A research group led by Dr. Takehiko Ogawa of the Department of Urology (Prof. Yoshinobu Kubota) at the university has succeeded in producing functional sperm in vitro from murine spermatogenic stem cells. This method can be further refined to apply to human spermatogenesis, which will be useful for new diagnostic and therapeutic advancements in male infertility. The research was performed in collaboration with the group of Prof. Atsuo Ogura at the RIKEN Bioresource Centre.
Main results of the research:
Summary of the research
Spermatogenesis is a process of cell proliferation and differentiation taking a long period, 64 and 35 days in humans and mice, respectively. It is therefore very difficult to reproduce it in vitro, and there has been no success with mammalian specimens even though such trials started a century ago. This lack of an in vitro system for spermatogenesis has hindered our understanding of the process and development of therapeutic modalities for male infertility due to spermatogenic failure.
The research group started studying in vitro spermatogenesis several years ago using mice as a model animal with the belief that a system of in vitro spermatogenesis, if feasible, would markedly help to elucidate the molecular mechanisms of spermatogenesis as well as reveal the mechanism of spermatogenic failures.
They started with a traditional organ culture method because they thought that testicular somatic cells, such as Sertoli cells*1, are indispensable for proper spermatogenesis and that the structure of testis tissue should be used en bloc. They modified a classic gas-liquid interphase method*2 to a simpler form which uses agarose gel half-soaked in media on which testis tissues were placed (Figure 1). In order to make evaluation simple and convenient, they used two lines of transgenic mice*3 whose germ cells express GFP*4 at particular stages of spermatogenesis. Those GFP expressions tell us how far spermatogenesis is progressing during culture experiments (Figure 2). After testing various culture conditions, especially regarding the medium composition, they found that the completion of meiosis is possible with the system. They further improved the results and produced sperm with a neonatal mouse testis which contains only primitive spermatogenic cells (Figure 3). The resultant sperm were used to successfully produce offspring by micro-insemination (ICSI*5) (Figure 4).
Those pup mice grew normally and become fertile, producing the next generation by natural mating. In addition, researchers froze testis tissues and cryopreserved them. After thawing, the tissues also supported spermatogenesis up to sperm formation. This cryopreservation might be useful in the future to preserve testis tissues of cancer patients and applicable in clinical treatments of male infertility.
This is the first report of reproducing mammalian spermatogenesis completely in vitro, and successfully producing offspring with the resultant sperm. Refinements and the individualization of the method to animals, including humans, other than mice must be possible. Application of the culture method to basic and clinical research would clarify the molecular mechanism of spermatogenesis and contribute to the advancement of reproductive medicine for male infertility.
*1 Sertoli cells: Cells in the testis forming seminiferous tubules. They support many aspects of spermatogenesis: nutritional support, secrete several important growth factors for the proliferation and differentiation of germ cells, protection from immunoreactions, phagocytosis of apoptotic cells, structural support for the movement of germ cells, etc.
*2 Gas-liquid interphase method: A culture method placing tissues between gas (air) and liquid (medium) phases in order to make oxygen and nutrient supplies easily accessible.
*3 Transgenic mice: Genetically modified mice which incorporate exogenous genes.
*4 GFP (Green fluorescence protein): A protein originally isolated from jellyfish, which exhibits bright green fluorescence when exposed to blue elicitation light.
*5 ICSI (Intra-cytoplasmic sperm injection): A technique used to fertilize oocytes by injecting sperm directly into them with a glass needle.
Source: Yokohama City University
