Two activators of in vitro fertilization in mice from licorice

AbstractSystems for artificial insemination have been established in some animals. However, due to limited availability of sperm and oocytes, more effective treatment methodologies are required. Recently, it was demonstrated that the rate of in vitro fertilization (IVF) in mice was improved by adding a water extract of licorice (Glycyrrhiza uralensis), but not glycyrrhizic acid, to the artificial insemination culture medium. In this study, we examined licorice extract for active compounds using bioassay-guided separation. The results indicated that isoliquiritigenin and formononetin were the active molecules in licorice that contributed to the improved rate of IVF

In Vitro Fertilization Activators for Future

Artificial insemination is an indispensable technology for cattle breeding and is used for treating infertility in humans. Thus, new or improved methods are needed to increase the efficiency of artificial insemination. In vitro fertilization (IVF) has been developed in mice. Although many mouse lines produced using IVF have been preserved by freezing embryos and/or fertilized eggs, the more efficient IVF using freezing preservation or long‐term refrigeration of sperm is expected to preserve mouse lines more easily. In this chapter, we introduce the active compounds in licorice to improve the rate of IVF. We previously reported that the rate of IVF in mice was improved by adding a water extract of licorice (Glycyrrhiza uralensis), but not glycyrrhizin, to the artificial insemination culture medium. Recently, we analyzed the active ethyl acetate fraction containing high levels of flavonoids. This fraction was further purified by bioassay‐guided separation to isolate isoliquiritigenin and formononetin, which contributed to the improved rate of IVF. Isoliquiritigenin and formononetin may be useful therapeutic agents for infertility treatment

Genetic Variation in the Testis-Specific GSG3/CAPZA3 Gene Encoding for the Actin Regulatory Protein in Infertile Males

ｱｸﾁﾝｷｬｯﾋﾟﾝｸﾞ蛋白質 GSG3/CAPZA3 は､受精可能な精子の形態形成に重要な役割を担っている｡GSG3/CAPZA3 遺伝子は､ｲﾝﾄﾛﾝﾚｽ遺伝子として哺乳類に保存され､ﾏｳｽにおいてﾐｯｾﾝｽ変異が精子の減少と形態異常を誘導し､雄性不妊症の原因となることが知られている｡日本人において男性不妊症と GSG3/CAPZA3 遺伝子多型の関係を調べるため､261人の男性不妊症患者と139人の妊孕性が確認された男性ﾎﾞﾗﾝﾃｨｱの遺伝子多型を調べた｡その結果､妊孕性が確認された男性ﾎﾞﾗﾝﾃｨｱの一人にｱﾐﾉ酸置換を伴う一塩基多型がﾍﾃﾛ接合型として検出された｡このことから､GSG3/CAPZA3 遺伝子には､他の精子細胞特異的ｲﾝﾄﾛﾝﾚｽ遺伝子に比べて遺伝子多型が極めてわずかしか存在しないことが示唆された｡The actin capping protein GSG3/CAPZA3 plays a physiologically important role in maintaining sperm architecture for fertilization. The GSG3/CAPZA3 gene is conserved in mammals and lacks introns. A missense mutation in the CAPZA3 gene in mice causes male infertility by reducing the concentration and changing the shape of sperm. To investigate possible associations between GSG3/CAPZA3 gene variations and impaired spermatogenesis in Japanese males, we screened for mutations in GSG3/CAPZA3 using DNA from 261 sterile male patients and 139 male volunteers who were fertile. A single nucleotide polymorphism(SNP)was found in one sample in the heterozygous state in the fertile male volunteers. The results indicate that compared with other germ-cell-specific intronless genes the change was restricted in GSG3/CAPZA3 protein

Discovery of a TEKTIN-t/TEKT2 Gene Variant Encoding Sperm Flagellar Protein in Japanese Males

ﾃｸﾁﾝは､ﾀﾞｲﾆﾝとともに精子の鞭毛や繊毛の形成に関与したﾀﾝﾊﾟｸ質である｡ﾃｸﾁﾝには､ﾋﾄにおいて少なくとも6種類の遺伝子の存在が報告されている｡ﾃｸﾁﾝ遺伝子のうち､Tektin-t/Tekt2､Tekt3､もしくは Tekt4 が欠失することによって､精子の鞭毛が機能不全を起こし､なかでも Tektin-t/Tekt2 遺伝子の欠失は､雄性不妊症になることがﾏｳｽで示されている｡男性不妊症の原因にﾃｸﾁﾝ遺伝子の機能不全が関与することが予想される｡私たちは､ﾋﾄ TETIN-t/TEKT2 遺伝子の遺伝子多型と男性不妊症との関係について調べるため､282人の原因不明の男性不妊症患者と89人の妊孕性が確認された男性ﾎﾞﾗﾝﾃｨｱの遺伝子の解析を行った｡その結果､日本人男性不妊症患者に有意に検出される TETIN-t/TEKT2 遺伝子の変化は見られなかった｡これらの結果は､TETIN-t/TEKT2 は､日本人男性不妊症の原因遺伝子となる割合は非常に低くいことを示すとともに､今後の大規模な男性不妊症の原因となる遺伝子の解析に役に立つものと考えられる｡TEKTIN proteins contribute to the formation of cilia and flagella together with dynein. At least six types of TEKTIN genes have been reported in humans. The disruption of Tektin-t/Tekt2, Tekt3, or Tekt4 in mice causes sperm flagellar dysfunction, and Tektin-t/Tekt2 null male mice are infertile. To investigate the possible association between variations in TEKTIN-t/TEKT2 and impaired spermatogenesis in Japanese males, we screened for mutations in TEKTIN-t/TEKT2 using DNA from 282 sterile male patients and 89 proven-fertile male volunteers. Six polymorphisms were found in the open reading frame of TEKTIN-t/TEKT2, but no significant differences in genotype frequency were identified in the infertile subjects(P>0.05). We also did not detect a previously reported TEKTIN-t/TEKT2 gene variant in our subjects. These data may be applied to future large-scale genetic analyses of the association between genetic background and male infertility

SNPs within the Intron-less TAF7 Gene Encoding a General Transcription Factor in Japanese Males

TAF7 は転写開始と伸長に必須な転写開始前複合体に含まれる｡精巣において､TAF7 は細胞の増殖を伴う精原細胞と第一精母細胞の核に局在し､また､TAF7 の精巣特異的ｱｲｿｻﾞｲﾑである TAF7L は､精子細胞分化過程で核に局在する｡このことから､雄性生殖細胞では､細胞増殖を伴う精原細胞では TAF7 が､その後の精子分化過程では TAF7L が転写調節に機能していることが考えられる｡今回私たちは､細胞増殖をともなう精原細胞で発現する TAF7 ｲﾝﾄﾛﾝﾚｽ遺伝子について､男性不妊症と TAF7 の遺伝子多型の関係を調べるため､282人の日本人男性不妊症患者と96人の妊孕性が確認された日本人男性ﾎﾞﾗﾝﾃｨｱの遺伝子多型を調べた｡その結果､男性不妊症性患者において､5\u27 非翻訳領域に CTC の3塩基欠失と､ｱﾐﾉ酸置換を伴う一塩基多型がそれぞれﾍﾃﾛ接合型として各々一人づつ検出された｡ The National Center for Biotechnology Information には TAF7 に関して多くの一塩基多型が登録されているが､日本人男性に関してそれらの遺伝子多型はほとんどが検出されなかった｡新たに同定されたこれらの遺伝子多型は､体質と遺伝子多型の大規模な解析に役立つものと考えられる｡TAF7 is a part of the protein complex that is indispensable for the start of transcription. In the testis, TAF7 localizes on nuclei in spermatogonia and primary spermatocytes during proliferation. To examine whether genetic polymorphisms of the intron-less TAF7 gene are associated with male infertility, we screened TAF7 for genetic polymorphisms using DNA from 282 sterile and 96 fertile male volunteers. We identified 11 genetic polymorphisms in the TAF7 region. Although many single nucleotide polymorphisms (SNPs) have been reported in the SNP database of the National Center for Biotechnology Information, we found a novel CTC deletion and one SNP with an amino acid substitution in the TAF7 genomic region in infertile patients. These genetic polymorphisms might be causes of male sterility. These results will be useful for analyzing the association of traits and genetic polymorphisms in further large-scale genetic analyses

Gene Knockout and Metabolome Analysis of Carnitine/Organic Cation Transporter OCTN1

金沢大学医薬保健研究域薬学系Purpose: Solute carrier OCTN1 (SLC22A4) is an orphan transporter, the physiologically important substrate of which is still unidentified. The aim of the present study was to examine physiological roles of OCTN1. Methods: We first constructed octn1 gene knockout (octn1-/-) mice. Metabolome analysis was then performed to identify substrates in vivo. The possible association of the substrate identified with diseased conditions was further examined. Results: The metabolome analysis of blood and several organs indicated complete deficiency of a naturally occurring potent antioxidant ergothioneine in octn1-/- mice among 112 metabolites examined. Pharmacokinetic analyses after oral administration revealed the highest distribution to small intestines and extensive renal reabsorption of [3H]ergothioneine, both of which were much reduced in octn1-/- mice. The octn1-/- mice exhibited greater susceptibility to intestinal inflammation under the ischemia and reperfusion model. The blood ergothioneine concentration was also much reduced in Japanese patients with Crohn\u27s disease, compared with healthy volunteers and patients with another inflammatory bowel disease, ulcerative colitis. Conclusions: These results indicate that OCTN1 plays a pivotal role for maintenance of systemic and intestinal exposure of ergothioneine, which could be important for protective effects against intestinal tissue injuries, providing a possible diagnostic tool to distinguish the inflammatory bowel diseases. © 2010 Springer Science+Business Media, LLC

A Single Nucleotide Polymorphism within the Novel Sex-Linked Testis-Specific Retrotransposed PGAM4 Gene Influences Human Male Fertility

The development of novel fertilization treatments, including in vitro fertilization and intracytoplasmic injection, has made pregnancy possible regardless of the level of activity of the spermatozoa; however, the etiology of male-factor infertility is poorly understood. Multiple studies, primarily through the use of transgenic animals, have contributed to a list of candidate genes that may affect male infertility in humans. We examined single nucleotide polymorphisms (SNPs) as a cause of male infertility in an analysis of spermatogenesis-specific genes.We carried out the prevalence of SNPs in the coding region of phosphoglycerate mutase 4 (PGAM4) on the X chromosome by the direct sequencing of PCR-amplified DNA from male patients. Using RT-PCR and western blot analyses, we identified that PGAM4 is a functional retrogene that is expressed predominantly in the testes and is associated with male infertility. PGAM4 is expressed in post-meiotic stages, including spermatids and spermatozoa in the testes, and the principal piece of the flagellum and acrosome in ejaculated spermatozoa. A case-control study revealed that 4.5% of infertile patients carry the G75C polymorphism, which causes an amino acid substitution in the encoded protein. Furthermore, an assay for enzymatic activity demonstrated that this polymorphism decreases the enzyme's activity both in vitro and in vivo.These results suggest that PGAM4, an X-linked retrogene, is a fundamental gene in human male reproduction and may escape meiotic sex chromosome inactivation. These findings provide fresh insight into elucidating the mechanisms of male infertility

OAZ-t/OAZ3 Is Essential for Rigid Connection of Sperm Tails to Heads in Mouse

Polyamines are known to play important roles in the proliferation and differentiation of many types of cells. Although considerable amounts of polyamines are synthesized and stored in the testes, their roles remain unknown. Ornithine decarboxylase antizymes (OAZs) control the intracellular concentration of polyamines in a feedback manner. OAZ1 and OAZ2 are expressed ubiquitously, whereas OAZ-t/OAZ3 is expressed specifically in germline cells during spermiogenesis. OAZ-t reportedly binds to ornithine decarboxylase (ODC) and inactivates ODC activity. In a prior study, polyamines were capable of inducing a frameshift at the frameshift sequence of OAZ-t mRNA, resulting in the translation of OAZ-t. To investigate the physiological role of OAZ-t, we generated OAZ-t–disrupted mutant mice. Homozygous OAZ-t mutant males were infertile, although the polyamine concentrations of epididymides and testes were normal in these mice, and females were fertile. Sperm were successfully recovered from the epididymides of the mutant mice, but the heads and tails of the sperm cells were easily separated in culture medium during incubation. Results indicated that OAZ-t is essential for the formation of a rigid junction between the head and tail during spermatogenesis. The detached tails and heads were alive, and most of the headless tails showed straight forward movement. Although the tailless sperm failed to acrosome-react, the heads were capable of fertilizing eggs via intracytoplasmic sperm injection. OAZ-t likely plays a key role in haploid germ cell differentiation via the local concentration of polyamines