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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

Semi-quantitative PCR analysis of mtDNA with the 4866bp deletion using serial dilution method in human spermatozoa.A. Lanes 1-8 represent the PCR products amplified from totalmtDNA serially diluted 28, 29, 210, 211, 212, 213, 214, 215-fold, respectivelywith primer pair LF2-HR2. B. Lanes 9-14 represent thePCR products amplified from 4866-bp deleted mtDNA seriallydiluted to 21, 22, 23, 24, 25, 26-fold, respectively with primer pairLF4–HR3. Lane M indicates the 1-kb DNA marker.
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Figure 3: Semi-quantitative PCR analysis of mtDNA with the 4866bp deletion using serial dilution method in human spermatozoa.A. Lanes 1-8 represent the PCR products amplified from totalmtDNA serially diluted 28, 29, 210, 211, 212, 213, 214, 215-fold, respectivelywith primer pair LF2-HR2. B. Lanes 9-14 represent thePCR products amplified from 4866-bp deleted mtDNA seriallydiluted to 21, 22, 23, 24, 25, 26-fold, respectively with primer pairLF4–HR3. Lane M indicates the 1-kb DNA marker.

Mentions: Direct sequencing of the 534 bp PCR product revealedthat it was amplified from the mtDNA with anovel 4866 bp deletion. This deletion is located betweennucleotide position (np) 8270 and np 13136 andflanked by a seven-nucleotide direct repeat of 5΄-ACCCCCT-3΄ within the deleted area, between np 8271-8277 and np 13127-13133 (Fig 3). DNA sequencingwas also performed on a 432 bp PCR product frommtDNA. As expected, the analysis of the nucleotidesequences flanking the break points of the 4977 bpdeletion revealed a 13 bp direct repeat (5΄-ACCTCCCTCACCA-3΄) associated with this common deletion(data not shown). These two deletions wereshown in both normal and abnormal motility groups.Also, 13 samples had both deletions of mtDNA (Table3). The frequency of occurrence of mtDNA withthe 4866 bp-deleted mtDNA (dmtDNA4866) and 4977bp-deleted mtDNA (dmtDNA4977) was different inboth groups. The abundance of the these deletions inabnormal motility group was 56% for dmtDNA4866and 52% for dmtDNA4977 in comparison with normalmotility group with 24% for dmtDNA4866 and 28%for dmtDNA4977, respectively (Table 3). Overall, theincidence of deleted mtDNA in the abnormal motilitygroup was higher than the normal motility group.


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)

Semi-quantitative PCR analysis of mtDNA with the 4866bp deletion using serial dilution method in human spermatozoa.A. Lanes 1-8 represent the PCR products amplified from totalmtDNA serially diluted 28, 29, 210, 211, 212, 213, 214, 215-fold, respectivelywith primer pair LF2-HR2. B. Lanes 9-14 represent thePCR products amplified from 4866-bp deleted mtDNA seriallydiluted to 21, 22, 23, 24, 25, 26-fold, respectively with primer pairLF4–HR3. Lane M indicates the 1-kb DNA marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Semi-quantitative PCR analysis of mtDNA with the 4866bp deletion using serial dilution method in human spermatozoa.A. Lanes 1-8 represent the PCR products amplified from totalmtDNA serially diluted 28, 29, 210, 211, 212, 213, 214, 215-fold, respectivelywith primer pair LF2-HR2. B. Lanes 9-14 represent thePCR products amplified from 4866-bp deleted mtDNA seriallydiluted to 21, 22, 23, 24, 25, 26-fold, respectively with primer pairLF4–HR3. Lane M indicates the 1-kb DNA marker.
Mentions: Direct sequencing of the 534 bp PCR product revealedthat it was amplified from the mtDNA with anovel 4866 bp deletion. This deletion is located betweennucleotide position (np) 8270 and np 13136 andflanked by a seven-nucleotide direct repeat of 5΄-ACCCCCT-3΄ within the deleted area, between np 8271-8277 and np 13127-13133 (Fig 3). DNA sequencingwas also performed on a 432 bp PCR product frommtDNA. As expected, the analysis of the nucleotidesequences flanking the break points of the 4977 bpdeletion revealed a 13 bp direct repeat (5΄-ACCTCCCTCACCA-3΄) associated with this common deletion(data not shown). These two deletions wereshown in both normal and abnormal motility groups.Also, 13 samples had both deletions of mtDNA (Table3). The frequency of occurrence of mtDNA withthe 4866 bp-deleted mtDNA (dmtDNA4866) and 4977bp-deleted mtDNA (dmtDNA4977) was different inboth groups. The abundance of the these deletions inabnormal motility group was 56% for dmtDNA4866and 52% for dmtDNA4977 in comparison with normalmotility group with 24% for dmtDNA4866 and 28%for dmtDNA4977, respectively (Table 3). Overall, theincidence of deleted mtDNA in the abnormal motilitygroup was higher than the normal motility group.

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