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A novel large-scale deletion of the mitochondrial DNA of spermatozoa of men in north iran.

Gholinezhad Chari M, Hosseinzadeh Colagar A, Bidmeshkipour A - Int J Fertil Steril (2015)

Bottom Line: After total DNA extraction, a long-range polymerase chain reaction (PCR) technique was used to determine the mtDNA deletions in human spermatozoa.The products of PCR analysis showed a common 4977 bp deletion and a novel 4866 bp deletion (flanked by a seven-nucleotide direct repeat of 5΄-ACCCCCT-3΄ within the deleted area) from the mtDNA of spermatozoa in both groups.However, the frequency of mtDNA deletions in abnormal motility group was significantly higher than the normal motility group (56, and 24% for 4866 bp-deleted mtDNA and, 52, and 28% for 4977 bp-deleted mtDNA, respectively).

View Article: PubMed Central - PubMed

Affiliation: Fatemehzahra Infertility and Reproductive Health Research Center, Babol University of Medical Sciences, Babol, Iran ; Department of Biology, Faculty of Basic Sciences, Razi University, Kermanshah, Iran.

ABSTRACT

Background: To investigate the level of correlation between large-scale deletions of the mitochondrial DNA (mtDNA) with defective sperm function.

Materials and methods: In this analytic study, a total of 25 semen samples of the nor- mozoospermic infertile men from North of Iran were collected from the IVF center in an infertility clinic. The swim-up procedure was performed for the separation of spermatozoa into two groups; (normal motility group and abnormal motility group) by 2.0 ml of Ham's F-10 medium and 1.0 ml of semen. After total DNA extraction, a long-range polymerase chain reaction (PCR) technique was used to determine the mtDNA deletions in human spermatozoa.

Results: The products of PCR analysis showed a common 4977 bp deletion and a novel 4866 bp deletion (flanked by a seven-nucleotide direct repeat of 5΄-ACCCCCT-3΄ within the deleted area) from the mtDNA of spermatozoa in both groups. However, the frequency of mtDNA deletions in abnormal motility group was significantly higher than the normal motility group (56, and 24% for 4866 bp-deleted mtDNA and, 52, and 28% for 4977 bp-deleted mtDNA, respectively).

Conclusion: It is suggested that large-scale deletions of the mtDNA is associated with poor sperm motility and may be a causative factor in the decline of fertility in men.

No MeSH data available.


Related in: MedlinePlus

Detection of large-scale deletions of mtDNA from human washed sperm by long-range PCR method. A: The 5860 bpband represents the PCR product of normal mtDNA with primer pair LF3-HR4. Lane M is the 1-kb DNA size. B: Using theprimer sets LF3-HR4, the 5860 bp band was amplified from the wild-type mtDNA, the 994 bp and 883 bands were amplifiedfrom the 4866 bp and 4977 bp-deleted mtDNA, respectively. Spermatozoa in lanes 1-4 had the motility scores of 5.0, 20.0, 30.0,40.0% respectively. Lane 5 is the blank, in which the sperm DNA was omitted from the reaction mixture. Lane M is the1-kb DNAsize marker. C. The arrow indicates the band of 994 bp produced with primer pair LF3-HR4. Using a short extension time of 1minute at 72˚C, the longer DNA product from wild-type mtDNA could not be produced and only mtDNA with 4866 bp-deletionwas amplified. Lanes N and A normal and abnormal groups, respectively.
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Figure 1: Detection of large-scale deletions of mtDNA from human washed sperm by long-range PCR method. A: The 5860 bpband represents the PCR product of normal mtDNA with primer pair LF3-HR4. Lane M is the 1-kb DNA size. B: Using theprimer sets LF3-HR4, the 5860 bp band was amplified from the wild-type mtDNA, the 994 bp and 883 bands were amplifiedfrom the 4866 bp and 4977 bp-deleted mtDNA, respectively. Spermatozoa in lanes 1-4 had the motility scores of 5.0, 20.0, 30.0,40.0% respectively. Lane 5 is the blank, in which the sperm DNA was omitted from the reaction mixture. Lane M is the1-kb DNAsize marker. C. The arrow indicates the band of 994 bp produced with primer pair LF3-HR4. Using a short extension time of 1minute at 72˚C, the longer DNA product from wild-type mtDNA could not be produced and only mtDNA with 4866 bp-deletionwas amplified. Lanes N and A normal and abnormal groups, respectively.

Mentions: To detect the common mtDNA deletion (4977 bp),a desired large segment of mtDNA (5.8 kb) was amplifiedfrom 20 ng of DNA in a 50 μl reaction mixturecontaining 200 μM of each dNTP, 0.5 μM of LF3 andHR4 primers (Fig 1, Table 2), 2 units of HLTaq DNApolymerase (Bioneer, Seoul, Korea), 40 mM KCl,1.5 mM MgCl2 and 10 mM Tris-HCl, (pH=9.0) PCRwas carried out for 35 cycles using the thermal profileof denaturation at 94˚C for 1 minute, annealingat 56˚C for 1 minute, and primer extension at 72˚Cfor 5 minutes. The PCR products were separated on1% agarose gel electrophoresis, stained with ethidiumbromide (1 μg/ml) and visualized by transilluminationunder UV light.


A novel large-scale deletion of the mitochondrial DNA of spermatozoa of men in north iran.

Gholinezhad Chari M, Hosseinzadeh Colagar A, Bidmeshkipour A - Int J Fertil Steril (2015)

Detection of large-scale deletions of mtDNA from human washed sperm by long-range PCR method. A: The 5860 bpband represents the PCR product of normal mtDNA with primer pair LF3-HR4. Lane M is the 1-kb DNA size. B: Using theprimer sets LF3-HR4, the 5860 bp band was amplified from the wild-type mtDNA, the 994 bp and 883 bands were amplifiedfrom the 4866 bp and 4977 bp-deleted mtDNA, respectively. Spermatozoa in lanes 1-4 had the motility scores of 5.0, 20.0, 30.0,40.0% respectively. Lane 5 is the blank, in which the sperm DNA was omitted from the reaction mixture. Lane M is the1-kb DNAsize marker. C. The arrow indicates the band of 994 bp produced with primer pair LF3-HR4. Using a short extension time of 1minute at 72˚C, the longer DNA product from wild-type mtDNA could not be produced and only mtDNA with 4866 bp-deletionwas amplified. Lanes N and A normal and abnormal groups, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4355932&req=5

Figure 1: Detection of large-scale deletions of mtDNA from human washed sperm by long-range PCR method. A: The 5860 bpband represents the PCR product of normal mtDNA with primer pair LF3-HR4. Lane M is the 1-kb DNA size. B: Using theprimer sets LF3-HR4, the 5860 bp band was amplified from the wild-type mtDNA, the 994 bp and 883 bands were amplifiedfrom the 4866 bp and 4977 bp-deleted mtDNA, respectively. Spermatozoa in lanes 1-4 had the motility scores of 5.0, 20.0, 30.0,40.0% respectively. Lane 5 is the blank, in which the sperm DNA was omitted from the reaction mixture. Lane M is the1-kb DNAsize marker. C. The arrow indicates the band of 994 bp produced with primer pair LF3-HR4. Using a short extension time of 1minute at 72˚C, the longer DNA product from wild-type mtDNA could not be produced and only mtDNA with 4866 bp-deletionwas amplified. Lanes N and A normal and abnormal groups, respectively.
Mentions: To detect the common mtDNA deletion (4977 bp),a desired large segment of mtDNA (5.8 kb) was amplifiedfrom 20 ng of DNA in a 50 μl reaction mixturecontaining 200 μM of each dNTP, 0.5 μM of LF3 andHR4 primers (Fig 1, Table 2), 2 units of HLTaq DNApolymerase (Bioneer, Seoul, Korea), 40 mM KCl,1.5 mM MgCl2 and 10 mM Tris-HCl, (pH=9.0) PCRwas carried out for 35 cycles using the thermal profileof denaturation at 94˚C for 1 minute, annealingat 56˚C for 1 minute, and primer extension at 72˚Cfor 5 minutes. The PCR products were separated on1% agarose gel electrophoresis, stained with ethidiumbromide (1 μg/ml) and visualized by transilluminationunder UV light.

Bottom Line: After total DNA extraction, a long-range polymerase chain reaction (PCR) technique was used to determine the mtDNA deletions in human spermatozoa.The products of PCR analysis showed a common 4977 bp deletion and a novel 4866 bp deletion (flanked by a seven-nucleotide direct repeat of 5΄-ACCCCCT-3΄ within the deleted area) from the mtDNA of spermatozoa in both groups.However, the frequency of mtDNA deletions in abnormal motility group was significantly higher than the normal motility group (56, and 24% for 4866 bp-deleted mtDNA and, 52, and 28% for 4977 bp-deleted mtDNA, respectively).

View Article: PubMed Central - PubMed

Affiliation: Fatemehzahra Infertility and Reproductive Health Research Center, Babol University of Medical Sciences, Babol, Iran ; Department of Biology, Faculty of Basic Sciences, Razi University, Kermanshah, Iran.

ABSTRACT

Background: To investigate the level of correlation between large-scale deletions of the mitochondrial DNA (mtDNA) with defective sperm function.

Materials and methods: In this analytic study, a total of 25 semen samples of the nor- mozoospermic infertile men from North of Iran were collected from the IVF center in an infertility clinic. The swim-up procedure was performed for the separation of spermatozoa into two groups; (normal motility group and abnormal motility group) by 2.0 ml of Ham's F-10 medium and 1.0 ml of semen. After total DNA extraction, a long-range polymerase chain reaction (PCR) technique was used to determine the mtDNA deletions in human spermatozoa.

Results: The products of PCR analysis showed a common 4977 bp deletion and a novel 4866 bp deletion (flanked by a seven-nucleotide direct repeat of 5΄-ACCCCCT-3΄ within the deleted area) from the mtDNA of spermatozoa in both groups. However, the frequency of mtDNA deletions in abnormal motility group was significantly higher than the normal motility group (56, and 24% for 4866 bp-deleted mtDNA and, 52, and 28% for 4977 bp-deleted mtDNA, respectively).

Conclusion: It is suggested that large-scale deletions of the mtDNA is associated with poor sperm motility and may be a causative factor in the decline of fertility in men.

No MeSH data available.


Related in: MedlinePlus