Low sperm motility due to mitochondrial DNA multiple deletions
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《中华医药杂志》英文版
Low sperm motility due to mitochondrial DNA multiple deletions
1 Department of Biology, Faculty of Science, Razi University, Baghabrisham, Kermanshah, Iran
2 Department of Medical Genetic, National Institute for Genetic Engineering and Biotechnology, Tehran, Iran
Corresponding to Massoud Houshmand, Department of Medical Genetics, National Institute for Genetic Engineering and Biotechnology, Tehran, Iran
Tel: +98 21 44580390,Fax: +98 21 44580399,E-mail:massoudh@nrcgeb.ac.ir
[Abstract] There is increasing evidence that mitochondrial DNA (mtDNA) anomalies in sperm may lead to infertility. Point mutations, deletions and depletion have been associated with decline of fertility and motility of human sperm. It has been proposed that mtDNA genetic alterations can also be responsible for sperm dysfunction. Sperm motility is one of the major determinants of male fertility and is required for successful fertilization.
It is becoming increasingly evident that both point mutations and large-scale deletions may have an impact on sperm motility and morphology. In this study we investigated association between occurrence of mtDNA△4977 bp deletion with diminished fertility and motility of human spermatozoa. The possible relationship between multiple deletions of mtDNA and the decline of fertility and motility in human spermatozoa was further explored in 50 subjects including sub-fertile and infertile males. Our results showed that the ratio of the deleted mtDNA in the spermatozoa with poor motility and diminished fertility were significantly higher than those in the spermatozoa with good motility and fertility. Our findings suggest that mutation and deletion may play an important role in some pathophysiological conditions of human spermatozoa.
[Key words] deletion； infertility；mitochondrial DNA；spermatozoa
INTRODUCTION
Human male infertility is a major health problem worldwide. Anatomic defects, endocrinopathies, immunologic problems, ejaculatory failures and environmental exposures are significant causes of infertility. Frequently, however, no clear cause for the observed infertility could be diagnosed coining the term “idiopathic infertility”.
Infertility in humans is an issue that affects 10%~15% of couples, and approximately half of these cases of infertility are attributable to men. Untreatable sub-fertility caused by poor semen quality accounts for 75% of patients consulting for fertility problems. It is assumed that in about 30 % of cases male infertility is caused by chromosome aberrations or mutations in genes functioning in the male germ line[1~3].
A general overview of genetic defects causing male infertility is therefore restricted to only a subgroup of infertile man, although this subgroup is large with about 40%. However, it is unknown whether a general genetic analysis of all infertile men would not also influence the therapeutic protocol of some other patient groups.
Defective sperm function is now recognized as one of the most important causes of human infertility. Sperm motility can be affected by a wide range of conditions, including abnormalities of the flagellar movement[4,5].
Massive amounts of energy are consumed by the fast swimming of spermatozoa during fertilization. There is increasing evidence showing that sperm motility is strongly dependent on the ATP supplied by the mitochondrial oxidative phosphorylation system (OXPHOS) [6,7].Four of the five enzymatic complexes that constitute the OXPHOS system are partially encoded by mitochondrial DNA (mtDNA). Thus, mutations in mtDNA genes that impair the expression of one or more proteins encoded in the mtDNA can promote diseases in humans[1,8~10].Accordingly, it has been proposed that mutations in the mtDNA affecting the performance of mitochondria ATP production could cause reduced sperm motility and, therefore, asthenozoospermia[11~13].
Some evidence of mitochondrial involvement in male infertility has been found. First, male infertility, associated with asthenozoospermia[12] or oligoasthenozoospermia[14], has been reported in patients suffering from typical mtDNA diseases, involving point mutations or multiple deletions of mtDNA. Secondly, sperm have been shown to be particularly prone to develop deletions of mtDNA[15~19]. Some studies have shown that, in human sperm, these deletions are associated with a decline of motility and fertility[13,20].Thirdly, a correlation has been found between the quality of the semen and the functionality of the respiratory chain in sperm mitochondria[7,21]. Moreover, it has been shown that mtDNA point mutations and single nucleotide polymorphisms can greatly influence semen quality[21]. The high rate of deletions or substitutions observed in sperm could be due to impaired mitochondrial maintenance or result from the deleterious effects of oxidative stress. Lastly, sperm treatment with extracellular ATP has been shown to induce a significant increase in the fertilizing potential of sperm, demonstrating the importance of the mitochondrial function in male fertility[22].
MATERIALS AND METHODS
Semen Collection
54 semen samples were collected from 50 patients and 4 normal controls by masturbation under hygienic conditions after a 3- to 5-day period of sexual abstinence. Samples were allowed to liquefy for 30 min at 37 ℃ and were analyzed according to World Health Organization recommendations within a period of 2 h recruited from couples who presented for semen analysis or assisted reproductive technology at the In Vitro Fecundation Laboratory of the Hospital of 4th Shahid Mehrab, Kermanshah, Iran. These couples suffered from either male or female infertility, or infertility of unknown etiology.
The volume of the ejaculate was measured, and the number and percentage of motile spermatozoa was determined. A total of 54 semen samples were classified to 4 as blank, 24 as normal, 21 as abnormal, 5 as azospermic. Characteristics of patients and blanks used in our study has been shown in Table 1 and Table 2.
Table 1 Characteristics of Patients in Our Study
Table 2 Properties of Blanks in Our Study
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Sperm and Extraction
Total DNA of human spermatozoa was extracted which performed using a DNA extracting kit (Diatom DNA prep 100, Genfanavaran, Iran). Total DNA was extracted from 0.1 ml of total semen and were subjected to agarose gel electrophoresis followed by staining with ethidium bromide (1 mg/ml) and visualized under UV illumination.
Multiplex PCR
The reaction mixture for multiplex PCR contained 2 pmol of each primer, 1 unit Taq polymerase, each dNTP at a final concentration of 200 μm, and 2 μl PCR buffer at a final volume of 50 μl. The PCR reactions were performed in a thermal cycler for 35 cycles with denaturation at 94 ℃ for 1′ (min), primer annealing at 55 ℃ for 1′ (min) and primer extension at 72 ℃ for 35″. The amplified fragments were separated by gel electrophoresis in 1 % agarose. We investigated all patients using the primers ONPF 86, 5461-5480 (ND2) ;ONPR 89, 5740-5721 (OL); ONPR 10, 15000-14981 (Cyt b) ; ONPR 74, 13640-13621 (ND5); ONPF 25, 8161-8180 (COX П); and ONPR 99, 16150-16131 (D-loop)(Table 3).The distances between the primers were long enough to allow amplification only if a part of the DNA between respective primers was deleted. Primer pair ONP 86, 89 was used to amplify a normal internal mtDNA fragment in a region, which is seldom afflicted by deletions and served as a control of the preparation and PCR analysis.
Primer pattern of human mtDNA and extented deletion with 99, 74,25,10,89 and 86 primers.
Deletion break points were analyzed by direct sequencing of DNA fragments amplified by the PCR.
Table 3 Oligonucleotide Primers Used for the Analysis of the Deletions in the mtDNA of Human
RESULTS
In this study, we investigated large-scale deletions of sperm mtDNA in 50 sub-fertile and infertile males. We screened for large-scale deletions of mtDNA in spermatozoa with poor motility by using multiplex PCR techniques. We found 4 977 bp deletion of mtDNA in the spermatozoa with low motility scores.
The rate of observed deletion was less than 15 % of consideration subjects. We confirmed the 4 977 bp deletion and identified some other deletions like 5 kbp, 7 kbp, 7.5 kbp and 8 kbp (Table 4).
Table 4 Large-scale Deletions in Our Patients
The results showed that the ratio of the deleted mtDNA in the spermatozoa with poor motility and diminished fertility were significantly higher than those in the spermatozoa with good motility and fertility. There was also a relationship between age, morphology and motility in our study (Figure 2).
Figure 1 Primer pattern of human mtDNA and extented deletion with 99, 74,25,10,89 and 86 primers
TP6
Figure 2 Relationship between age, morphology and motility in our study
DISCUSSION
In recent years, male infertility has increased in the industrialized countries due to a decline in sperm counts and a rise in testicular and sperm pathologies. Defective sperm function has been identified as the largest defined cause of male infertility[3]. Sperm dysfunction may be caused by a wide range of conditions, including abnormalities of flagellar movement[24,4], failure in sperm-zona recognition[25], and inability to carry out sperm-oocyte fusion[4].In order to understand the etiology of the decreased motility and diminished fertility of the spermatozoa and to develop an appropriate therapeutic strategy, the molecular basis of these defects must be elucidated.
Sperm mitochondria play an important role in spermatozoa functionality; therefore, genetic alterations of mitochondrial DNA (mtDNA) may have consequences for normal fertilization. Since sperm require a substantial amount of energy to swim fast enough to reach the oviduct during fertilization, the appropriatebioenergetics function of mitochondria is critical for male fertility[26,27]. Spermatozoa need a great deal of energy to support their rapid movement after ejaculation. While producing ATP by normal aerobic metabolism, sperm mitochondria also generate ROS and free radicals as byproducts. In the active respiration state, as much as 5% of the oxygen utilized by mitochondria is converted to superoxide anions and other ROS. MtDNA molecules, which are transiently attached to the inner membrane of mitochondria, are thus extremely vulnerable to oxidative damage under active respiration of spermatozoa[28]. Aitken and coworkers (1989) showed that excess amounts of ROS and free radicals in spermatozoa and seminal plasma have adverse effects on sperm motility and fertility.
As mtDNA is extensively damaged by ROS or free radicals, DNA strand breaks and large-scale deletions may be induced[29,30]. Defective respiratory enzymes containing protein subunits encoded by the deleted mtDNA may further enhance free radical production, resulting in more profound oxidative damage. Spermatozoa are especially liable to oxidative damage because their plasma membranes are rich in poly-unsaturated fatty acids. It has been established that mitochondrial dysfunction may be caused by mtDNA mutation and oxidative damage caused by endogenous and exogenous free radicals[31~34]. As reactive oxygen species (ROS) are continuously generated by the respiratory chain, they may cause significant oxidative damage to mtDNA if not efficiently eliminated[35,36].
In the present study, we investigated the possible role of mtDNA deletions in the pathophysiology of human sperm dysfunction. Our results showed that the frequency of occurrence and the proportion of deleted mtDNA were higher in sperm with poor motility than in sperm with good motility. We found 4 large-scale deletions with different sizes. Our previous study on 100 infertile men using multiple PCR and Expanded Long Range PCR methods revealed 5~6 kb deletions in three infertile men who had about 78%, 50% and 57% immobility of sperm (Unpublished data). These large-scale deletions result in complete removal or truncation of some structural genes and tRNA genes of mtDNA. These genetic alterations could be pathological mutations or common mtDNA variants that only affect male fertility because mtDNA is maternally inherited. Thus, mutations responsible for encephalomyopathies decrease sperm motility and single and multiple mtDNA deletions have been associated with sperm dysfunction[37,13,20].The cumulative effects of large scale deletions in total have also been associated with male infertility. There is relationship between large-scale deletions of mtDNA in spermatozoa with poor motility and those from patients with infertility and oligospermia or asthenospermia[38,39].
Among the mtDNA deletions observed, the so-called “common deletion” of 4 977 bp was the most prevalent and abundant one[13,40]. We detected the common 4 977 bp deletion in mtDNA from spermatozoa with poor motility and diminished fertility. The 4 977 bp deletion of mtDNA causes the removal or truncation of multiple structural genes (ATPase 6/8, COIII, ND3, ND4L, and ND4) and five tRNA genes.
In addition, there has been reported 2 novel deletions of 7 345 and 7 599 bp of mtDNA in spermatozoa with poor motility[13,20]. These 3 large-scale deletions all occur at the hot regions[30] in a large area between 2 replication origins of mtDNA. These large-scale deletions result in complete removal or truncation of some structural genes and tRNA genes of mtDNA. Our findings suggest that mtDNA deletion may play an important role in some pathophysiological condition of human infertility.
Acknowledgement
This project was supported by National Institute for Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
REFERENCES
1. Vogt PH, Chandley AC, Hargreave TB, Keil R, Ma K, Sharkey A. Microdeletions in interval 6 of the Y chromosome of males with idiopathic sterility point to disruption of AZF, a human spermatogenesis gene. Hum Genet，1992，9: 491-496.
2. Vogt Peter H. Molecular Genetic of Human Male Infertility: From Genes to New Therapeutic Perspectives, Section Molecular Genetics & Infertility, Dept. Gynecol. Endocrinol. & Reproductive Medicine, University of Heidelberg, D-69115 Heidelberg, Germany, 2000.
3. Vogt Peter H. Molecular genetic of human male infertility: from genes to new therapeutic perspectives. Current Pharmaceutical，2004.
4. Aitken, R.J., Clarkson, J.S. and Fisel, S. Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol. Reprod，1989, 40, 183-197.
5. Tredway, D.R., Settlage, D.S.F., Nakamura, R.M. et al. Significance of timing for the post-coital evaluation of cervical mucus. Am. J. Obstet. Gynecol，1975, 121, 287-293.
6. Ford WC, Harrison A.The role of oxidative phosphorylation in the generation of ATP in human spermatozoa. J Reprod Fertil，1981，271-278.
7. Ruiz-Pesini E, Diez C, Lapen a AC, Pe′rez-Martos A, Montoya J, Alvarez E, Arenas J, Lopez-Perez M.Correlation of sperm motility with mitochondrial enzymatic activities. Clin Chem,1998,4:1616-1620.
8. Enriquez JA, Chomyn A, Attardi G.MtDNA mutation in MERRF syndrome causes defective aminoacylation of tRNA(Lys) and premature translation termination. Nat Genet,1995,10(1):47-55.
9. Larsson, N.G., Oldfors, A., Garman, J.D., Barsh, G.S. and Clayton, D.A. Down-regulation of mitochondrial transcription factor A during spermatogenesis in humans. Hum. Mol. Genet,1997,6:185-191.
10. Wallace, D.C. Mitochondrial DNA sequence variation in human evolution and disease. Proc. Natl. Acad. Sci. USA, 1994,91, 8739-8746.
11. Zeviani M and Antozzi C.Mitochondrial disorders. Mol Hum Reprod,1997,3:133-148.
12. Folgero T, Torbergsen T, Oian P. The 3243-MELAS mutation in a pedigree with MERRF. Eur Neurol,1995,35:168-171.
13. Kao SH, Chao HT, Wei YH.Mitochondrial deoxyribonucleic acid 4977-bp deletion is associated with diminished fertility and motility of human sperm. Biol Reprod,1995,52: 729-736.
14. Lestienne, P, Reynier, P, Chretien, M.F., Penisson-Besnier, I., Malthiery, Y. and Rohmer, V. Oligoasthenospermia associated with multiple mitochondrial DNA rearrangements. Mol. Hum. Reprod,1997, 3, 811-814.
15. Cummins, J. Mitochondrial DNA in mammalian reproduction. Rev. Reprod,1998, 3, 172-182.
16. Cummins, J.M., Jequier, A.M., Martin, R., Mehmet, D. and Goldblatt, J.Semen levels of mitochondrial DNA deletions in men attending an infertility clinic do not correlate with phenotype. Int. J. Androl., 1998,21, 47-52.
17. Reynier P, Chretien MF, Savagner F, Larcher G, Rohmer V, Barriere P, Malthiery Y. Long PCR analysis of human gamete mtDNA suggests defective mitochondrial maintenance in spermatozoa and supports the bottleneck theory for oocytes. Biochem Biophys Res Commun,1998,252:373-377.
18. O'Connell, M., McClure, N. and Lewis, S.E. A comparison of mitochondrial and nuclear DNA status in testicular sperm from fertile men and those with obstructive azoospermia. Hum. Reprod,2002, 17, 1571-1577.
19. O'Connell, M., McClure, N. and Lewis, S.E.Mitochondrial DNA deletions and nuclear DNA fragmentation in testicular and epididymal human sperm. Hum. Reprod,2002b, 17, 1565-1570.
20. Kao S H., Chao H T and Y H Wei. Multiple deletions of mitochondrial DNA are associated with the decline of motility and fertility of human spermatozoa. Molecular Human Reproduction,1998,4:7: 657-666.
21. Ruiz-Pesini E, Lapena A, Diez-Sanchez C, Perez-Martos A, Enriquez J, Diaz M, Urries A, Montoro L, Lopez-Perez M, Enriquez J.Human mitochondrial DNA haplogroups associated with high or reduced spermatozoa motility. Am J Hum Genet,2000,67:682-696.
22. Matzuk MM, Lamb DJ. Genetic dissection of mammalian fertility pathways. Nat Cell Biol,2002,4: Suppl s41-49.
23. Holt, I.J., Harding, A.E., Cooper, J.M,et al. Mitochondrial myopathies: clinical and biochemical features of 30 patients with major deletions of muscle mitochondrial DNA. Ann. Neurol,1989, 29, 699-710.
24. Mortimer D., Pandy, I.J. and Sawers, R.S. Relationship between human sperm motility characteristics and sperm penetration into cervical mucus in vitro. J. Reprod. Fertil,1986, 78, 93-102.
25. Overstreet, T.W. Transport of gametes in the reproductive tract of female mammal. In Hartman, J.F.(ed.), Mechanism and Control of Animal Fertilization. Academic Press, New York, USA, 1983,499-543.
26. Copeland WC, Ponamarev MV, Nguyen D,et al. Mutations in DNA polymerase gamma cause error prone DNA synthesis in human mitochondrial disorders.Acta Biochim Pol.2003,50(1):155-167.
27. Spiropoulos J., Turnbull D M., and P F Chinnery. Can mitochondrial DNA mutations cause sperm dysfunction? Molecular Human Reproduction,2002,18,719-721.
28. Fraga, C.G., Motchnik, P.A., Wyrobek, A.J. et al. Smoking and low antioxidant levels increase oxidative damage to sperm DNA. Mutat. Res,1996, 351, 199-203.
29. Hou, J.H. and Wei, Y.H. The unusual structures of the hot-regions flanking large-scale deletions in human mitochondrial DNA. Biochem. J,1996, 318, 1065-1070.
30. Lee, H.C. and Wei, Y.H. Mutation and oxidative damage of mitochondrial DNA and defective turnover of mitochondria in human aging. J. Formos. Med. Assoc,1997, 96, 770-718.
31. Yakes, F.M. and van Houton, B.Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Proc. Natl. Acad. Sci. USA,1997,94, 514-519.
32. Bakker HD, Scholte HR, Dingemans KP, Spelbrink JN, Wijburg FA, Van den Bogert C. Depletion of mitochondrial deoxyribonucleic acid in a family with fatal neonatal liver disease. J Pediatr,1996,128:683- 687.
33. Cooke HJ, Saunders PT. Mouse models of male infertility. Nat Rev Genet,2002,3: 790-801.
34. Rossignol R., Faustin B., Rocher C., Malgat M., Mazat J P., and Thierry Letellier. Mitochondrial Threshold Effects. Biochemical Journal Immediate Publication. BJ20021594.
35. Shoffner, J.M., and Wallace, D.C. Oxidative phosphorylation disease and mitochondrial DNA mutations: diagnosis and treatment. Ann. Rev. Nutr,1994, 14, 535-568.
36. Rao, J.C.B. and Martin, S.M. Lipid peroxidation in human spermatozoa are related to midpiece abnormalities and motility. Gamete Res,1989, 24, 127-134.
37. Frederick L., Renee A., Reijo-Pera. Male sperm motility dictated by mother's mtDNA. Am. J. Hum. Genet,2000,67:543-54.
38. William J., Ballard O and M C Whitlock. The incomplete natural history of mitochondria. Molecular Ecology,2004,13, 729-744.
39. Houshmand M, Holme E, Hanson C, Wennerholm UB, Hamberger L. Is paternal mitochondrial DNA transferred to the offspring following intracytoplasmic sperm injection? J Assist Reprod Genet,1997,14:223-7.
40. D′ez-Sa′nchez C., Ruiz-Pesini E., Cristina Lapen a A., Montoya J., Pe′rez-Martos A.,Enr′quez J A., and M J. Lo′pez-Pe′rez. Mitochondrial DNA content of human spermatozoa. Biology of Reproduction, 2003,68, 180-185.
41. MITOMAP. A human mitochondrial genome database. Center for Molecular Medicine, Emory University, Atlanta,USA. http://www.gen.emory.edu/mitomap.html (2000).
42. World Health Organization. World Health Organization Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. U.K.: Press Syndicate of Cambridge,Cambridge,1992.
(Editor Jaque)(Mohammad Hossein Salehi1,)
1 Department of Biology, Faculty of Science, Razi University, Baghabrisham, Kermanshah, Iran
2 Department of Medical Genetic, National Institute for Genetic Engineering and Biotechnology, Tehran, Iran
Corresponding to Massoud Houshmand, Department of Medical Genetics, National Institute for Genetic Engineering and Biotechnology, Tehran, Iran
Tel: +98 21 44580390,Fax: +98 21 44580399,E-mail:massoudh@nrcgeb.ac.ir
[Abstract] There is increasing evidence that mitochondrial DNA (mtDNA) anomalies in sperm may lead to infertility. Point mutations, deletions and depletion have been associated with decline of fertility and motility of human sperm. It has been proposed that mtDNA genetic alterations can also be responsible for sperm dysfunction. Sperm motility is one of the major determinants of male fertility and is required for successful fertilization.
It is becoming increasingly evident that both point mutations and large-scale deletions may have an impact on sperm motility and morphology. In this study we investigated association between occurrence of mtDNA△4977 bp deletion with diminished fertility and motility of human spermatozoa. The possible relationship between multiple deletions of mtDNA and the decline of fertility and motility in human spermatozoa was further explored in 50 subjects including sub-fertile and infertile males. Our results showed that the ratio of the deleted mtDNA in the spermatozoa with poor motility and diminished fertility were significantly higher than those in the spermatozoa with good motility and fertility. Our findings suggest that mutation and deletion may play an important role in some pathophysiological conditions of human spermatozoa.
[Key words] deletion； infertility；mitochondrial DNA；spermatozoa
INTRODUCTION
Human male infertility is a major health problem worldwide. Anatomic defects, endocrinopathies, immunologic problems, ejaculatory failures and environmental exposures are significant causes of infertility. Frequently, however, no clear cause for the observed infertility could be diagnosed coining the term “idiopathic infertility”.
Infertility in humans is an issue that affects 10%~15% of couples, and approximately half of these cases of infertility are attributable to men. Untreatable sub-fertility caused by poor semen quality accounts for 75% of patients consulting for fertility problems. It is assumed that in about 30 % of cases male infertility is caused by chromosome aberrations or mutations in genes functioning in the male germ line[1~3].
A general overview of genetic defects causing male infertility is therefore restricted to only a subgroup of infertile man, although this subgroup is large with about 40%. However, it is unknown whether a general genetic analysis of all infertile men would not also influence the therapeutic protocol of some other patient groups.
Defective sperm function is now recognized as one of the most important causes of human infertility. Sperm motility can be affected by a wide range of conditions, including abnormalities of the flagellar movement[4,5].
Massive amounts of energy are consumed by the fast swimming of spermatozoa during fertilization. There is increasing evidence showing that sperm motility is strongly dependent on the ATP supplied by the mitochondrial oxidative phosphorylation system (OXPHOS) [6,7].Four of the five enzymatic complexes that constitute the OXPHOS system are partially encoded by mitochondrial DNA (mtDNA). Thus, mutations in mtDNA genes that impair the expression of one or more proteins encoded in the mtDNA can promote diseases in humans[1,8~10].Accordingly, it has been proposed that mutations in the mtDNA affecting the performance of mitochondria ATP production could cause reduced sperm motility and, therefore, asthenozoospermia[11~13].
Some evidence of mitochondrial involvement in male infertility has been found. First, male infertility, associated with asthenozoospermia[12] or oligoasthenozoospermia[14], has been reported in patients suffering from typical mtDNA diseases, involving point mutations or multiple deletions of mtDNA. Secondly, sperm have been shown to be particularly prone to develop deletions of mtDNA[15~19]. Some studies have shown that, in human sperm, these deletions are associated with a decline of motility and fertility[13,20].Thirdly, a correlation has been found between the quality of the semen and the functionality of the respiratory chain in sperm mitochondria[7,21]. Moreover, it has been shown that mtDNA point mutations and single nucleotide polymorphisms can greatly influence semen quality[21]. The high rate of deletions or substitutions observed in sperm could be due to impaired mitochondrial maintenance or result from the deleterious effects of oxidative stress. Lastly, sperm treatment with extracellular ATP has been shown to induce a significant increase in the fertilizing potential of sperm, demonstrating the importance of the mitochondrial function in male fertility[22].
MATERIALS AND METHODS
Semen Collection
54 semen samples were collected from 50 patients and 4 normal controls by masturbation under hygienic conditions after a 3- to 5-day period of sexual abstinence. Samples were allowed to liquefy for 30 min at 37 ℃ and were analyzed according to World Health Organization recommendations within a period of 2 h recruited from couples who presented for semen analysis or assisted reproductive technology at the In Vitro Fecundation Laboratory of the Hospital of 4th Shahid Mehrab, Kermanshah, Iran. These couples suffered from either male or female infertility, or infertility of unknown etiology.
The volume of the ejaculate was measured, and the number and percentage of motile spermatozoa was determined. A total of 54 semen samples were classified to 4 as blank, 24 as normal, 21 as abnormal, 5 as azospermic. Characteristics of patients and blanks used in our study has been shown in Table 1 and Table 2.
Table 1 Characteristics of Patients in Our Study
Table 2 Properties of Blanks in Our Study
sh
Sperm and Extraction
Total DNA of human spermatozoa was extracted which performed using a DNA extracting kit (Diatom DNA prep 100, Genfanavaran, Iran). Total DNA was extracted from 0.1 ml of total semen and were subjected to agarose gel electrophoresis followed by staining with ethidium bromide (1 mg/ml) and visualized under UV illumination.
Multiplex PCR
The reaction mixture for multiplex PCR contained 2 pmol of each primer, 1 unit Taq polymerase, each dNTP at a final concentration of 200 μm, and 2 μl PCR buffer at a final volume of 50 μl. The PCR reactions were performed in a thermal cycler for 35 cycles with denaturation at 94 ℃ for 1′ (min), primer annealing at 55 ℃ for 1′ (min) and primer extension at 72 ℃ for 35″. The amplified fragments were separated by gel electrophoresis in 1 % agarose. We investigated all patients using the primers ONPF 86, 5461-5480 (ND2) ;ONPR 89, 5740-5721 (OL); ONPR 10, 15000-14981 (Cyt b) ; ONPR 74, 13640-13621 (ND5); ONPF 25, 8161-8180 (COX П); and ONPR 99, 16150-16131 (D-loop)(Table 3).The distances between the primers were long enough to allow amplification only if a part of the DNA between respective primers was deleted. Primer pair ONP 86, 89 was used to amplify a normal internal mtDNA fragment in a region, which is seldom afflicted by deletions and served as a control of the preparation and PCR analysis.
Primer pattern of human mtDNA and extented deletion with 99, 74,25,10,89 and 86 primers.
Deletion break points were analyzed by direct sequencing of DNA fragments amplified by the PCR.
Table 3 Oligonucleotide Primers Used for the Analysis of the Deletions in the mtDNA of Human
RESULTS
In this study, we investigated large-scale deletions of sperm mtDNA in 50 sub-fertile and infertile males. We screened for large-scale deletions of mtDNA in spermatozoa with poor motility by using multiplex PCR techniques. We found 4 977 bp deletion of mtDNA in the spermatozoa with low motility scores.
The rate of observed deletion was less than 15 % of consideration subjects. We confirmed the 4 977 bp deletion and identified some other deletions like 5 kbp, 7 kbp, 7.5 kbp and 8 kbp (Table 4).
Table 4 Large-scale Deletions in Our Patients
The results showed that the ratio of the deleted mtDNA in the spermatozoa with poor motility and diminished fertility were significantly higher than those in the spermatozoa with good motility and fertility. There was also a relationship between age, morphology and motility in our study (Figure 2).
Figure 1 Primer pattern of human mtDNA and extented deletion with 99, 74,25,10,89 and 86 primers
TP6
Figure 2 Relationship between age, morphology and motility in our study
DISCUSSION
In recent years, male infertility has increased in the industrialized countries due to a decline in sperm counts and a rise in testicular and sperm pathologies. Defective sperm function has been identified as the largest defined cause of male infertility[3]. Sperm dysfunction may be caused by a wide range of conditions, including abnormalities of flagellar movement[24,4], failure in sperm-zona recognition[25], and inability to carry out sperm-oocyte fusion[4].In order to understand the etiology of the decreased motility and diminished fertility of the spermatozoa and to develop an appropriate therapeutic strategy, the molecular basis of these defects must be elucidated.
Sperm mitochondria play an important role in spermatozoa functionality; therefore, genetic alterations of mitochondrial DNA (mtDNA) may have consequences for normal fertilization. Since sperm require a substantial amount of energy to swim fast enough to reach the oviduct during fertilization, the appropriatebioenergetics function of mitochondria is critical for male fertility[26,27]. Spermatozoa need a great deal of energy to support their rapid movement after ejaculation. While producing ATP by normal aerobic metabolism, sperm mitochondria also generate ROS and free radicals as byproducts. In the active respiration state, as much as 5% of the oxygen utilized by mitochondria is converted to superoxide anions and other ROS. MtDNA molecules, which are transiently attached to the inner membrane of mitochondria, are thus extremely vulnerable to oxidative damage under active respiration of spermatozoa[28]. Aitken and coworkers (1989) showed that excess amounts of ROS and free radicals in spermatozoa and seminal plasma have adverse effects on sperm motility and fertility.
As mtDNA is extensively damaged by ROS or free radicals, DNA strand breaks and large-scale deletions may be induced[29,30]. Defective respiratory enzymes containing protein subunits encoded by the deleted mtDNA may further enhance free radical production, resulting in more profound oxidative damage. Spermatozoa are especially liable to oxidative damage because their plasma membranes are rich in poly-unsaturated fatty acids. It has been established that mitochondrial dysfunction may be caused by mtDNA mutation and oxidative damage caused by endogenous and exogenous free radicals[31~34]. As reactive oxygen species (ROS) are continuously generated by the respiratory chain, they may cause significant oxidative damage to mtDNA if not efficiently eliminated[35,36].
In the present study, we investigated the possible role of mtDNA deletions in the pathophysiology of human sperm dysfunction. Our results showed that the frequency of occurrence and the proportion of deleted mtDNA were higher in sperm with poor motility than in sperm with good motility. We found 4 large-scale deletions with different sizes. Our previous study on 100 infertile men using multiple PCR and Expanded Long Range PCR methods revealed 5~6 kb deletions in three infertile men who had about 78%, 50% and 57% immobility of sperm (Unpublished data). These large-scale deletions result in complete removal or truncation of some structural genes and tRNA genes of mtDNA. These genetic alterations could be pathological mutations or common mtDNA variants that only affect male fertility because mtDNA is maternally inherited. Thus, mutations responsible for encephalomyopathies decrease sperm motility and single and multiple mtDNA deletions have been associated with sperm dysfunction[37,13,20].The cumulative effects of large scale deletions in total have also been associated with male infertility. There is relationship between large-scale deletions of mtDNA in spermatozoa with poor motility and those from patients with infertility and oligospermia or asthenospermia[38,39].
Among the mtDNA deletions observed, the so-called “common deletion” of 4 977 bp was the most prevalent and abundant one[13,40]. We detected the common 4 977 bp deletion in mtDNA from spermatozoa with poor motility and diminished fertility. The 4 977 bp deletion of mtDNA causes the removal or truncation of multiple structural genes (ATPase 6/8, COIII, ND3, ND4L, and ND4) and five tRNA genes.
In addition, there has been reported 2 novel deletions of 7 345 and 7 599 bp of mtDNA in spermatozoa with poor motility[13,20]. These 3 large-scale deletions all occur at the hot regions[30] in a large area between 2 replication origins of mtDNA. These large-scale deletions result in complete removal or truncation of some structural genes and tRNA genes of mtDNA. Our findings suggest that mtDNA deletion may play an important role in some pathophysiological condition of human infertility.
Acknowledgement
This project was supported by National Institute for Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
REFERENCES
1. Vogt PH, Chandley AC, Hargreave TB, Keil R, Ma K, Sharkey A. Microdeletions in interval 6 of the Y chromosome of males with idiopathic sterility point to disruption of AZF, a human spermatogenesis gene. Hum Genet，1992，9: 491-496.
2. Vogt Peter H. Molecular Genetic of Human Male Infertility: From Genes to New Therapeutic Perspectives, Section Molecular Genetics & Infertility, Dept. Gynecol. Endocrinol. & Reproductive Medicine, University of Heidelberg, D-69115 Heidelberg, Germany, 2000.
3. Vogt Peter H. Molecular genetic of human male infertility: from genes to new therapeutic perspectives. Current Pharmaceutical，2004.
4. Aitken, R.J., Clarkson, J.S. and Fisel, S. Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol. Reprod，1989, 40, 183-197.
5. Tredway, D.R., Settlage, D.S.F., Nakamura, R.M. et al. Significance of timing for the post-coital evaluation of cervical mucus. Am. J. Obstet. Gynecol，1975, 121, 287-293.
6. Ford WC, Harrison A.The role of oxidative phosphorylation in the generation of ATP in human spermatozoa. J Reprod Fertil，1981，271-278.
7. Ruiz-Pesini E, Diez C, Lapen a AC, Pe′rez-Martos A, Montoya J, Alvarez E, Arenas J, Lopez-Perez M.Correlation of sperm motility with mitochondrial enzymatic activities. Clin Chem,1998,4:1616-1620.
8. Enriquez JA, Chomyn A, Attardi G.MtDNA mutation in MERRF syndrome causes defective aminoacylation of tRNA(Lys) and premature translation termination. Nat Genet,1995,10(1):47-55.
9. Larsson, N.G., Oldfors, A., Garman, J.D., Barsh, G.S. and Clayton, D.A. Down-regulation of mitochondrial transcription factor A during spermatogenesis in humans. Hum. Mol. Genet,1997,6:185-191.
10. Wallace, D.C. Mitochondrial DNA sequence variation in human evolution and disease. Proc. Natl. Acad. Sci. USA, 1994,91, 8739-8746.
11. Zeviani M and Antozzi C.Mitochondrial disorders. Mol Hum Reprod,1997,3:133-148.
12. Folgero T, Torbergsen T, Oian P. The 3243-MELAS mutation in a pedigree with MERRF. Eur Neurol,1995,35:168-171.
13. Kao SH, Chao HT, Wei YH.Mitochondrial deoxyribonucleic acid 4977-bp deletion is associated with diminished fertility and motility of human sperm. Biol Reprod,1995,52: 729-736.
14. Lestienne, P, Reynier, P, Chretien, M.F., Penisson-Besnier, I., Malthiery, Y. and Rohmer, V. Oligoasthenospermia associated with multiple mitochondrial DNA rearrangements. Mol. Hum. Reprod,1997, 3, 811-814.
15. Cummins, J. Mitochondrial DNA in mammalian reproduction. Rev. Reprod,1998, 3, 172-182.
16. Cummins, J.M., Jequier, A.M., Martin, R., Mehmet, D. and Goldblatt, J.Semen levels of mitochondrial DNA deletions in men attending an infertility clinic do not correlate with phenotype. Int. J. Androl., 1998,21, 47-52.
17. Reynier P, Chretien MF, Savagner F, Larcher G, Rohmer V, Barriere P, Malthiery Y. Long PCR analysis of human gamete mtDNA suggests defective mitochondrial maintenance in spermatozoa and supports the bottleneck theory for oocytes. Biochem Biophys Res Commun,1998,252:373-377.
18. O'Connell, M., McClure, N. and Lewis, S.E. A comparison of mitochondrial and nuclear DNA status in testicular sperm from fertile men and those with obstructive azoospermia. Hum. Reprod,2002, 17, 1571-1577.
19. O'Connell, M., McClure, N. and Lewis, S.E.Mitochondrial DNA deletions and nuclear DNA fragmentation in testicular and epididymal human sperm. Hum. Reprod,2002b, 17, 1565-1570.
20. Kao S H., Chao H T and Y H Wei. Multiple deletions of mitochondrial DNA are associated with the decline of motility and fertility of human spermatozoa. Molecular Human Reproduction,1998,4:7: 657-666.
21. Ruiz-Pesini E, Lapena A, Diez-Sanchez C, Perez-Martos A, Enriquez J, Diaz M, Urries A, Montoro L, Lopez-Perez M, Enriquez J.Human mitochondrial DNA haplogroups associated with high or reduced spermatozoa motility. Am J Hum Genet,2000,67:682-696.
22. Matzuk MM, Lamb DJ. Genetic dissection of mammalian fertility pathways. Nat Cell Biol,2002,4: Suppl s41-49.
23. Holt, I.J., Harding, A.E., Cooper, J.M,et al. Mitochondrial myopathies: clinical and biochemical features of 30 patients with major deletions of muscle mitochondrial DNA. Ann. Neurol,1989, 29, 699-710.
24. Mortimer D., Pandy, I.J. and Sawers, R.S. Relationship between human sperm motility characteristics and sperm penetration into cervical mucus in vitro. J. Reprod. Fertil,1986, 78, 93-102.
25. Overstreet, T.W. Transport of gametes in the reproductive tract of female mammal. In Hartman, J.F.(ed.), Mechanism and Control of Animal Fertilization. Academic Press, New York, USA, 1983,499-543.
26. Copeland WC, Ponamarev MV, Nguyen D,et al. Mutations in DNA polymerase gamma cause error prone DNA synthesis in human mitochondrial disorders.Acta Biochim Pol.2003,50(1):155-167.
27. Spiropoulos J., Turnbull D M., and P F Chinnery. Can mitochondrial DNA mutations cause sperm dysfunction? Molecular Human Reproduction,2002,18,719-721.
28. Fraga, C.G., Motchnik, P.A., Wyrobek, A.J. et al. Smoking and low antioxidant levels increase oxidative damage to sperm DNA. Mutat. Res,1996, 351, 199-203.
29. Hou, J.H. and Wei, Y.H. The unusual structures of the hot-regions flanking large-scale deletions in human mitochondrial DNA. Biochem. J,1996, 318, 1065-1070.
30. Lee, H.C. and Wei, Y.H. Mutation and oxidative damage of mitochondrial DNA and defective turnover of mitochondria in human aging. J. Formos. Med. Assoc,1997, 96, 770-718.
31. Yakes, F.M. and van Houton, B.Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Proc. Natl. Acad. Sci. USA,1997,94, 514-519.
32. Bakker HD, Scholte HR, Dingemans KP, Spelbrink JN, Wijburg FA, Van den Bogert C. Depletion of mitochondrial deoxyribonucleic acid in a family with fatal neonatal liver disease. J Pediatr,1996,128:683- 687.
33. Cooke HJ, Saunders PT. Mouse models of male infertility. Nat Rev Genet,2002,3: 790-801.
34. Rossignol R., Faustin B., Rocher C., Malgat M., Mazat J P., and Thierry Letellier. Mitochondrial Threshold Effects. Biochemical Journal Immediate Publication. BJ20021594.
35. Shoffner, J.M., and Wallace, D.C. Oxidative phosphorylation disease and mitochondrial DNA mutations: diagnosis and treatment. Ann. Rev. Nutr,1994, 14, 535-568.
36. Rao, J.C.B. and Martin, S.M. Lipid peroxidation in human spermatozoa are related to midpiece abnormalities and motility. Gamete Res,1989, 24, 127-134.
37. Frederick L., Renee A., Reijo-Pera. Male sperm motility dictated by mother's mtDNA. Am. J. Hum. Genet,2000,67:543-54.
38. William J., Ballard O and M C Whitlock. The incomplete natural history of mitochondria. Molecular Ecology,2004,13, 729-744.
39. Houshmand M, Holme E, Hanson C, Wennerholm UB, Hamberger L. Is paternal mitochondrial DNA transferred to the offspring following intracytoplasmic sperm injection? J Assist Reprod Genet,1997,14:223-7.
40. D′ez-Sa′nchez C., Ruiz-Pesini E., Cristina Lapen a A., Montoya J., Pe′rez-Martos A.,Enr′quez J A., and M J. Lo′pez-Pe′rez. Mitochondrial DNA content of human spermatozoa. Biology of Reproduction, 2003,68, 180-185.
41. MITOMAP. A human mitochondrial genome database. Center for Molecular Medicine, Emory University, Atlanta,USA. http://www.gen.emory.edu/mitomap.html (2000).
42. World Health Organization. World Health Organization Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. U.K.: Press Syndicate of Cambridge,Cambridge,1992.
(Editor Jaque)(Mohammad Hossein Salehi1,)