التحري الجزيئي عن بعض التغيرات في الدنا المايتوكونديري للنطف لمرضى يعانون من وهن حركة النطف == Molecular Screening of Some Changes In Sperm Mitochondrial DNA In Asthenozoospermic Patients
Author name:
عدي عدنان مهدي
Supervisor name:
اسماعيل عبد الرضا عبد الحسن
General topic:
Biology
Specific topic:
Biotechnologies
Degree:
Master
University:
University of Baghdad - Institute Of Genetic Engineering And Biotechnology - Department Of Biotechnology
Language:
English
University location:
Baghdad
First pages:
24T2706 - p.pdf
Abstract:
من خلال البحوث والدراسات التي اجريت على اسباب وهن حركة النطف لدى الرجال الا انه هناك عدد من المسببات لم تحدد والى الوقت الحاضر. الا انه بالرغم من ذلك بعض الدراسات تشيربان العامل الوراثي يلعب دور في ذلك والمتمثل بعضيات المايتوكوندريا والحاوية على الدنا الم | Through researches and studies done on the causes of the asthenozoospermia in men, although there was no specific reason so far responsible for the infertility in people, but some of the studies were indicated that the genetic factors plays a role in the sperm dysmotility through the mitochondria that contained mitochondrial DNA responsible for providing the energy required for the sperm motility by production of ATP, which serve as the fuel that is consumed by sperm during motility. This study is conducted to determine the changes that could occur in the sperm mtDNA which included (common deletion, mtDNA copy number per cell and mutations in the ATPase8 and ATPase6 genes).This study was consisted of two parts as follow : The first part was consisted of two steps : The first step conducted on 71 samples from subjects suffering from the weakness of the movement of sperm and 12 samples from subjects who have a normal movement of sperm, total samples were 83 samples were divided patient samples into 5 groups depending on the percentage of the sperm motility under the classification adopted by the World Health Organization as well as a control group, that it has become 6 groups, these samples were subjected for investigation about the mitochondrial DNA deletions and mitochondrial DNA copy number by using real time PCR.On the other hand was taking 66 sample dispersed sample 56 patients and 10 healthy samples were subjected to the tests of ATPase6 and ATPase8 genes sequences for detection about mtDNA mutation.The second step was included the use of discontinuous gradient centrifugation (percoll) , which are 40 % and 80 % which represent both progressive and non progressive motile sperms, bringing the total samples 166 samples that collected and fractionated in a hospital, Kamal Al - Samarrai to treat infertility and IVF in Baghdad and examined for further molecular investigations for the period from February 2012 to October 2013.The second part was included the molecular tests that carried out after DNA extraction from the cells and this part include three steps : The first step was to investigate the common deletion in mtDNA where the results showed that the total number of deleted samples in the group (0 - 5) in the nonprogressive sperm was 81.25 %, which ranged between 6.8 - 74.7% while the ratio between the other groups 6 - 10, 11 - 15 , 16 - 20, 21 - 25 where were 75, 35.72, 28.5 , 36.3, and 12% for control group respectively. The second step was to investigate the sperm mtDNA copy number. The same groups where the results showed that the content of DNA in subjects with impaired movement of sperm was high compared with the control group where scored in some samples the highest level of gene expression 246.9 copies per cell in the group (0 - 5%) either other groups have shown lower levels were observed in groups (6 - 10) , (11 - 15) , (16 - 20) and (21 - 25) were 203.1, 180.7, 133.5 and 128.3 copies per cell, either the highest level of DNA content of the control group was 94.7 copies per cell, there was also a significant difference (P>0.05) between the normospermic motile isolated from 80% class and poor sperm movement isolated from 40 % class. The third step was included the detection of mutations by analyzing the sequences of the MT - ATAase8 and MT - ATPase6 genes which documented 59 mutations that were 23 missense and 36 silent mutations where all of them were heteroplasmic mutation except for a single mutation of the type missense (A8860G) which was homoplasmic mutation was noticed in all mtDNA copies of patients and control subjects also in progressive and non progressive sperm cells. Thought this current study novel nucleotides changes in MT - ATAase8 and MT - ATPase6 genes among groups where 3 novel missense mutations in MT - ATAase8 gene at positions (8378, 8483 and 8558), the changes of nucleotides bases were A>G, A>C, A>C and A>C respectively, replacing Asparagine to Aspartic acid, Leucine to Proline and Proline to Alanine respectively. Also two novel missense mutations were observed in MT - ATPase6 gene at positions nt (8822 and 9055) where the changes nucleotides were (C >T and G>A) that replaced amino acids (Serine to Phenylalanine and Alanine to Threonine) respectively. Silent novel heteroplasmic change as a transition substitutions in ATPase8 gene at position nt (8371) by replacing C>T without any changing in amino acid of protein. Results of this present study showed novel heteroplasmic silent mutations in ATPase6 gene at positions nt (8899, 9048 and 9060) nucleotides changes (C>T, T>C and C>A) respectively, without changing in amino acid of protein which were observed in infertile group
