Mast cells reside in various tissues, such as skin, stomach, mesentery and peritoneal cavity. Stem cell factor is an important growth factor for mast cells, and the expression level of its receptor, KIT, is correlated with the number of mast cells. We examined the number of mast cells in several kinds of homozygous mutant mice at the mi locus, at which a transcription factor named MITF is encoded. MITF regulated the expression of KIT gene, and most homozygous mutants at the mi locus showed a reduced KIT expression. The number of mast cells in these mutants decreased in the skin, and no mast cells were present in stomach, mesentery and peritoneal cavity. In contrast to MITF mutants showing decreased expression of KIT, Mi^wh/ Mi^wh mice were normal in KIT expression. In consistent with the previous observation, Mi^wh/ Mi^wh mice contained normal number of mast cells in the skin, stomach and mesentery. However, no mast cells were detected in the peritoneal cavity of Mi^wh/Mi^wh mice. This indicated that the number of peritoneal mast cells was not correlated with the KIT expression level. Recently, we found that a newly identified mast cell adhesion molecule, SgIGSF, was not expressed in Mi^wh/ Mi^wh mice. This suggested the possibility that SgIGSF expression was related to the number of peritoneal mast cells. Here, we examined the number of peritoneal mast cells in a mild homozygous mutant, mi^vit/mi^vit In mi^vit/mi^vit mice, SgIGSF expression was reduced but apparently detected and KIT expression was normal. The number of peritoneal mast cells was one fifth that of wild-type mice. The number of mast cells in the skin, stomach and mesentery was comparable to that of wild-type mice. These results suggested that the adhesion molecule, SgIGSF, regulated the number of mast cells in the peritoneal cavity, and that KIT regulated the number of mast cells in other tissues, such as skin, stomach and mesentery.

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Although the possible involvement of neurotrophic factors in itchy skins of atopic dermatitis has been predicted, the exact mechanism by which itch is induced remains unclear. Since nerve growth factor (NGF) has crucial effects on development and functions of sensory nerves, we analyzed a correlation between NGF production at the affected site and scratching behavior in atopic NC/Nga mice. NC/Nga mice spontaneously develop atopic dermatitis-like skin lesions when they are raised in air-unregulated conventional circumstances. We quantified scratching behavior of NC/Nga mice with a novel scratch analyzer (SCLABA^®; NOVELTEC INC., Kobe, Japan) during the development of atopic dermatitis and compared to clinical skin severity scores. We found that there was a strong correlation between the severity of dermatitis and the increase in the number of scratches, indicating that scratching behavior may exacerbate clinical skin conditions. NGF contents in the skin lesion of conventional NC/Nga mice were significantly higher than those of SPF NC/Nga mice. Positive reaction for NGF was observed in keratinocytes and fibroblasts in affected skins of conventional NC/Nga mice. Immunohistochemical analysis showed the extension of PGP 9.5 positive nerve fibers from dermis toward epidermis at the affected skins. The protease-activated receptor (PAR) 2 has been reported to be expressed on peripheral nerve fibers and mediate neuronal activation. Thus, we attempted to produce a model with neurogenic itch by PAR2 stimulation. Seven days after intra-dermal injection of NGF, we injected a PAR2 agonist into the same sites of SPF NC/Nga mice. Scratching behavior was significantly increased in mice injected with NGF, but not in mice injected with diluent alone. These results suggest that sensory nerves induced by NGF may contribute to the appearance of itch through PAR2 activation. NGF produced at the affected skin may induce excessive extension of sensory nerves, resulting in abnormal skin sensitivity in atopic dermatitis.

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Generation of mutant mice with the conventional gene-targeting method is useful approach in reverse genetics. However, the desired phenotype may not be observed easily because this approach is labor-intensive and the only one gene can be mutated at a time. In contrast, phenotype-driven forward genetics provide great advantages to analyze the mutated functional genes. Although, large-scale production of mutant mice by chemical mutagen has been reported, detection of induced point mutation responsible for phenotype is difficult and time-consuming. The Sleeping Beauty (SB) transposon is a mobile DNA element jumping from one location to another, and has been developed as a tool for insertional mutagenesis. Determination of the insertion site is facilitated by using the transposon sequence as a molecular tag. The efficient activity of the SB transposase in the mouse germline provides us with a useful system for random germline insertional mutagenesis. We have tested SB transposon vector for use in mouse germline insertional mutagenesis. The offsprings generated from mating transgenic males harboring transposon and transposase with wild-type females demonstrated efficient transposition in mouse germline. The newly developed transposon vector combined with gene trap method and GFP reporter enabled us to select for mutant mice rapidly and non-invasively. The homozygous mutant mice generated with this vector showed apparent phenotype, indicating that our transposon vector was highly mutagenic. Taken together, these results indicate that the SB transposon system has promise as a new powerful tools for large-scale genetic screening in mice.

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In most multicellular organisms, fertilized eggs differentiate into somatic and germ cells. They become separated during the early developmental stages and have quite different roles. Germ cells maintain species continuity, whereas somatic cells ensure the constitution and activity of the individual. In other words, somatic cells help the germ cells to ensure survival and continuity of the species. Thus, germ cells play a fundamental role in multi-cellular organisms.
To understand the unique mechanism of germ cell differentiation, the most straightforward strategy is to identify and characterize differentiation-specific molecules and their associated genes in germ cells. We have cloned haploid germ-cell-specific cDNAs from a subtracted cDNA library that was generated by subtracting the mRNA of 17-day-old mouse testes (before haploid germ cells develop) from the cDNA of 35-day-old mouse testes. Detailed mRNA expression analysis revealed that the genes corresponding to the cloned cDNAs were exclusively expressed in germ cells at all steps of differentiation, at specific steps of differentiation, or at specific steps in the development of haploid germ cells. The expression of all of these genes was developmentally controlled. The products of germ-cell-specific genes included various proteins having roles in spermatogenesis. Some germ-cell-specific isozymes of previously known enzymes for energy metabolism are interesting examples.
We also isolated the genomic DNA of haploid-specific genes and identified a number of regulatory motifs in the gene-promoter regions that were essential for transcription. One of these motifs, the cyclic AMP response element, was present in the promoter regions of several testis-specific genes, and was deemed to be functionally important. However, the haspin gene-promoter region did not contain a CRE motif. Similarly, the promoter regions of the MMP-28 (Illman et al. 2001), Hormone-sensitive lipase (Blaise et al. 2001), ldhc (Jethanandani and Goldberg 2001), SP-10 (Reddi et al. 1999) genes, which were specifically expressed in haploid germ cells, did not have CRE motifs. These findings suggest the existence of haploid germ-cell-specific regulatory proteins specifically regulate the expression of haploid germ-cell-specific genes. Here, we describe our recent findings of germ-cell-specific genes and gene products, and discuss the relevance of our approaches to the study of germ cell differentiation mechanisms.

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Approximately 10% of couples attempting to conceive over a period of 2 year are unable to become pregnant and 20% of those cases are attributable to a male factor alone. Testicular sperm extraction (TESE) with ICSI (TESE-ICSI) is becoming the first-line treatment for male infertility, especially for azoospermia (non obstructive azoospermia: NOA or obstructive azoospermia: OA) . Recently, the sperm retrieval rate (SRR) by microdissection TESE was reported to be approximately 45% and higher than by conventional TESE. However, still more than 50% of NOA cases are unable to obtain testicular sperm and even if they get testicular sperm, the fertilization rate, clinical implantation rate and clinical pregnancy rate were not enough high and lower in the NOA compared with the OA. Thus further breakthrough is required for higher SRR and subsequent high clinical pregnancy rate. To gain insight into the genetic factors implicated in male infertility, we have studied single nucleotide polymorphisms of the various spermiogenic genes in human male. Protamines, which are the major DNA-binding proteins in the sperm nucleus, package the DNA into the sperm head. Analysis of the human protamine-1 (PRM1) and -2 (PRM2) gene sequences in 226 sterile male patients and in 270 proven-fertile male volunteers revealed four single nucleotide polymorphisms (SNPs) in the PRM1 coding region, which did not cause any amino acid substitutions, and one SNP in the PRM2 gene, which produced translation termination. We also observed one SNP in the 3' non-coding region of the PRM1 gene, and two SNPs within the intron of the PRM2 gene. The prevalence of these SNPs was similar in both infertile patients and in proven-fertile volunteers, except that the c248t alteration in the PRM2 gene induced a nonsense colon under conditions of heterozygosity in one infertile patient. Although the PRM1 and PRM2 genes are highly conserved, the single SNP in the PRM2 gene that induces translation termination may result in male infertility due to haploinsufficiency of PRM2. Further investigation of spermiogenic genes will reveal novel causative genes for the different type of idiopathic male infertility.

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Poly (A) tails of eukaryotic mRNAs are implicated in various aspects of mRNA metabolism, including transport into the cytoplasm, stability, and translational control. Thus, the control of poly (A) tail length is one of the posttranscriptional regulations of gene expression. Spermatogenesis is a highly specialized process of differentiation of male germ cells to produce spermatozoa. This differentiation process requires a controlled program of stage-specific gene expression, which is precisely regulated at the transcriptional, posttranscriptional, and translational levels. We have identified a novel, testis-specific cytoplasmic poly (A) polymerase, TPAP (PAPβ), as a candidate molecule responsible for additional extension of poly (A) tails of specific mRNAs in haploid, round spermatids. The TPAP gene was most abundantly expressed coincident with the additional elongation of mRNA poly (A) tails in round spermatids. TPAP shared a high degree of amino acid sequence identity (86%) with a constitutively expressed nuclear poly (A) polymerase, PAPII. Despite the sequence conservation of functional elements, including three catalytic Asp residues, an ATP-binding site, and an RNA-binding domain, TPAP lacked an approximately 100-residue C-terminal sequence carrying one of two bipartite-type nuclear localization signals, and part of a Ser/Thr-rich domain found in PAPII. Indeed, TPAP was localized in the cytoplasm of spermatogenic cells as an enzymatically active 70-kDa protein. To elucidate the role of TPAP in spermatogenesis, we produced mutant mice lacking the functional TPAP gene, using homologous recombination in embryonic stem cells. The absence of TPAP resulted in the arrest of spermiogenesis at step 7. TPAP-deficient mice displayed impaired expression of haploid-specific genes that are required for the morphogenesis of spermatozoa. The TPAP deficiency also caused incomplete elongation of poly (A) tails of specific transcription factor mRNAs. Although the overall cellular level of the transcription factor TAF10 was unaffected, TAF10 was insufficiently transported into the nucleus of germ cells. These data demonstrate that TPAP affects the transport of at least TAF10 in the nucleus, possibly by additional polyadenylation-dependent translational activation of dormant mRNA encoding a transporter protein or possibly by TPAP itself. Our study shows a direct link between the deficiency of a cytoplasmic poly (A) polymerase, TPAP, and the arrest of mouse spermiogenesis, providing new insights into the regulation of haploid-specific gene expression by cytoplasmic polyadenylation in male germ cells.

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Several Phospholipase C (PLC) isof orms have been found in male and female mammalian gametes and splicing isoforms of PLCδ4 are predominantly expressed in testis. To understand a physiological function of PLCδ4, we generated PLCδ4 gene-deficient mice. PLCδ4 gene-disrupted male mice either produced few small litters or were sterile. In vivo and in vitro fertilization studies showed that PLCδ4 has a functional role in sperm at the early step of fertilization. We also observed that the calcium transients in eggs associated with fertilization were absent or delayed when we used PLCδ4^-/- sperm. PLCδ4^-/- sperm were unable to initiate the acrosome reaction, an exocytotic event required for fertilization and induced by interaction with the egg coat, the zona pellucida (ZP) . Next we have monitored Ca²⁺ responses in single sperm, and we found that the [Ca²⁺] _i increase in response to ZP is absent in PLCδ4^-/- sperm. Progesterone, another physiological inducer of the acrosome reaction, failed to induce sustained [Ca²⁺] _i increases in PLCδ^-/- sperm and consequently the acrosome reaction was partially inhibited. Calcium imaging studies revealed that the [Ca²⁺] _i increases induced by exposure to ZP and progesterone started at different sites within the sperm head, indicating that these agonists induce the acrosome reaction via different Ca²⁺ mechanisms. Furthermore, storeoperated channel (SOC) activity was severely impaired in PLCδ4^-/- sperm. These results support that PLCδ4 is an essential protein for calcium mobilization in sperm that drives acrosome reactions.

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Microinsemination is good tool for analyzing the fertilizing ability of germ. And it has also become practical for conserving genetic resources, generating transgenic animals, and treating male-factor infertility. Microinsemination is not need sperm-specific properties, such as sperm motility, acrosome reaction, or sperm-oocyte membrane fusion. So we can obtain normal offspring not only with mature (epididymal) and immature (testicular) spermatozoa, but also with immature spermatogenic cells. However, we have to product normal offspring with spermatogenic cells inapplicable to natural mating or in vitro fertilization (IVF) . To obtain many zygotes and offspring, we have to select the best protocols with consideration the biochemical genetic conditions of spermatogenic cells. Mature spermatozoa carry factor (s) that activate oocytes. In mice, this sperm-borne oocyte-activating factor (SOAF) is present in testicular spermatozoa, but not in round spermatids and spermatocytes. Therefore, one method of microinsemination with mouse round spermatids, is to activate the oocytes first, and then to inject round spermatids at telophase II stage. The fertilization rate was similar when microinseminaion was carried out with mature spermatozoa, elongated spermatids, or round spermatids. However, the rate of development to normal offspring depended on the maturity of these germ cells. The high degree of skill is required for microinsemination techniques, and it also needs to be master the knowledge, the handling, and the preservation method about spermatogenic cells. Successful fertilization with round spermatid in humans been reported from a few clinics. The rapidity of human application has raised some concern about potential health hazards for the progeny. More general use of these techniques and significant advances in germ cell biology will require technical improvements.

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