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BOR - Papers in Press, published online ahead of print March 21, 2007.
Biol Reprod 2007, 10.1095/biolreprod.106.056879
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BIOLOGY OF REPRODUCTION 77, 127–137 (2007)
DOI: 10.1095/biolreprod.106.056879
© 2007 by the Society for the Study of Reproduction, Inc.

Identification, Isolation, and In Vitro Culture of Porcine Gonocytes1

Sandeep Goel 3, Miki Sugimoto 4, Naojiro Minami 3, Masayasu Yamada 3, Shinichi Kume 4, and Hiroshi Imai 2 3

Laboratories of Reproductive Biology3 and Anatomy and Cell Biology,4 Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

ABSTRACT

Gonocytes are primitive germ cells that reside in the seminiferous tubules of neonatal testes and give rise to spermatogonia, thereby initiating spermatogenesis. Due to a lack of specific markers, the isolation and culture of these cells has proven to be difficult in the pig. In the present study, we show that a lectin, Dolichos biflorus agglutinin (DBA), which has specific affinity for primordial germ cells (PCGs) in the genital ridge, binds specifically to gonocytes in neonatal pig testes. The specific affinity of DBA for germ cells was progressively lost with age. This suggests that DBA binds strongly to primitive germ cells, such as gonocytes, weakly to primitive spermatogonia, and not at all to spermatogonia. The presence of alkaline phosphatase (AP) activity in the germ cells of neonatal pig testis confirmed the existence of primitive germ cells. Gonocytes from neonatal pig testis were purified, and a cell population that consisted of approximately 70% gonocytes was obtained, as indicated by the DBA binding assay. Purified gonocytes were cultured in DMEM/F12 supplemented with 10% FBS in the absence of any specific growth factors for 7 days. The cells remained viable and proliferated actively in culture. Initially, the gonocytes grew as focal colonies that transformed to three-dimensional colonies by 7 days of culture. Cultured germ cells expressed SSEA-1, a marker for embryonic stem (ES) cells, and were negative for the expression of somatic cell markers. These results should help to establish a male germ cell line that could be used for studying spermatogenesis in vitro and for genetic modification of pigs.

developmental biology, gamete biology, gametogenesis, spermatogenesis, testis

FOOTNOTES

1Supported in part by the grant (17658123) from the Ministry of Education, Science and Culture (to H.I.).

Correspondence: 2FAX: 81 75 753 6329; e-mail: imai{at}kais.kyoto-u.ac.jp






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Copyright © 2007 by the Society for the Study of Reproduction.