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Human sperm chromatin epigenetic potential: genomics, proteomics, and male infertility.

Castillo J, Estanyol JM, Ballescá JL, Oliva R - Asian J. Androl. (2015 Jul-Aug)

Bottom Line: This present work reviews the available information on the composition of the human sperm chromatin and its epigenetic potential, with a particular focus on recent results derived from high-throughput genomic and proteomic studies.As a complement, we provide experimental evidence for the detection of phosphorylations and acetylations in human protamine 1 using a mass spectrometry approach.The available data indicate that the sperm chromatin is much more complex than what it was previously thought, raising the possibility that it could also serve to transmit crucial paternal epigenetic information to the embryo.

View Article: PubMed Central - PubMed

Affiliation: Human Genetics Research Group, IDIBAPS, Faculty of Medicine, University of Barcelona, Casanova 143; Biochemistry and Molecular Genetics Service, Biomedical Diagnostic Centre, Hospital Clinic, Villarroel 170, 08036 Barcelona, Spain.

ABSTRACT
The classical idea about the function of the mammalian sperm chromatin is that it serves to transmit a highly protected and transcriptionally inactive paternal genome, largely condensed by protamines, to the next generation. In addition, recent sperm chromatin genome-wide dissection studies indicate the presence of a differential distribution of the genes and repetitive sequences in the protamine-condensed and histone-condensed sperm chromatin domains, which could be potentially involved in regulatory roles after fertilization. Interestingly, recent proteomic studies have shown that sperm chromatin contains many additional proteins, in addition to the abundant histones and protamines, with specific modifications and chromatin affinity features which are also delivered to the oocyte. Both gene and protein signatures seem to be altered in infertile patients and, as such, are consistent with the potential involvement of the sperm chromatin landscape in early embryo development. This present work reviews the available information on the composition of the human sperm chromatin and its epigenetic potential, with a particular focus on recent results derived from high-throughput genomic and proteomic studies. As a complement, we provide experimental evidence for the detection of phosphorylations and acetylations in human protamine 1 using a mass spectrometry approach. The available data indicate that the sperm chromatin is much more complex than what it was previously thought, raising the possibility that it could also serve to transmit crucial paternal epigenetic information to the embryo.

No MeSH data available.


Related in: MedlinePlus

Analysis of the intact human sperm protamine sequences by MS. After following the indicated workflow and LC-MS/MS analysis, the registers obtained demonstrate the presence of the intact unmodified Protamine 1 mass and masses of the protamine 1 containing different combinations of phosphorylation and acetylation. Methods: human sperm cells from normozoospermic sperm donors were collected and processed as described92 following World Health Organization guidelines125 and in accordance with the ethical and internal review board guidelines. Seminal plasma was discarded and sperm were treated with acid and reducing agents,92 in order to obtain a protein extract constituted mostly by a mixture of purified protamine 1 (P1) and protamine 2 (P2) (top). The correct protamine purification was confirmed through acid-urea polyacrylamide gel electrophoresis, as previously described,61 prior to the analysis by LC-MS/MS. Spectra corresponding to the liquid chromatography elution time 4–6 min obtained after MS analysis of nondigested proteins (top-down proteomics) is shown in the middle of the image. Mass peaks corresponding to nonmodified P1, mono-phosphorylated P1, di-phosphorylated and mono-acetylated P1, and tri-phosphorylated P1 were observed (note that each peak is showing a mass/charge (m/z) value corresponding to a specific “m,” since “z” is set as 1). A maximum error of 200 ppm between theoretical and experimental masses was considered to give a match as valid. At the bottom of the figure, the human P1 amino-acid sequence is indicated together with its theoretical “m.” Potential phosphorylation and acetylation sites are highlighted with an asterisk. DTT: dithiothreitol; MS: mass spectrometry; LC-MS/MS: liquid chromatography followed by tandem mass spectrometry.
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Figure 2: Analysis of the intact human sperm protamine sequences by MS. After following the indicated workflow and LC-MS/MS analysis, the registers obtained demonstrate the presence of the intact unmodified Protamine 1 mass and masses of the protamine 1 containing different combinations of phosphorylation and acetylation. Methods: human sperm cells from normozoospermic sperm donors were collected and processed as described92 following World Health Organization guidelines125 and in accordance with the ethical and internal review board guidelines. Seminal plasma was discarded and sperm were treated with acid and reducing agents,92 in order to obtain a protein extract constituted mostly by a mixture of purified protamine 1 (P1) and protamine 2 (P2) (top). The correct protamine purification was confirmed through acid-urea polyacrylamide gel electrophoresis, as previously described,61 prior to the analysis by LC-MS/MS. Spectra corresponding to the liquid chromatography elution time 4–6 min obtained after MS analysis of nondigested proteins (top-down proteomics) is shown in the middle of the image. Mass peaks corresponding to nonmodified P1, mono-phosphorylated P1, di-phosphorylated and mono-acetylated P1, and tri-phosphorylated P1 were observed (note that each peak is showing a mass/charge (m/z) value corresponding to a specific “m,” since “z” is set as 1). A maximum error of 200 ppm between theoretical and experimental masses was considered to give a match as valid. At the bottom of the figure, the human P1 amino-acid sequence is indicated together with its theoretical “m.” Potential phosphorylation and acetylation sites are highlighted with an asterisk. DTT: dithiothreitol; MS: mass spectrometry; LC-MS/MS: liquid chromatography followed by tandem mass spectrometry.

Mentions: Similar to histones, it is also worth-considering in detail the protamine amino-acid sequences, as protamines are considered critical in the maintenance of sperm chromatin status. Protamines are the most abundant proteins in the mammalian sperm nucleus (Figure 1) and their distinctive physicochemical characteristics (such as extreme basicity and high proportion of arginines and cysteines; Figure 2) confer elevated protein stability,27285758 at least in Eutherian mammals. For this reason, the identification of potential posttranslational modification (PTM) sites in the sequence of protamine 1 (P1) or protamine 2 (P2) is an intriguing field of study which could increase our knowledge of the sperm chromatin epigenetic landscape.


Human sperm chromatin epigenetic potential: genomics, proteomics, and male infertility.

Castillo J, Estanyol JM, Ballescá JL, Oliva R - Asian J. Androl. (2015 Jul-Aug)

Analysis of the intact human sperm protamine sequences by MS. After following the indicated workflow and LC-MS/MS analysis, the registers obtained demonstrate the presence of the intact unmodified Protamine 1 mass and masses of the protamine 1 containing different combinations of phosphorylation and acetylation. Methods: human sperm cells from normozoospermic sperm donors were collected and processed as described92 following World Health Organization guidelines125 and in accordance with the ethical and internal review board guidelines. Seminal plasma was discarded and sperm were treated with acid and reducing agents,92 in order to obtain a protein extract constituted mostly by a mixture of purified protamine 1 (P1) and protamine 2 (P2) (top). The correct protamine purification was confirmed through acid-urea polyacrylamide gel electrophoresis, as previously described,61 prior to the analysis by LC-MS/MS. Spectra corresponding to the liquid chromatography elution time 4–6 min obtained after MS analysis of nondigested proteins (top-down proteomics) is shown in the middle of the image. Mass peaks corresponding to nonmodified P1, mono-phosphorylated P1, di-phosphorylated and mono-acetylated P1, and tri-phosphorylated P1 were observed (note that each peak is showing a mass/charge (m/z) value corresponding to a specific “m,” since “z” is set as 1). A maximum error of 200 ppm between theoretical and experimental masses was considered to give a match as valid. At the bottom of the figure, the human P1 amino-acid sequence is indicated together with its theoretical “m.” Potential phosphorylation and acetylation sites are highlighted with an asterisk. DTT: dithiothreitol; MS: mass spectrometry; LC-MS/MS: liquid chromatography followed by tandem mass spectrometry.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492051&req=5

Figure 2: Analysis of the intact human sperm protamine sequences by MS. After following the indicated workflow and LC-MS/MS analysis, the registers obtained demonstrate the presence of the intact unmodified Protamine 1 mass and masses of the protamine 1 containing different combinations of phosphorylation and acetylation. Methods: human sperm cells from normozoospermic sperm donors were collected and processed as described92 following World Health Organization guidelines125 and in accordance with the ethical and internal review board guidelines. Seminal plasma was discarded and sperm were treated with acid and reducing agents,92 in order to obtain a protein extract constituted mostly by a mixture of purified protamine 1 (P1) and protamine 2 (P2) (top). The correct protamine purification was confirmed through acid-urea polyacrylamide gel electrophoresis, as previously described,61 prior to the analysis by LC-MS/MS. Spectra corresponding to the liquid chromatography elution time 4–6 min obtained after MS analysis of nondigested proteins (top-down proteomics) is shown in the middle of the image. Mass peaks corresponding to nonmodified P1, mono-phosphorylated P1, di-phosphorylated and mono-acetylated P1, and tri-phosphorylated P1 were observed (note that each peak is showing a mass/charge (m/z) value corresponding to a specific “m,” since “z” is set as 1). A maximum error of 200 ppm between theoretical and experimental masses was considered to give a match as valid. At the bottom of the figure, the human P1 amino-acid sequence is indicated together with its theoretical “m.” Potential phosphorylation and acetylation sites are highlighted with an asterisk. DTT: dithiothreitol; MS: mass spectrometry; LC-MS/MS: liquid chromatography followed by tandem mass spectrometry.
Mentions: Similar to histones, it is also worth-considering in detail the protamine amino-acid sequences, as protamines are considered critical in the maintenance of sperm chromatin status. Protamines are the most abundant proteins in the mammalian sperm nucleus (Figure 1) and their distinctive physicochemical characteristics (such as extreme basicity and high proportion of arginines and cysteines; Figure 2) confer elevated protein stability,27285758 at least in Eutherian mammals. For this reason, the identification of potential posttranslational modification (PTM) sites in the sequence of protamine 1 (P1) or protamine 2 (P2) is an intriguing field of study which could increase our knowledge of the sperm chromatin epigenetic landscape.

Bottom Line: This present work reviews the available information on the composition of the human sperm chromatin and its epigenetic potential, with a particular focus on recent results derived from high-throughput genomic and proteomic studies.As a complement, we provide experimental evidence for the detection of phosphorylations and acetylations in human protamine 1 using a mass spectrometry approach.The available data indicate that the sperm chromatin is much more complex than what it was previously thought, raising the possibility that it could also serve to transmit crucial paternal epigenetic information to the embryo.

View Article: PubMed Central - PubMed

Affiliation: Human Genetics Research Group, IDIBAPS, Faculty of Medicine, University of Barcelona, Casanova 143; Biochemistry and Molecular Genetics Service, Biomedical Diagnostic Centre, Hospital Clinic, Villarroel 170, 08036 Barcelona, Spain.

ABSTRACT
The classical idea about the function of the mammalian sperm chromatin is that it serves to transmit a highly protected and transcriptionally inactive paternal genome, largely condensed by protamines, to the next generation. In addition, recent sperm chromatin genome-wide dissection studies indicate the presence of a differential distribution of the genes and repetitive sequences in the protamine-condensed and histone-condensed sperm chromatin domains, which could be potentially involved in regulatory roles after fertilization. Interestingly, recent proteomic studies have shown that sperm chromatin contains many additional proteins, in addition to the abundant histones and protamines, with specific modifications and chromatin affinity features which are also delivered to the oocyte. Both gene and protein signatures seem to be altered in infertile patients and, as such, are consistent with the potential involvement of the sperm chromatin landscape in early embryo development. This present work reviews the available information on the composition of the human sperm chromatin and its epigenetic potential, with a particular focus on recent results derived from high-throughput genomic and proteomic studies. As a complement, we provide experimental evidence for the detection of phosphorylations and acetylations in human protamine 1 using a mass spectrometry approach. The available data indicate that the sperm chromatin is much more complex than what it was previously thought, raising the possibility that it could also serve to transmit crucial paternal epigenetic information to the embryo.

No MeSH data available.


Related in: MedlinePlus