Mitochondrial variations in the MT-ND4 and MT-TL1 genes are associated with male infertility

ABSTRACT

Mitochondrial gene mutations have been reported to be associated with sperm motility and the quality of semen. The aim of this study was to investigate whether the two mitochondrial genes (MT-ND4 and MT-TL1) are involved in Chinese male infertility. A total of 97 asthenospermia patients and 80 fertile controls were recruited in this case-control study. Genomic DNA were extracted from the sperm of all participants. Two mitochondrial DNA genes (MT-ND4 and MT-TL1) were amplified by using polymerase chain reaction (PCR) with the gene-specific primers and sequenced on an ABI 3730XL DNA sequencer. For the MT-ND4 gene, we found a total of 64 and 54 nucleotide substitutions in patients and controls, respectively, with no discrepancy in the mutation rates (66.0% vs. 67.5%, p>0.05). However, one mutation (g.11084A>G, p.T109A) leading to an amino acid substitution in a highly conserved residue and predicted to be deleterious was detected only in the cases. For another gene MT-TL1, a novel mutation (g.3263C>T) near the anticodon TAA was identified in an asthenospermia patient and was absent from normal controls. However, the mutation positions in the cases varied from the controls and one highly conserved mutation (g.11084A>G, p.T109A) which was not found in the controls and probably caused damage to the protein structure might contribute to asthenospermia. For another gene MT-TL1, a highly conservative novel mutation which is located closely next to the anticodon also might contribute to asthenospermia. Our result suggests that the MT-ND4 and MT-TL1 genes might be associated with Chinese male infertility.

Abbreviations: MT-ND4: mitochondrially encoded NADH dehydrogenase 4; MT-TL1: mitochondrially encoded tRNA leucine 1 (UUA/G); PCR: polymerase chain reaction; OXPHOS: mitochondrial oxidative phosphorylation; ATP: adenosine triphosphate; mtDNA: mitochondrial DNA; SNPs: single nucleotide substitutions; AD: alzheimer’s disease; PD: parkinson’s disease; MELAS: mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes; ROS: reactive oxygen species

KEYWORDS:

• Male infertility
• MT-ND4
• MT-TL1

Introduction

In the world, about 15% of married couples suffer from infertility [De Kretser 1997] and male factor accounts for half of the causes. Male infertility represents a variety of conditions, including low sperm motility (asthenozoospermia) or/and few sperm cells (oligospermia) [Nakada et al. 2006]. Mitochondria are primary organelles in the sperm and the most important function of the mitochondria is to produce energy for sperm activity. Sperm mitochondria are located in the mid piece and produce the energy to support sperm motility [O’Connell et al. 2002]. Human mitochondrial DNA (mtDNA) is a naked double-stranded circular molecule, which encodes two rRNA genes, 22 tRNA genes, and 13 proteins [Anderson et al. 1981]. It encodes many essential components of four respiratory enzyme complexes that have a profound impact on sperm motility. It has no efficient protection by histones or DNA-binding proteins and also lacks an efficient repair mechanism [DiMauro and Schon 2003]. As sperm cells need a great amount of adenosine triphosphate (ATP) to function properly, sperm mitochondria are essential to spermatozoa [Diez-Sánchez et al. 2003]. In recent years, mtDNA mutations and male infertility, particularly asthenospermia have aroused people’s wide attention. A growing body of evidence points to the importance of mtDNA variations with low sperm motility and male infertility. Mitochondrial DNA variants have been suggested to be associated with asthenozoospermia or oligoasthenozoospermia [Folgero et al. 1993; Lestienne et al. 1997]. Mutations at the mitochondrial DNA polymerase (POLG) locus, for instance, have been reported to be associated with male infertility [Rovio et al. 2001]. High incidence of single nucleotide substitutions (SNPs) of mtDNA attributes to poor semen quality [Holyoake et al. 2001].

In this study, we performed a case-control analysis of two mitochondrial genes involved in oxidative phosphorylation and ATP synthesis (mitochondrially encoded NADH dehydrogenase 4 (MT-ND4) and mitochondrially encoded tRNA leucine 1 (UUA/G) (MT-TL1)) in the sperm from 97 Chinese male infertile patients and 80 fertile controls. The aim of our study was to investigate whether these two mitochondrial genes are associated with male infertility in the Chinese population.

Results

We sequenced the two mitochondrial DNA genes (MT-ND4 and MT-TL1) in the sperm of 97 Chinese male infertility patients and 80 fertile controls. As for the MT-ND4 gene, we found a total of 64 nucleotide substitutions which were located in 35 different loci in the cases and a total of 54 nucleotide substitutions which were located in 37 different loci in the controls. All of these variants have been reported in the human mitochondrial DNA database (www.mitomap.org). There was no difference in the mutation rates between the case and control group (66.0% vs. 67.5%, p>0.05). Among these variants, we found seven mutations introducing an amino acid substitution, three of which were absent from controls (NC_012920.1:g.11061C>T, p.S101F; g.11084A>G, p.T109A; g.12123C>T, p.T455I; Table 1). Two non-synonymous mutations were predicted to be benign or not evolutionary conserved in silico except g.11084A>G. The mutation g.11084A>G, altering the amino acid residue from theronine to alanine at position 109, was previously linked to Alzheimer’s disease (AD), Parkinson’s disease (PD), and mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) [Levin et al. 2013; Pereira et al. 2011], which implies an important role in the maintenance of normal mitochondrial function. Bioinformatic analysis indicated this mutation was highly conserved among several species (Figure 1) and probably damaging to the protein function and structure (PolyPhen2 score 0.994).:

Table 1. The non-synonymous mtDNA variations detected in the mitochondrial MT-ND4 gene.

Position	Nucleotide Change	No. of Cases	Amino Acid Change	Found in Control	Reported in Mitomap	Reported Diseases	Reference
11061	C-T	1	non-syn:S-F	N	Y	N	None
11084	A-G	1	non-syn:T-A	N	Y	AD, PD; MELAS	Levin et al. 2013; Pereira et al. 2011
11150	G-A	1	non-syn:A-T	Y	Y	N	None
11696	G-A	2	non-syn:V-I	Y	Y	LHON/LDYT/DEAF/hypertension helper mut.	Pereira et al. 2011
11969	G-A	2	non-syn:A-T	Y	Y	N	None
12026	A-G	4	non-syn:I-V	Y	Y	DM	Mitchell et al. 2006
12123	C-T	1	non-syn:T-I	N	Y	N	None

mtDNA: mitochondrial DNA.

Figure 1. Sequence alignment of ND4 proteins among several species. The conservation analysis through CLC Main Workbench Software indicated that the mutation g.11084A>G, p.T109A was conservative among five species (Homo sapiens, Homo heidelbergensis, Mus musculus, Rattus norvegicus, and Danio rerio).

For another gene MT-TL1, we found two mutations in the cases (g.3263C>T and g.3277G>A, shown in Table 2) and there were no mutations found in the controls. The novel mutation g.3263C>T was highly conserved among several species (Figure 2) and located at the position 34 which was next to the anticodon TAA. Another less conservative mutation g.3277G>A was reported in Mitomap and located in the extra loop of this tRNA. To see whether these two mutations could alter the secondary structure of tRNAs, we made an online prediction on the MFE secondary structure of the wild and mutant types of this mt-tRNA using RNAfold web server [Gruber et al. 2008]. However, no significant change was observed:

Table 2. The mtDNA variations detected in the mitochondrial MT-TL1 gene.

Position	Nucleotide Change	No. of cases	Amino Acid Change	Found in Control	Reported in Mitomap	Reported Diseases	Reference
3263	C-T	1	tRNA	N	N	N	None
3277	G-A	1	tRNA	N	Y	Poss. hypertension factor	Zhu et al. 2009

mtDNA: mitochondrial DNA.

Figure 2. Sequence alignment of MT-TL1 genes among several species. The conservation analysis through CLC Main Workbench Software indicated that the mutation g.3263C>T was conservative among five species (Homo sapiens, Homo heidelbergensis, Mus musculus, Rattus norvegicus, and Danio rerio).

Discussion

The aim of this study was to investigate whether the two mitochondrial genes (MT-ND4 and MT-TL1) are involved in Chinese male infertility. Sperm mitochondrial DNA is a naked, loosely structured double-stranded circular molecule and exposed to reactive oxygen species (ROS), is easily damaged. Thus, its variation rate is higher than that of the nuclear genome and this may affect sperm motility [Yakes and Houten 1997]. Mitochondrial oxidative phosphorylation (OXPHOS) is a major metabolic pathway which is necessary for sperm to function properly by producing ATP and any changes may affect its normal activity [Ferramosca et al. 2008; Ferramosca and Zara 2014]. These two mitochondrial genes play an essential role in the process of oxidative phosphorylation and it has been reported that they are associated with sperm motility [Holyoake et al. 2001; Rani et al. 2006; Spiropoulos et al. 2002].

MT-ND4 is one of the core mitochondrial-encoded subunits of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I) which is the largest and most complicated mitochondrial respiratory-chain enzyme [Yagi and Matsuno 2003; Hirst et al. 2003]. MT-TL1 is a small 75 nucleotide transfer RNA (human mitochondrial map position 3230-3304) which is encoded by the mitochondrial MT-TL1 gene. It is also known as mitochondrially encoded tRNA leucine 1 (UUA/G) that transfers the amino acid leucine to the growing polypeptide chain in the progress of protein synthesis [Anderson et al. 1981].

Recent studies have suggested that some sequence variants in these two genes were related to sperm quality. Holyoake et al. found a remarkably higher frequency of nucleotide substitution at 11719 of MT-ND4 in infertile male patients while Rani et al. identified the missense mutation C11994T as a cause of low sperm motility in the Indian population [Holyoake et al. 2001; Rani et al. 2006]. Another study pointed out that the A3243G mutation of MT-TL1 was associated with lower sperm motility in a UK population [Spiropoulos et al. 2002].

Our study revealed that the mutation g.11084A>G of MT-ND4 gene and g.3263C>T of MT-TL1 gene might be risk factors for asthenospermia and Chinese male infertility. For the three non-synonymous mtDNA variations which were not found in the controls, only the mutation g.11084A>G probably damaged the protein structure of ND4 and was also the only mutation being associated with other diseases caused by mitochondrial dysfunction. Previous studies suggested g.11084A>G was a disease-causing variant that contributes to AD, PD, and MELAS which may be caused by an aberrant function of mitochondria [Levin et al. 2013; Pereira et al. 2011]. Therefore, the mutation g.11084A>G, p.T109A might be a new risk factor. The novel mutation g.3263C>T was highly conserved and closely located next to the anticodon which was the key to the identification of mRNA codon and protein synthesis. Thus, this mutation might affect the efficient synthesis of leucine and also might be a risk factor. Though another mutation g.3277G>A had been reported to be a possible hypertension factor [Zhu et al. 2009], it might not be a risk factor, owing to it had less conservation and was located in the extra loop of the tRNA.

Through this case-control study, our result suggests that the MT-ND4 and MT-TL1 genes might contribute to Chinese male infertility. However, genetic analysis of larger samples and functional analysis will be needed to confirm the role of the MT-ND4 and MT-TL1 genes in Chinese male infertility.

Materials and methods

Semen collection

A total of 97 infertile males from the Reproductive Medicine Center of the 105^th PLA were diagnosed with asthenospermia based on semen analyses. The patients were aged between 20 and 35 y and the mean age was 29.32±4.25 y. All patients screened for the detection of AZFa, AZFb, and AZFc microdeletions previously and had normal karyotypes. Semen samples were obtained by masturbation after 2-7 d abstinence. Routine semen analysis was performed after liquefaction of the semen within one h. The diagnosis of asthenospermia was according to three consecutive semen analyses results being strictly in accordance with the World Health Organization guidelines (concentration of spermatozoa >15 ×10⁶/ml, A+ B sperm motility less than 32% in fresh ejaculate) [WHO 2010]. Semen samples from 80 fertile controls were also collected. There was no significant age difference between the case and control groups. Exclusion criteria were: genital trauma, reproductive tract infections, varicocele, Y chromosome microdeletions. Informed consent was obtained from all subjects in the study. Ethical approval was formally obtained from the Ethics Committee of the Hospital of 105^th PLA (China).

DNA extraction from semen

Genomic DNAs from the sperm of azoospermia patients and fertile controls were extracted by the QIAamp Tissue DNA kit (Qiagen, Germany) according to the manufacturer’s methods. The concentration and quality of all DNA samples were examined by Nano-Drop 2000 spectrophotometer (Thermo, USA) and agarose gel electrophoresis.

PCR and sequencing of mitochondrial genes

The two genes (MT-ND4 and MT-TL1) from the sperm of the patients and fertile controls were amplified and sequenced. Primer sequences for the above two genes: MT-ND4, 5’-TCCTCCCTACTATGCCTAG-3’ and 5’-AGCATTCGGAGACAACAG-3’; MT-TL1, 5’-AATCCAGGTCGGTTTCT-3’ and 5’-TACATCTCCCACTACCATCT-3’. PCR products were sequenced on ABI 3730XL DNA sequencer by using proper PCR primers and the BigDye Terminator Cycle Sequencing kit (Life Technology, USA). The novelty of variant found in sequencing was verified by searching from the controls and the human mitochondrial DNA database (www.mitomap.org).

Conservation analysis and bioinformatics prediction

The conservation analysis was performed by CLC Main Workbench Software (Aarhus, Denmark). The possible functional impact of amino acid change was predicted using the PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) and RNAfold web server was used to predict the MFE secondary structure of the wild and mutant type of this mt-tRNA (http://nhjy.hzau.edu.cn/kech/swxxx/jakj/dianzi/Bioinf4/miRNA/miRNA1.htm).

Declaration of interest

This work was supported by the Key program from the Medical Science and Technique of Nanjing Military Command Region (No. 10Z010). The authors declare no conflicts of interest.

Acknowledgments

The authors thank all of the subjects who participated in this study.

Additional information

Notes on contributors

Feng Ni

Conceived and designed the experiments: FN HJ. Performed the experiments and analyzed the data: FN YZ WXZ XMW XMS. Wrote the paper: FN. All authors read and approved the final version.

Yun Zhou

Conceived and designed the experiments: FN HJ. Performed the experiments and analyzed the data: FN YZ WXZ XMW XMS. Wrote the paper: FN. All authors read and approved the final version.

Wen-xiang Zhang

Conceived and designed the experiments: FN HJ. Performed the experiments and analyzed the data: FN YZ WXZ XMW XMS. Wrote the paper: FN. All authors read and approved the final version.

Xue-mei Wang

Conceived and designed the experiments: FN HJ. Performed the experiments and analyzed the data: FN YZ WXZ XMW XMS. Wrote the paper: FN. All authors read and approved the final version.

Xiao-min Song

Conceived and designed the experiments: FN HJ. Performed the experiments and analyzed the data: FN YZ WXZ XMW XMS. Wrote the paper: FN. All authors read and approved the final version.

Hong Jiang

Conceived and designed the experiments: FN HJ. Performed the experiments and analyzed the data: FN YZ WXZ XMW XMS. Wrote the paper: FN. All authors read and approved the final version.