Human Reproduction Update Advance Access originally published online on January 25, 2006
Human Reproduction Update 2006 12(3):293-301; doi:10.1093/humupd/dmk004
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N-ethyl-N-nitrosourea (ENU) mutagenesis and male fertility research
The Centre for Reproduction and Development, Monash Institute of Medical Research and the ARC Centre of Excellence in Biotechnology and Development, Monash University, Melbourne, Australia
1 To whom correspondence should be addressed at: The Centre for Reproduction and Development and The ARC Centre of Excellence in Biotechnology and Development, Monash Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton 3168, Victoria, Australia. E-mail: moira.obryan{at}med.monash.edu.au
Submitted on October 3, 2005; resubmitted on December 8, 2005; accepted on December 20, 2005
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Abstract |
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 TOP Abstract N-ethyl-N-nitrosourea...Screening ENU-mutated mice for...ENU mutagenesis: a work...Clinical applications of ENU...ConclusionsReferences |
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Male infertility affects about 1 in 25 men in the western world. Conversely, there is an urgent requirement for additional male-based contraceptives, yet progress in both areas has been severely hampered by a lack of knowledge of the biochemistry and physiology of male reproductive function. It is only through a thorough knowledge of these processes that we can hope to insightfully regulate male reproductive function. Without doubt, mouse models will form an important foundation in any future process. In recent years, the chemical mutagen N-ethyl-N-nitrosourea (ENU) has been used widely to identify genes essential for a range of biological systems including male infertility. These studies have shown random mutagenesis is an attractive means of identifying key genes for male fertility. This technique has distinct, but complementary advantages compared to knockout technologies. Specifically, it allows the removal of researcher bias whereby only pre-conceived genes are tested for function; it produces mice with a guaranteed phenotype and allows for the production of allelic series of mice to dissect all aspects of gene function. ENU mouse mutagenesis programs will enable advances in the diagnosis and treatment of human male infertility and ultimately aid in the development of novel male-based contraceptives.
Key words: ENU mutagenesis / infertility / spermatogenesis / testis
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N-ethyl-N-nitrosourea mutagenesis |
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TOPAbstractN-ethyl-N-nitrosourea...Screening ENU-mutated mice for...ENU mutagenesis: a work...Clinical applications of ENU...ConclusionsReferences |
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Due to the similarity between the mouse and human genomes, their developmental and biochemical pathways and physiology, the mouse has become the major model system to study the genetics and pathogenesis of human disease. Although the current mouse mutant database, which is composed largely of knockout animals, has been a valuable source of models, only a small proportion of the likely total number of mammalian genes is represented. This gap is being partially filled by the establishment of new mutagenesis programs using potent mammalian mutagens and carcinogens such as N-ethyl-N-nitrosourea (ENU).
In recent years, several laboratories have utilized ENU mutagenesis to identify novel genes that are involved in human disease. With the development of genomic technologies that facilitate mapping and positional cloning of mutations, there has been a surge of re-interest in random mutagenesis programs including those using ENU (Beier, 2000
). ENU mutagenesis followed by linkage analysis provides a non-biased, phenotype-driven (or forward genetic) approach as opposed to a genotype-driven (reverse genetic) approach, whereby a favoured gene is altered using knockout or transgenic technologies. ENU is an ideal mutagen for phenotype-driven studies due to the high rate of induction of point mutations it produces, which potentially lead to a wide range of disease phenotypes (Balling, 2001). The single base pair alterations induced by ENU can be invaluable for dissecting the fine structure of a protein. The advantages of using phenotype-driven mutagenesis include speed of generation, a guaranteed phenotype and the removal of investigator bias (Justice, 2000b). The disadvantage of forward genetic approaches, such as ENU mutagenesis, is that setup costs are usually large because of the animal housing costs involved and that research does not follow the hypothesis-driven paradigm favoured by some granting agencies. Similar work to that described herein has been done using ethylmethanesulphonate (EMS) on cultured embryonic stem cells (Munroe et al., 2000) or random transgene insertion (Overbeek et al., 2001). This review will, however, focus on work using ENU.
ENU is a laboratory-synthesized compound that causes random, single base pair mutations, acting directly on nucleic acids without any metabolic processing required for its activation (Singer and Dosanjh, 1990; Justice, 2000a; Noveroske et al., 2000). It is a yellowish-pink crystal that is highly sensitive to humidity, pH and light and has a molecular weight of 117.1 (Justice, 2000a). ENU is an alkylating agent that can transfer its ethyl group to oxygen or nitrogen radicals in DNA which, if not repaired, results in mis-pairing and base substitution (Justice et al., 1999). There are numerous potential alkylation reaction sites (nucleophilic sites) that have been identified for all four nucleotide bases, though not all of these have equal reactivity (Friedberg et al., 1995). ENU predominantly induces point mutations, although a few small deletions or insertions have been reported. The most common mutations are AT to TA transversions and AT to GC transitions (>82% of sequenced mutations) (Popp et al., 1983; Noveroske et al., 2000). As such, there are a number of factors that affect the rate of mutation in any one gene. As ENU preferentially alters AT base pairs, GC-rich genes will not be mutated as often as those with a lower GC content. Similarly, the size of the gene will also affect the rate at which it will be mutated i.e. larger genes will provide a larger target for random mutagenesis than a smaller one (Russell et al., 1979; Noveroske et al., 2000). Also, the size of the functionally important domains within a protein will also affect the rate at which functional mutations (those affecting protein function and producing a phenotype) are generated.
ENU is considered to be the most effective chemical mutagen on mice, with a mutation rate of 0.0015 per locus per gamete in commonly used treatment regimes (Rinchik, 1991). Functionally, what this means is that screening a 1000 gametes should theoretically identify a mutation at any given locus (Hitotsumachi et al., 1985; Rinchik, 1991; Nolan et al., 2000b). Because ENU predominantly causes point mutations, the effect on a gene is to produce many different types of alleles (known as an allelic series). Each may have a different effect on the protein product (Table I). The generation of allelic series of a particular gene can enable the identification of the critical domains and residues within a protein.
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ENU is primarily used as an in vivo mutagen of the male germ line. To initiate a screen, male mice are injected intraperitoneally with ENU to cause random mutations that affect the spermatogonial stem cells. Spermatogonia experience the highest rate of mutation of any cell type examined (Russell et al., 1979
). Much lower rates of mutation have been seen in female germ cells (Russell and Russell, 1992). Following treatment with ENU, male mice usually undergo a sterile period due to a temporary depletion of spermatogonial stem cells. Treated mice also become particularly susceptible to pathogens as a consequence of a reduction in other stem cell types including hematopoietic stem cells (Weber et al., 2000). As such, ENU screens are usually carried out in pathogen-free animal houses in an effort to minimize loss from infections. The susceptibility of different strains to ENU toxicity varies, but for most of the commonly used laboratory strains this is now well characterized (Justice et al., 2000). The mutagenized spermatogonia that survive the treatment repopulate the testis, undergo mitosis and meiosis in the seminiferous tubules and eventually give rise to clones of mutagenized sperm that can be passed on to future generations (Justice et al., 2000). Not surprisingly, ENU is also a carcinogen at the doses used. As a result, many mice succumb to different types of cancer, shortening the life span of the treated mice, with treated males living an average of 1 year rather than the expected 18 months to 2 years (Justice et al., 2000). The treatment protocol must therefore stay within a narrow range of dose and exposure times to achieve the optimal balance between a high mutation frequency, the re-establishment of fertility and animal loss from infection or cancer (Balling, 2001).
Following an initial sterile period, breeding programs specifically designed to identify dominant or recessive mutations will produce mice displaying a range of abnormalities. Any individual ENU-treated mouse (G0) will carry many different mutations that are simultaneously induced i.e. silent, loss-of-function, gain-of-function (Table I). Most of the functional gene defects that are induced by ENU will be loss-of-function variants including the complete loss of a protein or the deletion/inactivation of a critical domain that renders the protein non-functional. Such mutations behave as recessive traits and are only revealed after breeding to homozygosity or in mice carrying large deletions or chromosomal inversions (Justice et al., 1997; Kile et al., 2003). There are now several large-scale ENU projects worldwide that focus on different biologically or clinically relevant phenotypes (Balling, 2001; Nelms and Goodnow, 2001; Kile et al., 2003; Inoue et al., 2004; Reinholdt et al., 2004). All of these screens will be producing models relevant to all forms of human pathology including male infertility. The challenge remains for scientists to firstly look for these models, then to map the causal mutation and finally to relate the phenotype and gene alterations to human fertility. By accessing established programs, researchers will avoid many of the regime problems outlined above and will add value to the enormous investment used to set such programs up.
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Screening ENU-mutated mice for male infertility phenotypes |
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TOPAbstractN-ethyl-N-nitrosourea...Screening ENU-mutated mice for...ENU mutagenesis: a work...Clinical applications of ENU...ConclusionsReferences |
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Forward genetics (phenotype to genotype) screens for dominant mutations
Logistically, a dominant screen is the most simple to perform, as only one generation is required before a phenotype should be observed. For dominant screens, the ENU-treated male is mated to a wild-type female and the resulting offspring (the G1s) are examined for a phenotype. As the treated male will carry several dominant mutations, his offspring may display a variety of phenotypes with each G1 animal heterozygous for a number of unique induced mutations. This breeding scheme provides rapid genome-wide coverage for dominant mutations, with published figures indicating that up to 2% of progeny will carry a heritable mutant phenotype for conditions such as deafness, immunological or behavioural phenotypes (de Angelis et al., 2000; Nolan et al., 2000a). To identify the causal gene, the mutation must be bred onto a genetically unrelated mouse strain to enable mapping, this is known as outbreeding. Obviously this type of screen for dominant mutations is usually not suitable for male fertility phenotypes. The possibility remains, but is yet to be realized, that dominant mutations affecting male-specific genes could be identified via transmission through the female germ line from female G1 mice.
Forward genetics screens for Y-linked mutations
Similar to screens for dominant mutations, Y-linked mutations critically affecting fertility are not feasible as the affected males would not be able to sire offspring.
Forward genetics screens for recessive (including X-linked) mutations
Most of the functional gene defects that are induced by ENU will be loss-of-function variants behaving as recessive traits that can only be identified after breeding to homozygosity (Justice et al., 1999). This is the most complex and logistically exhaustive ENU-based screen, requiring three generations of crosses and large numbers of animals (Figure 1). Depending on the strain of mouse and dose of ENU used, the number of induced mutations per spermatogonia will vary. The G1 progeny (produced by mating a wild-type female to the ENU-treated male) pass on mutant alleles to the heterozygous G2 generation. The mutations are bred to homozygosity by inter- or backcrossing the G2 generation to produce the G3 offspring that will be screened for abnormal phenotypes (Figure 1). The breeding scheme described above enables the identification of only autosomal mutations. To allow for the possibility of including X-chromosome mutations, G1 male can be mated to an unrelated G1 female. This enables the transmission of X-linked mutations through the female germline. Such mutations can be identified in males at the G3 stage (Figure 1) and will affect 50% of males within a carrier pedigree, where a pedigree is defined as a lineage/family arising from any particular G1 mouse.
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The Monash/Australian Phenomics Facility (The Australian National University) screen used three to four injections, 1 week apart, of 85–100 mg/kg ENU, we expect each G0 ENU-treated male to carry approximately 60 loss-of-function mutations. Approximately 30 of these will be passed on to his G1 offspring. With the assumption that each G1 male (or pedigree founder) carries 30 loss-of-function mutations, the resulting G3 offspring will be homozygous for three to four different loss-of-function mutations. In addition, all G3 mice will be heterozygous for several other mutations. To screen a large fraction of genes in the genome for their effect on a particular biological process, hundreds of pedigrees must be examined for a phenotype of interest.
If within a pedigree, affected G3 animals appear in a Mendelian frequency (one in four showing a phenotype), it is a good indication that the phenotype is heritable and recessive. Recurring frequencies <1 in 4 are indicative of a phenotype caused by the effects of multiple gene mutations (a compound phenotype) (Russ et al., 2002). For mice produced using strategies similar to those outlined in Figure 1, it should be remembered that 2/3 of the remaining apparently normal/fertile G3 mice will be heterozygous and 1/3 will be genetically wild type. The identification of heterozygous siblings or the retention of the heterozygous father is the key to successful linkage analysis and causal mutated gene identification (see below).
In addition to the standard screen using wild-type background mice described above, it is also possible to carry out screens on a ‘sensitized’ background. Such mice often carry transgenes, or a pre-defined mutation, pre-disposing them to a particular type of pathology. This has not as yet been published for any fertility-related pathology but has been very productive for complex traits such as epigenetic regulation using agouti viable yellow mice (Blewitt et al., 2005) and cancer (Shima et al., 2003).
ENU screens for infertility mutants currently underway
A handful of laboratories worldwide are now utilizing ENU to identify mutants that display male (and female) infertility. These large-scale screens have shown ENU to be highly effective for the production of lines with disruptions at different stages of male fertility. Monash University and the Australian Phenomics Facility (The Australian National University) are undertaking genome-wide screens to identify recessive mutations affecting male infertility (Kennedy et al., in press). This screen utilizes a number of parameters to assess testicular function including testicular and epididymal histology, blood serum hormone analysis for key fertility-related hormones and immunological status and more recently breeding experiments (Kennedy and O’Bryan, personal communication). Further, sperm have been cryopreserved from ENU mutant lines to facilitate rapid regeneration of mouse lines of interest (Kennedy et al., in press).
A large scale, and significantly more developed screen, is currently underway at the Jackson Laboratories, where infertile mouse lines are identified through natural mating to wild-type animals (Lessard et al., 2004). When no offspring are produced the male and female mice are put into an ‘infertility clinic’ through which the block in fertility is identified and analysed (Ward et al., 2003; Lessard et al., 2004; Reinholdt et al., 2004). Some mouse line phenotypes identified in this screen include abnormal spermatogonia, meiosis arrest and abnormal sperm morphology. This list of phenotypes is continually updated and can be accessed through http://reprogenomics.jax.org/index.html. Groups with particular interest in a phenotype can contact the Jackson Laboratories directly. Assistance with linkage is also provided.
A group at the Department of Molecular and Human Genetics, Baylor College of Medicine in Texas is using a different approach to the three generations and outbreeding strategy described in Figure 1. Theirs is a more rapid screen for recessive mutations using mice with chromosome deletions (Figure 2A). Mice have been engineered to have a small region completely deleted from a chromosome. Following the breeding of the ENU-treated males to females homozygous for a dominant coat colour marker, the G1 founders are mated to mice carrying the specific chromosomal deletion and marked with a different coat colour marker. Four different classes of offspring are produced that can be distinguished by coat colour, from which it can be determined which will be useful (Figure 2) (Rinchik et al., 1990; Rinchik and Carpenter, 1999). The resultant G2 mice can be screened for infertility (or any other phenotype).
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A similar but alternative approach is the use of mice with chromosome inversions in which mice with a balanced inversion are used. In ‘balancer’ mice, a chromosome segment is removed, flipped and re-inserted (Figure 2B). The chromosome is also marked with a coat colour marker and is lethal in the homozygous state. The ENU-treated male is mated to mice carrying an inversion. The G1 founders are then crossed with mice that are heterozygous for the balancer and a different coat marker that allows selection of mice at the G2 stage. Only mice known to be carrying the balancer and the mutagenized are inter-crossed to produce three classes of G2 mice that are readily distinguishable based on coat type (Figure 2B). One class will carry two copies of the inversion and will have died, the other two classes will form the carrier class and the ‘test’ class that can be screened for new phenotypes (Figure 2B) (Brown and Balling, 2001). An ENU screen using a 24cM inversion on chromosome 11 has identified, among other abnormalities, three independent mutations affecting male fertility (Kile et al., 2003; Clark et al., 2004). Each independent mutation affected epididymal sperm morphology with distinct and different defects in sperm and/or tail morphology for each line. In addition to the 88 genes that this group mapped to the inverted region on chromosome 11, a further 142 recessive mutant phenotypes were found that were not linked to the inverted region (Pask et al., 2005). One of the non-linked mutations was subsequently identified as being within the GnRH receptor gene and resulted in hypogonadotrophic hypogonadism (Pask et al., 2005).
For screens using balancer chromosomes or chromosome deletions, only mutations occurring within the inverted or deleted regions can be rapidly defined. These strategies offer the advantage that the mutation is immediately mapped to a relatively small pre-defined region of the genome. Specific mutations are usually identified by the sequencing of candidate genes from within the region. This strategy is currently limited by the poor, but improving, availability of chromosomal deletions (Russ et al., 2002).
Mapping the phenotype causing mutation
Once a phenotype of interest had been identified the next step is to localize the causal mutation. To do this, a known carrier of the mutation needs to be outbred to a wild-type mouse of a different genetically inbred strain; e.g. if the mutation was originally identified on a C57Bl/6J background, it could then be outbred to a NOD background. With each generation of subsequent breeding, the chromosomes become more and more of a mix of the two strains (e.g. C57Bl/6J and NOD) along the length of the chromosome due to the requirement that each chromosome cross-over at least once during meiosis (Figure 3A) (Cobb and Handel, 1998; Pittman and Schimenti, 1998). All outbred mice displaying the abnormal phenotype will carry the causal mutation embedded within a region of chromosome derived from the original mutant strain i.e. C57Bl/6J in the example described in Figure 3. The region carrying the causal mutation can be identified using a number of mapping techniques (see below and Figure 3). A large number of mice are, however, usually required to map the mutation to a manageable chromosomal region e.g. a 10cM chromosomal region, which in the mouse can contain 
300 genes. It is of particular note that as the phenotype of interest is infertility, homozygous males cannot be used for breeding. Generating enough ‘affected’ animals can take considerable time. In some instances, homozygous females could be used for breeding and will produce 50% affected and 50% normal offspring.
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An alternative to using ‘pure breed’ mice for the screening, then outbreeding onto a second mouse strain (Figure 1), is to start the outbreeding at the G0 stage. This involves breeding the ENU-treated male to a wild-type mouse of a different mouse strain rather than a wild-type mouse of the same strain. For male infertility phenotypes, this should save two generations of breeding compared to the strategy described in Figure 1. This approach can, however, be complicated by modifier genes in the second strain leading to phenotype variation.
Microsatellite mapping
Microsatellites are simple, tandemly repeated di- to tetra-nucleotide sequence motifs flanked by unique sequences. They are valuable as genetic markers, because they are easily and economically assayed by the PCR (McCouch et al., 1997). By selecting markers that differ in length between two mouse strains, a simple PCR amplification can be done to identify the strain origin of a particular chromosome region within a mixed background mouse (Figure 3B). Markers that differ between the two mouse strains (e.g. between C57Bl/6J and NOD) can be found using databases such as the Jackson Laboratories Mouse Genome Informatics website (http://www.informatics.jax.org) or the Entrez SNP database (http://www.ncbi.nlm.nih.gov/entrez).
By scanning the entire autosome using PCR amplification of selected microsatellites that differ in length between the two mouse strains used and comparing mice displaying the abnormal phenotype to unaffected sibling controls, a bias should be seen towards chromosomal regions of the original mouse strain e.g. C57Bl/6J in the example shown in Figure 3. The mutation will be contained within one of the regions showing a strong bias to the original mutation strain. By identifying a large chromosomal region initially, then refining the linkage region using more closely spaced markers followed by the sequencing of candidate genes, the causal gene can be identified. In reality, researchers often map the region to an approximately 10 cM region, then with the aide of databases such as the NCBI mouse genome resources (http://www.ncbi.nlm.nih.gov/genome/guide/mouse/) will select candidate genes for sequencing. Factors to consider when selecting candidate genes include tissue expression, known biochemistry related to the affected cell/process and the existence of other mouse models with a similar phenotype.
Single-nucleotide polymorphism mapping
Similar to mapping using microsatellite markers, single-nucleotide polymorphisms (SNPs) can be used to identify the chromosomal region in which the ENU-induced mutation resides. SNPs are a sequence polymorphism that differs in a single base pair between mouse strains or individuals. SNPS can be identified using a number of techniques, such as denaturing high-performance liquid chromatography (DHPLC) (Kwok and Chen, 2003), matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF) (Jurinke et al., 2004), Taqman genotyping (Shinohara et al., 1997) and single-nucleotide primer extension (SNuPE) (Rautanen et al., 2005). By analysing SNPs distributed throughout the entire genome that differ between the original mutated mouse strain and the outbreding mouse strain, as with the above technique, a chromosomal region can be identified that shows a strong bias to SNPs from the original mutated mouse strain. Additional SNP mapping to further narrow down this region can then be done using more closely spaced polymorphisms, followed by the sequencing of candidate genes.
SNP genotyping has the advantage over microsatellite markers as there are usually many more SNP variations between strains than there are microsatellite length differences. Their disadvantage is however, that their analysis usually requires expensive and specialized equipment, as opposed to microsatellite markers that require only PCR. These two techniques are often used in combination, beginning with microsatellite mapping to crudely link the chromosomal region then further narrowing of the region using SNPs.
Reverse genetics (genotype to phenotype) screens for ENU mutations
Once established on a large scale, ENU mutagenesis also provides an alternative to gene knockout/knockin technologies and can be very useful in deducing a genes full function through the generation of an allelic series of mice. As outlined in Table I, there are many effects a single point mutation caused by ENU can have on the gene product. To determine the function of a particular gene, G1 (or G2) mouse DNA can be screened for mutations within a gene of interest using a range of mutation detection techniques including denaturing DHPLC, direct sequencing and chemical cleavage. With the high mutation load in the G1 mice, this approach is feasible and may produce mice with phenotypes more reflective of human disease than traditional knockouts i.e. point mutations. Several different mutations within the same gene should be found if a sufficiently large number of G1 mice can be obtained. For example, assuming there are 30 functional mutations within a G1 mouse and there are approximately 30 000 genes within the genome (Claverie, 2001), screening 1000 mice should on average identify one mutation in every gene. As previously mentioned, an allelic series of mutations is extremely valuable in understanding the function of a gene. One such successful example using ENU was the generation of an allelic series of mice for the Kit ligand (KitL) by Rajaraman et al. (2002a,b). Seven new ENU-induced mutations were identified and analysed in combination with two previously known mutant lines. Five of the lines carried mis-sense mutations, one carried a nonsense mutation and resulted in exon skipping and the other affected a splice site. Each of these mutations provided important information on the structural requirements for the function of KitL and its function in several processes, including male fertility (Rajaraman et al., 2002a,b).
Both Ingenium Pharmaceuticals (Augustin et al., 2005) and the RIKEN Genomic Sciences Center (Sakuraba et al., 2005) have used this reverse genetics strategy and now make it available as a commercial service. They have generated large numbers of ENU-mutant G1 mice and archived both sperm and DNA to enable the rapid identification and regeneration of mouse lines of interest. The RIKEN group have screened 9224 G1 mice for 63 target loci and have identified 148 ENU-induced mutations within this region using temperature gradient capillary electrophoresis followed by direct sequencing (Sakuraba et al., 2005). Ingenuim Pharmaceuticals have estimated that their archive of over 17,000 mice contains approximately 340,000 independent alleles and estimate a 99% success rate in the discovery of five allelic variants for any given average-sized gene (Augustin et al., 2005). Large archives such as these two provide a complementary and cost-competitive alternative to gene-knockout technologies and hold the potential to generate allelic series for the dissection of gene function.
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ENU mutagenesis: a work in progress |
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TOPAbstractN-ethyl-N-nitrosourea...Screening ENU-mutated mice for...ENU mutagenesis: a work...Clinical applications of ENU...ConclusionsReferences |
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Although ENU mutagenesis programs worldwide have been effective in identifying interesting mutants followed by the successful mapping of the causal gene, it must be noted that this process requires large colony numbers and personnel. Additionally, the identification of these mutated genes can be somewhat challenging. These large-scale screens are capable of producing many mutant mouse lines, of which it would not be possible for interested investigators to follow up on every single one at the time of identification. This highlights the importance of an efficient and effective sperm archiving system by which mouse lines of interest can be generated at a later date. Such archiving will ensure that these identified mutants will produce many results in the years to come. Additionally, researchers must be encouraged to use established programs. This will not only increase publication output but will drive down the per mouse costs for the whole project.
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Clinical applications of ENU mutagenesis: male infertility and the development of new male-based contraceptives |
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TOPAbstractN-ethyl-N-nitrosourea...Screening ENU-mutated mice for...ENU mutagenesis: a work...Clinical applications of ENU...ConclusionsReferences |
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About 10–15% of couples have difficulty falling pregnant, and for approximately half of these cases the basis of the infertility is with the male (Holden et al., 2005
). The majority of infertile men demonstrate a defect in sperm production and, for most, the cause of their abnormal semen patterns are unknown (idiopathic infertility) (de Kretser and Baker, 1999). Increasingly, it is recognized that the aetiology of the infertility will be genetic (McLachlan et al., 1998). In addition to the knowledge that can be gained from the identification of new genes involved in male fertility, identification of new genes that cause infertility when mutant may aid in the development and treatment of male infertility and also the development of new male-based contraceptives.
Studies into the mechanisms of spermatogenesis have uncovered many genetic determinants for male infertility. It has been estimated that there are >2000 genes involved in specific male development and reproduction (Hargreave, 2000; Hochstenbach and Hackstein, 2000) and it could be assumed that mutations within a large number of these genes would result in abnormalities. Over 150 different genetically modified mouse lines have shown abnormalities in male fertility, with more being published weekly (O’Bryan and de Kretser, in press). The establishment of male fertility is long and complex, beginning with germ-cell colonization, mitosis, meiosis, the transformation of round haploid germ cells into highly polarized sperm (spermiogenesis), epididymal maturation, capacitation and culminating in fertilization (O’Bryan and de Kretser, in press). Disruptions at any of these stages can result in infertility.
With the exception of vasectomy and condoms, there are currently no male-based contraceptives that are widely available. Of the different experimental methods of male contraception, a hormonal approach is the closest to being implemented with the pharmaceutical companies, Schering and Organon, recently beginning clinical trials using testosterone and progestin which temporarily suppress the hypothalamus–hypophyseal axis that stimulates sperm production (Mandavilli, 2004). As a consequence of the suppression of testicular steroidogenesis, hormonal approaches can have a variety of side effects, [reviewed in (Lyttle and Kopf, 2003)].
An immunological approach has also been employed in the development of male-based contraceptives using sperm-specific antigens which lead to the development of circulating or reproductive tract anti-sperm antibodies. The criteria for selection of a suitable antigen are important and include the localization of the antigen on the sperm surface (or oocyte), and the ability of bound antibodies to interfere with function e.g. motility or zona pellucida binding [reviewed in Lyttle and Kopf, (2003)]. The efficiency of this immunological approach in humans has been varied (Moudgal et al., 1997). There have been a wide range of undesirable side effects reported and efficacy appears to be significantly influenced by racial origin. This approach is now widely considered as unsuitable for humans (Primakoff et al., 1997; Tung et al., 1997; Anderson and Baird, 2002), although it shows promise for several pest animal species including wild mice (Singleton et al., 2002), the grey squirrel (Lurz et al., 2002), rabbits and foxes (Seamark, 2001).
Most recently, researchers have focused on identifying targets for non-hormonal contraception so as to take advantage of the cellular and physiological processes unique to the reproductive organs [reviewed in Lyttle and Kopf, (2003)]. The main goal of this approach is to interfere in a highly specific manner in key processes involved in spermatogenesis, epididymal sperm maturation or sperm function. This approach requires the identification of many unknown genes. With the exception of the spermatogonial stem cells, the disruption of germ-cell function/survival is attractive as upon removal of the treatment, spermatogenesis would presumably resume and fertility would be restored. Theoretically at least, this goal is achievable because of the high number of male reproductive tract-specific genes or isoforms of somatic genes. Clinically, we are yet to see the outcomes of this approach.
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Conclusions |
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TOPAbstractN-ethyl-N-nitrosourea...Screening ENU-mutated mice for...ENU mutagenesis: a work...Clinical applications of ENU...ConclusionsReferences |
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ENU mutagenesis has already been proven by many laboratories as an efficient and successful way to produce mice displaying any phenotype of interest, including abnormal male fertility. Additionally, with advances being made in mapping techniques and the continued identification of markers (particularly SNPs), linking the causal gene will only get easier. The advantages of using ENU screens is the removal of an investigator bias and possession of a guaranteed phenotype (O’Bryan and de Kretser, in press). Additionally, with the ability to generate allelic series of any gene, the functional and critical regions of a protein can be studied in a way not easily available using traditional knockouts/knockins. As a result of the identification of new genes and mutations using ENU-induced mouse models, significant advances can be made in the diagnosis and treatment of infertile men and the development of new male gamete-based contraceptives.
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