Association of sperm oxidative stress and mitochondrial DNA mutation in iraqi infertile men

189 Pages·2014·1.92 MB·English

Ministry of Higher Education and Scientific Research
University of Baghdad
College of Science
Biotechnology Department

Association of sperm oxidative
stress and mitochondrial DNA
mutation in iraqi infertile men

A thesis submitted to the College of Science, University of

Baghdad as partial fulfillment of the requirements for the

degree of Master of Science in Biotechnology

By
Naeem Mohammed.Mohsen

B. Sc. Biology /University of Wassit
College of science (2008)

Supervised by
Prof.Dr. Hasan Al- Azzawie
Ass .Dr.prof Ehab D.Salman

2013 1435

ﻢﻴﺣﺮﱠ ﻟﺍ ﻦﻤﺣْ ﺮﱠ ﻟﺍ ﷲ ﻢﺴْ ﺑ
ِ ِ ِ ِ ِ ِ

ُ ْ َ ْ ْ ﱠ
ﺎﻣَ ﻖُ ﻠﺨَﻳ ﺽﺭْ ﻷﺍﻭَ ﺕِ ﺍﻭَ ﺎﻤَ ﺴﱠ ﻟﺍ ﻚُ ﻠﻣُ ِﻟِ
ِ
ً
ﻦﻤَ ِﻟ ﺐُ َﻬَﻳﻭَ ﺎﺛﺎَﻧِﺇ ءُ ﺎﺸَ َﻳ ﻦْ ﻤَ ِﻟ ﺐُ َﻬَﻳ ءُ ﺎﺸَ َﻳ
٤۹ ﺭَ ﻮﻛُ ﺬﱡ ﻟﺍ ءُ ﺎﺸَ َﻳ
﴾ ﴿
ً ً ْ ُ َ
ﻦﻣَ ﻞُ ﻌَ ﺠْ َﻳﻭَ ﺎﺛﺎَﻧِﺇﻭَ ﺎﻧﺍﺮَ ﻛﺫ ﻢْ ُﻬﺟُ ﻭﱢ ﺰَ ُﻳ ﻭْ ﺃ
﴾٥۰﴿ ﺮٌ ﻳﺪِ َﻗ ﻢٌ ﻴِﻠﻋَ ُﻪﱠﻧِﺇ ًﺎﻤﻴِﻘ ﻋَ ءُ ﺎﺸَ َﻳ

ﻢُ ﻴﻈﻌَ ﻟﺍ ﻲُ ﻠﻌﻟﺍ ُﷲ ﻕَ ﺪَ ﺻَ

(ﻯﺭﻮﺸﻟﺍ)
Dedication

To memory of my father….

NAEEM
Acknowledgement

First of all, I would like to thank the grace of Allah has blessing and mercy
surrounded me during the completing this work and to write this thesis.
Sincerely, I feel a great urge to present my deepest affection and gratitude to
supervisor, Professor Dr. Hassan Al-Azzawi for his scientific my
discussions and encouragement.
My profound thanks to my supervisor Assistant Professor Dr Ehab .
D.Salman for her scientific guidance kind advice, patience, generosity.
Special thanks are due to the Dean of the College of Science/ University of
Baghdad. Also, I would like to thank the head Assistant Prof. Dr. Abdul
Kareem A. Al-Kazaz and all the staff members of the Department of
Biotechnology /College of Science / University of Baghdad.
My deepest gratitude to my friend Mr. Sabah Al-Kaabi and Mr. Haider
Karim for their faithful support and attendant through the survey of this
study. Special thank to all consultants and other staff Laboratory at / Al-
Karama and Al-Zahra Teaching Hospitals in Kut city. And Special thank to
Dr. Mohammed Naji, Mr. Muhannad , Mr. Riad Wali ,Mrs. Eman and
all of my colleagues in the Department of Biotechnology.

Finally, special loving thanks go to my family for their endless support,
encouragement, without their patience, this work would never been finished.
Thank for all of my friends who support and encourage me.

Naeem
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Committee Certification

We, the examining committee, certify that we have read this thesis and
examined the student in its contents and that, according to our opinion, is
accepted as a thesis for the Master of Science degree in Biotechnology.
Signature
Name: Dr .Sabah Nasr Al. Alwachi
Title: Professor
Date: / / 2014
(Chairman)
Signature Signature
Name: Dr. Abdul Kareem A.AL-Kazaz Name: Dr.Yahya.Y.Z. Farid
Title: Assistant Professor Title: Professor
Date: / / 2014 Date: / / 2014
(Members) (Members)
Signature Signature
Name: Dr.Hasan Al-Azzawie Name: Dr.Ehab.D.Salman
Title: Professor Title: Assistant Professor
Date: / / 2014 Data: / /2014
(Supervisor) (Supervisor)
I here by certify upon the decision of the examining committee
Signature:
Name :
Address:
Date: / / 2014
Certification
We certify that this thesis was prepared under our supervision at the
department of Biotechnology, College of science, University of Baghdad,
as a partial requirement for the degree of Master of Science in
biotechnology
.

Signature: Signature:
Name: Dr Hassan F. Al-Azzawie Name: Dr Ehab . D.Salman
Title: Professor Title: Assist .Professor
Date: Date:

In view of available recommendation, I forward this thesis for debate
by the Examination Committee

Signature:
Name: Assistant Prof. Dr. Abdul Kareem A. Al-Kazaz
Title: chairman of Biotechnology
Date:
Summary

Summary
This study was conducted on infertile subjects attending to Al-Karama and Al-Zahra
Teaching Hospitals in Kut city during the period October 2012 to June 2013. Sperm

analysis and hormone assays were conducted, as part of their investigations for infertility.
Semen specimens of 27 asthenospermic subjects and 23 oligospermic in addition, 30
healthy fertile men with normal semen parameters according to World Health
Organization (WHO) criteria were enrolled as controls. The three groups were similar as
regards mean age (21-45 years of age).subjects had no systemic diseases were non
smokers and had no alcohol dependence, and none were taking an oral antioxidant
supplement for three months prior to the study. All semen samples were collected by
masturbation following 3 days of abstinence. After liquefaction, semen volume, sperm
concentration, total sperm count, morphology , motility grades were determined using
WHO standard procedures .All major determinations were carried out in duplicate.

Determination of sperm DNA damage by comet assay using alkaline method revealed
that sperm cell of control showed normal DNA integrity with no tail. Oligospermic group
showed very less tail in cells while, in asthenospermic group showed typical comet with
long tail indicating heavy damage to the DNA. These data indicated that male infertility
was associated with oxidative stress leading to oxidative sperm DNA damage. Therefore,
sperm DNA damage could be a suitable early bio-marker to monitor the environmental or
occupational infertility toxicity.
By using the long-range PCR techniques and the primer sets L3–H6, the existence of
large-scale deletions of mtDNAs in human spermatozoa from 50 infertile males and 30
fertile men was for the first time screened in Iraq peoples and results revealed four major
bands (with lengths of ~8360, 3383, 1015, and 761 bp, respectively on the gel after
ethidium bromide staining. The 8360 bp band was the full-length PCR product from the
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Summary

wild type mtD NAs, the 3383 bp band was generated from the 4977 bp-deleted mtDNAs,
the 1015 bp band was obtained from the 7345 bp-deleted mtDNAs, and finally the 761
bp band was amplified from the 7599 bp-deleted mtDNAs.

By using the primer-shift PCR technique, the PCR products with sizes in agreement
with the shift in the distance of different primer pairs were obtained by using the primer
pairs L6–H5, L6–H6, and L5–H4, the PCR products of 295, 354, and 555 bp respectively
was observed from the 7345 bp-deleted mtDNAs. Similarly, the PCR products of 406 and
702 bp from the 7599 bp-deleted mtDNAs using primer sets of L4–H3, L3–H5
respectively were obtained and nothingness the PCR products by using primer L2–H3
were noticed.
The 4977, 7345, and the 7599 bp deletions of mtDNAs were found more frequently
in the spermatozoa with low motility. These three large-scale deletions were found to
occur either alone or in different combinations. Among them, the 7345 bp deletion was
the most frequently seen in spermatozoa with poor motility ,in addition 4977 and 7599
bp-deleted mtDNAs in the spermatozoa from Iraqi infertile males.
Analysis of the nucleotide sequences flanking the breakpoints of the 7599 bp deletion
revealed a seven nucleotide direct repeat, which was generally found in the large-scale
deletions of mtDNAs. The seven nucleotide repeat (5-CATCAAC-3) was located in the
breakpoints at np 8643/ 8637(ATPase6). The breakpoint of 7599 bp deletions was located
in the hot-regions that were prone to large-scale deletions. The nucleotide sequences
within these regions were found to assume unusual structures, such as bent DNA and
anti-bent DNA. These regions may be thus more vulnerable to attack by free radicals or
serve as recognition motifs for certain recombination machinery involved in the large-
scale deletions. Analysis of the human mtDNAs revealed five CATCAAC direct repeats,
which include np 5617–5611, np 8643–8637, np 13296–13290, np 16079–16073, and np
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