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Review

Infertility Caused by Polymorphisms or Mutations in Spermatogenesis-Specific Genes

From the Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, Suita City, Osaka, Japan.

Correspondence to: Hiromitsu Tanaka, Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita City, Osaka 565-0871, Japan (e-mail: tanaka{at}biken.osaka-u.ac.jp).

Received for publication September 15, 2005; accepted for publication December 19, 2005.

In male germ cells, many dramatic morphological changes occur during spermatogenesis, especially in haploid spermatids after meiotic division (Russell et al, 1990). In various mammals, male germ cell differentiation proceeds actively and continuously in the testis after puberty, and sperm are produced throughout adulthood (Russell et al, 1990). One month of male cell differentiation in mice (2 months in humans) is required for completion of spermatogonial stem cell proliferation and differentiation, meiosis, generation of haploid germ cells, and morphogenesis of the developing sperm in somniferous tubules (Russell et al, 1990). After meiotic division (during the process of haploid germ cell differentiation, or spermiogenesis), the rounded spermatids undergo marked morphological changes to become sperm: the nucleus assumes a compact shape, the mitochondria are rearranged, the flagellum forms, and the acrosome is generated. During this period of differentiation, which takes about 5 to 6 weeks in humans (Clermont, 1963; Heller and Clermont, 1963) and 2 to 3 weeks in mice (Oakberg, 1956), haploid germ cells do not divide, but morphogenesis occurs, indicating that some regulatory mechanism arrests the cell cycle. Searching for functional changes in genes and gene products involved in male infertility would increase our understanding of the causes of this problem, and perhaps lead to treatment in some cases. Comprehensive isolation and analysis of haploid germ cell-specific genes showed that a large number of specific molecules are involved in spermiogenesis (Tanaka et al, 1994, 2002b; Fujii et al, 2002). The most straightforward strategy for elucidating the mechanism of spermatogenesis is to identify and characterize differentiation-specific molecules and their associated genes in germ cells.

Isolation and Characterization of Germ Cell-Specific Molecules

Germ cell-specific molecules were isolated and identified using immunological, biochemical, and molecular biological techniques. Identification of interesting molecules in specific stages of differentiation was performed by a variety of techniques, including two-dimensional gel electrophoresis (Padma et al, 2003), isolation of polyclonal or monoclonal antibodies recognizing specific stages of germ cells (Tanaka et al, 1998) (Figure 1), cDNA cloning from testicular cDNA libraries or cDNA databases in silico (Rajkovic et al, 2001; Hong et al, 2005), and microarray techniques (Shima et al, 2004). Isolation of cDNAs and characterization of encoded proteins that are specifically expressed at different steps in germ cell development should help elucidate mechanisms of spermatogenesis. We cloned and characterized germ cell-specific genes of mice using antibodies and cDNA subtraction methods (Tanaka et al, 2002b) (Figure 2), together with human orthologs (Tanaka et al, 1994; Fujii et al, 2002).

View larger version (9K): [in this window] [in a new window]   Figure 1. Monoclonal antibodies isolated in our laboratory, which recognized specific antigens of mouse germ cells. The bars indicate the expression periods of each antigen. M.W. indicates the molecular weight of each antigen on sodium dodecyl sulfate polyacrylamide gel electrophoresis.

View larger version (33K): [in this window] [in a new window]   Figure 2. Germ-cell-specific genes that have been cloned and characterized in our laboratory. The numbers in parentheses are the DDBJ/GenBank/EMBL accession numbers. The bars indicate the expression periods of each gene. Translation was regulated in some genes. Shadowed bars indicate the expression of mRNA only. All of these genes show expression that is controlled developmentally.

The identification of germ cell-specific genes showed that many genes are expressed in differentiated spermatids, although the nuclei are compacted and most of the cytoplasm is lost in spermiogenesis. The chromatin status can be distinguished by using different covalent modifications of histones or histone variants in spermiogenesis. The chromatin associated with transcriptionally active loci becomes enriched in histones with lysine modifications. Because haploid germ cells lack general histones, they may be difficult to regulate using a conventional histone code in which histone modification of the chromatin regulates gene expression (Fischle et al, 2003). It is likely that some specific mechanisms act to conserve an unusually large number of intronless genes (described later) in haploid germ cell-specific expression. Further detailed studies of other haploid germ cell-specific genes may resolve these particular issues. Germ cell-specific genes have proven to be an extremely interesting genomic feature of the evolutionary process, and it would be very rewarding to elucidate the relationship between the frequency of male infertility and the genomic characteristics of germ cell-specific genes.

Genomic Construction of Germ Cell-Specific Genes

The identity and genomic structures of germ cell-specific genes have been clarified, and many haploid germ cell-specific genes have been found to lack introns. The reported intronless germ cell-specific genes are shown in Table 1. The 5' upstream and 3' downstream noncoding genomic regions of some of these genes have been sequenced (Yoshimura et al, 1999, 2001; Tanaka et al, 2002a). The results indicated that intronless germ cell-specific genes were derived from ancestral genes via the movement of retroposons. Retroposition is an important mechanism for copying genes, and it has produced a large number of functional genes during mammalian genome evolution. There are approximately 100 functional retrotransposed genes on human and mouse chromosomes (Emerson et al, 2004), and gene loss and gain from X chromosomes via retroposition occurs more readily than on autosomes (Emerson et al, 2004). Furthermore, many genes located on the X chromosome are expressed in germ cells (Wang et al, 2001). The X chromosome plays a prominent role in the premeiotic stages of mammalian spermatogenesis. There are three Y-linked and ten X-linked genes among 25 expressed specifically in mouse spermatogonia (Wang et al, 2001). However, mouse haploid germ cell-specific genes are scattered throughout the genome, and are present on autosomes as well as on sex chromosomes. Thus, the candidate causal genes of male infertility may occur on both autosomes and sex chromosomes.

Phosphoglycerate kinases (PGK) are important for maintenance of metabolism in many cell types. Intronless genes on autosomes, such as phosphoglycerate kinase 2 (PGK-2), are expressed specifically in testicular germ cells (McCarrey and Thomas, 1987). PGK-2 is believed to be derived from the transposon-mediated reverse transcription of ancestral genes on the X chromosome. Retrotransposition from the X chromosome provides a means of escaping X chromosome inactivation during spermatogenesis (McCarrey and Thomas, 1987). Recently, the UTP14b gene (Rohozinski and Bishop, 2004) on mouse chromosome 1 was identified as a derivative of mUTP14a. Further examination and comparison of the genomic sequences of human and other mammals have revealed how retroposition occurred in germ cells at some point after the phylogenetic separation of mammals.

Single-Nucleotide Polymorphisms (SNPs) or Mutation Analyses of Germ Cell-Specific Genes

Approximately 15% of couples attempting to conceive over a two-year period are unsuccessful (de Kretser and Baker, 1999). The causal frequency of male and female infertility is equal. More than half of male infertility has uncertain causes and is thus idiopathic. In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are used to treat infertility, even when the cause is uncertain. Recently, testicular sperm extraction (TESE) with ICSI (TESE = ICSI) has become the first-line treatment for azoospermic male infertility in both nonobstructive and obstructive azoospermia (Tsujimura et al, 2002). However, knowing the cause of infertility is important in order to develop better remedies.

The so-called Azoospermia Factor gene (AZF) is located on the Y chromosome. The long arm of the Y chromosome contains at least 3 distinct deletion intervals that have been named AZFa, AZFb, and AZFc (Vogt et al, 2005). Molecular techniques are much better able to identify these microdeletions than cytogenetic approaches. In nature, an infertility mutation on the Y chromosome is not transmitted to the next generation, because pregnancy does not occur. However, these mutations can be transmitted to male offspring through the use of assisted reproductive technologies (ART), either IVF or ICSI, and would cause male infertility in the offspring. If the effects of an autosomal mutation are not dominant, the recessive mutation can be inherited by the next generation, and under certain conditions can produce infertility. Even a dominant negative autosomal mutation that affects spermiogenesis can, in theory, be transferred to the next generation via females. The function and expression of the genes and gene products might be changed by polymorphisms on chromosomes. It may also be possible to induce infertility by combining polymorphisms in different genes. Moreover, polymorphisms of genes expressed ubiquitously in somatic cells could also contribute to infertility. However, to be an effective cause of infertility, this mechanism requires that there be no defects in the somatic cells, and we consider this to be an extremely rare circumstance. Thus, when seeking male infertility genes, the first step should be to examine polymorphisms or mutations in male germ cell-specific genes.

Since almost all mouse male germ cell-specific genes have human orthologs, it is possible to isolate and characterize the counterparts of human genes. Mutations or loss of function in some of male germ cell-specific genes in men should give rise to male infertility. One strategy is to examine and compare the whole DNA sequences of these genes in fertile and infertile men. Mutations in housekeeping genes would result in a loss of function, thereby affecting embryogenesis or somatic cell functions, which could lead to defects or some illness. In contrast, even if the germ cell-specific genes were totally defective, the phenotype would be limited to germ cells, and the only repercussion would be infertility. Thus, we examined SNPs or detect mutations by direct DNA sequencing of the PCR products of chromosomal DNA from blood samples.

As a starting point, we examined the relationship between variation in nuclear proteins specifically expressed in spermiogenesis and human male infertility. During spermiogenesis, round spermatids undergo complex morphological, biochemical, and physiological modifications that result in the formation of mature spermatozoa. The sperm nucleus also undergoes marked rearrangement, which involves the removal of histones and their replacement by various nuclear proteins. Finally, the DNA of human male gametes is highly condensed in the sperm head through the activity of highly positively charged protamines (PRMs). The replacement of histones and the deposition of protamines are supported by different nuclear proteins, including transition nuclear proteins, which have a major role in remodeling chromatin. Almost all of these basic nuclear proteins, including PRMs, are derived from histone H1 and have undergone complex modifications in the evolution of mammals (Tanaka and Baba, 2005).

Azoospermia and oligospermia patients were examined for SNPs through analyses of PRMs, TNPs, and HANP1 (Figure 3). Analysis of human PRM1 and PRM2 gene sequences of 226 sterile male patients and 270 proven-fertile male volunteers revealed 4 single nucleotide polymorphisms (SNPs) in the PRM1 coding region, which did not cause any amino acid substitutions, and 1 SNP in the PRM2 gene, which produced translation termination (Tanaka et al, 2003a). Although the PRM1 and PRM2 genes are highly conserved, the single SNP in the PRM2 gene inducing translation termination may result in male infertility, due to haploinsufficiency of PRM2 (Cho et al, 2001).

View larger version (31K): [in this window] [in a new window]   Figure 3. Schematic representation of the genomic construction of spermatogenic nuclear proteins and their major mutations. The upper bar indicates the PRM1, RPM2, and TNP2 genes that are clustered within approximately 10 kb of genomic DNA on 16p13.13 (Engel et al, 1992). The middle bar indicates the TNP1 gene on genomic DNA of 2q36-36 (Luerssen et al, 1990). The lower bar indicates the HANP1 gene on genomic DNA of 12q13.11 (Tanaka et al, 2006). Boxes indicate regions transcribed as mRNA. Shadowed boxes indicate introns. The DNA sequences of infertile patients are shown in the middle boxes (Tanaka et al, 2003a; Miyagawa et al, 2005). The C248T alteration in PRM2 induced a nonsense codon under conditions of heterozygosity (right box). A deletion of 15 nucleotides, which encompassed the recognition site for the CRE transcription factor, was found in the 5' promoter region of the TNP1 gene under conditions of heterozygosity (left box).

Analysis of the human TNP1 and TNP2 gene sequences in 282 sterile male patients and 270 (TNP1) and 266 (TNP2) proven-fertile male volunteers revealed 5 amino acid substitutions caused by single nucleotide polymorphisms in the open reading frame (ORF) of TNP2. In addition, a 15-nucleotide deletion, which encompasses the CRE motif, was found in the 5' promoter region of TNP1 in infertile men (Miyagawa et al, 2005). This deletion mutation should introduce much depression in TNP1 gene expression. In general, loss of the TNP1 or TNP2 gene does not lead to complete male infertility in mice (Yu et al, 2000; Zhao et al, 2001), although some TNP1-deficient mice showed infertility (Meistrich et al, 2003). The loss of both TNP1 and TNP2 results in reduced litter sizes (Zhao et al, 2004). Although TP1 protein has been shown to play some important roles in mouse spermiogenesis, its functions in human sperm formation are not yet fully understood. The 15-nucleotide deletion in the promoter region would cause reduction of TNP1 expression, and should cause human male infertility. Even though some single nucleotide polymorphism would not cause a deletions effect on fertility, it is possible to induce infertility by combining some additional polymorphisms in other genes.

Hanp1 encodes a histone H1-like protein. Homozygous Hanp1 mutant male mice are infertile, while females are fertile. Although a substantial number of sperm can be recovered from the epididymides of these infertile males, the shape and function of sperm are abnormal. HANP1 protein is essential for nuclear formation in functional spermatozoa, and is specifically involved in the replacement of histones with protamines during spermiogenesis (Martianov et al, 2005; Tanaka et al, 2005). If 1 allele of the haploid germ cell-specific Hanp1 has a mutation, the phenotype of sperm is normal and functional. The allele of the Hanp1 mutation in the heterozygous condition is inherited in a Mendelian manner (since haploid cells are connected to each other by a cytoplasmic bridge to exchange macromolecules such as protein and mRNA; Morales et al, 1998). Analysis of human Hanp1 sequences in 226 sterile male patients and 270 proven-fertile male volunteers revealed 5 SNPs in the coding region that introduce amino acid changes (Tanaka et al, 2006). However, the prevalence of these 5 SNPs did not differ significantly between our infertile and proven-fertile clinical cases. Some human cases with deficient HANP1 will likely suffer teratozoospermia in the same way as KO mice.

Many forms of partial deletion occur on the Y chromosome in male infertility cases (Hopps et al, 2003). These deletions associated with infertility in nature cannot be inherited (Silber and Repping, 2002). Most infertile males do not show any mutations or deletions on the Y chromosome relating to infertility. Mutations of the PRM and TNP genes located on autosomes were found to be the cause of male sterility in 1 of 200 to 300 Japanese male infertility cases we examined. Although the frequency of a single SNP is lower than the rate of mutation on the Y chromosome in infertile males, changes in many haploid germ cell-specific genes should be considered as causes of genomic male infertility (Tanaka et al, 2003a; Miygawa et al, 2005; Singh et al, 2005). If haploid germ cell-specific genes are located on autosomes, sterile mutations can be transferred to progeny via the mothers. Alternatively, autosomal mutations may disappear during gametogenic meiosis, creating an interesting and delicate balance. These considerations are of important concern when treating infertility by IVF or ICSI. Although a few SNPs associated with male infertility have been found in some clinical samples, the data are too few for explaining all male infertility. Further study is needed to clarify the frequency of these mutations and genomic changes in other genes in larger human samples.

We cloned the haploid germ cell-specific genes group using cDNA subtraction methods and have been characterizing these genes (Fujii et al, 2002). They take part not only in haploid germ cell-specific differentiation, such as formation of the sperm flagellum, but also in various maintenance functions of the cell, such as signal transduction and energy metabolism. Also, these genes are scattered over all chromosomes, and their construction resembles that of intronless genes. It is generally understood that mutation of these genes causes human infertility. Finally, we need to deepen our understanding of the roles of specific molecules in spermatogenesis and of the phenotypes that result from loss of function in these molecules.

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Tanaka H, Iguchi N, Nakamura Y, Kohroki J, Egydio de Carvalho C, Nishimune Y. Cloning and characterization of human haspin gene encoding haploid germ cell-specific nuclear protein kinase. Mol Hum Reprod. 2001a;7: 211 -218.[Abstract/Free Full Text]

Tanaka H, Koga M, Iguchi N, Nozaki M, Onishi M, Egydio de Carvalho C, Nakamura Y, Miyagawa Y, Takeyama M, Matsumiya K, Okuyama A, Nishimune Y. Isolation and characterization of haploid germ cell-specific OXTC cDNAs; testis-specific succinyl CoA: 3-oxo acid CoA transferases (scot-t1 and scot-t2). In: Robaire B, Chemes H, Morales RC, eds. Andrology in the 21st Century: Proceedings of the VIIth International Congress of Andrology. Englewood, NJ: Medimond Medical Publications; 2001b : 157-161.

Tanaka H, Kohroki J, Iguchi N, Onishi M, Nishimune Y. Cloning and characterization of a human orthologue of testis-specific succinyl CoA: 3-oxo acid CoA transferase (Scot-t) cDNA. Mol Hum Reprod. 2002a; 8: 16 -23.[Abstract/Free Full Text]

Tanaka H, Matsuoka Y, Onishi M, Kitamura K, Miyagawa Y, Nishimura H, Tsujimura A, Okuyama A, Nishimune Y. Expression profiles and single-nucleotide polymorphism analysis of human HANP1/H1T2 encoding a histone H1-like protein. Int J Andro. 2006 . In press.

Tanaka H, Miyagawa Y, Tsujimura A, Matsumiya K, Okuyama A, Nishimune Y. Single nucleotide polymorphisms in the protamine-1 and -2 genes of fertile and infertile human male populations. Mol Hum Reprod. 2003a;9: 69 -73.[Abstract/Free Full Text]

Tanaka H, Nozaki M, Yomogida K, Nishimune Y. Cloning and characterization of male germ-cell-specific genes. In: Rommerts FFG, Teerds KJ, eds. Testicular Tangrams. Heidelberg, Germany: Springer-Verlag; 2002b.

Tanaka H, Okabe M, Ikawa M, Tsuchida J, Yoshimura Y, Yomogida K, Nishimune Y. Studies on the mechanism of sperm production. In: Stefanini M, Boitani C, Galdieri M, Geremia R, Palombi F, eds. Testicular Function: From Gene Expression to Genetic Manipulation. Heidelberg, Germany: Springer-Verlag; 1998: 235 -251.

Tanaka H, Pereira LA, Nozaki M, Tsuchida J, Sawada K, Mori H, Nishimune Y. A germ cell-specific nuclear antigen recognized by a monoclonal antibody raised against mouse testicular germ cells. Int J Androl. 1997;20: 361 -366.[CrossRef][Medline]

Tanaka H, Yoshimura Y, Nishina Y, Nozaki M, Nojima H, Nishimune Y. Isolation and characterization of cDNA clones specifically expressed in testicular germ cells. FEBS Lett. 1994; 355: 4 -10.[CrossRef][Medline]

Tanaka H, Yoshimura Y, Nozaki M, Yomogida K, Tsuchida J, Tosaka Y, Habu T, Nakanishi T, Okada M, Nojima H, Nishimune Y. Identification and characterization of a haploid germ cell-specific nuclear protein kinase (Haspin) in spermatid nuclei and its effects on somatic cells. J Biol Chem. 1999;274: 17049 -17057.[Abstract/Free Full Text]

Tian XC, Xu J, Yang X. Normal telomere lengths found in cloned cattle. Nat Genet. 2005; 26: 272 -273.

Tosaka Y, Tanaka H, Yano Y, Masai K, Nozaki M, Yomogida K, Otani S, Nojima H, Nishimune Y. Identification and characterization of testis specific ornithine decarboxylase antizyme (OAZ-t) gene: expression in haploid germ cells andpolyamine-induced frameshifting. Genes Cells. 2000; 5: 265 -276.[Abstract]

Tsuchida J, Nishina Y, Wakabayashi N, Nozaki M, Sakai Y, Nishimune Y. Molecular cloning and characterization of meichroacidin (male meiotic metaphase chromosome-associated acidic protein). Dev Biol. 1998;197: 67 -76.[CrossRef][Medline]

Tsujimura A, Matsumiya K, Miyagawa Y, Tohda A, Miura H, Nishimura K, Koga M, Takeyama M, Fujioka H, Okuyama A. Conventional multiple or microdissection testicular sperm extraction: a comparative study. Hum Reprod. 2002; 17: 2924 -2929.[Abstract/Free Full Text]

Uchida K, Tsuchida J, Tanaka H, Koga M, Nishina Y, Nozaki M, Yoshinaga K, Toshimori K, Matsumiya K, Okuyama A, Nishimune Y. Cloning and characterization of a complementary deoxyribonucleic acid encoding haploid-specific alanine-rich acidic protein located on chromosome-X. Biol Reprod. 2000; 63: 993 -999.[Abstract/Free Full Text]

Ventura-Holman T, Seldin MF, Li W, Maher JF. The murine fem1 gene family: homologs of the Caenorhabditis elegans sex-determination protein FEM-1. Genomics. 1998; 54: 221 -230.[CrossRef][Medline]

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Wang PJ, McCarrey JR, Yang F, Page DC. An abundance of X-linked genes expressed in spermatogonia. Nat Genet. 2001; 27: 422 -426.[CrossRef][Medline]

Watanabe D, Yamada K, Nishina Y, Tajima Y, Koshimizu U, Nagata A, Nishimune Y. Molecular cloning of a novel Ca(2+)-binding protein (calmegin) specifically expressed during male meiotic germ cell development. J Biol Chem. 1994;269: 7744 -7749.[Abstract/Free Full Text]

Yamanaka M, Koga M, Tanaka H, Nakamura Y, Ohta H, Yomogida K, Tsuchida J, Iguchi N, Nojima H, Nozaki M, Matsumiya K, Okuyama A, Toshimori K, Nishimune Y. Molecular cloning and characterization of phosphatidylcholine transfer protein-like protein gene expressed in murine haploid germ cells. Biol Reprod. 2000; 62: 1694 -1701.[Abstract/Free Full Text]

Yan W, Ma L, Burns KH, Matzuk MM. HILS1 is a spermatid-specific linker histone H1-like protein implicated in chromatin remodeling during mammalian spermiogenesis. Proc Natl Acad Sci U S A. 2003; 100: 10546 -10551.[Abstract/Free Full Text]

Yoshida K, Kondoh G, Matsuda Y, Habu T, Nishimune Y, Morita T. The mouse RecA-like gene Dmc1 is required for homologous chromosome synapsis during meiosis. Mol Cell. 1998; 1: 707 -718.[CrossRef][Medline]

Yoshimura Y, Tanaka H, Nozaki M, Yomogida K, Shimamura K, Yasunaga T, Nishimune Y. Genomic analysis of male germ cell-specific actin capping protein alpha. Gene. 1999; 237: 193 -199.[CrossRef][Medline]

Yoshimura Y, Tanaka H, Nozaki M, Yomogida K, Yasunaga T, Nishimune Y. Nested genomic structure of haploid germ cell specific haspin gene. Gene. 2001;267: 49 -54.[CrossRef][Medline]

Yu YE, Zhang Y, Unni E, Shirley CR, Deng JM, Russell LD, Weil MM, Behringer RR, Meistrich ML. Abnormal spermatogenesis and reduced fertility in transition nuclear protein 1-deficient mice. Proc Natl Acad Sci U S A. 2000;97: 4683 -4688.[Abstract/Free Full Text]

Zhao M, Shirley CR, Hayashi S, Marcon L, Mohapatra B, Suganuma R, Behringer RR, Boissonneault G, Yanagimachi R, Meistrich ML. Transition nuclear proteins are required for normal chromatin condensation and functional sperm development. Genesis. 2004; 38: 200 -213.[CrossRef][Medline]

Zhao M, Shirley CR, Yu YE, Mohapatra B, Zhang Y, Unni E, Deng JM, Arango NA, Terry NH, Weil MM, Russell LD, Behringer RR, Meistrich ML. Targeted disruption of the transition protein 2 gene affects sperm chromatin structure and reduces fertility in mice. Mol Cell Biol. 2001; 21: 7243 -7255.[Abstract/Free Full Text]

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