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 941430720, USA
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Abstract |
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Key words: DNA repair/male infertility/meiosis/mismatch repair/nucleotide excision repair
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Introduction |
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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, 1979). 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., 1970
). 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., 1995; Wind et al., 1995
). 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, 1999
). Basebase 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, 1997
). In addition, genetic evidence also suggests that a pathway yet to be genetically dissected exists in yeast to repair DNA during meiosis (Kirkpatrick, 1999
). 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., 1998
).
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, 1982; Baker et al., 1995
, 1996
; Wind et al., 1995
; Edelmann et al., 1996
; Tishkoff et al., 1997
)? 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?
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Materials and methods |
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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 #H85341458602.
DNA preparation
Genomic blood DNA was prepared by salt precipitation from peripheral blood leukocytes as previously described (Gilbert, 1997). 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 0.30.6 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., 1988; Yu et al., 1996
). Reactions contained 10 mmol/l TrisHCl, 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, 1998).
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Results |
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Discussion |
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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, 1998). 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., 1995a
,b
). 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., 1996
; Jeffreys et al., 1998
) 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., 1999). Based on the observed mutation rate for Pfu polymerase (Andre et al., 1999
), 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, 1996). 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., 1998
). 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.
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Acknowledgments |
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Notes |
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References |
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Submitted on November 5, 1999; accepted on March 8, 2000.