Increased frequency of mutations in DNA from infertile men with meiotic arrest

David Nudell1,2, Michael Castillo1,2, Paul J. Turek2 and Renee Reijo Pera1,2,3,4

1 Department of Obstetrics, Gynecology and Reproductive Sciences, 2 Department of Urology and 3 Department of Physiology, University of California at San Francisco, San Francisco, CA 94143–0720, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In diverse organisms from yeast to mice, mutations in numerous genes required for DNA repair may lead to defects in meiosis. Although it is likely that meiosis is conserved throughout evolution, little is known about the genetics of meiosis in humans even though meiotic arrest associated with azoospermia is common. In this work, we compared the sequence fidelity of a polymorphic marker amplified from DNA of two groups of patients: those with testis biopsy suggesting meiotic arrest and those with normal spermatogenesis who were obstructed. We demonstrated that mutations are more common in DNA from testicular tissue derived from men with meiotic arrest than in DNA from testicular tissue derived from men with normal spermatogenesis and physical obstruction (P < 0.05). No mutations were observed in blood tissue from either group of men. This suggests the possibility that defects in genes required in DNA repair could contribute to meiotic arrest in men just as has been observed in other organisms.

Key words: DNA repair/male infertility/meiosis/mismatch repair/nucleotide excision repair


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nearly half of the causes of infertility in men is linked to faulty gametogenesis characterized as non-obstructed azoospermia or oligozoospermia (deKretser, 1997Go). These disorders could result from defects in crucial events in germ cell development. Recently, many genes required to complete meiosis in other organisms have been identified (Roeder, 1997Go). Some of these genes encode proteins that tightly regulate the recombination of genetic material and the repair of DNA should mutations occur during replication. In diverse organisms from yeast to mice, mutations in genes required for DNA repair lead to infertility characterized by arrest in meiosis I at a meiotic checkpoint (Baker et al., 1995Go, 1996Go; Roeder, 1997Go; Pittman et al., 1998Go; Yoshida et al., 1998Go).

Genetic evidence that infertility in humans may be caused by the arrest of germ cells in meiosis was first presented in the 1970s when Chaganti and German reported a family in which human male infertility appeared to be genetically determined (Chaganti and German, 1979Go). In this family, three out of eight brothers in one generation were azoospermic or oligozoospermic and four out of six brothers in the subsequent generation were infertile. Histological examination of biopsies of two of the infertile men indicated complete meiotic arrest in one man and nearly complete arrest in the other. Based on these observations, the authors suggested that infertility might run in this family due to mutations in genes that regulate meiotic progression. In another report, Pearson et al. observed a reduced ability to repair induced DNA damage in lymphocytes of an azoospermic man with desynaptic germ cells that progressed no further than meiosis (Pearson et al., 1970Go). Taken together, these observations suggest the possibility that human meiotic arrest could be genetic and possibly linked to mutations in genes required for the repair of DNA.

Later, when the homologues of two DNA repair genes, that were genetically mapped in HNPCC (hereditary non-polyposis colon cancer) patients were disrupted in mice, unexpectedly, meiotic arrest was found in the testes of all progenitor mice (Baker et al., 1995Go; Wind et al., 1995Go). The genes that were disrupted were required for mismatch repair. Other genes are also likely to be required for the repair of mismatched bases and the repair of double strand breaks during meiosis. Several pathways of DNA repair are required to complete meiosis in yeast (Kirkpatrick, 1999Go). Base–base mismatches and small loops are repaired by the MMR (mismatch repair) system. A second pathway repairs large loops and involves nucleotide excision repair (NER) and a number of genes have been identified that are required to repair double strand breaks (Roeder, 1997Go). In addition, genetic evidence also suggests that a pathway yet to be genetically dissected exists in yeast to repair DNA during meiosis (Kirkpatrick, 1999Go). The identity and the role of most of the genes likely to be required to repair DNA during mammalian spermatogenesis is not known since some of the genes are required for embryonic development and disruptions cannot be assessed for their role in meiosis. However, it is clear that many DNA-repair genes are upregulated during meiosis in mammals, and therefore may be required not only for embryonic development but also for meiosis (Tebbs et al., 1998Go).

The similarity of the testis histology of transgenic mice with mismatch repair mutations to that observed in infertile men with meiotic arrest has prompted us to ask: do defects in genes involved in DNA repair cause infertility due to meiotic arrest in men as they do in other organisms (Baker and Lindsley, 1982Go; Baker et al., 1995Go, 1996Go; Wind et al., 1995Go; Edelmann et al., 1996Go; Tishkoff et al., 1997Go)? If so, could defective DNA repair genes possibly be transmitted to the offspring of men who use assisted reproductive techniques such as ICSI (intracytoplasmic sperm injection)? In order to begin to address these questions we sequenced a polymorphic marker from testis and blood DNA obtained from both infertile men with meiotic arrest and infertile men with normal spermatogenesis who were obstructed. If defective DNA repair might underlie some cases of infertility characterized by meiotic arrest, then we expected to observe increased numbers of mutations in DNA sequences from testicular DNA from men with meiotic arrest as compared to men with normal spermatogenesis. Alternatively, if we observed the same frequency of mutations in testicular DNA from infertile men with meiotic arrest as compared to that of men with normal spermatogenesis, then we would have less reason to suspect that DNA repair might be defective in some infertile men. Here we report an increased frequency of one type of DNA sequence mutation, base pair substitutions, in sequences derived from testis DNA obtained from infertile men with meiotic arrest as compared to infertile men with normal spermatogenesis who are obstructed. This suggests that we should follow up these results to determine: are the mutations that we observed the primary cause of infertility in some men with meiotic arrest? If so, then what genes might be defective in these men?


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient selection
Patients were selected to compare the germ cell findings from a cohort of men with decreased spermatogenesis that might be more likely due to a genetic aetiology to that from men with normal spermatogenesis. For this reason, men with spermatogenic failure and a complete early meiotic arrest pattern at the primary spermatocyte stage were chosen. To ensure that study biopsies came from men with a global pattern of meiotic arrest, the spermatogenic defect observed histologically was confirmed with cytology findings obtained by systematic fine needle aspiration `mapping' of all testicles (Turek et al., 1997Go). Men with mixed histology or mixed cytology findings in different samples from the testis, suggestive of more than one pattern of spermatogenic failure, were excluded from the study with the exception of AZF17, who was found to have some spermatids in some spermatogenic tubules upon re-examination of the biopsy results.

Eleven men were selected in this study to provide germ cell and blood tissue. Men were diagnosed as azoospermic (no spermatozoa in the ejaculate) by standard semen analysis on two occasions and by pelleting of semen to detect very low numbers of spermatozoa. The control group contained five men diagnosed with reproductive tract ductal obstruction and normal spermatogenesis that was confirmed by histological and/or cytological assessment. The experimental group contained six men with abnormal spermatogenesis and histology that indicated meiotic arrest. The average FSH concentration was determined and then at the time that diagnostic testis biopsy was performed for therapeutic reasons, such as sperm retrieval for ICSI, a 50 mg specimen of testis tissue was submerged in liquid nitrogen and stored at –70°C until further analysis. The protocol for these studies was approved under IRB (Institutional Review Board) protocol #H8534–14586–02.

DNA preparation
Genomic blood DNA was prepared by salt precipitation from peripheral blood leukocytes as previously described (Gilbert, 1997Go). DNA was stored at –20°C in water containing 1 IU RNase per ml. DNA concentrations were obtained by spectroscopy at absorbance 260/280 nm. DNA was prepared from testis tissue by homogenization of up to 100 mg of tissue in 1 ml of TRIzol as per manufacturer's instructions (Gibco, BRL; Rockville, MD, USA) and resuspended in 0.3–0.6 ml of sterile water.

DNA amplification
The polymerase chain reaction (PCR) was used to amplify genomic DNA from testis or blood with primers that flank a polymorphic microsatellite (CA repeat containing) marker on chromosome 19q12, (marker number D19S49 Genome DataBase (GDB) accession number GDB:171162 at the internet address, http://www.gdb.org). These sequences were chosen based on previous reports indicating that this marker is highly polymorphic and can be amplified from small quantities of DNA (Li et al., 1988Go; Yu et al., 1996Go). Reactions contained 10 mmol/l Tris–HCl, 50 mmol/l KCl, 2.5 mmol/l MgCl2, 50 mmol/l of each dNTP, 0.2 µmol/l of each primer, 1 IU Pfu (Pyrococcus furiosis) polymerase, and 100 ng of DNA in a 100 µl reaction. Two rounds of amplification were performed: a first round with a pair of outside primers (5'-AAAGTGCAGGGATTAGAGGCGTGAGCTACC and 5'-GAAGGTGACAGTTCCTCAGGCCCACAGTAA) and a second round with two inside primers. (5'-GAAGGTGACAGTTCCTCAGGCCCACAGTAA and 5'-GCCCCTAGGGTTTTAGATTGAGTGTTGTTGACC). PCR conditions for the first round consisted of a single initial denaturation of 94°C for 4 min, followed by 11 cycles of 94°C for 45 s and 60°C for 3 min. Finally, 14 cycles of 94°C for 45 s and 60°C for 2 min preceded a second round of PCR using 2 µl of the first-round product as the template for 25 cycles (94°C, 45 s; 60°C, 1 min).

DNA cloning and sequencing
PCR samples were run on 1% agarose gels and products were excised from the gel. The agarose gel was digested with ß-agarase (New England Biolabs, Beverly, MA, USA) and the products in solution were cloned into the TOPO TA Cloning vector as per manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). Plasmids were transfected into Escherichia coli and bacterial colonies with inserts were identified. Plasmids were isolated using the R.E.A.L. Prep 96 Plasmid Kit (Qiagen, Inc., Valencia, CA, USA) and sequenced using an ABI 377 automated sequencer (Perkin Elmer, Inc., Foster City, CA, USA).

Sequence analysis
Plasmid clones were sequenced using fluorescently labelled M13 forward and M13 reverse primers (Perkin Elmer). Sequences were analysed only when both forward and reverse sequence was obtained to confirm that each nucleotide had been properly sequenced in both directions; the number of sequences analysed in all groups was 25. Sequences were aligned using the LaserGene SeqMan program (DNA-Star Inc., Madison, WI, USA). Normal allele length was examined by standard microsatellite genotyping on 6% acrylamide gels using 32P-labelled primers as outlined (Hudson, 1998Go).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Clinical characteristics
The clinical characteristics of the six study patients with meiotic arrest testis histology and the five control patients with normal spermatogenesis are shown in Table IGo. Note that, as expected, patients with arrested germ cells had a significantly higher FSH concentration (10.2 versus 3.1 mIU/ml) consistent with global spermatogenic failure. Similarly, patients considered to have normal spermatogenesis with obstruction had a significantly lower semen volume than those with meiotic arrest (1.3 versus 4.2 ml) consistent with obstruction as the primary cause for their infertility.


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Table I. Clinical parameters of infertile men in DNA repair study
 
All patients underwent either formal testis biopsy or testis cytology for confirmation of spermatogenesis. Representative testis morphologies from patients in this study are illustrated in Figure 1Go. Figure 1a and bGo show seminiferous tubules that exhibit normal spermatogenesis and meiotic arrest respectively in two patients. Figure 1cGo demonstrates the fine needle aspiration findings (cytology) in the same patient as in Figure 1bGo.



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Figure 1. Testicular histology of meiotic arrest. (a) testis morphology from a study patient with obstruction and normal spermatogenesis (arrows denote mature spermatids); (b) testis morphology from a study patient with meiotic arrest pattern of spermatogenesis. Arrows indicate primary spermatocytes; (c) representative view of human testis cytology from FNA (fine needle aspiration) mapping showing primary spermatocytes (arrows) as the most mature spermatogenic cell type. Note the similarity of the histology of the maturation arrest testis to that of a DNA-repair deficient mouse as shown previously (Baker et al., 1996Go). Bars represent 25 µm in length; staining was by haematoxylin and eosin (Dorfman et al., 1999Go).

 
Point mutations in testis DNA but not blood DNA in infertile men with meiotic arrest
DNA was prepared from blood and testis tissue from each man described in Table IGo. Note that the FSH concentrations shown are consistent with meiotic arrest in the azoospermic men who did not produce spermatozoa; the low semen volume in the control group may indicate obstruction and/or dysfunctional ejaculation. After PCR of marker D19S49, products were cloned and sequenced. Each clone corresponds to one copy of marker D19S49 from genomic DNA. The sequence analyses of D19S49 from two infertile men with meiotic arrest, AZF2 and AZF11, along with one man with normal spermatogenesis (AZFN1) are shown in Figure 2Go. For simplicity, only the region of the dinucleotide `CA' repeats from the polymorphic marker D19S49 is shown. The sequence analysis indicates that the most common mutation is A->G (68%) followed by C->T, C->G, A->T and A->C. Similar data were obtained by repeating the sequence analysis on another 30 clones from AZF2 and AZFN1 that were prepared independently. For comparison, blood DNA from all patients was also analysed. None of the clones obtained from blood DNA contained point mutations.



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Figure 2. Examples of mutations observed in infertile men with meiotic arrest (AZF2 and AZF11) and a single mutation observed in a man with normal spermatogenesis (AZFN1). The normal alleles are shown for each man at the top and the number of mutant sequences. Percentage of mutant sequences in each man is shown in Figure 3Go. Note that reduction of CA repeat number cannot be scored in this assay since the products are cloned and reduction in size may represent the stutter bands or truncated products arising from normal alleles.

 
The results of sequence analysis of the D19S49 marker in testis and blood DNA from patients with meiotic arrest and from patients with obstruction are summarized in Figure 3Go. As shown, three of six patients with meiotic arrest had significantly more mutations in testis DNA within the dinucleotide repeats. Point mutations were not found in the blood DNA of either group of men, those with meiotic arrest or those with obstruction. Taken together, these results suggest that the mutation frequency in testis tissue from infertile men with meiotic arrest is higher than the mutation frequency in testis tissue from obstructed men, 9 ± 3% of clones contained a mutation versus 1 ± 1 (mean ± SE) respectively. Note that these values are similar to those obtained when testis tissue enriched for germ cells from Mlh1 –/– mice were analysed for DNA repair defects. Whereas tissue from wildtype mice contained no mutations, 14% of PCR products from testis tissue from Mlh1 –/– mice had evidence of DNA repair defects in a polymorphic marker, D9Mit67 (Baker et al., 1996Go).



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Figure 3. The percentage of clones with point mutations in DNA cloned from the D19S49 marker of men with early MA (meiotic arrest), incomplete MA, or normal spermatogenesis. *This man produced some spermatids and underwent intracytoplasmic sperm injection (ICSI).

 
Defects in DNA repair may exist in men with incomplete meiotic arrest
The results shown in Figure 3Go prompted us to consider whether potential defects in DNA repair might also be found in men with incomplete meiotic arrest who produce spermatids in testis tissue. One such patient, AZF17, had spermatozoa extracted from a biopsy and underwent ICSI. Sequence analysis of the D19S49 marker in AZF17 DNA indicated that 25% of the cloned DNA from his testis tissue carried mutations similar to those shown in Figure 2Go. The couple underwent testicular sperm extraction and ICSI. Eight mature oocytes were collected from his 32 year old partner and five exhibited normal 2-pronuclear fertilization. Although viable embryos were produced, no pregnancy was achieved. This clinical scenario illustrated two points. First, potential DNA repair defects were not limited to those with complete meiotic arrest. Limited spermatogenesis could be completed despite potential DNA repair defects. Second and more importantly, some men undergoing ICSI may therefore harbour potential defects in the DNA repair pathways.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Use of sequence analysis to assay DNA repair in infertile men
In 1979, it was suggested (Chaganti and German, 1979Go) that meiotic defects in infertile men in one family might be caused by an autosomal dominant mutation in a gene required for DNA repair. Advances since that work have allowed us to test directly whether the fidelity of DNA repair is impaired in some infertile men by sequence analysis to detect mutations in a polymorphic microsatellite repeat, a short repeat of DNA that is prone to proof reading errors.

Defective DNA repair may lead to several types of mutations. Mutations in the genes PMS1, MLH1, MSH2, MSH3 and MSH5 result more often in deletion or insertion of nucleotides into CA repeats than in base pair substitutions, whereas mutations in the genes MSH6 or EXO1 most commonly lead to base pair substitutions. Usually, microsatellite DNA instability is examined using labelled PCR primers and polyacrylamide gel separation of amplified DNA from polymorphic markers (Hudson, 1998Go). By this method, several studies have demonstrated that expansions and contractions of microsatellite repeat lengths occur when DNA repair processes have gone awry. In humans, there is a direct link between microsatellite instability and tumorigenesis, especially in patients with HNPCC syndrome (Liu et al., 1995aGo,bGo). In addition to creating novel allele sizes, defective DNA repair can also lead to the introduction of more subtle mutations in microsatellite DNA (Edelmann et al., 1996Go; Jeffreys et al., 1998Go) just as we have observed in testis biopsy tissue. In contrast to observations on DNA repair in HNPCC, however, we do not expect the introduction of a mutation in a germ cell to lead to clonal expansion of that cell's progeny as would be expected from a tumour tissue arising from a single mutation. Instead, we expect that the introduction of mutations in germ cells may lead to meiotic arrest as it does in other organisms. Experimentally, this means that in order to detect defects in DNA repair in testis tissue, the DNA sequence from microsatellites must be analysed individually. Thus, we did not analyse DNA from bulk populations of cells by gel electrophoresis. Instead, we analysed sequences from single clones. We found that the mutations we observed in men with meiotic arrest are consistent with potential defects in either MMR or NER.

Evidence that mutations arise in germ cells
Our study relied on testis biopsy material which consisted of a pooled sample of cells containing both germ cell precursors and somatic support cells. In order to reduce the risk that observed mutations came from somatic cells, we compared mutations found in testis tissue to those found in blood. We expect that mitotically active blood tissue should approximate any background level of mutations contributed by the non-germ cell components within testis tissue. In contrast to our findings with testis tissue, we found no mutations in any of the cloned blood DNA samples from either meiotic arrest or normal men in the study.

Given that we used a testis tissue sample that also contained somatic cells, we elected to amplify and analyse several clones in order to represent the diverse cell population present in any given biopsy. This allows the possibility that mutations are introduced during the experimental procedure. To maximize the likelihood that the mutations observed were germ cell mutations rather than mutations introduced by the experiment, we took two precautions. First, we used Pfu polymerase for amplification of D19S49. Pfu is known to have the most efficient proof reading capability of any polymerase suitable for PCR (Andre et al., 1999Go). Based on the observed mutation rate for Pfu polymerase (Andre et al., 1999Go), we do not expect to observe any mutations in our entire study. Indeed, we observed no mutations in blood DNA. In contrast we observed mutations in testis DNA from some men with meiotic arrest in as many as 25% of the clones. Clearly some men with meiotic arrest have greater numbers of sequence mutations than expected. However, it is important to note that two men, AZF 6 and AZF19, did not have mutations even though they had a similar histology to other men with mutations. Second, we compared cloned samples from testis biopsies from meiotic arrest patients to those from patients with normal spermatogenesis that were prepared, amplified, cloned and analysed in an identical manner. Any background level of mutations introduced by experimental techniques should have been evident in both the normal patient testis DNA and in blood DNA from either group of patients.

Does the increase in sequence mutations suggest that DNA repair is defective?
Obviously, we must address whether the apparent DNA repair defects caused meiotic arrest in those men who show increased number of sequence mutations or are merely a consequence of meiotic arrest due to induction of unknown factors in the testicular microenvironment of men with meiotic arrest. We know that in other organisms, such as mice, deficiencies in some DNA repair enzymes cause meiotic arrest but only when the mice are homozygous for null mutations. Thus, if we are to extend these findings to humans, we expect that if in humans meiotic arrest is caused by DNA repair deficiencies associated with mutations in genes such as PMS1 or MLH1, then some men are mosaic for mutations in these genes. Perhaps, they have one mutant copy in all cells and sustain a second mutation in an early population of spermatogonia. Alternatively, defective DNA repair could be associated with genes that are only required to repair DNA at meiosis and may be inherited in an autosomal or X-linked dominant or recessive manner. To address these issues, the inheritance of infertility in those families with men showing evidence of defective DNA repair should be assessed.

What are the consequences of defective DNA repair to children conceived by ICSI?
Can we expand these results to develop tests to predict the consequences of defective DNA repair to the outcome of pregnancies and/or to the health of children conceived via assisted reproductive techniques such as ICSI? At this time, it is not clear what the consequences of potentially defective DNA repair in germ cells might be. In part, this is because we know that DNA repair defects can arise from mutations in genes required for both germline and somatic cell repair or in genes required only for germline repair (Kleckner, 1996Go). Thus risks associated with DNA repair defects could differ between a child conceived and his or her infertile father. In part, our limited ability to predict the consequences of defective DNA repair is also illustrated by a recent demonstration that defective DNA repair is commonly found in spontaneously aborted embryos (Spandidos et al., 1998Go). This suggests that perhaps potential DNA repair defects will not be translated into the birth of viable offspring. Alternatively, however, children conceived via ICSI with spermatozoa carrying potential DNA defects may survive to term. If so, depending on the genetic basis for potential DNA repair defects, they may incur an increased risk of infertility and possibly increased risks of somatic defects later in life. At this time, it is not clear which of these scenarios will be observed. In any case, these observations suggest the need for increased analysis of family history of infertility. In addition, perhaps increased testing of infertile fathers to determine the role of defective DNA repair in infertility and longer follow-up of children born through ICSI are warranted.


    Acknowledgments
 
We thank Lauri Black for co-ordinating patient information and acquisition of samples, Douglas Lee, Leslie Woo and Dana Kostiner for preparation of DNA samples, and Victoria Carleton for assistance with sequencing. This study was supported by grants from the National Institutes of Health (R.R.P.), the University of California Campus Laboratory Collaboration (P.J.T., R.R.P.), UCSF-Stanford Healthcare (P.J.T.) and the California Urological Foundation (D.N.). D.N. was a recipient of a Bank of America – Giannini Foundation fellowship and R.R.P. was the recipient of Searle Scholar and Sandler Family Foundation Awards.


    Notes
 
4 To whom correspondence should be addressed at: Department of Obstetrics, Gynecology and Reproductive Sciences, University of California at San Francisco, San Francisco, CA 94143–0720, USA.E-mail: reijo{at}itsa.ucsf.edu Back


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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on November 5, 1999; accepted on March 8, 2000.