Oxidative Stress Causes Masculinization of Genetically Female Medaka Without Elevating Cortisol.

Medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system. Sex reversal from female-to-male (masculinization of XX fish) can be induced through cortisol elevation from exposure to environmental stress such as high temperature during sexual differentiation. However, the effects of oxidative stress, generated via metabolic reactions and biological defense mechanisms, on the sexual differentiation of medaka are unclear. Here, we investigated the effect of oxidative stress on medaka sexual differentiation using hydrogen peroxide (H2O2), which induces oxidative stress in vertebrates. H2O2 treatment from 0 to 5 days post-hatching induced masculinization of wild-type XX medaka, but not of gonadal soma-derived growth factor (gsdf) or peroxisome proliferator-activated receptor alpha-a (pparaa) knockout XX fish. Co-treatment with an oxidative stress inhibitor caused masculinization recovery but co-treatment with a cortisol synthesis inhibitor did not. H2O2 treatment significantly upregulated gsdf and pparaa expression in XX medaka. However, H2O2 did not elevate cortisol levels in medaka larvae during sexual differentiation. These results strongly indicate that oxidative stress induces masculinization of XX medaka without causing elevation of cortisol.

Soy Isoflavones Induce Feminization of Japanese Eel (Anguilla japonica).

Under aquaculture conditions, Japanese eels (Anguilla japonica) produce a high percentage of males. However, females gain higher body weight and have better commercial value than males, and, therefore, a high female ratio is required in eel aquaculture. In this study, we examined the effects of isoflavones, genistein, and daidzein on sex differentiation and sex-specific genes of eels. To investigate the effects of these phytoestrogens on the gonadal sex, we explored the feminizing effects of soy isoflavones, genistein, and daidzein in a dose-dependent manner. The results showed that genistein induced feminization more efficiently than daidzein. To identify the molecular mechanisms of sex-specific genes, we performed a comprehensive expression analysis by quantitative real-time PCR and RNA sequencing. Phenotypic males and females were produced by feeding elvers a normal diet or an estradiol-17ß- or genistein-treated diet for 45 days. The results showed that female-specific genes were up-regulated and male-specific genes were down-regulated in the gonads, suggesting that genistein induces feminization by altering the molecular pathways responsible for eel sex differentiation.

The mutual history of Schlegel's Japanese gecko (Reptilia: Squamata: Gekkonidae) and humans inscribed in genes and ancient literature.

Knowing how the present distribution of organisms was formed is an essential issue in evolutionary ecology. Recently, the distribution of organisms on Earth has been significantly changed by human-mediated dispersal due to globalization. Therefore, significant attention has been paid to such processes. However, although humankind has taken considerable time to achieve modernization, the impact of ancient human activity on ecosystems has not yet been thoroughly studied. We hypothesized that ancient urban development and transitions had a non-negligible effect on species distribution. Inferring the impact of past human activity on ecosystems from ancient literature and verifying that impact by genetic analysis and human history is an effective means of tackling this problem. As geckos, a popular neighbor of human dwellings, are good material for this model, we performed this combination approach using Schlegel's Japanese gecko, Gekko japonicus. We show that G. japonicus migrated from China to the western Japanese archipelago before Christ. The gecko species dispersed itself from western to eastern the archipelago on a time scale of thousands of years. There are many synchronizations between the dispersal history of G. japonicus and the historical development of human society. It is suggested by such synchronizations that humans have influenced the distribution of G. japonicus many times throughout its dispersal history.

Transcriptional Regulation of Müllerian Inhibiting Substance (MIS) and Establishment of a Gonadal Somatic Cell Line Using mis-GFP Transgenic Medaka (Oryzias latipes).

In vertebrate germ cell differentiation, gonadal somatic cells and germ cells are closely related. By analyzing this relationship, it has recently been reported in mammals that primordial germ cells (PGCs), induced from pluripotent stem cells and germline stem cells, can differentiate into functional gametes when co-cultured in vitro with fetal gonadal somatic cells. In some fish species, differentiation into functional sperm by reaggregation or co-culture of gonadal somatic cells and germ cells has also been reported; however, the relationship between gonadal somatic cells and germ cells in these species is not well-understood. Here, we report the transcriptional regulation of Müllerian inhibiting substance (MIS) and the establishment of a gonadal somatic cell line using mis-GFP transgenic fish, in medaka (Oryzias latipes)-a fish model which offers many advantages for molecular genetics. MIS is a glycoprotein belonging to the transforming growth factor ß superfamily. In medaka, mis mRNA is expressed in gonadal somatic cells of both sexes before sex differentiation, and MIS regulates the proliferation of germ cells during this period. Using luciferase assays, we found that steroidogenic factor 1 (SF1) and liver receptor homolog 1 (LRH1) activate medaka mis gene transcription, probably by binding to the mis promoter. We also report that mis-GFP transgenic medaka emit GFP fluorescence specific to gonadal somatic cells in the gonads. By fusing Sertoli cells from transgenic medaka with a cell line derived from medaka hepatoma cancer, we produced a hybridoma cell line that expresses gonadal somatic cell-specific markers, including Sertoli and Leydig cell markers. Moreover, embryonic PGCs co-cultured with the established hybridoma, as feeder cells, proliferated and formed significant colonies after 1 week. PGCs cultured for 3 weeks expressed a germ cell marker dnd, as well as the meiotic markers sycp1 and sycp3. Thus, we here provide the first evidence in teleosts that we have successfully established a gonadal somatic cell-derived hybridoma that can induce both the proliferation and meiosis of germ cells.

Peroxisome proliferator-activated receptor alpha is involved in the temperature-induced sex differentiation of a vertebrate.

Medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system, similar to that of mammals. However, under high temperature conditions, XX medaka is masculinised by elevation of cortisol, the major teleost glucocorticoid. In this study, to identify novel factors in the gonads acting downstream from cortisol during sexual differentiation, we performed RNA sequencing (RNA-seq) analysis using the gonadal regions of larvae reared at normal temperature with and without cortisol, and at high temperature. The RNA-seq and real-time PCR analyses showed that expression of some peroxisome proliferator-activated receptor α (PPARα) signalling-targeted genes was increased by cortisol. PPARα agonist treatment induced masculinisation of XX medaka in some cases, and co-treatment of the agonist with cortisol further induced masculinisation, whereas treatment of pparaa knockout medaka with cortisol or the agonist did not induce masculinisation. This study provides the first evidence that PPARα is involved in environmental sex determination in vertebrates.

Involvement of Heat Shock Proteins on the Transcriptional Regulation of Corticotropin-Releasing Hormone in Medaka.

Medaka (Oryzias latipes) are teleost fish with a XX/XY sex determination system. Recently, it was reported that high temperature (HT) induced the masculinization of XX medaka by increasing the levels of cortisol, a major glucocorticoid produced by interrenal cells in teleosts. Cortisol secretion is regulated by adrenocorticotropic hormone (ACTH) secreted from the pituitary gland, which is partly regulated by corticotropin-releasing hormone (CRH) secreted from the hypothalamus. In teleosts, two crh paralogs, named crha and crhb, have been identified. Recently, the expression of crhb but not crha was upregulated by HT during gonadal sex differentiation period in medaka and loss-of-functions of its receptors under HT suppressed masculinization of XX medaka and increase of cortisol levels, suggesting that crhb is involved in masculinization induced by HT. However, the transcriptional regulation of crhb under HT has not been elucidated. We analyzed the gene expression pattern in the hypothalamus of medaka embryos incubated under HT using DNA microarray. The expressions of heat shock protein (hsp) genes, such as hsp70.1 and hsp30, were increased. Overexpression of hsp70.1 or hsp30 in cultured rat hypothalamic 4B cells significantly induced crh gene expression. Moreover, hypothalamic hsp70.1-overexpressing transgenic medaka also showed increased crhb gene expression that increased cortisol levels compared with fish incubated at a normal temperature. These results provide the first evidence that HSPs induce cortisol levels by elevating crhb gene expression in the hypothalamus.

Heat shock factor 1 protects germ cell proliferation during early ovarian differentiation in medaka.

The heat shock response is important for the viability of all living organisms. It involves the induction of heat shock proteins whose expression is mainly regulated by heat shock factor 1 (HSF1). Medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system. High water temperature (HT) inhibits the female-type proliferation of germ cells and induces the masculinisation of XX medaka in some cases during gonadal sex differentiation. Here, we investigated the roles of HSF1 on the proliferation of germ cells using HSF1 knockout medaka. Loss of HSF1 function under HT completely inhibited the female-type proliferation of germ cells, induced the expression of the anti-Mullerian hormone receptor type 2 (amhr2) and apoptosis-related genes, and suppressed that of the dead end (dnd) and heat shock protein-related genes. Moreover, the loss of HSF1 and AMHR2 function under HT recovered female-type proliferation in germ cells, while loss of HSF1 function under HT induced gonadal somatic cell apoptosis during early sex differentiation. These results strongly suggest that HSF1 under the HT protects the female-type proliferation of germ cells by inhibiting amhr2 expression in gonadal somatic cells. These findings provide new insights into the molecular mechanisms underlying environmental sex determination.