Selection Committee for the 2016 Academic Awards and Other Honors (Held on May 21, 2016)
\nKoji Akasaka, Takahiro Asami, Hiroshi Iida, Taisen Iguchi, Shigeru Kuratani, Kenji Tomioka, Toshiya Matsushima
\nChairperson Taisen Iguchi<\/p>\n\n\n\n
All four applicants for this award are outstanding researchers representing diverse fields of zoology, and they met the criteria outlined in the selection regulations for \"researchers who have achieved academically highly beneficial results and made important and remarkable contributions to the progress and development of zoology.\" As a result of detailed deliberations on the applicants' research content, research achievements, and degree of contribution to the progress and development of zoology, it was decided to recommend the following one individual to the Board of Directors.<\/p>\n\n\n\n
Member Masahisa Nakamura clarified that the androgen receptor (AR) gene and steroid hormone synthesis enzyme genes are involved in the masculinization of the gonads of wild wrinkled frogs, which possess two sex-determination systems (the XY and ZW systems). At the same time, he performed the first chromosomal mapping in amphibians and discovered DNA markers for sex determination in the wrinkled frog. Furthermore, he revealed that the AR gene is located on the sex chromosomes; while the Z-AR on the Z chromosome is expressed normally, the W-AR on the W chromosome is hardly expressed due to numerous mutations in the transcription region, making the Z-AR gene advantageous for the masculinization of the wrinkled frog. Subsequently, as a result of introducing the Z-AR gene into female (ZW) embryos, the female sex was incompletely converted to form ovotestes, demonstrating for the first time in the world that the AR gene is deeply involved in vertebrate sex determination. He also discovered that three-dimensional reconstruction is essential for elucidating the mechanisms of gonadal differentiation. It was determined that the achievements of Member Masahisa Nakamura, who has consistently conducted world-leading research in the study of amphibian gonadal differentiation and made immense contributions to the development of the field of reproductive endocrinology, make him well-suited for the Zoological Society of Japan Award.<\/p>\n\n\n\n
The animals I used for my research are not model animals such as mice, Xenopus laevis, medaka, or nematodes. At the Amphibian Research Center of Hiroshima University, where I was appointed, I encountered a wonderful animal: the wild-type Daruma pond frog (Glandirana rugosa). Although belonging to the same species, this frog has two sex-determination systems (the XY and ZW types). The East Japan and West Japan populations have XY types with homomorphic sex chromosomes, although the centromere positions differ; the Central Japan population has an XY type with heteromorphic sex chromosomes; and the North Japan population has a ZW type with heteromorphic sex chromosomes. In other words, there are four regional populations of the Daruma pond frog with different sex chromosome shapes. I became interested in why the same species possesses such diverse sex chromosomes. Furthermore, the sex of this frog can be relatively easily reversed from male to female, or vice versa, using steroid hormones. Therefore, it is an optimal animal for understanding the mechanisms of male and female gonad differentiation. There are no other vertebrates with such characteristics. I chose the Daruma pond frog as the animal to study the mechanisms of sex determination and gonad differentiation in amphibians. However, I had no prior experience using amphibians for research, the breeding season of the wild species was only two months, and furthermore, it was necessary to understand the characteristics of the research material, so it took a lot of time before the research got on track.<\/p>\n\n\n\n
As anyone who uses wild animals as research material will experience, there was a complete lack of molecular-level information for the wrinkled frog (Glandirana rugosa) as well. Therefore, we isolated many genes, including steroid hormone synthesis enzyme genes and other genes thought to be involved in gonad differentiation. This included many genes isolated for the first time in amphibians. Among them, we found that the androgen receptor (AR) gene and the steroid hormone-synthesizing enzyme gene CYP17 are deeply involved in the masculinization of the undifferentiated gonads of the wrinkled frog. These genes are not involved in the masculinization of gonads in mammals or fish. We also confirmed the expression of steroid hormone-synthesizing enzyme genes in differentiating male and female gonads, and verified enzyme activity at the protein level as well. Furthermore, we revealed that Sox3 promotes the transcription of the CYP19 aromatase gene, which is involved in estrogen synthesis, and plays a role in feminization. Recently, we isolated the anti-M\u00fcllerian hormone (AMH) gene for the first time in an amphibian and reported that the regulation of AMH gene expression differs from that in mammals, birds, and reptiles. These results indicate that steroid hormones are deeply involved in the gonad differentiation of the wrinkled frog, and that the mechanisms of gonad differentiation in vertebrates are diverse. Furthermore, we produced antibodies against wrinkled frog proteins\u2014such as Vasa, AR, CYP17, SF1, Sox9, Dmrt1, and Pat1a\u2014to conduct detailed histological analyses of male and female gonads, thereby clarifying the timing of sex determination and morphological changes during development in the gonads of the wrinkled frog. These achievements were made possible by starting the isolation of wrinkled frog genes and the production of antibodies from scratch. Undifferentiated male and female gonads cannot be distinguished morphologically. Determining whether there are sex-based differences in gene expression in undifferentiated gonads is only possible by identifying the sex of the gonads at the molecular level. My laboratory has established a molecular-level sex identification method for XY and ZW types of wrinkled frogs.<\/p>\n\n\n\n
Next, cytogenetic techniques revealed that two independent inversions have occurred in the sex chromosomes of the brownish frog. Using the FISH method, we demonstrated at the molecular level where these inversions occurred on the Y and W chromosomes. This is a first for amphibians. Subsequently, using cDNAs of 13 genes thought to be involved in gonad differentiation, we performed chromosome mapping for the first time in amphibians. Since the brownish frog has 26 chromosomes (n=13), statistically, one gene should be located on a sex chromosome. Fortunately, we found that three genes (AR, SF1, and Sox3) are located on the sex chromosomes, and furthermore, AR and SF1 are located within the inverted region of the W chromosome. Generally, genes located in inverted (non-homologous recombination) regions degenerate. Therefore, to clarify whether AR and SF1 are in the process of degeneration, we produced gynogenetic diploid embryos (ZZ and WW embryos) and examined the expression of these three genes. We found that the AR gene on the W chromosome (W-AR) is hardly expressed at all. In other words, in the brownish frog, the W-AR gene had degenerated due to an inversion occurring on the sex chromosome. We also discovered that the lack of W-AR expression is caused by mutations in the base sequence of the W-AR transcriptional regulatory region, and that the expression ratio of AR between wild-type ZZ male and ZW female embryos is 2:1. This 2:1 expression ratio of AR supports the degeneration of the W-AR gene on the sex chromosomes of the brownish frog. Because AR is strongly expressed in the genetically male gonads even before sex determination in the brownish frog, I concluded that the degeneration of the W-AR gene on the W chromosome created a sexual dimorphism in AR expression (female 1: male 2), which consequently led to AR becoming involved in gonadal sex determination. In mammals, knocking out the AR gene on the X chromosome does not alter the phenotype. This is a first-time discovery in vertebrates.<\/p>\n\n\n\n
The next goal was to create transgenic Japanese wrinkled frogs introduced with the AR gene to demonstrate that AR is actually involved in the masculinization of the gonads. Therefore, when the Z-AR gene was introduced into wild-type ZW female embryos to overexpress AR, the females did not form ovaries, but instead formed ovotestes. Partial sex reversal was caused by introducing the Z-AR gene. At this time, it was also verified at the protein level by tissue immunostaining using antibodies against the two proteins that the expression of CYP17 and AR genes increased similarly to the testicular level. Furthermore, it was found that androgens are necessary for the complete sex reversal from female to male. In ZW-type Japanese wrinkled frogs, the AR gene was involved in the sex determination of the gonads. Creating transgenic frogs of wild species involved many difficulties due to the short spawning season and the difficulty of obtaining high-quality artificially fertilized eggs, but it was successful.<\/p>\n\n\n\n
Finally, sex change is induced by steroid hormones in many animals, and wrinkled frogs undergo sex reversal as well. I believed that conventional two-dimensional analysis was insufficient to elucidate the mechanisms of sex reversal and gonad differentiation, and that three-dimensional tissue reconstruction was necessary. Therefore, I undertook the three-dimensional reconstruction of gonads, which no one had attempted before, and after three years of effort, successfully reconstructed sex-reversed gonads in 3D. This method allowed for multifaceted observation of the internal structure of sex-reversed tissues, yielding a wealth of information, such as the critical importance of the mesorchium\/mesovarium connecting the gonads and kidneys for sex reversal. I believe that three-dimensional reconstruction will be immensely useful in elucidating the mechanisms of gonad differentiation in other vertebrates.<\/p>\n\n\n\n
Thus, I have proceeded with research on the mechanisms of gonadal differentiation in amphibians using wild species. Through this research, I have experienced many difficulties as well as the joy of overcoming them. This is thanks to the many researchers and graduate and undergraduate students who cooperated with my research.<\/p>","protected":false},"excerpt":{"rendered":"\u5e73\u621028\u5e74\u5ea6\u5b66\u4f1a\u8cde\u7b49\u9078\u8003\u59d4\u54e1\u4f1a\u3000\uff08\u5e73\u621028\u5e745\u670821\u65e5\u958b\u50ac\uff09 \u3000\u8d64\u5742\u7532\u6cbb\u3001\u6d45\u898b\u5d07\u6bd4\u5442\u3001\u98ef\u7530\u3000\u5f18\u3001\u4e95\u53e3\u6cf0\u6cc9\u3001\u5009\u8c37\u3000\u6ecb\u3001\u5bcc\u5ca1\u61b2\u6cbb\u3001\u677e\u5cf6\u4fca\u4e5f \u3000\u59d4\u54e1\u9577\u3000\u4e95\u53e3\u6cf0\u6cc9 \u672c\u8cde\u306e\u51684\u540d\u306e\u5fdc\u52df\u8005\u306f\u3001\u52d5\u7269\u5b66\u306e\u591a\u69d8\u306a\u9818\u57df\u3092\u4ee3\u8868\u3059\u308b\u512a\u308c\u305f\u7814\u7a76\u8005 […]","protected":false},"author":5,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_jetpack_newsletter_access":"","_jetpack_dont_email_post_to_subs":false,"_jetpack_newsletter_tier_id":0,"_jetpack_memberships_contains_paywalled_content":false,"_jetpack_feature_clip_id":0,"_jetpack_memberships_contains_paid_content":false,"footnotes":"","jetpack_post_was_ever_published":false},"categories":[42,12],"tags":[],"class_list":["post-2021","post","type-post","status-publish","format-standard","hentry","category-topics","category-zs-award"],"acf":[],"jetpack_sharing_enabled":true,"jetpack_shortlink":"https:\/\/wp.me\/pb55P2-wB","jetpack_featured_media_url":"","_links":{"self":[{"href":"https:\/\/www.zoology.or.jp\/english\/wp-json\/wp\/v2\/posts\/2021","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.zoology.or.jp\/english\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.zoology.or.jp\/english\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.zoology.or.jp\/english\/wp-json\/wp\/v2\/users\/5"}],"replies":[{"embeddable":true,"href":"https:\/\/www.zoology.or.jp\/english\/wp-json\/wp\/v2\/comments?post=2021"}],"version-history":[{"count":1,"href":"https:\/\/www.zoology.or.jp\/english\/wp-json\/wp\/v2\/posts\/2021\/revisions"}],"predecessor-version":[{"id":2022,"href":"https:\/\/www.zoology.or.jp\/english\/wp-json\/wp\/v2\/posts\/2021\/revisions\/2022"}],"wp:attachment":[{"href":"https:\/\/www.zoology.or.jp\/english\/wp-json\/wp\/v2\/media?parent=2021"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.zoology.or.jp\/english\/wp-json\/wp\/v2\/categories?post=2021"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.zoology.or.jp\/english\/wp-json\/wp\/v2\/tags?post=2021"}],"curies":[{"name":"WP","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}