Culture-space control is effective in promoting haploid cell formation and spermiogenesis in vitro in neonatal mice

Author:

Hashimoto Kiyoshi,Odaka Hisakazu,Ishikawa-Yamauchi Yu,Nagata Shino,Nakamura Hiroko,Kimura Hiroshi,Sato Takuya,Makiyama Kazuhide,Ogawa Takehiko

Abstract

AbstractThe classical organ culture method, in which tissue is placed at the gas‒liquid interphase, is effective at inducing mouse spermatogenesis. However, due to reginal variations in the supply of oxygen and nutrients within a tissue, the progress of spermatogenesis was observed only in limited areas of a tissue. In addition, haploid cell formation and its differentiation to spermatozoon, i.e. spermiogenesis, were infrequent and inefficient. Here, we show that the polydimethylsiloxane (PDMS)-chip ceiling (PC) method, which ensures a uniform supply of nutrients and oxygen throughout the tissue by pressing it into a thin, flat shape, can provide control over the culture space. We used this method to culture testis tissue from neonatal mice, aged 1 to 4 days, and found that modulating the culture space during the experiment by replacing one chip with another that had a higher ceiling effectively increased tissue growth. This adjustment also induced more efficient spermatogenesis, with the process of spermiogenesis being particularly promoted. Meiotic cells were observed from culture day 14 onward, and haploid cells were confirmed at the end of each experiment. This technique was also shown to be a sensitive assay for testicular toxicity. Culture-space control will be a critical regulation parameter for sophisticated tissue culture experiments.

Funder

Japan science and technology agency

Japan Society for the Promotion of Science

Yokohama City University

Publisher

Springer Science and Business Media LLC

Subject

Multidisciplinary

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