Searching for culture condition optimal for proliferation of spermatogonial stem cells

2004 Fiscal Year Final Research Report Summary

• Project/Area Number: 15591702
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Urology
• Research Institution: YOKOHAMA CITY UNIVERSITY
• Principal Investigator: OGAWA Takehiko Yokohama City University, School of Medicine, Associate Professor, 医学部, 講師 (50254222)
• Project Period (FY): 2003 – 2004
• Keywords: spermatogenesis / stem cell / cell culture

Research Abstract

Culturing of Spermatogonial Stem Cells
In April, 2003, it was reported that male germline stem cells, collected from neonatal mouse testes (strains of DBA/2 and ICR), were cultured for long period on feeder layer of mouse embryonic fibroblasts with 4 growth factors (EGF, bFGF, LIF, GDNF). We immediately repeated and confirmed results of the report and progressed it one step beyond by establishing germline stem cells (GS cells) from not only neonates but also from adult mice. The GS cells expanded exponentially in vitro. They produce sperm when transplanted recipient mouse testes. They did not produce tumor nor showed abnormal proliferation in the body of recipient mice. The GS cells was re-derived from recipient mouse testes, which showed that GS cells in vitro and spermatogonial stem cells in the testis were identical or mutually convertible.
Fertilization of recipient mice with spermatogonial transplantation
In order to make recipient mice fertile after spermatogonial transplantation, immature mice (10-15 days old) were treated with irradiation before transplantation. The aim of irradiation was to make empty niche in the testis by removing endogenous germ cells for efficient colonization of donor stem cells. The dose of irradiation turned out to be optimal at around 12Gy for that purpose. One to Three days after the irradiation, the recipient mice received spermatogonial transplantation. Two months after the transplantation onward, the recipient mice were mated with females to test their fertility. It was confirmed that mice became fertile fathered pups of donor germ cell origin at high rate (about 70%). When we used GS cells, the fertility rate of recipient mice became higher, around 80%. The important factors for making recipient fertile with donor germ cells therefore include age of recipient, stem cell concentration of donor cell suspension.

Research Products

• [Journal Article] Oligo-astheno-teratozoospermia in mice lacking Cnot7, a regulator of retinoid X receptor beta (2004)
  • Author(s): Nakamura T, Yao R, Ogawa T, et al.
  • Journal Title: Nature Genetics 36・5 Pages: 528-533
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Derivation and Morphological Characterization of Mouse Spermatogonial Stem Cell Lines. (2004)
  • Author(s): Ogawa T, Ohmura M, Tamura Y, Kita K, et al.
  • Journal Title: Arch.Histol.Cytol. 67・4 Pages: 297-306
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Abnormal sperm morphology caused by defects in Sertoli cells of Cnot7 knockout mice. (2004)
  • Author(s): Ogawa T, Ito C, Nakamura T, et al.
  • Journal Title: Arch.Histol.Cytol. 67・4 Pages: 307-314
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Oligo-astheno-teratozoospermia in mice lacking Cnot7, a regulator of retinoid X receptor beta (2004)
  • Author(s): Nakamura, T., Yao, R., Ogawa, T., Suzuki, T., Ito, C., Tsunekawa, N., Ajima, R., Miyasaka, T., Yoshida, Y., Toshimori, K., Noce, T., Yamamoto, T., Noda, T.
  • Journal Title: Nature Genetics 36 Pages: 528-533
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Derivation and Morphological Characterization of Mouse Spermatogonial Stem Cell Lines (2004)
  • Author(s): Ogawa, T., Ohmura, M., Tamura, Y., Kita, K., Ohbo, K., Suda, T., Kubota, Y.
  • Journal Title: Arch. Histol. Cytol. 67 Pages: 297-306
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Abnormal sperm morphology caused by defects in Sertoli cells of Cnot7 knockout mice (2004)
  • Author(s): Ogawa, T., Ito, C., Nakamura, T., Tamura, Y., Yamamoto, T., Noda, T., Kubota, Y., Toshimori, K.
  • Journal Title: Arch. Histol. Cytol. 67 Pages: 307-314
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Increment of murine spermatogonial cell number by gonadotropin- releasing hormone analogue is independent of stem cell factor - c-kit signal. (2003)
  • Author(s): Ohmura M, Ogawa T, et al.
  • Journal Title: Biology of Reproduction 68 Pages: 2304-2313
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Identification and characterization of stem cells in prepubertal spermatogenesis in mice. (2003)
  • Author(s): Ohbo K, Yoshida S, Ohmura M, Ohneda O, Ogawa T, et al.
  • Journal Title: Developmental Biology 258 Pages: 209-225
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Increment of murine spermatogonial cell number by Gonadotropin-releasing hormone analogue is independent of stem cell factor-c-kit signal (2003)
  • Author(s): Ohmura, M., Ogawa, T., Ono, M., Dezawa, M., Hosaka, M., Kubota, Y., Sawada, H.
  • Journal Title: Biol Reprod. 68 Pages: 2304-2313
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Identification and characterization of stem cells in prepubertal spermatogenesis in mice (2003)
  • Author(s): Ohbo, K., Yoshida, S., Ohmura, M., Ohneda, O., Ogawa, T., Tsuchiya, H., Kuwana, T., Kehler, J., Abe, K., Scholer, H.R., Suda, T.
  • Journal Title: Dev Biol. 258 Pages: 209-225
  • Description: 「研究成果報告書概要(欧文)」より
• [Book] 精子学「第17章 精細管内移植」 (2005)
  • Author(s): 小川毅彦 (分担執筆)
  • Publisher: 東京大学出版(印刷中)(未定)
  • Description: 「研究成果報告書概要(和文)」より