Transcriptional Regulation of Müllerian Inhibiting Substance (MIS) and Establishment of a Gonadal Somatic Cell Line Using mis-GFP Transgenic Medaka (Oryzias latipes)
In vertebrate germ cell differentiation, gonadal somatic cells and germ cells are closely related. By analyzing this relationship, it has recently been reported in mammals that primordial germ cells (PGCs), induced from pluripotent stem cells and germline stem cells, can differentiate into functiona...
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Frontiers Media S.A.
2020-09-01
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| Series: | Frontiers in Endocrinology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fendo.2020.578885/full |
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| author | Toshiaki Kawabe Toshiaki Kawabe Hiroyuki Kariya Seiji Hara Tsuyoshi Shirozu Eri Shiraishi Koki Mukai Takashi Yazawa Seiya Inoue Takeshi Kitano |
| author_facet | Toshiaki Kawabe Toshiaki Kawabe Hiroyuki Kariya Seiji Hara Tsuyoshi Shirozu Eri Shiraishi Koki Mukai Takashi Yazawa Seiya Inoue Takeshi Kitano |
| author_sort | Toshiaki Kawabe |
| collection | DOAJ |
| description | In vertebrate germ cell differentiation, gonadal somatic cells and germ cells are closely related. By analyzing this relationship, it has recently been reported in mammals that primordial germ cells (PGCs), induced from pluripotent stem cells and germline stem cells, can differentiate into functional gametes when co-cultured in vitro with fetal gonadal somatic cells. In some fish species, differentiation into functional sperm by reaggregation or co-culture of gonadal somatic cells and germ cells has also been reported; however, the relationship between gonadal somatic cells and germ cells in these species is not well-understood. Here, we report the transcriptional regulation of Müllerian inhibiting substance (MIS) and the establishment of a gonadal somatic cell line using mis-GFP transgenic fish, in medaka (Oryzias latipes)—a fish model which offers many advantages for molecular genetics. MIS is a glycoprotein belonging to the transforming growth factor β superfamily. In medaka, mis mRNA is expressed in gonadal somatic cells of both sexes before sex differentiation, and MIS regulates the proliferation of germ cells during this period. Using luciferase assays, we found that steroidogenic factor 1 (SF1) and liver receptor homolog 1 (LRH1) activate medaka mis gene transcription, probably by binding to the mis promoter. We also report that mis-GFP transgenic medaka emit GFP fluorescence specific to gonadal somatic cells in the gonads. By fusing Sertoli cells from transgenic medaka with a cell line derived from medaka hepatoma cancer, we produced a hybridoma cell line that expresses gonadal somatic cell-specific markers, including Sertoli and Leydig cell markers. Moreover, embryonic PGCs co-cultured with the established hybridoma, as feeder cells, proliferated and formed significant colonies after 1 week. PGCs cultured for 3 weeks expressed a germ cell marker dnd, as well as the meiotic markers sycp1 and sycp3. Thus, we here provide the first evidence in teleosts that we have successfully established a gonadal somatic cell-derived hybridoma that can induce both the proliferation and meiosis of germ cells. |
| first_indexed | 2024-12-10T23:59:16Z |
| format | Article |
| id | doaj.art-8d2f7350372e40e6aaf6257989cee6e1 |
| institution | Directory Open Access Journal |
| issn | 1664-2392 |
| language | English |
| last_indexed | 2024-12-10T23:59:16Z |
| publishDate | 2020-09-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Endocrinology |
| spelling | doaj.art-8d2f7350372e40e6aaf6257989cee6e12022-12-22T01:28:30ZengFrontiers Media S.A.Frontiers in Endocrinology1664-23922020-09-011110.3389/fendo.2020.578885578885Transcriptional Regulation of Müllerian Inhibiting Substance (MIS) and Establishment of a Gonadal Somatic Cell Line Using mis-GFP Transgenic Medaka (Oryzias latipes)Toshiaki Kawabe0Toshiaki Kawabe1Hiroyuki Kariya2Seiji Hara3Tsuyoshi Shirozu4Eri Shiraishi5Koki Mukai6Takashi Yazawa7Seiya Inoue8Takeshi Kitano9Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto, JapanARK Resource Co., Ltd., Kumamoto, JapanARK Resource Co., Ltd., Kumamoto, JapanDepartment of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto, JapanDepartment of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto, JapanDepartment of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto, JapanDepartment of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto, JapanDepartment of Biochemistry, Asahikawa Medical University, Asahikawa, JapanARK Resource Co., Ltd., Kumamoto, JapanDepartment of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto, JapanIn vertebrate germ cell differentiation, gonadal somatic cells and germ cells are closely related. By analyzing this relationship, it has recently been reported in mammals that primordial germ cells (PGCs), induced from pluripotent stem cells and germline stem cells, can differentiate into functional gametes when co-cultured in vitro with fetal gonadal somatic cells. In some fish species, differentiation into functional sperm by reaggregation or co-culture of gonadal somatic cells and germ cells has also been reported; however, the relationship between gonadal somatic cells and germ cells in these species is not well-understood. Here, we report the transcriptional regulation of Müllerian inhibiting substance (MIS) and the establishment of a gonadal somatic cell line using mis-GFP transgenic fish, in medaka (Oryzias latipes)—a fish model which offers many advantages for molecular genetics. MIS is a glycoprotein belonging to the transforming growth factor β superfamily. In medaka, mis mRNA is expressed in gonadal somatic cells of both sexes before sex differentiation, and MIS regulates the proliferation of germ cells during this period. Using luciferase assays, we found that steroidogenic factor 1 (SF1) and liver receptor homolog 1 (LRH1) activate medaka mis gene transcription, probably by binding to the mis promoter. We also report that mis-GFP transgenic medaka emit GFP fluorescence specific to gonadal somatic cells in the gonads. By fusing Sertoli cells from transgenic medaka with a cell line derived from medaka hepatoma cancer, we produced a hybridoma cell line that expresses gonadal somatic cell-specific markers, including Sertoli and Leydig cell markers. Moreover, embryonic PGCs co-cultured with the established hybridoma, as feeder cells, proliferated and formed significant colonies after 1 week. PGCs cultured for 3 weeks expressed a germ cell marker dnd, as well as the meiotic markers sycp1 and sycp3. Thus, we here provide the first evidence in teleosts that we have successfully established a gonadal somatic cell-derived hybridoma that can induce both the proliferation and meiosis of germ cells.https://www.frontiersin.org/article/10.3389/fendo.2020.578885/fullMISgerm cellgonadal somatic cellmedaka (Oryzias lapites)hybridoma |
| spellingShingle | Toshiaki Kawabe Toshiaki Kawabe Hiroyuki Kariya Seiji Hara Tsuyoshi Shirozu Eri Shiraishi Koki Mukai Takashi Yazawa Seiya Inoue Takeshi Kitano Transcriptional Regulation of Müllerian Inhibiting Substance (MIS) and Establishment of a Gonadal Somatic Cell Line Using mis-GFP Transgenic Medaka (Oryzias latipes) Frontiers in Endocrinology MIS germ cell gonadal somatic cell medaka (Oryzias lapites) hybridoma |
| title | Transcriptional Regulation of Müllerian Inhibiting Substance (MIS) and Establishment of a Gonadal Somatic Cell Line Using mis-GFP Transgenic Medaka (Oryzias latipes) |
| title_full | Transcriptional Regulation of Müllerian Inhibiting Substance (MIS) and Establishment of a Gonadal Somatic Cell Line Using mis-GFP Transgenic Medaka (Oryzias latipes) |
| title_fullStr | Transcriptional Regulation of Müllerian Inhibiting Substance (MIS) and Establishment of a Gonadal Somatic Cell Line Using mis-GFP Transgenic Medaka (Oryzias latipes) |
| title_full_unstemmed | Transcriptional Regulation of Müllerian Inhibiting Substance (MIS) and Establishment of a Gonadal Somatic Cell Line Using mis-GFP Transgenic Medaka (Oryzias latipes) |
| title_short | Transcriptional Regulation of Müllerian Inhibiting Substance (MIS) and Establishment of a Gonadal Somatic Cell Line Using mis-GFP Transgenic Medaka (Oryzias latipes) |
| title_sort | transcriptional regulation of mullerian inhibiting substance mis and establishment of a gonadal somatic cell line using mis gfp transgenic medaka oryzias latipes |
| topic | MIS germ cell gonadal somatic cell medaka (Oryzias lapites) hybridoma |
| url | https://www.frontiersin.org/article/10.3389/fendo.2020.578885/full |
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