Skip to main content

Successful vitrification of whole juvenile testis in the critically endangered cyprinid honmoroko (Gnathopogon caerulescens)

  • Shogo Higaki (a1), Natsue Kuwata (a1), Kotaro Tanaka (a1), Ikuo Tooyama (a2), Yasuhiro Fujioka (a3), Noriyoshi Sakai (a4) and Tatsuyuki Takada (a5)...

Sperm cryopreservation is a valuable conservation method for endangered fish species. Here we report an easy and efficient cryopreservation method for juvenile whole testis by vitrification and successful sperm production from the vitrified whole testis via in vitro spermatogenesis in the critically endangered cyprinid honmoroko (Gnathopogon caerulescens). Juvenile testis (approximately 10 mm in length and 1 mm in width), consisting predominantly of spermatogonia, were aseptically dissected out and adherent fatty and non-testicular tissues were subsequently removed. Then, the testes were rapidly cooled on a nylon mesh by direct immersion in liquid nitrogen after serial exposures to pretreatment solution (PS), containing 2 M ethylene glycol (EG) and 1 M dimethyl sulfoxide (DMSO), for 20 or 30 min and vitrification solution (VS), containing 3 M EG, 2 M DMSO, and 0.5 M sucrose, for 5, 10, or 20 min. The highest survival rate of testicular cells (84.0%) was obtained from testes vitrified by immersion in PS for 20 min and in VS for 10 min. Spermatogonia were recovered from the vitrified testis by dissociation and cell culture produced many haploid sperm. Fertility and developmental competence were confirmed by in vitro fertilization assays. These results indicate that the vitrification of juvenile whole testis provides a new strategy to preserve the genetic resources of endangered fishes without affecting their reproductive population.

Corresponding author
All correspondence to: Tatsuyuki Takada. Laboratory of Cell Engineering, Department of Pharmaceutical Sciences, Ritsumeikan University, Nojihigashi 1-1-1, Kusatsu, Shiga 525–8577, Japan. Tel: +81 77 561 2569. Fax: +81 77 561 2569. E-mail:
Hide All
Amorim, C.A., Curaba, M., Van Langendonckt, A., Dolmans, M.-M. & Donnez, J. (2011). Vitrification as an alternative means of cryopreserving ovarian tissue. Reprod. Biomed. Online 23, 160–86.
Brockbank, K.G. & Taylor, M.J. (2006). Tissue preservation. In Advances in Biopreservation (ed. Baust, J.), pp. 157–96. London: CRC Press.
Fowler, A. & Toner, M. (2005). Cryo-injury and biopreservation. Ann. N. Y. Acad. Sci. 1066, 119–35.
Fujioka, Y. (1993). Induction of gynogenetic diploids and cytological studies in honmoroko Gnathopogon caerulescens. Nippon Suisan Gakkaishi 59, 493500.
Fujioka, T., Yasuchika, K., Nakamura, Y., Nakatsuji, N. & Suemori, H. (2004). A simple and efficient cryopreservation method for primate embryonic stem cells. Int. J. Dev. Biol. 48, 1149–54.
Higaki, S., Kawakami, Y., Eto, Y., Yamaha, E., Nagano, M., Katagiri, S., Takada, T. & Takahashi, Y. (2013). Cryopreservation of zebrafish (Danio rerio) primordial germ cells by vitrification of yolk-intact and yolk-depleted embryos using various cryoprotectant solutions. Cryobiology 67, 374–82.
Higaki, S., Shimada, M., Kawamoto, K., Todo, T., Kawasaki, T., Tooyama, I., Fujioka, Y., Sakai, N. & Takada, T. (2017). In. vitro differentiation of fertile sperm from cryopreserved spermatogonia in endangered endemic cyprinid honmoroko (Gnathopogon caerulescens). Sci. Rep. 7, 42852.
Leal, M.C., Cardoso, E.R., Nóbrega, R.H., Batlouni, S.R., Bogerd, J., França, L.R. & Schulz, R.W. (2009). Histological and stereological evaluation of zebrafish (Danio rerio) spermatogenesis with an emphasis on spermatogonial generations. Biol. Reprod. 81, 177–87.
Lee, S., Iwasaki, Y., Shikina, S. & Yoshizaki, G. (2013). Generation of functional eggs and sperm from cryopreserved whole testes. Proc. Natl. Acad. Sci. USA 110, 1640–5.
Luyet, B. & Hodapp, E. (1938). Revival of frog's spermatozoa vitrified in liquid air. Exp. Biol. Med. 39, 433–4.
Maki, I. (1968). Studies on the population dynamics of Gnathopogon. caerulescens sauvage (pisces), in Lake Biwa, Japan. Jap. J. Ecol. 18, 158–66 [in Japanese].
Mazur, P. (1984). Freezing of living cells: mechanisms and implications. Am. J. Physiol. Cell. Physiol. 247, 125–42.
Ministry of the Environment Government of Japan, Red List of Japanese Brackish and Freshwater Fishes 2015, (accessed 28 May 2017)
Nakamura, M. (1969). Honomoroko. In Cyprinid Fishes of Japan: Studies on the Life History of Cyprinid Fishes of Japan (ed. Nakamura, M.) pp. 117–25. Tokyo: Research Institute for Natural Resources, [in Japanese].
Okutsu, T., Shikina, S., Kanno, M., Takeuchi, Y. & Yoshizaki, G. (2007). Production of trout offspring from triploid salmon parents. Science 317, 1517.
Pšenička, M., Saito, T., Rodina, M. & Dzyuba, B. (2016). Cryopreservation of early stage Siberian sturgeon Acipenser baerii germ cells, comparison of whole tissue and dissociated cells. Cryobiology 72, 119–22.
Raz, E. (2000). The function and regulation of vasa-like genes in germ-cell development. Genome Biol. 1, 1017.
Sakai, N. (2002). Transmeiotic differentiation of zebrafish germ cells into functional sperm in culture. Development 129, 3359–65.
Sakai, N (2006). In vitro male germ cell cultures of zebrafish. Methods 39, 239–45.
Saragusty, J. & Arav, A. (2011). Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification. Reproduction 141, 119.
Scheffen, B. (1986). A simple and efficient procedure for preservation of mouse embryos by vitrification. Cryo-letters 7, 260–9.
Seki, S., Kusano, K., Lee, S., Iwasaki, Y., Yagisawa, M., Ishida, M., Hiratsuka, T., Sasado, T., Naruse, K. & Yoshizaki, G. (2017). Production of the medaka derived from vitrified whole testes by germ cell transplantation. Sci. Rep. 3, 43185.
Tiersch, T. (2001). Cryopreservation in aquarium fishes. Mar. Biotechnol. 3, S212–23.
Westerfield, M. (2007). Recipes-embryo extract. In The Zebrafish Book (ed. Westerfield, M.), p. 10.14. Eugene, Oregon, USA: The University of Oregon Press.
Yang, H. & Tiersch, T. (2009). Current status of sperm cryopreservation in biomedical research fish models: Zebrafish, medaka & Xiphophorus . Comp. Biochem. Physiol. C. Pharmacol. Toxicol. Endocrinol. 149, 224–32.
Yavin, S. & Arav, A. (2007). Measurement of essential physical properties of vitrification solutions. Theriogenology 67, 81–9.
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

  • ISSN: 0967-1994
  • EISSN: 1469-8730
  • URL: /core/journals/zygote
Please enter your name
Please enter a valid email address
Who would you like to send this to? *


Type Description Title
Supplementary materials

Higaki et al. supplementary material
Higaki et al. supplementary material 1

 Unknown (3.0 MB)
3.0 MB
Supplementary materials

Higaki et al. supplementary material
Higaki et al. supplementary material 2

 Video (738 KB)
738 KB
Supplementary materials

Higaki et al. supplementary material
Higaki et al. supplementary material 4

 Word (247 KB)
247 KB
Supplementary materials

Higaki et al. supplementary material
Higaki et al. supplementary material 3

 Video (463 KB)
463 KB


Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed