The mechanism to maintain stem cell properties and to exit into differentiation pathways is a pivotal question in stem cell research. Spermatogonia are the adult stem cells of the male germ line, which are used in biomedical research as a source of undifferentiated cells. The communication between germ line stem cells and specialized somatic cells (Sertoli cells and Leydig cells) plays important roles in stem cell maintenance, germ cell proliferation, and differentiation. With regard to the biology of stem cells and spermatogenesis, the medaka (Oryzias latipes) is used as a teleost model organism, and it is also used to assess the effects of endocrine disruptors on reproductive phenomena. However, the lack of suitable molecular markers hampers the detection, isolation and analysis of different testis cells including gonial stem cells and Leydig cells. Therefore, oct4, sox2 and cyp11b were chosen to create transgenic reporter lines for the labeling of stem cells and Leydig cells, respectively. The present study had the aim to examine the temporal and spatial expression of the respective genes during embryonic development and in adult gonads of the medaka, and to describe the application of these transgenic lines in stem cell biology and reproductive biology. The mCherry expression in transgenic fish of the line FSI-Tg(sox2-mCherry)17 marks embryonic stem cells, Leydig cells and interstitial cells in adult testis. Faithful EGFP and DsRed expression in transgenic reporters strains for oct4 and cyp11b mimics the endogenous expression of oct4/pou2 and cyp11b-protein, respectively. The reporter gene expression in the strains FSI-Tg(oct4-EGFP)9 and FSI-Tg(oct4-EGFP)A allows the visualization of oct4 positive cells during embryonic development, PGCs, early germ cells and adult gonial cells. The Leydig cells express brightly green or red fluorescence in the medaka strains FSI-Tg(cyp11b-EGFP)20 and FSI-Tg(cyp11b-DsRed)1434, respectively, allowing the easy identification of Leydig cells in adult testis. The oct4-EGFP reporter labels medaka embryonic and spermatogonial stem cells, in which the spermatogonial stem cells at the ends of the testicular lobules show brightly green fluorescence. The transgenic expression in stem cells is also shown in the flow plot of primary testis cells. The spermatogonia are the largest cells and have the strongest fluorescence, which decreased upon differentiation. Therefore, the oct4-EGFP reporter strains will provide an opportunity to detect and to isolate the EGFP expressing cells for transplantation. These strains will also facilitate further experiments on the effects of drugs or hypoxia on these cells, because the strongest EGFP expressing cells can be easily detected in transgenic lines. Labeling of Leydig cells in cyp11b reporter lines opens a new area to study the seasonal variation of spermatogenesis. The medaka is a seasonal breeder in its natural habitat and the simulation of seasonal changes allows the simultaneous quantitative analysis of oct4-EGFP and cyp11b-DsRed expressing cells under such conditions.

The mechanism to maintain stem cell properties and to exit into differentiation pathways is a pivotal question in stem cell research. Spermatogonia are the adult stem cells of the male germ line, which are used in biomedical research as a source of undifferentiated cells. The communication between germ line stem cells and specialized somatic cells (Sertoli cells and Leydig cells) plays important roles in stem cell maintenance, germ cell proliferation, and differentiation. With regard to the biology of stem cells and spermatogenesis, the medaka (Oryzias latipes) is used as a teleost model organism, and it is also used to assess the effects of endocrine disruptors on reproductive phenomena. However, the lack of suitable molecular markers hampers the detection, isolation and analysis of different testis cells including gonial stem cells and Leydig cells. Therefore, oct4, sox2 and cyp11b were chosen to create transgenic reporter lines for the labeling of stem cells and Leydig cells, respectively. The present study had the aim to examine the temporal and spatial expression of the respective genes during embryonic development and in adult gonads of the medaka, and to describe the application of these transgenic lines in stem cell biology and reproductive biology. The mCherry expression in transgenic fish of the line FSI-Tg(sox2-mCherry)17 marks embryonic stem cells, Leydig cells and interstitial cells in adult testis. Faithful EGFP and DsRed expression in transgenic reporters strains for oct4 and cyp11b mimics the endogenous expression of oct4/pou2 and cyp11b-protein, respectively. The reporter gene expression in the strains FSI-Tg(oct4-EGFP)9 and FSI-Tg(oct4-EGFP)A allows the visualization of oct4 positive cells during embryonic development, PGCs, early germ cells and adult gonial cells. The Leydig cells express brightly green or red fluorescence in the medaka strains FSI-Tg(cyp11b-EGFP)20 and FSI-Tg(cyp11b-DsRed)1434, respectively, allowing the easy identification of Leydig cells in adult testis. The oct4-EGFP reporter labels medaka embryonic and spermatogonial stem cells, in which the spermatogonial stem cells at the ends of the testicular lobules show brightly green fluorescence. The transgenic expression in stem cells is also shown in the flow plot of primary testis cells. The spermatogonia are the largest cells and have the strongest fluorescence, which decreased upon differentiation. Therefore, the oct4-EGFP reporter strains will provide an opportunity to detect and to isolate the EGFP expressing cells for transplantation. These strains will also facilitate further experiments on the effects of drugs or hypoxia on these cells, because the strongest EGFP expressing cells can be easily detected in transgenic lines. Labeling of Leydig cells in cyp11b reporter lines opens a new area to study the seasonal variation of spermatogenesis. The medaka is a seasonal breeder in its natural habitat and the simulation of seasonal changes allows the simultaneous quantitative analysis of oct4-EGFP and cyp11b-DsRed expressing cells under such conditions.