Animal models of genetic causes of male infertility

Asian J Androl 2002 Sep; 4: 213-219 

Keywords: animal models; genes; genetics; knockout; male infertility

Introduction

In the past 10 years, more than 100 different genetically engineered mice have been developed with an abnormality in spermatogenesis. More than half of these male infertility animal models have been reported in the past three years alone. Spermatogenic defects in these models vary considerably, ranging from the inappropriate migration of primordial germ cells to the inability of mature spermatozoa to bind the zona pellucida. Many of these genetically engineered mice have been reviewed previously [1-4]. Rather than discussing all of the genes that have been connected with male infertility, the purpose of this review will be highlighting the genes that seem to have the greatest potential for being clinically relevant, based on a main phenotype of male infertility in the knockout mouse model.

Our laboratory is actively evaluating infertility candidate genes in infertile humans. Because of our interests it is important to identify genes for which the gene structure, including exons, introns, and their boundaries can be established. Therefore, each time a candidate murine gene was identified, a literature search was conducted and the NCBI databases searched to determine if a human homologue had been reported or published. In several cases there was no information currently available on the nucleotide or amino acid sequence in humans. If an amino acid sequence was available, it was used to conduct a translated BLAST search of the HTGS (high throughput genomic sequences) database of the human genome. If the structure of the gene in humans could be derived from the BLAST analysis or a published account it was included in this review. If the structure could not be determined, it was excluded.

Male Infertility candidate genes

Genes can be divided into many different groups based on the function or characteristics of the mRNAs or proteins that they produce. Examples include transcription regulators, growth factors, apoptosis regulators, enzymes, hormones, and receptors. For the purposes of this review, the genes are grouped into categories related to the defect produced in spermatogenesis when they are disrupted, or the physical location of the proteins the genes code for in the developing spermatozoa.

Genes related to defects in Meiosis

Meiotic cell division is unique to the production of mature gametes. Meiosis proceeds differently from mitotic cell division in several ways, most notably in how the chromosomes pair and divide. Many of the genes involved with meiosis have been disrupted in mice, and some have produced very good models of male infertility. It is likely that at least some of these models may be relevant to human infertility as well.

During prophase and metaphase I of meiosis, sister chromatids are linked together by the synaptonemal complex. SCP3 forms the lateral elements of the synaptonemal complex, and is necessary for homologous chromosome pairing. When SCP3 is disrupted, the synaptonemal complex does not form. Male mutant mice undergo spermatogenic arrest during early meiosis, and no post-meiotic germ cells can be identified in the testes [5]. Females are still capable of reproduction, but there is an increase in aneuploidy and embryo death [6].

Another gene that results in abnormal synaptonemal complex formation is disrupted meiotic cDNA (DMC1). DMC1 was isolated from a screen for meiosis-specific, prophase-induced genes. Disruption of the DMC1 locus results in meiotic arrest in early prophase 1, with no cells progressing past the zygotene stage, and a malformed synaptonemal complex. Both males and females with a null mutation are infertile [7]. Heat shock protein 70-2 (HSP 70-2) also associates with the synaptonemal complex during meiotic prophase. Synaptonemal complexes in mutant mice assemble and develop through the final pachytene substage, but fail to desynapse, resulting in an absence of normal spermatocytes and an increase in apoptotic cells [8].

The translocated in liposarcoma (TLS) gene encodes a RNA binding protein that plays a role as an oncoprotein in the development of human liposarcomas and leukemias. The exact role of TLS during meiosis is still unclear, but male mice homozygous for an induced mutation in the TLS gene are sterile and have an increase in unpaired and mispaired chromosomes in premeiotic spermatocytes. Chromosomal pairing appears delayed, occurring after the axial elements of the synaptonemal complex have been formed, rather than concurrently, which occurs in the wild type [9].

Several DNA mismatch repair genes have been identified that negatively impact meiosis. MutS homolog 4 and 5 (MSH4, MSH5) are mismatch repair genes expressed predominantly in the testes. Mutant MSH4 males are infertile and lose the majority of their spermatogonia by adulthood. They exhibit abnormalities in chromosomal pairing at the zygotene stage of prophase 1. Female mutants are subfertile, having a depleted number of oocytes and a decreased ovary size [10]. In MSH5 mutants, approximately 98 % of chromosomes fail to synapse, and the few chromosomes that do synapse appear to pair incorrectly. Deletion of the MSH5 locus affects female fertility even more severely, resulting in degeneration of the ovary by adulthood [11].

Two additional mismatch repair genes are MLH1 and PMS2, which form a heterodimer in vivo. They also play a role in chromosomal pairing and meiotic crossover events, and mutations in these genes are associated with a predisposition for hereditary non-polyposis colon cancer (HNPCC). Normal chromosomal pairing during meiosis is detected in MLH1 mice, but post-pachytene stages are rarely observed, with a complete absence of spermatozoa. Females are also infertile [12]. Despite functioning with MLH1 as a heterodimer, disruption of the PMS2 locus produces a different phenotype. Chromosomal pairing is disrupted, but spermatids and spermatozoa are observed. The spermatids are round, abnormal and multinucleated, and the spermatozoa have grossly abnormal head shapes with truncated, irregular tails. The epididymal sperm concentration is only 25 % of that in controls. Female fertility is unaffected [13].

Several families of cyclin proteins exist that bind cyclin-dependant-kinases and regulate cell division and growth. Cyclin A1 is a member of the A-type cyclin family and is expressed exclusively in the germ cell lineages of mice, and in the testis and some myeloid leukemia tumors in humans. Cyclin A1 is encoded by the CcnA1 locus, and mice bearing a null mutation for the CcnA1 gene were sterile due to a block of spermatogenesis before the first meiotic division, while female fertility was normal. Arrest of meiosis was associated with increased germ cell apoptosis and desynapsis abnormalities [14]. Another cyclin, cyclin D2, which is activated by FSH and causes female infertility, has also been listed as a male infertility candidate. While male mice with this gene disrupted are still fertile, they do experience a two-to-three-fold decrease in sperm concentration and have a testicular weight approximately 40% of normal [15].

Several more genes have been identified, in addition to those with a specific role in meiosis, which result in spermatogenic arrest when mutated. Dazla is an autosomal homologue to the DAZ gene found on the Y chromosome. It is a cytoplasmic factor and anti-Dazla antibody stains intensely in pachytene spermatocytes. Male and female knockout mice are sterile, and there is an almost complete absence of germ cells beyond the spermatogonial stage, indicating that arrest takes place prior to the onset of meiosis. Interestingly, though they are still fertile, male mice heterozygous for the mutation have 60% of their sperm visibly abnormal, compared with 15% in control groups. Heterozygotes also have sperm concentrations approximately half of that in the control group population [16].

A gene that has seen a great deal of attention for its role in spermatogenesis is the nuclear transcription factor cAMP responsive element modulator (CREM). Through variable splicing, several isoforms of the gene are expressed. Repressor forms of CREM are expressed in pre-meiotic germ cells while activator forms are expressed in post-meiotic germ cells. Spermatogenic arrest in CREM mutant mice occurs at the round spermatid stage and males are azoospermic. Mice heterozygous for the mutation are subfertile [17, 18].

Genes in the myb family encode nuclear proteins that function as regulators of transcription. One of these is A-myb, which is expressed primarily in male germ cells and to a lesser degree in the developing mammary tissue of pregnant females. The primary spermatocytes of mutant males degenerate, and there is a complete absence of post-meiotic cells [19]. The Bcl-2 gene family includes several members that either promote or inhibit apoptosis. Bcl-w is an apoptosis-inhibiting member of the family. Targeted disruption of the gene produce healthy mice with azoospermic males; females are unaffected [20]. The Microchidia gene (Morc) was identified when it was inadvertently disrupted by the insertion of an unrelated transgene, which resulted in complete male sterility and extensive germ cell apoptosis. Morc is expressed in a testis-specific fashion in both mice and humans, and spermatogenesis in mice arrests in late prophase 1 [21].

Spermine synthase is an enzyme that converts spermidine to spermine, a highly conserved polyamine important for cell growth and division. A mutation in this gene results in complete spermatogenic arrest and sterility [22]. TATA box binding protein (TBP) functions as the central core of the general transcription factors that initiate transcription. TBP-related factor 2 (TRF2), is a testis-specific form of TBP that is able to take its place in the transcription core. TRF2 is expressed in the testes of mice and humans from the late pachytene to round spermatid stage. Mice with the TRF2 gene disrupted are healthy, but azoospermic [23]. VASA is a conserved, ATP-dependant RNA helicase with germ cell-specific expression. Mice with the VASA gene knocked out appear and develop normally, but males have a depletion of post-meiotic germ cells and are sterile [24].

During spermiogenesis approximately 85% of sperm histones are replaced with protamines 1 and 2 (P1, P2), assisting in the condensation of the sperm nucleus through the more compact packaging of the sperm DNA [25]. The quantity of P1 and P2 in the nucleus is approximately equal in fertile controls, but several reports have documented infertile or subfertile populations of men with abnormalities in P1 and P2 expression [26]. The obvious importance of the nuclear core proteins in spermatogenesis has led to the investigation of the associated genes.

The transition proteins (TNP1, TNP2) are intermediaries that play a role in the conversion from histones to protamines. Mice with a disruption of the TNP1 gene develop and appear normal, but have increased abnormal sperm head shapes, including an increased number of decondensed heads and severe asthenozoospermia [27]. A knockout of TNP2 also resulted in highly abnormal morphology of spermatozoa and reduced fertility [28]. A transgenic model that translated P1 prematurely resulted in precocious nuclear condensation, teratozoo-spermia, and sterile males, some with complete spermatogenic arrest [29]. Another recent report, in which both the P1 and P2 loci were separately disrupted, indicates that deletion of either the P1 or P2 gene is sufficient to cause infertility [30].

Two additional male infertility candidate gene connected with protamine replacement are calmodulin dependant protein kinase (CamK4) and Tarbp2. CamK4 is a testis-specific protein kinase that phosphoralates P2 and is expressed in pachytene spermatocytes. A knockout of the gene resulted in prolonged expression of TNP 2, a lack of P2, sperm concentrations only 4 % of normal, and complete male infertility [31, 32]. Tarbp2 is a translational regulator believed to be essential for translation of the protamine mRNAs. A knockout of the gene produced mice with severe oligozoospermia and sterility [33].

Genes related to sperm structure

A number of genes have produced mice with structural sperm abnormalities and partial or complete infertility as a result of their disruption. EGR4, a zinc finger transcription factor, is expressed at low levels in the brain and testis. Disruption of the EGR4 gene results in a partial maturation arrest of germ cells during the pachytene stage, and oligozoospermia. Spermatozoa isolated from EGR4 mutants show multiple structural abnormalities including separated heads and tails, heads bent back sharply on the tail, and tails that are fragmented, kinked or tightly coiled at the distal end [34]. JunD is one of three Jun proteins contributing to the AP-1 transcription factor complex. A knockout of the C-jun or JunB proteins is lethal, but JunD disruption produces healthy, viable, infertile male mice. Male mutants can be separated into two categories; those with oligo-astheno-teratozoospermia, and ones with normal concentration but decreased motility. During elongation there are severe tail malformations and the microtubules are highly disorganized [35].

THEG is a testis-specific gene with a nuclear signaling domain that is expressed in the round spermatids of mice and humans. Male mutants are infertile and exhibit abnormal sperm elongation and either absent or severely defective tails [36]. E-MAP-115 is a microtubule-associated protein with high levels of expression in the Sertoli cells and the manchette and flagellum of spermatids. Male mutants were infertile, and exhibited deformed spermatid nuclei and a gradual loss of germ cells. Microtubule associations within the manchette of spermatids and in the Sertoli cells were highly abnormal [37].

Another gene contributing to tail malformations upon mutation is C-ros, which is expressed in cells of the epididymis, rather than functioning directly in sperm production. C-ros mutants have a high percentage of sperm with decreased motility and bent tails, which are unable to pass through the utero-tubal junction and tangle in a mass at that location. The mass of sperm apparently blocks the junction from being navigated by sperm with normal morphology and motility [38, 39]. Protein C inhibitor (PCI) is a plasma protein that also has high expression in the male reproductive organs. Disruption of the PCI locus results in male, but not female, infertility. The sperm concentration in these mutants is normal, but 95 % of the sperm have abnormal morphology and they are incapable of fertilization with or without the zona pellucida in in vitro and in vivo studies [40].

Casein kinase II (CK2) is a serine-threonine kinase with a couple of different isoforms. The Csnk2a2 isoform is expressed in the germ cells during the late stages of spermatogenesis. Disruption of the Csnk2a2 gene produces mice with male infertility, exhibiting oligozoospermia and globozoospermia [41]. The final gene in this category is Nectin-2, a cell adhesion molecule that is expressed only during the late stages of spermatogenesis. Nectin-2 mutant males have a normal concentration of motile sperm, but highly misshapen heads and thin midpieces. Electron microscopy of the sperm reveals that there are mitochondria in the head, the mitochondria in the middle piece are disorganized, the nucleus is condensed but distorted, and the outer dense fibers are jumbled and extended into the head. Male mice mate normally, but are infertile; female fertility is not affected [42].

A small group of genes have been identified that result in male factor defects of fertilization, despite the production of sperm with normal concentration, motility, and morphology, when they are disrupted. Two genes that result in a very similar phenotype are fertilin b and calmegin. Fertilin b is an integrin protein localized to the mammalian sperm plasma membrane. Calmegin is a testis-specific, endoplasmic reticulum chaperone protein. When either gene is disrupted, it results in a model with a deficiency in sperm-egg membrane adhesion and fusion in vitro, and poor migration of sperm from the uterus through the oviduct and zona binding in vivo [43, 44]. Recently it was discovered that fertilin b binds specifically to calmegin, which likely acts as its chaperone, explaining the common phenotype [45].

One additional gene that shares a similar phenotype to calmegin and fertilin beta upon disruption is Cyritestin. Like fertilin b, Cyritestin is a plasma membrane protein and a member of the ADAM family. Cyritestin -/- mice also exhibit an inability to bind and penetrate the zona pellucida, although they fertilize normally when the zona pellucida is removed. Unlike fertilin b and calmegin knockouts, the sperm from cyritestin deficient mice are able to move without difficulty from the uterus through the oviduct [46].

In the course of this review we have attempted to present the genes identified from animal studies that appear to be the most likely candidates for human male infertility. Less than half of the total genes that have been identified in the literature as resulting in male infertility when altered were actually reviewed, since many had other associated complications that were considered too severe to be consistent with human infertility types observed clinically. While several of the genes that have been presented in this review do have some additional characteristics in the mutant males besides infertility, they are generally mild or do not occur until later in life.

As the knowledge gained from animal models is applied in the screening of genetic infertility in humans, it will be important to remember a couple of the lessons learned from the animal studies. First, in almost all cases, heterozygous mutant mice retained fertility, despite severe phenotypes in homozygous mutants. This would imply that single allele mutations alone are not likely to be a cause of infertility in humans. Second, in several cases genes that had expression patterns that were not testis-specific produced infertility phenotypes with few, if any, additional complications.

This suggests that gene redundancy in the somatic tissues can often provide substitutes for missing gene products while spermatogenesis, with fewer redun-dancies, is more vulnerable. Taking this a step further, as we search for candidate infertility genes we should not limit the search to only those genes having testis-specific expression.

Despite advances in diagnoses, a large percentage of male infertility remains unexplained. It has been estimated that 2000 genes regulate the process of spermato-genesis, only 30 of which are located on the Y chromosome [47, 48]. It is highly probable that alterations in some of these genes, including the relative handful presented in this paper may be a cause for some cases of idiopathic male infertility. Determining which of these many genes are clinically relevant will require the close cooperation of both geneticists and clinicians. With the steady advances in molecular biology, including the increased rate at which candidate genes can be identified and evaluated, we are certain to see significant progress in this area of research.

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Correspondence to: Dr Douglas T. Carrell, Andrology Department, University of Utah School of Medicine, 50 North Medical Drive, Suite 3B208, Salt Lake City, Utah 84132, USA.
Tel: +1-801-581 3740, Fax: +1-801-581 6127
E-mail: dcarrell@med.utah.edu
Received 2002-06-24      Accepted 2002-08-07