-PCR analysis. The gene was silenced at the zygotic stage using antisense oligonucleotide gapmer technology. The gapmer oligonucleotide is comprised of modified locked nucleic acids (LNA) which flank DNA monomers specific to a target mRNA of interest [27, 28]. Gene silencing is mediated when the gapmer DNA monomers bind to the target mRNA and upon the formation of the DNA:RNA heteroduplex, RNase H will cleave the RNA target strand [27, 28]. Cell culture experiments have demonstrated effective uptake of gapmers from culture media in the absence of transfection agents and efficient repression of gene expression within cells [29, 38]. Following supplementation of 1 μM TFB2M-specific gapmer to the culture media of zygotes, the blastocyst rate of supplemented embryos was significantly reduced by 10.92% (P < 0.001), which was about 70% of the control embryos (Table 4). Similarly, blastocyst rate was reduced by 9.58% with 5 μM gapmer (P < 0.05) in comparison to control non-supplemented zygotes (Table 4). Further examination of the mRNA expression revealed a significant reduction in gene expression using 1 μM TFB2M gapmer supplemented blastocysts in comparison to controls (P < 0.05; Fig. 2). The relative expression of the 5 μM TFB2M gapmer was also greatly reduced and tended towards significance, however, expression was variable across the 2 IVF replicates (P = 0.11; Fig. 2).Table 4

View Article: PubMed Central - PubMed

ABSTRACT

Background: Infertility in dairy cattle is a concern where reduced fertilization rates and high embryonic loss are contributing factors. Studies of the paternal contribution to reproductive performance are limited. However, recent discoveries have shown that, in addition to DNA, sperm delivers transcription factors and epigenetic components that are required for fertilization and proper embryonic development. Hence, characterization of the paternal contribution at the time of fertilization is warranted. We hypothesized that sire fertility is associated with differences in DNA methylation patterns in sperm and that the embryonic transcriptomic profiles are influenced by the fertility status of the bull. Embryos were generated in vitro by fertilization with either a high or low fertility Holstein bull. Blastocysts derived from each high and low fertility bulls were evaluated for morphology, development, and transcriptomic analysis using RNA-Sequencing. Additionally, DNA methylation signatures of sperm from high and low fertility sires were characterized by performing whole-genome DNA methylation binding domain sequencing.

Results: Embryo morphology and developmental capacity did not differ between embryos generated from either a high or low fertility bull. However, RNA-Sequencing revealed 98 genes to be differentially expressed at a false discovery rate&thinsp;&lt;&thinsp;1%. A total of 65 genes were upregulated in high fertility bull derived embryos, and 33 genes were upregulated in low fertility derived embryos. Expression of the genes CYCS, EEA1, SLC16A7, MEPCE, and TFB2M was validated in three new pairs of biological replicates of embryos. The role of the differentially expressed gene TFB2M in embryonic development was further assessed through expression knockdown at the zygotic stage, which resulted in decreased development to the blastocyst stage. Assessment of the epigenetic signature of spermatozoa between high and low fertility bulls revealed 76 differentially methylated regions.

Conclusions: Despite similar morphology and development to the blastocyst stage, preimplantation embryos derived from high and low fertility bulls displayed significant transcriptomic differences. The relationship between the paternal contribution and the embryonic transcriptome is unclear, although differences in methylated regions were identified which could influence the reprogramming of the early embryo. Further characterization of paternal factors delivered to the oocyte could lead to the identification of biomarkers for better selection of sires to improve reproductive efficiency.

Electronic supplementary material: The online version of this article (doi:10.1186/s12864-017-3673-y) contains supplementary material, which is available to authorized users.

No MeSH data available.