Mouse models in male fertility research

Abstract

Limited knowledge of the genetic causes of male infertility has resulted in few treatment and targeted therapeutic options. Although the ideal approach to identify infertility causing mutations is to conduct studies in the human population, this approach has progressed slowly due to the limitations described herein. Given the complexity of male fertility, the entire process cannot be modeled in vitro. As such, animal models, in particular mouse models, provide a valuable alternative for gene identification and experimentation. Since the introduction of molecular biology and recent advances in animal model production, there has been a substantial acceleration in the identification and characterization of genes associated with many diseases, including infertility. Three major types of mouse models are commonly used in biomedical research, including knockout/knockin/gene-trapped, transgenic and chemical-induced point mutant mice. Using these mouse models, over 400 genes essential for male fertility have been revealed. It has, however, been estimated that thousands of genes are involved in the regulation of the complex process of male fertility, as many such genes remain to be characterized. The current review is by no means a comprehensive list of these mouse models, rather it contains examples of how mouse models have advanced our knowledge of post-natal germ cell development and male fertility regulation.

Figure 1

Reverse and forward genetic approaches for in vivo gene functional analyses in mouse models. The reverse approach begins by selecting a candidate gene of interest followed by evaluating its in vivo function using gene manipulation techniques and animal model production. Once generated, animal models are used for phenotypic characterization to define pathological abnormalities. In many cases, candidate genes have multiple roles in different tissues/organs; thus, the animal models often give rise to more than one phenotypic defect. By contrast, the forward genetic approach is initiated by the creation of animal models exhibiting a phenotypic defect of interest followed by defining the genetic alteration responsible for the phenotypic defects.

Figure 2

Gene targeting strategy for the generation knockout mice. A targeting cassette is designed to contain homology arms flanking a drug selectable marker (for example, kanamycin/neomycin) to facilitate the identification of targeted ES clones. Depending upon the design of the targeting cassette, a null allele can be generated by: (i) elimination of sequences critical for translational initiation; (ii) creation of a frame shift in the coding region and premature stop codon (indicated by *); and (iii) creation of unstable mRNA. Untranslated regions are depicted by unfilled boxes; protein-coding regions are depicted by filled boxes; ATG: initiation codon. ES, embryonic stem.

Figure 3

A high-throughput gene trapping approach. A gene trap cassette is designed to contain the features necessary for its integration into the ES cell genome and termination of transcription of the trapped allele. These features include SA site and poly A signal. If inserted within an intron of a gene, the native splicing pattern is affected by the SA, resulting in fusion of the upstream exons with the trapped cassette. Transcription of the trapped mRNA is subsequently terminated at the poly A site. If inserted within the 5′ UTR, the trapped mRNA will produce no functional protein. If insertion occurs within an intron downstream of the translational start site of a gene, a truncated protein (fused with the trapped cassette) may be produced depending upon the stability of the trapped mRNA. ES, embryonic stem; poly A, polyadenylation; SA, slicing acceptor; UTR, untranslated region.

Figure 4

Transgenesis strategy. A transgene cassette is designed to contain features necessary for gene expression, for example, promoter and polyadenylation signal. Additional markers such as fluorescent protein tags (for example, GFP and YFP) can be included to facilitate the detection of transgene expression. The transgene is introduced into the genome of fertilized mouse oocytes. Integration of the transgene occurs randomly and multiple copies of the transgene may be integrated in the same chromosomal location or on different chromosomes. GFP, green fluorescent protein; YFP, yellow fluorescent protein.

Figure 5

Example of a three-generation breeding scheme used to identify recessive mutations. To screen for recessive mutations, a three-generation breeding scheme is required. Founder male mouse (referred to as generation 0 (G₀)) of an inbred strain (for example, C57BL/6) is injected with ENU. The ENU-treated male is subsequently mated with wild-type females of a different inbred strain (for example, CBA) to produce G₁ offspring. G₂ progeny can be produced from G₁ littermate intercrosses or from G₁×wild-type CBA crosses (as shown here). Finally, G₃ progeny are generated from G₂ littermate intercrosses and/or G₂ females×G₁ fathers. During each step of crossing, each progeny will have different combinations of chromosomes from the ENU-treated mouse strain and the strain used for subsequent outcrossing. These differences enable researchers to map the region containing the ENU-induced mutation causing phenotypic defects of interest (indicated by *). In this case, mutations are introduced into the C57BL/6 genome. CBA, cytometric bead array; ENU, N-ethyl-N-nitrosourea.