Thyroid axis participates in high-temperature-induced male sex reversal through its activation by the stress response.

Environmental changes alter the sex fate in about 15% of vertebrate orders, mainly in ectotherms such as fish and reptiles. However, the effects of temperature changes on the endocrine and molecular processes controlling gonadal sex determination are not fully understood. Here, we provide evidence that thyroid hormones (THs) act as co-players in heat-induced masculinization through interactions with the stress axis to promote testicular development. We first demonstrated that the thyroid axis (through thyroid-related genes and T3 levels) is highly active in males during the gonadal development in medaka (Oryzias latipes). Similarly, T3 treatments promoted female-to-male sex reversal in XX embryos. Subsequently, embryonic exposure to temperature-induced stress up-regulated the genes related to the thyroid and stress axes with a final increase in T3 levels. In this context, we show that blocking the stress axis response by the loss of function of the corticotropin-releasing hormone receptors suppresses thyroid-stimulating hormone expression, therefore, heat-induced activation of the thyroid axis. Thus, our data showed that early activation of the stress axis and, in consequence, the TH axis, too, leaves us with that both being important endocrine players in inducing female-to-male reversal, which can help predict possible upcoming physiological impacts of global warming on fish populations.

Paternal methotrexate exposure affects sperm small RNA content and causes craniofacial defects in the offspring.

Folate is an essential vitamin for vertebrate embryo development. Methotrexate (MTX) is a folate antagonist that is widely prescribed for autoimmune diseases, blood and solid organ malignancies, and dermatologic diseases. Although it is highly contraindicated for pregnant women, because it is associated with an increased risk of multiple birth defects, the effect of paternal MTX exposure on their offspring has been largely unexplored. Here, we found MTX treatment of adult medaka male fish (Oryzias latipes) causes cranial cartilage defects in their offspring. Small non-coding RNA (sncRNAs) sequencing in the sperm of MTX treated males identify differential expression of a subset of tRNAs, with higher abundance for specific 5' tRNA halves. Sperm RNA methylation analysis on MTX treated males shows that m5C is the most abundant and differential modification found in RNAs ranging in size from 50 to 90 nucleotides, predominantly tRNAs, and that it correlates with greater testicular Dnmt2 methyltransferase expression. Injection of sperm small RNA fractions from MTX-treated males into normal fertilized eggs generated cranial cartilage defects in the offspring. Overall, our data suggest that paternal MTX exposure alters sperm sncRNAs expression and modifications that may contribute to developmental defects in their offspring.

The complete mitochondrial genome of the fine flounder Paralichthys adspersus revealed by next-generation sequencing.

The complete mitochondrial genome of the fine flounder Paralichthys adspersus, was determined for the first time through Next Generation Sequencing (NGS) approach. The mitogenome (GenBank accession no. MW288827) has 17,060 bp in length and consisted of the well-known 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and the control region. The overall nucleotide composition of the whole mitogenome was A: 27.5%, C: 29.5%, G: 17.1%, and T: 25.9%. Phylogenetic analyses based on 12 protein-coding genes clustered P. adspersus in the monophyletic Paralichthyidae clade, showing the closest phylogenetic relationship with its congeneric species P. olivaceus.

Cystic proliferation of germline stem cells is necessary to reproductive success and normal mating behavior in medaka.

The production of an adequate number of gametes is necessary for normal reproduction, for which the regulation of proliferation from early gonadal development to adulthood is key in both sexes. Cystic proliferation of germline stem cells is an especially important step prior to the beginning of meiosis; however, the molecular regulators of this proliferation remain elusive in vertebrates. Here, we report that ndrg1b is an important regulator of cystic proliferation in medaka. We generated mutants of ndrg1b that led to a disruption of cystic proliferation of germ cells. This loss of cystic proliferation was observed from embryogenic to adult stages, impacting the success of gamete production and reproductive parameters such as spawning and fertilization. Interestingly, the depletion of cystic proliferation also impacted male sexual behavior, with a decrease of mating vigor. These data illustrate why it is also necessary to consider gamete production capacity in order to analyze reproductive behavior.

Stress and sex determination in fish: from brain to gonads.

Fish present remarkable malleability regarding gonadal sex fate. This phenotypic plasticity enables an organism to adapt to changes in the environment by responding with different phenotypes. The gonad and the brain present this extraordinary plasticity. These organs are involved in the response to environmental stressors to direct gonadal fate, inducing sex change or sex reversal in hermaphroditic and gonochoristic fish, respectively. The presence of such molecular and endocrine plasticity gives this group a large repertoire of possibilities against a continuously changing environment, resulting in the highest radiation of reproduction strategies described in vertebrates. In this review, we provide a broad and comparative view of tremendous radiation of sex determination mechanisms to direct gonadal fate. New results have established that the driving mechanism involves early response to environmental stressors by the brain plus high plasticity of gonadal differentiation and androgens as by-products of stress inactivation. In addition to the stress axis, two other major axes - the hypothalamic-pituitary-gonadal axis and the hypothalamic-pituitary-thyroid axis, which are well known for their participation in the regulation of reproduction - have been proposed to reinforce brain-gonadal interrelationships in the fate of the gonad.

High temperature stress response is not sexually dimorphic at the whole-body level and is dependent on androgens to induce sex reversal.

The understanding of the molecular and endocrine mechanisms behind environmentally-induced sex reversal in fish is of great importance in the context of predicting the potential effects of climate change, especially increasing temperature. Here, we demonstrate the global effects of high temperature on genome-wide transcription in medaka (Oryzias latipes) during early development. Interestingly, data analysis did not show sexual dimorphic changes, demonstrating that thermal stress is not dependent on genotypic sex. Additionally, our results revealed significant changes in several pathways under high temperature, such as stress response from brain, steroid biosynthesis, epigenetic mechanisms, and thyroid hormone biosynthesis, among others. These microarray data raised the question of what the exact molecular and hormonal mechanisms of action are for female-to-male sex reversal under high temperatures in fish. Complementary gene expression analysis revealed that androgen-related genes increase in females (XX) experiencing high water temperature. To test the involvement of androgens in thermal-induced sex reversal, an androgen antagonist was used to treat XX medaka under a high-temperature setup. Data clearly demonstrated failure of female-to-male sex reversal when androgen action is inhibited, corroborating the importance of androgens in environmentally-induced sex reversal.

Steroid hormones and estrogenic activity in the wastewater outfall and receiving waters of the Chascomús chained shallow lakes system (Argentina).

Natural and synthetic steroid hormones, excreted by humans and farmed animals, have been considered as important sources of environmental endocrine disruptors. A suite of estrogens, androgens and progestogens was measured in the wastewater treatment plant outfall (WWTPO) of Chascomús city (Buenos Aires province, Argentina), and receiving waters located downstream and upstream from the WWTPO, using solid phase extraction and high-performance liquid chromatography mass spectrometry. The following natural hormones were measured: 17ß-estradiol (E2), estrone (E1), estriol (E3), testosterone (T), 5α-dihydrotestosterone (DHT), progesterone (P), 17-hydroxyprogesterone (17OHP) and the synthetic estrogen 17α-ethinylestradiol (EE2). Also, in order to complement the analytical method, the estrogenic activity in these surface water samples was evaluated using the in vitro transactivation bioassay that measures the estrogen receptor (ER) activity using mammalian cells. All-natural steroid hormones measured, except 17OHP, were detected in all analyzed water samples. E3, E1, EE2 and DHT were the most abundant and frequently detected. Downstream of the WWTPO, the concentration levels of all compounds decreased reaching low levels at 4500 m from the WWTPO. Upstream, 1500 m from the WWTPO, six out of eight steroid hormones analyzed were detected: DHT, T, P, 17OHP, E3 and E2. Moreover, water samples from the WWTPO and 200 m downstream from it showed estrogenic activity exceeding that of the EC50 of the E2 standard curve. In sum, this work demonstrates the presence of sex steroid hormones and estrogenic activity, as measured by an in vitro assay, in superficial waters of the Pampas region. It also suggests the possibility of an unidentified source upstream of the wastewater outfall.

The Duplicated Y-specific amhy Gene Is Conserved and Linked to Maleness in Silversides of the Genus Odontesthes.

Sex-determining genes have been successively isolated in several teleosts. In Odontesthes hatcheri and O. bonariensis, the amhy gene has been identified as a master sex-determining gene. However, whether this gene is conserved along related species is still unknown. In this study, the presence of amhy and its association with phenotypic sex was analyzed in 10 species of Odontesthes genus. The primer sets from O. hatcheri that amplify both amhs successfully generated fragments that correspond to amha and amhy in all species. The full sequences of amhy and amha isolated for four key species revealed higher identity values among presumptive amhy, including the 0.5 Kbp insertion in the third intron and amhy-specific insertions/deletions. Amha was present in all specimens, regardless of species and sex, whereas amhy was amplified in most but not all phenotypic males. Complete association between amhy-homologue with maleness was found in O. argentinensis, O. incisa, O. mauleanum, O. perugiae, O. piquava, O. regia, and O. smitti, whereas O. humensis, O. mirinensis, and O. nigricans showed varied degrees of phenotypic/genotypic sex mismatch. The conservation of amhy gene in Odontesthes provide an interesting framework to study the evolution and the ecological interactions of genotypic and environmental sex determination in this group.

The central nervous system acts as a transducer of stress-induced masculinization through corticotropin-releasing hormone B.

Exposure to environmental stressors, such as high temperature (HT), during early development of fish induces sex reversal of genotypic females. Nevertheless, the involvement of the brain in this process is not well clarified. In the present work, we investigated the mRNA levels of corticotropin-releasing hormone b (crhb) and its receptors (crhr1 and crhr2), and found that they were upregulated at HT during the crucial period of gonadal sex determination in medaka. In order to clarify their roles in sex reversal, biallelic mutants for crhr1 and crhr2 were produced by CRISPR/Cas9 technology. Remarkably, biallelic mutants of both loci (crhr1 and crhr2) did not undergo female-to-male sex reversal upon exposure to HT. Inhibition of this process in double corticotropin-releasing hormone receptor mutants could be successfully rescued through the administration of the downstream effector of the hypothalamic-pituitary-interrenal axis, cortisol. Taken together, these results reveal for the first time that the CNS acts as a transducer of masculinization induced by thermal stress.

Sex hormone binding globulin during an annual reproductive cycle in the hepatopancreas and ovary of pejerrey (Odontesthes bonariensis).

In the present study, we determined the hepatopancratic shbg transcript abundance and ovarian immunoreactive Shbg (ir-Shbg) localization in pejerrey females at different gonadal stages during an annual ovarian cycle. In the hepatopancreas, shbg expression remains was constant in pre-vitellogenic stages, decreased at final vitellogenesis to increase again in final maturation and atretic stages to previous levels at post-vitellogenic stages. Related to this, also we found a negative significant relation between sex steroid and shbg expression. On the other hand, in the ovary we found ir-Shbg inside of cortical alveoli, from previtellogenic stages to final maturation. This localization of Shbg in a teleost fish ovary suggests a new role for Shbg in oocytes, that may also extend the oocyte fertilization/development process.

Folate deficiency prevents neural crest fate by disturbing the epigenetic Sox2 repression on the dorsal neural tube.

Folate deficiency has been known to contribute to neural tube and neural crest defects, but why these tissues are particularly affected, and which are the molecular mechanisms involved in those abnormalities are important human health questions that remain unanswered. Here we study the function of two of the main folate transporters, FolR1 and Rfc1, which are robustly expressed in these tissues. Folate is the precursor of S-adenosylmethionine, which is the main donor for DNA, protein and RNA methylation. Our results show that knockdown of FolR1 and/or Rfc1 reduced the abundance of histone H3 lysine and DNA methylation, two epigenetic modifications that play an important role during neural and neural crest development. Additionally, by knocking down folate transporter or pharmacologically inhibiting folate transport and metabolism, we observed ectopic Sox2 expression at the expense of neural crest markers in the dorsal neural tube. This is correlated with neural crest associated defects, with particular impact on orofacial formation. By using bisulfite sequencing, we show that this phenotype is consequence of reduced DNA methylation on the Sox2 locus at the dorsal neural tube, which can be rescued by the addition of folinic acid. Taken together, our in vivo results reveal the importance of folate as a source of the methyl groups necessary for the establishment of the correct epigenetic marks during neural and neural crest fate-restriction.

Sex hormone binding globulin: Expression throughout early development and adult pejerrey fish, Odontesthes bonariensis.

Sex hormone binding globulin (Shbg) is a plasma glycoprotein that binds and transports steroids in the blood of all vertebrate classes apart from birds. In the present study we characterized shbg from pejerrey, a fish species with a well characterized temperature-dependent sex determination. The pejerrey shbg mRNA comprises 1185bp encoding for a 395 amino acid Shbg precursor protein that includes a leader sequence for secretion. Relative quantification of shbg transcript abundance revealed expression early in development coinciding with the sex-determining period and probably in association with temperature leading to male determination. The hepatopancreas was the main site of shbg expression, which varied according to the sex cycle in females. It was also expressed in gills, gonads, gut and taste buds during both larval stages and in adult fish. The presence of Shbg in organs in close contact with the environment such as gills, pseudobranchs, gut and taste buds suggests that these are potential sources of uptake or release of steroids/xenosteroids to and from the aquatic environment.

Effects of 5α-dihydrotestosterone on expression of genes related to steroidogenesis and spermatogenesis during the sex determination and differentiation periods of the pejerrey, Odontesthes bonariensis.

Sex steroid hormones are important players in the control of sex differentiation by regulating gonadal development in teleosts. Although estrogens are clearly associated with the ovarian differentiation in teleosts, the effects of androgens on early gonadal development are still a matter of debate. Traditionally, 11-ketotestosterone (11-KT) is considered the major androgen in fish; however, 5α-dihydrotestosterone (5α-DHT), the most potent androgen in tetrapods, was recently found in fish testis and plasma, but its physiological role is still unknown. In this context, the expression of genes associated with steroidogenesis and spermatogenesis, body growth and sex differentiation were assessed in Odontesthes bonariensis larvae fed with food supplemented with two doses of 5α-DHT (0.1 and 10µg/g of food) from hatching to 6weeks of age. At the lowest dose, 5α-DHT treated larvae showed an estrogenic gene expression pattern, with low hsd11b2 and high cyp19a1a and er2 expression levels with no differences in sex ratio. At the highest dose, 5α-DHT produced a male-shifted sex ratio and the larvae exhibited a gene expression profile characteristic of an advancement of spermatogenesis, with inhibition of amh and stimulation of ndrg3. No differences were observed in somatic growth. These results suggest that in this species, 5α-DHT could have a role on sex differentiation and its effects can differ according to the dose.

Crossover of the hypothalamic pituitary-adrenal/interrenal, -thyroid, and -gonadal axes in testicular development.

Besides the well-known function of thyroid hormones (THs) for regulating metabolism, it has recently been discovered that THs are also involved in testicular development in mammalian and non-mammalian species. THs, in combination with follicle stimulating hormone, lead to androgen synthesis in Danio rerio, which results in the onset of spermatogenesis in the testis, potentially relating the hypothalamic-pituitary-thyroid (HPT) gland to the hypothalamic-pituitary-gonadal (HPG) axes. Furthermore, studies in non-mammalian species have suggested that by stimulating the thyroid-stimulating hormone (TSH), THs can be induced by corticotropin-releasing hormone. This suggests that the hypothalamic-pituitary-adrenal/interrenal gland (HPA) axis might influence the HPT axis. Additionally, it was shown that hormones pertaining to both HPT and HPA could also influence the HPG endocrine axis. For example, high levels of androgens were observed in the testis in Odonthestes bonariensis during a period of stress-induced sex-determination, which suggests that stress hormones influence the gonadal fate toward masculinization. Thus, this review highlights the hormonal interactions observed between the HPT, HPA, and HPG axes using a comparative approach in order to better understand how these endocrine systems could interact with each other to influence the development of testes.

Feminization and altered gonadal gene expression profile by ethinylestradiol exposure to pejerrey, Odontesthes bonariensis, a South American teleost fish.

In pejerrey (Odontesthes bonariensis), ovarian differentiation has been associated with gonadal aromatase expression. It is also known that exposure of pejerrey larvae to estradiol (E(2)) produces all female populations. During the last few years, the presence of ethinylestradiol (EE(2)), a synthetic E(2) analogue, has been reported in water reservoirs of different parts of the world. In the present study, the effects of EE(2) were assessed on sex ratio bias and gene expression levels of gonadal aromatase (cyp19a1a), 11ß-hydroxysteroid dehydrogenase type 2 (hsd11b2), estrogens (erα, erß1), and androgen receptors (arα, arß). Pejerrey larvae were fed with commercial food containing EE(2) (0.1 and 1 µg/g) and E(2 ) (50 µg/g) as a positive control for six weeks after hatching. The gonadal histological analysis showed that 42 to 46% of the fish had clearly differentiated ovaries in both the EE(2) - and E(2) -treated groups, compared with 27% in the control group. Moreover, in the EE(2) - (1 µg/g) and E(2) -treated groups, no fish presented signs of testicular development compared with controls. In addition, expression of cyp19a1a and hsd11b2 was significantly up- and downregulated, respectively, by EE(2) and E(2) . The authors' results suggested that the feminization process driven by EE(2) depends on the positive balance of cyp19a1a in relation to hsd11b2. Thus, these genes can be used as early indicators of exposure to xenoestrogens in this species.

Molecular characterization of cyp11a1 and cyp11b1 and their gene expression profile in pejerrey (Odontesthes bonariensis) during early gonadal development.

Sex steroids are known to be involved in gonadal differentiation in fish, but whether androgens are early mediators of testis differentiation remains unclear. We studied the sex-related developmental variations in the gene expression of two key enzymes involved in steroids and androgen synthesis (cyp11a1 and cyp11b1) in trunks and isolated gonads of pejerrey (Odontesthes bonariensis) larvae during and after the sex determination period. Also, and in order to have a better characterization of this process we studied the expression of Sertoli (dmrt1, amh, sox9) and Leydig (nr5a1 or sf-1) cell markers as well as a gene with higher expression in females (cyp19a1a). No clear differences were observed in the expression of cyp11a1 and cyp11b1 during the temperature-sensitive window in the trunk of pejerrey larvae. Nevertheless, a clear increase of cyp11b1 was observed in isolated gonads taken from fish reared at the male producing temperature. In these gonads we also confirmed the trends of genes with higher expression in males (dmrt1, amh) and females (cyp19a1a) as previously described in larval trunks of pejerrey. Our results showed that the expression of cyp11b1 was positively associated with the morphological differentiation of the testis. Nevertheless the involvement of 11-oxygenated androgens during the temperature-sensitive window could not be clearly established.

Brain aromatase (Cyp19A2) and estrogen receptors, in larvae and adult pejerrey fish Odontesthes bonariensis: Neuroanatomical and functional relations.

Although estrogens exert many functions on vertebrate brains, there is little information on the relationship between brain aromatase and estrogen receptors. Here, we report the cloning and characterization of two estrogen receptors, alpha and beta, in pejerrey. Both receptors' mRNAs largely overlap and were predominantly expressed in the brain, pituitary, liver, and gonads. Also brain aromatase and estrogen receptors were up-regulated in the brain of estradiol-treated males. In situ hybridization was performed to study in more detail, the distribution of the two receptors in comparison with brain aromatase mRNA in the brain of adult pejerrey. The estrogen receptors' mRNAs exhibited distinct but partially overlapping patterns of expression in the preoptic area and the mediobasal hypothalamus, as well as in the pituitary gland. Moreover, the estrogen receptor alpha, but not beta, were found to be expressed in cells lining the preoptic recess, similarly as observed for brain aromatase. Finally, it was shown that the onset expression of brain aromatase and both estrogen receptors in the head of larvae preceded the morphological differentiation of the gonads. Because pejerrey sex differentiation is strongly influenced by temperature, brain aromatase expression was measured during the temperature-sensitive window and was found to be significantly higher at male-promoting temperature. Taken together these results suggest close neuroanatomical and functional relationships between brain aromatase and estrogen receptors, probably involved in the sexual differentiation of the brain and raising interesting questions on the origin (central or peripheral) of the brain aromatase substrate.

Characterization of the cDNAs encoding three GnRH forms in the pejerrey fish Odontesthes bonariensis (Atheriniformes) and the evolution of GnRH precursors.

Most vertebrates express two gonadotropin releasing hormone (GnRH) variants in brain tissue but there is an increasing number of fish species for which a third GnRH form has been detected. We characterized the precursors (cDNAs) of all three forms expressed in the brain of the pejerrey (silverside) fish, Odontesthes bonariensis (Atheriniformes): type I (GnRH-I; 440 bp), type II (GnRH-II; 529 bp), and type III (GnRH-III; 515 bp). The expression of these GnRHs precursors was also observed in peripheral tissues related to reproduction (gonads), visual and chemical senses (eye and olfactory epithelium), and osmoregulation (gill), suggesting that in teleost fish and possibly other vertebrates GnRH mediates directly or indirectly many other functions besides reproduction. We also present a comprehensive phylogenetic analysis including representatives of all chordate GnRH precursors characterized to date that supports the idea of two main paralogous GnRH lineages with different function. A "forebrain lineage" separates evolutionarily from the "midbrain lineage" as a result of an ancient duplication (ca. 600 million years ago). A third, fish-only clade of GnRH genes seems to have originated before the divergence of fish and tetrapods but retained only in fish. Phylogenetic analyses of GnRH precursors (DNA and protein sequences) under different optimality criteria converge on this result. Although alternative scenarios could not be statistically rejected in this study due to the relatively short size of the analyzed molecules, this hypothesis also receives support from chromosomal studies of synteny around the GnRH genes in vertebrates.