Nonylphenol reduced the number of haploids in in vitro spermatogenesis of the endangered cyprinid Gnathopogon caerulescens.

Nonylphenol (NP), an endocrine disrupting chemical, is widely used in industrial and agricultural processes, causing NP influx into aquatic environments. NP induces hormonal imbalance, and male feminization, and reduces germ cell production during spermatogenesis; however, the mechanism by which it affects spermatogenesis remains unknown. Here, we investigated the effect of NP on spermatogenesis in honmoroko (Gnathopogon caerulescens), an endangered fish endemic to Lake Biwa, Japan, using an in vitro differentiation system. We collected spermatogonia from the testes of non-spawning G. caerulescens and subjected them to suspension culture. The spermatogonia differentiated into flagellated spermatozoa in 3 weeks, regardless of the presence of NP. NP concentrations as low as 1 nM caused a decrease in the number of germ cells in a dose-dependent manner, whereas the number of somatic cells decreased only at a high concentration of 1 µM. Flow cytometric analysis revealed that the decrease in germ cell number was attributed to haploids (spermatids and spermatozoa); the number of spermatogonia and spermatocytes was not affected by NP treatment. This result is consistent with the hypothesis that NP might repress the second meiosis or induce apoptosis in haploids. This study demonstrated that the combination of in vitro germ cell differentiation and flow cytometric analysis is useful for evaluating the direct effects of NP on germ cell differentiation in endangered endemic fish.

Germ cell-specific expression of Venus by Tol2-mediated transgenesis in endangered endemic cyprinid Honmoroko (Gnathopogon caerulescens).

Fishes expressing a fluorescent protein in germ cells are useful to perform germ cell transfer experiments for conservation study. Nonetheless, no such fish has been generated in endangered endemic fishes. In this study, we tried to produce a fish expressing Venus fluorescent protein in germ cells using Honmoroko (Gnathopogon caerulescens), which is one of the threatened small cyprinid endemic to the ancient Lake Biwa in Japan. To achieve germ cell-specific expression of Venus, we used piwil1 (formally known as ziwi) promoter and Tol2 transposon system. Following the co-injection of the piwil1-Venus expression vector and the Tol2 transposase mRNA into fertilized eggs, presumptive transgenic fish were reared. At 7 months of post-fertilization, about 19% (10/52) of the examined larvae showed Venus fluorescence in their gonad specifically. Immunohistological staining and in vitro spermatogenesis using gonads of the juvenile founder fish revealed that Venus expression was detected in spermatogonia and spermatocyte in male, and oogonia and stage I and II oocytes in female. These results indicate that the Tol2 transposon and zebrafish piwil1 promoter enabled gene transfer and germ cell-specific expression of Venus in G. caerulescens. In addition, in vitro culture of juvenile spermatogonia enables the rapid validation of temporal expression of transgene during spermatogenesis.

Daily growth increments in otoliths of wild-caught honmoroko Gnathopogon caerulescens.

Otolith growth increments in wild-caught alizarin complex one (ALC)-marked honmoroko Gnathopogon caerulescens were examined to verify the veracity of the age determination method in cyprinids. ALC-marked G. caerulescens recaptured from their natural environment had lapilli increment counts outside the ALC ring mark that had formed on a daily basis during the juvenile stage. This apparently being the first direct evidence of daily periodicity of otolith increment formation in wild-caught cyprinids.

Cryopreservation of male and female gonial cells by vitrification in the critically endangered cyprinid honmoroko Gnathopogon caerulescens.

We investigated the feasibility of cryopreservation of spermatogonia and oogonia in the critically endangered cyprinid honmoroko Gnathopogon caerulescens using slow-cooling (freezing) and rapid-cooling (vitrification) methods. Initially, we examined the testicular cell toxicities and glass-forming properties of the five cryoprotectants: ethylene glycol (EG), glycerol (GC), dimethyl sulfoxide (DMSO), propylene glycol (PG), and 1,3-butylene glycol (BG), and we determined cryoprotectant concentrations that are suitable for freezing and vitrification solutions, respectively. Subsequently, we prepared the freezing solutions of EG, GC, DMSO, PG, and BG at 3, 2, 3, 2, and 2 M and vitrification solutions at 7, 6, 5, 5, and 4 M, respectively. Following the cryopreservation of the testicular cells mainly containing early-stage spermatogenic cells (e.g., spermatogonia and primary spermatocytes), cells were cultured for 7 days and immunochemically stained against germ cell marker protein Vasa. Areas occupied by Vasa-positive cells indicated that vitrification led to better survival of germ cells than the freezing method, and the best result was obtained with 5 M PG, about 50% recovery of germ cells following vitrification. In the case of ovarian cells containing oogonia and stage I, II, and IIIa oocytes, vitrification with 5 M DMSO resulted the best survival of oogonia, with equivalent cell numbers to those cultured without vitrification. The present data suggest that male and female gonial cells of the endangered species G. caerulescens can be efficiently cryopreserved using suitable cryoprotectants for spermatogonia and oogonia, respectively.

Successful vitrification of whole juvenile testis in the critically endangered cyprinid honmoroko (Gnathopogon caerulescens).

Sperm cryopreservation is a valuable conservation method for endangered fish species. Here we report an easy and efficient cryopreservation method for juvenile whole testis by vitrification and successful sperm production from the vitrified whole testis via in vitro spermatogenesis in the critically endangered cyprinid honmoroko (Gnathopogon caerulescens). Juvenile testis (approximately 10 mm in length and 1 mm in width), consisting predominantly of spermatogonia, were aseptically dissected out and adherent fatty and non-testicular tissues were subsequently removed. Then, the testes were rapidly cooled on a nylon mesh by direct immersion in liquid nitrogen after serial exposures to pretreatment solution (PS), containing 2 M ethylene glycol (EG) and 1 M dimethyl sulfoxide (DMSO), for 20 or 30 min and vitrification solution (VS), containing 3 M EG, 2 M DMSO, and 0.5 M sucrose, for 5, 10, or 20 min. The highest survival rate of testicular cells (84.0%) was obtained from testes vitrified by immersion in PS for 20 min and in VS for 10 min. Spermatogonia were recovered from the vitrified testis by dissociation and cell culture produced many haploid sperm. Fertility and developmental competence were confirmed by in vitro fertilization assays. These results indicate that the vitrification of juvenile whole testis provides a new strategy to preserve the genetic resources of endangered fishes without affecting their reproductive population.

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.

Lack of hormonal stimulation prevents the landlocked Biwa salmon (Oncorhynchus masou subspecies) from adapting to seawater.

Landlocking of salmon relaxes selective pressures on hypoosmoregulatory ability (seawater adaptability) and may lead to the abandonment of its physiological system. However, little is known about the mechanism and consequence of the process. Biwa salmon is a strain/subspecies of Oncorhynchus masou that has been landlocked in Lake Biwa for an exceptionally long period (about 500,000 years) and has low ability to adapt to seawater. We compared activity of gill Na(+),K(+)-ATPase (NKA) of Biwa salmon with those of anadromous strains of the same species (masu and amago salmon) during downstream migration periods and after exogenous hormone treatment. Gill NKA activity in anadromous strains increased during their migration periods, while that in Biwa salmon remained low. However, treatments of Biwa salmon with growth hormone (GH) and cortisol increased gill NKA activity. Cortisol treatment also improved the whole body seawater adaptability of Biwa salmon. Receptors for GH and cortisol responded to hormonal treatments, whereas their mRNA levels during downstream migration period were essentially unchanged in Biwa salmon. Circulating levels of cortisol in masu salmon showed a peak during downstream migration period, while no such increase was seen in Biwa salmon. The present results indicate that Biwa salmon can improve its seawater adaptability by exogenous hormonal treatment, and hormone receptors are capable of responding to the signals. However, secretion of the endogenous hormone (cortisol) was not activated during the downstream migration period, which explains, at least in part, their low ability to adapt to seawater.

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.

Establishment of testicular and ovarian cell lines from Honmoroko (Gnathopogon caerulescens).

We succeeded to establish cell lines from endemic fish species Honmoroko Gnathopogon caerulescens, which inhabits Lake Biwa, the third oldest lake in the world. Two cell lines designated as RMT1 and RMO1 were established from testis and ovary of G. caerulescens, respectively. These cell lines were initially cultured in Leibovitz's L-15 medium supplemented with fetal bovine serum (FBS), fish embryo extract, epidermal growth factor, and basic fibroblast growth factor. Further addition of forskolin and ß-mercaptoethanol was required to establish and maintain these cell lines for more than 60 passages. RMT1 and RMO1 cells showed fibroblast- and epithelial-like morphology, respectively. From immunocytochemical staining and gene expression patterns, RMT1 cells showed a characteristic of testicular Sertoli cells and RMO1 cells did that of ovarian theca cells. Both RMT1 and RMO1 cells multiplied well in the medium supplemented with 10 % FBS at 28 °C and their minimum population doubling times were 24.4 and 28.8 h, respectively. At the 45th passage, most of the RMT1 and RMO1 cells had a hyperploid set of chromosomes (67.3 and 96.1 %, respectively). Cells with normal diploid chromosome set were not observed. RMT1 cells were transfected with an enhanced green fluorescent protein (EGFP) expression vector and human elongation factor 1 α promoter worked efficiently to express EGFP. In addition, EGFP-expressing cell lines were also established, suggesting that the cell lines could be utilized as an in vitro monitor system (biosensor) for the evaluation of endocrine disruptors which might affect gonadal function.