In vitro differentiation of fertile sperm from cryopreserved spermatogonia of the endangered endemic cyprinid honmoroko (Gnathopogon caerulescens).

Many endemic fish species are threatened with extinction. Conservation strategies and the restoration of endemic fish after extinction must therefore be investigated. Although sperm cryopreservation is indispensable for the conservation of endangered fishes, the limited number of mature fish and limited availability (volume and period) of sperm from small endemic fish hinders the optimization and practical use of this material. In this report, we demonstrate the in vitro differentiation of fertile sperm from cryopreserved spermatogonia of juveniles of the endangered small cyprinid honmoroko (Gnathopogon caerulescens), which is endemic to Lake Biwa in Japan. The entire process of spermatogenesis was recapitulated in vitro using cryopreserved spermatogonia of non-spawning adult and juvenile fish. The differentiation of sperm from spermatogonia was captured as a time-lapse video and confirmed by 5-ethynyl-2'-deoxyuridine (EdU) incorporation into sperm. Fertility was demonstrated by artificial insemination. These results suggest that the combination of cryopreservation of spermatogonia and in vitro sperm differentiation will provide a new and promising strategy for the preservation of paternal genetic materials.

Development and characterization of an embryonic cell line from endangered endemic cyprinid Honmoroko Gnathopogon caerulescens (Sauvage, 1883).

Establishing a cell line from endemic species facilitates the cell biological research of these species in the laboratory. In this study, an epithelium-like cell line RME1 was established from the blastula-stage embryos of the critically endangered cyprinid Honmoroko Gnathopogon caerulescens, which is endemic to ancient Lake Biwa in Japan. To the best of our knowledge, this is the first embryonic cell line from an endangered fish species. This cell line is well adapted to grow at 28°C in the culture medium, which was successfully used for establishing testicular and ovarian cell lines of G. caerulescens, and has displayed stable growth over 60 passages since its initiation in June 2011. Although RME1 did not express the genes detected in blastula-stage embryos, such as oct4, sox2, nanog, and klf4, it showed a high euploidy rate (2n = 50; 67.2%) with normal diploid karyotype morphology, suggesting that RME1 retains the genomic organization of G. caerulescens and can prove to be a useful tool to investigate the unique properties of endangered endemic fishes at cellular level.

Cynomolgus monkey induced pluripotent stem cells established by using exogenous genes derived from the same monkey species.

Induced pluripotent stem (iPS) cells established by introduction of the transgenes POU5F1 (also known as Oct3/4), SOX2, KLF4 and c-MYC have competence similar to embryonic stem (ES) cells. iPS cells generated from cynomolgus monkey somatic cells by using genes taken from the same species would be a particularly important resource, since various biomedical investigations, including studies on the safety and efficacy of drugs, medical technology development, and research resource development, have been performed using cynomolgus monkeys. In addition, the use of xenogeneic genes would cause complicating matters such as immune responses when they are expressed. In this study, therefore, we established iPS cells by infecting cells from the fetal liver and newborn skin with amphotropic retroviral vectors containing cDNAs for the cynomolgus monkey genes of POU5F1, SOX2, KLF4 and c-MYC. Flat colonies consisting of cells with large nuclei, similar to those in other primate ES cell lines, appeared and were stably maintained. These cell lines had normal chromosome numbers, expressed pluripotency markers and formed teratomas. We thus generated cynomolgus monkey iPS cell lines without the introduction of ecotropic retroviral receptors or other additional transgenes by using the four allogeneic transgenes. This may enable detailed analysis of the mechanisms underlying the reprogramming. In conclusion, we showed that iPS cells could be derived from cynomolgus monkey somatic cells. To the best of our knowledge, this is the first report on iPS cell lines established from cynomolgus monkey somatic cells by using genes from the same species.

Response to fish specific reproductive hormones and endocrine disrupting chemicals of a Sertoli cell line expressing endogenous receptors from an endemic cyprinid Gnathopogon caerulescens.

Fish Sertoli cells play a critical role in spermatogenesis by mediating androgen and progestogen signaling. Their hormonal response, however, considerably differ among species. Therefore it would be ideal to use Sertoli cells originated from the fish of interest to investigate the effects of hormones as well as endocrine disrupting chemicals (EDCs). The aim of this study was to investigate the responses to reproductive hormones and EDCs of a Sertoli cell line that we established from an endemic cyprinid Gnathopogon caerulescens. As the Sertoli cell line expressed endogenous androgen and progestogen receptors, we were able to detect hormone responses by transfecting only a reporter vector (pGL4.36) expressing luciferase under the control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR) promoter into the cell line. Unlike previous reporter gene assays using fish steroid hormone receptors expressed in mammalian cell lines, luciferase activities were induced by the fish specific androgen (11-ketotestosterone) and progestogen (17α,20ß-dihydroxy-4-pregnen-3-one), but not by testosterone and progesterone, at physiologically relevant concentrations. Furthermore, we found 4-nonylphenol (NP) but not bisphenol A showed strong anti-androgenic effects, implying that NP may have direct anti-androgenic effects on fish Sertoli cells in vivo. This is the first evidence, to the best of our knowledge, of anti-androgenic effects of NP in a fish Sertoli cell line. In addition, neither NP nor BPA showed anti-progestogenic effects. These results suggest that the Sertoli cell line established from the fish of interest can be a useful in vitro tool for investigating the mechanisms of reproductive hormones and EDCs in the specific fish.