Oligoasthenospermia associated with multiple mitochondrial DNA rearrangements

Molecular Human Reproduction vol.3 no.9 pp. 811–814, 1997

Oligoasthenospermia associated with multiple mitochondrial DNA rearrangements

Patrick Lestienne1,6, Pascal Reynier2, Marie-Franc¸oise Chre´tien3, Isabelle Penisson-Besnier4, Yves Malthie`ry2 and Vincent Rohmer5 1U 298 INSERM, 2Laboratoire de Biochimie et Biologie Moleculaire A, 3Laboratoire d’Histologie Embryologie Cytologie, ´ 4Service de Neurologie A, and 5Service de Medecine C, Centre Hospitalier Universitaire d’Angers, 49033 Angers Cedex,

´

France 6To

whom correspondence should be addressed

A patient who wished to be treated for infertility by intracytoplasmic sperm injection (ICSI) was referred to our group for assessment. Upon clinical examination, a ptosis (partial closure of the eyelid) was noted, and histology revealed ragged red fibres in the skeletal muscle. Southern blot analysis of spermatozoa and skeletal muscle revealed the presence of multiple mitochondrial DNA deletions. This kind of rearrangement may be of nuclear origin since three nuclear loci have been ascribed to multiple mitochondrial DNA deletions in humans. Since mitochondrial DNA is maternally transmitted, the use of ICSI was feasible. However, an alteration of nuclear gene product affecting the integrity of mitochondrial DNA, and thus sperm mobility, might be transmitted to the offspring with the risk of developing a mitochondrial DNA disease. Key words: hypofertility/ICSI/mitochondrial DNA/multiple deletions

Introduction Mitochondria are localized in the midpiece of the spermatozoon, and deliver ATP which is mainly used to provide flagellar propulsion. Each mitochondrion contains several copies of a covalently closed 16 569 bp DNA molecule (Anderson et al., 1981) encoding for 13, out of 83, subunits of the respiratory chain complexes. Human mitochondrial DNA is maternally inherited (Giles et al., 1980). A number of diseases have been ascribed to its genetic alteration, due to either point mutations segregating along with the maternal lineage such as in myoclonic epilepsy with ragged red fibres (MERRF), mitochondrial encephalomyopathy-lactic acidosis-stroke like episodes (MELAS), and Leber’s disease, or to sporadic large scale rearrangements, e.g. Kearns–Sayre syndrome (KSS), progressive external ophtalmoplegia (PEO), Pearson’s syndrome and other multisystemic disorders (Lestienne, 1992; Wallace, 1992). On other hand, nuclear driven multiple mitochondrial (mt)DNA deletions (Zeviani et al., 1989) and depletions (Moraes et al., 1991) have been observed in mitochondrial DNA diseases, pointing to nuclear defects responsible for dysfunction in mitochondrial DNA metabolism. Hence, there is the possibility of transmission by the male, of a mutation which might impair mitochondrial DNA structure in the descendants. That possibility must now be taken into account due to the increase in the practice of intracytoplasmic sperm injection (ICSI) (Van Steirteghem et al., 1996), which does not require mobile spermatozoa, and hence a functional respiratory chain, for fertilization. Here we report a study of the mtDNA of the spermatozoon of a patient who wished to undergo ICSI due to oligoasthenospermy, and found the presence of an © European Society for Human Reproduction and Embryology

abnormal structure potentially transmissible by a defective nuclear gene.

Materials and methods The patient A 34 year old patient was referred to us for investigation of infertility associated with an increase in follicule stimulating hormone (FSH 18 IU/l; normal range ,9 IU/l) in the absence of either a pathological increase in luteinizing hormone (LH) or reduction in plasma testosterone. Neither hypofertility nor mitochondrial disease were found in the patient’s family. Clinical examination, however, revealed an asymmetric ptosis, suggestive of mitochondrial disease, which had appeared at the age of ~20 years and which had progressively increased. Neither progressive external ophtalmoplegia nor any central neurological disorder were observed. Tendon reflexes were absent in the legs. Motor and sensory nerve conduction velocities were normal but sural nerve amplitudes were unrecordable. Serum creatine kinase was high at 335 IU/l (normal range ,105 IU/l) and histological studies on the deltoid muscle tissue revealed the presence of ragged red fibres and cytochrome C oxidase negative fibres. However, the enzymatic activities of the respiratory chain complexes in the deltoid muscle tissue were normal. The spermiogram revealed a 10-fold concentration increase (from 73105 to 73106 spermatozoa/ml; normal value 23107/ml) in the spermatozoa after 10 months treatment with human menopausal gonadotrophin (HMG 75 IU, three times a week), carnitine (4 g/day), coenzyme Q 10 (150 mg/day) and vitamin B2 (60 mg/day). These latter three treatments have been postulated to improve mitochondrial function. However, the few spermatozoa present were almost immotile, even after treatment. DNA extraction Spermatozoa obtained prior to any treatment were centrifuged for 10 min at 5000 g. DNA was extracted from the pellet with proteinase

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P.Lestienne et al. K, phenol extraction and ethanol precipitation (Maniatis et al., 1982). Muscle DNA was extracted by the same method. Polymerase chain reaction (PCR) analysis of mtDNA mutations were performed using 2 µl of blood collected in EDTA to look for the presence of Leber’s mutations 5244, 7444, 15812, 15257 (Gerbitz et al., 1992). The sample was diluted to 100 µl and the Taq reaction medium was heated for 10 min at 99°C. After the addition of 30 pmoles of each primer, 0.2 mM dNTP, 2.5 IU Taq DNA polymerase (Goldstar, Eurogentec, Seraing, Belgium), 2 min of denaturation at 95°C was followed by hybridization at 55°C for 1 min, primer extension for 1 min at 72°C, for 30 cycles. The mixture (10 µl) was digested by the appropriate restriction enzyme. Samples were subjected to agarose gel electrophoresis and stained with ethidium bromide (1 µg/ml) and visualized under UV illumination. The same procedure was used to investigate muscle tissue for the presence of Leber’s mutations at position 11778 (Wallace et al., 1988), 3460 (Howell and McCullough, 1990) and MELAS mutation 3243 (Goto et al., 1991), except that 1 µg of total DNA was used as template. One µg of total DNA from spermatozoa was used to look for the MELAS (3243) and MERRF (8344) mutations (Shoffner et al., 1990). Southern blot analysis was performed as described previously (Nelson et al., 1989) using 5 µg of total DNA digested with BamHI, and probed with mitochondrial DNA (Drouin, 1980) or nuclear ribosomal gene (Wilson et al., 1978). Long PCR was performed as described (Reynier and Malthie`ry, 1995) using primers D6 (59 TCTAGAGCCCACTGTAAAG 39 L strand sequence, position 8286–8304) and R10 (59 AGTGCATACCGCCAAAAGA 39, L strand sequence position 421–403). The standard PCR conditions were 1.5 mM MgCl2, 75 mM Tris–HCl (pH 9.0 at 25°C) 20 mM (NH4)2SO4, 0.01% (w:v) Tween 20, 50 pmol of each primer, 0.2 mM of each dNTP, 500 ng of total DNA and 2.5 IU of DNA polymerase (Goldstar, Eurogentec) in 50 µl reaction mixture. The reactions were performed using a minicycler (MJ Research, Watertown, MA, USA). The cycle time was as follows: denaturation 30 s at 95°C, annealing 30 s at 55°C, and primer extension 10 min at 72°C for 30 cycles. The reaction products were electrophoresed on a 0.8% agarose gel at 100 V for 30 min and stained with ethidium bromide.

Figure 1. Autoradiogram showing analysis of undigested mitochondrial DNA (lane 1: spermatozoa of a healthy donor; lane 2: spermatozoa from the patient; lane 3: muscle tissue from patient’s deltoid) and mitochondrial DNA digested with BamHI (lane 4: spermatozoa from a healthy donor; lane 5: spermatozoa from the patient; lane 6; muscle tissue from patient’s deltoid). CCSC 5 closed circular super coiled form; L 5 linear form.

Results Upon clinical examination of the patient, a ptosis, which is often associated with the presence of mitochondrial DNA disease, was noted. Histological examination of a muscle sample confirmed the possibility by the presence of RRF and some cox-negative fibres. Therefore, we searched for point mutations associated with MELAS (Goto et al., 1991), MERRF (Shoffner et al., 1990) and Leber’s disease. The results were all negative. Southern blot analysis of the mitochondrial DNA from the spermatozoa and from the deltoid muscle, shown in Figure 1, revealed a large number of linear molecules of 16.6 kb in the spermatozoa, as well as traces of molecules of lower molecular weight ranging down to 7 kb. Upon linearization with BamHI, which cleaves the mtDNA at position 14 258, most of the closed circular high molecular weight DNA molecules were transformed into a normal linear population of 16.6 kb, as well as abnormal higher and lower molecules down to ~7 kb (Figure 1, Lane 5). It is worth noting that the apparently normal sized mtDNA from the spermatozoa of the patient is 812

Figure 2. Autoradiogram showing the same amount of nuclear ribosomal RNA genes in the sample of the sperm of the healthy donor (lane 1) and of the patient (lane 2). M 5 molecular weight markers (λDNA digested with EcoRI and HindIII).

much more abundant, about 10-fold, than that of the control subject (compare lanes 1 and 2, and 4 and 5). The amount of DNA loaded on the gel was checked with a nuclear rRNA DNA probe, showing equivalent amounts of nuclear genes to

Oligoasthenospermy and mitochondrial DNA rearrangements

molecules spanning the region from the end of the D loop to the gene of CoxII (Nelson et al., 1989), confirmed the presence of smaller molecules ranging from 4 to 0.4 kb in size, as shown in Figure 3. Patient and control samples were analysed under the same conditions to exclude the possibility of contamination. These combined data strongly suggest the presence of multiple deletions in the large segment (between the heavy and light origins of replication) of the mtDNA of this patient, whose size may be estimated to vary up to 9 kb from the Southern blot, and from 8.3 to 4.7 kb from the long PCR experiment. These multiple deletions encompass those which are currently found in the majority of mitochondrial myopathies, namely cytochrome C oxidase, ATPase, NADH dehydrogenase, cytochrome b and up to nine tRNA genes (Figure 4), leading to impairment of the normal respiratory chain function. Figure 3. Ethidium bromide staining of long polymerase chain reaction (PCR) amplification products from the mitochondrial DNA of the spermatozoa of the patient (lane 1) and of a healthy donor (lane 3). Lane 2 is the amplification product of the patients’s deltoid muscle tissue DNA. M 5 molecular weight markers (λDNA digested by HindIII).

Figure 4. Schematic representation of mitochondrial DNA. D-Loop 5 displacement loop; ND 5 NADH dehydrogenase; CO 5 cytochrome C oxidase; ATPase 5 ATP synthetase; Cyt b 5 cytochrome b; OH and OL 5 heavy and light strand origins of replication. tRNA genes are in grey symbolized by a letter. The multiple DNA deletions are located between OH and OL. From the Southern blot (Figure 1), partial duplications cannot be excluded.

be present in these samples (Figure 2, Lanes 1 and 2). Taken together, these results indicate multiple deletions of the mtDNA in this patient, of an average size of 9 kb. Long PCR amplification of the mtDNA with primers D6 and R10, which are normally able to amplify linear 8.7 kb

Discussion Under normal conditions, spermatozoa require a large amount of energy for motility and subsequent fertilization. This may now be overcome by ICSI, which does not require mobile gametes for fertilization. Any paternal mutation of mtDNA would not be transmitted since it is maternally inherited (Giles et al., 1980). However, it has been recently shown that, in mice, one paternal mtDNA molecule may be detected among 10 000 maternal ones (Gyllensten et al., 1991), and that nuclear genes of the zygote, but not the mitochondrial DNA by itself, lead to that situation in mammals (Kaneda et al., 1995). Various mitochondrial myopathies have been found to be associated with mtDNA alterations associated with reduced fertility; Kao et al. (1995) observed a higher incidence of the common 4977 bp deletion in patients with primary infertility. However, Huang et al. (1994) found a patient with the MELAS mutation without altered sperm motility. Mitochondrial DNA provides the gene encoding the respiratory chain complexes. Cells such as spermatozoa which have a high rate of division require a lot of energy. These factors have two major implications: firstly, unlike other cells with rapid turnover, such as blood cells or cultivated fibroblasts, spermatozoa do not eliminate any rearrangement or mutation of their mitochondria. This may imply that mitochondria by themselves play a role in the differentiation of these cells. Secondly, Larsson et al. (1996) have recently reported the presence of a double exclusive localization (nuclear or mitochondrial) of the mitochondrial transcription factor activator (mtTFA) in the testis, which is the factor that triggers mtDNA transcription at the light strand promotor and hence initiation of replication (Dairaghi et al., 1995). These two phenomena may each have a bearing on the production of a functional mobile haploid cell. The most relevant point of our observation involves the possibility of transmitting a mitochondrial DNA myopathy by the father (although he was mildy affected) by ICSI, since at least three nuclear loci have been ascribed to multiple mtDNA deletions: 10 Q 23.3–24.3 (Suomalainen et al., 1995), 3p 14.1– 21.2 (Kaukonen et al., 1996) and 4p16 (Barrientos et al., 1996). In view of the increasing use of in-vitro fertilization 813

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(IVF), the importance of cytoplasmic inheritance (Willmut et al., 1997), and the critical role in development of nucleo– cytoplasmic interactions (Lestienne, 1989), the practice of IVF in humans should be re-evaluated, since epigenetics appears to play a major role during cell differentiation.

Acknowledgements We thank Dr M.F.Montfort for her contribution and the Association Franc¸aise pour la lutte contre les myopathies (AFM) for support.

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