Genome sequencing and analysis of black flounder (Paralichthys orbignyanus) reveals new insights into Pleuronectiformes genomic size and structure.

Black flounder (Paralichthys orbignyanus, Pleuronectiformes) is a commercially significant marine fish with promising aquaculture potential in Argentina. Despite extensive studies on Black flounder aquaculture, its limited genetic information available hampers the crucial role genetics plays in the development of this activity. In this study, we first employed Illumina sequencing technology to sequence the entire genome of Black flounder. Utilizing two independent libraries-one from a female and another from a male-with 150 bp paired-end reads, a mean insert length of 350 bp, and over 35 X-fold coverage, we achieved assemblies resulting in a genome size of ~ 538 Mbp. Analysis of the assemblies revealed that more than 98% of the core genes were present, with more than 78% of them having more than 50% coverage. This indicates a somehow complete and accurate genome at the coding sequence level. This genome contains 25,231 protein-coding genes, 445 tRNAs, 3 rRNAs, and more than 1,500 non-coding RNAs of other types. Black flounder, along with pufferfishes, seahorses, pipefishes, and anabantid fish, displays a smaller genome compared to most other teleost groups. In vertebrates, the number of transposable elements (TEs) is often correlated with genome size. However, it remains unclear whether the sizes of introns and exons also play a role in determining genome size. Hence, to elucidate the potential factors contributing to this reduced genome size, we conducted a comparative genomic analysis between Black flounder and other teleost orders to determine if the small genomic size could be explained by repetitive elements or gene features, including the whole genome genes and introns sizes. We show that the smaller genome size of flounders can be attributed to several factors, including changes in the number of repetitive elements, and decreased gene size, particularly due to lower amount of very large and small introns. Thus, these components appear to be involved in the genome reduction in Black flounder. Despite these insights, the full implications and potential benefits of genome reduction in Black flounder for reproduction and aquaculture remain incompletely understood, necessitating further research.

Developmental aspects of the hypothalamic-pituitary network related to reproduction in teleost fish.

The hypothalamic-pituitary-gonadal axis is the main system that regulates reproduction in vertebrates through a complex network that involves different neuropeptides, neurotransmitters, and pituitary hormones. Considering that this axis is established early on life, the main goal of the present work is to gather information on its development and the actions of its components during early life stages. This review focuses on fish because their neuroanatomical characteristics make them excellent models to study neuroendocrine systems. The following points are discussed: i) developmental functions of the neuroendocrine components of this network, and ii) developmental disruptions that may impact adult reproduction. The importance of the components of this network and their susceptibility to external/internal signals that can alter their specific early functions and/or even the establishment of the reproductive axis, indicate that more studies are necessary to understand this complex and dynamic network.

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.

Biogeography, habitat transitions and hybridization in a radiation of South American silverside fishes revealed by mitochondrial and genomic RAD data.

Rivers and lake systems in the southern cone of South America have been widely influenced by historical glaciations, carrying important implications for the evolution of aquatic organisms, including prompting transitions between marine and freshwater habitats and by triggering hybridization among incipient species via waterway connectivity and stream capture events. Silverside fishes (Odontesthes) in the region comprise a radiation of 19 marine and freshwater species that have been hypothesized on the basis of morphological or mitochondrial DNA data to have either transitioned repeatedly into continental waters from the sea or colonized marine habitats following freshwater diversification. New double digest restriction-site associated DNA data presented here provide a robust framework to investigate the biogeographical history of and habitat transitions in Odontesthes. We show that Odontesthes silversides originally diversified in the Pacific but independently colonized the Atlantic three times, producing three independent marine-to-freshwater transitions. Our results also indicate recent introgression of marine mitochondrial haplotypes into two freshwater clades, with more recurring instances of hybridization among Atlantic- versus Pacific-slope species. In Pacific freshwater drainages, hybridization with a marine species appears to be geographically isolated and may be related to glaciation events. Substantial structural differences of estuarine gradients between these two geographical areas may have influenced the frequency, intensity and evolutionary effects of hybridization events.

Multimodal hypothalamo-hypophysial communication in the vertebrates.

The vertebrate pituitary is arguably one of the most complex endocrine glands from the evolutionary, anatomical and functional perspectives. The pituitary plays a master role in endocrine physiology for the control of growth, metabolism, reproduction, water balance, and the stress response, among many other key processes. The synthesis and secretion of pituitary hormones are under the control of neurohormones produced by the hypothalamus. Under this conceptual framework, the communication between the hypophysiotropic brain and the pituitary gland is at the foundation of our understanding of endocrinology. The anatomy of the connections between the hypothalamus and the pituitary gland has been described in different vertebrate classes, revealing diverse modes of communication together with varying degrees of complexity. In this context, the evolution and variation in the neuronal, neurohemal, endocrine and paracrine modes will be reviewed in light of recent discoveries, and a re-evaluation of earlier observations. There appears to be three main hypothalamo-pituitary communication systems: 1. Diffusion, best exemplified by the agnathans; 2. Direct innervation of the adenohypophysis, which is most developed in teleost fish, and 3. The median eminence/portal blood vessel system, most conspicuously developed in tetrapods, showing also considerable variation between classes. Upon this basic classification, there exists various combinations possible, giving rise to taxon and species-specific, multimodal control over major physiological processes. Intrapituitary paracrine regulation and communication between folliculostellate cells and endocrine cells are additional processes of major importance. Thus, a more complex evolutionary picture of hypothalamo-hypophysial communication is emerging. There is currently little direct evidence to suggest which neuroendocrine genes may control the evolution of one communication system versus another. However, studies at the developmental and intergenerational timescales implicate several genes in the angiogenesis and axonal guidance pathways that may be important.

Kisspeptin and GnRH interactions in the reproductive brain of teleosts.

It is well known that gonadotropin-releasing hormone (Gnrh) has a key role in reproduction by regulating the synthesis and release of gonadotropins from the anterior pituitary gland of all vertebrates. About 25 years ago, another neuropeptide, kisspeptin (Kiss1) was discovered as a metastasis suppressor of melanoma cell lines and then found to be essential for mammalian reproduction as a stimulator of hypothalamic Gnrh and regulator of puberty onset. Soon after, a kisspeptin receptor (kissr) was found in the teleost brain. Nowadays, it is known that in most teleosts the kisspeptin system is composed of two ligands, kiss1 and kiss2, and two receptors, kiss2r and kiss3r. Even though both kisspeptin peptides, Kiss1 and Kiss2, have been demonstrated to stimulate gonadotropin synthesis and secretion in different fish species, their actions appear not to be mediated by Gnrh neurons as in mammalian models. In zebrafish and medaka, at least, hypophysiotropic Gnrh neurons do not express Kiss receptors. Furthermore, kisspeptinergic nerve terminals reach luteinizing hormone cells in some fish species, suggesting a direct pituitary action. Recent studies in zebrafish and medaka with targeted mutations of kiss and/or kissr genes reproduce relatively normally. In zebrafish, single gnrh mutants and additionally those having the triple gnrh3 plus 2 kiss mutations can reproduce reasonably well. In these fish, other neuropeptides known to affect gonadotropin secretion were up regulated, suggesting that they may be involved in compensatory responses to maintain reproductive processes. In this context, the present review explores and presents different possibilities of interactions between Kiss, Gnrh and other neuropeptides known to affect reproduction in teleost fish. Our intention is to stimulate a broad discussion on the relative roles of kisspeptin and Gnrh in the control of teleost reproduction.

Chondrichthyan research in South America: Endocrinology overview and research trends over 50 years (1967-2016) compared to the rest of the world.

The endocrine system plays a crucial role in regulating the activity of cells and organs among vertebrates, including the class Chondrichthyes. Accordingly, Chondrichthyan endocrinology publications have been steadily increasing in the global literature. However, while interest in South American Chondrichthyan research has been growing over the last 50 years, the field of endocrinology related to Chondrichthyans has been limited. Understanding the trajectory of a scientific discipline assists researchers and stakeholders in making decisions regarding which research areas require further attention. Further, visualisation techniques based on bibliometric analysis of scientific publications assist in understanding fluctuations in the trends of specific research fields over time. In this study, Chondrichthyan research publications over time were analysed by creating visualisation maps using VOSviewer bibliometric software. Trends in South America Chondrichthyan research with an emphasis on endocrinology were explored over a 50-year period (1967-2016). These trends were compared with Chondrichthyans research worldwide for the more recent 15-year period (2002-2016). The number of South America Chondrichthyan scientific publications increased from six during the 1967-1981 period to 112 in 2016. However, only eight papers were found published in the area of Chondrichthyan endocrinology research. Fisheries, reproduction and taxonomy were the dominate research areas in South America over the 50 years. For the more recent 15 years, South American publications comprised 11% of the total literature published globally. While South America research outputs fluctuated closely with global research trends, differences appeared when comparing areas of growth. This study describes the trends in Chondrichthyan research literature globally and more specifically in South America. Although South American countries may never contribute to the same scale as the wider international scientific community, the future of Chondrichthyans would strongly benefit from the contributions of the many diverse research groups around the world.

Effects of temperature on the reproductive physiology of female elasmobranchs: The case of the narrownose smooth-hound shark (Mustelus schmitti).

The knowledge of how temperature influences elasmobranchs reproductive physiology allows a better understanding of their reproductive patterns. This study describes the relationship between temperature fluctuations and the plasmatic changes of the sex steroids related to reproduction: testosterone (T), estradiol (E2) and progesterone (P4), throughout the female reproductive cycle of the shark Mustelus schmitti. A total of 123 adult females were bi-monthly sampled in Buenos Aires, Argentina, coastal waters. Bottom temperatures were recorded at each sampling point and blood samples were taken from each female for plasma sex steroids measurement. Sex steroid plasma levels were analyzed in relation with maximum follicular diameter (MFD), uterosomatic index (USI, as indicator of pregnancy) and temperature using Generalized Additive Models. Plasmatic E2 and T increased during follicular growth until MFD reached 1.34 and 1.46 cm, respectively. Peak of T occurred at the follicular stage associated with parturition (MFD, 1.4-1.6 cm), just prior to final maturation and ovulation (MFD, 1.6-2.0 cm). Progesterone significantly increased at this last ovarian phase, while T and E2 decreased. The increase of USI with pregnancy was associated to a decrease in T and mainly E2 levels, while P4 remained unaffected. Prior to ovulation, T plasma levels decreased with temperature below to 13 °C and then increased progressively with a pronounced elevation above 17 °C, while E2 presented an opposite pattern. Progesterone plasma levels changed with temperature showing a similar pattern to that observed for T. Using M. schmitti shark as model species, this study shows a clear picture of how seawater temperature variations can affect the reproductive physiology in elasmobranch females. A hypothetical mechanism (based on T elevation driven by temperature increase and its connection by feedback with a P4 rise and parturition/ovulation induction) is proposed as evidence to support that the increase in temperature can trigger reproductive events in elasmobranchs. In addition to its ecological scope, this work contributes to reinforce the relatively scarce general knowledge of elasmobranchs reproductive physiology.

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.

Morphological relationship between GnIH and GnRH neurons in the brain of the neotropical cichlid fish Cichlasoma dimerus.

Reproduction is regulated by the hypothalamic-pituitary-gonadal axis. The first neuropeptide identified that regulates this function was the decapeptide gonadotropin-releasing hormone (GnRH). Nowadays, in gnatostomates, a number of GnRH variants have been identified and classified into three different types: GnRH1, GnRH2, and GnRH3. Almost 30 years later, a new peptide that inhibits gonadotropin synthesis and secretion was discovered and thus named as gonadotropin-inhibitory hormone (GnIH). In avians and mammals, the interaction and regulation between GnRH and GnIH neurons has been widely studied; however, in other vertebrate groups there is little information about the relationship between these neurons. In previous works, three GnRH variants and a GnIH propeptide were characterized in Cichlasoma dimerus, and it was demonstrated that GnIH inhibited gonadotropins release in this species. Because no innervation was detected at the pituitary level, we speculate that GnIH would inhibit gonadotropins via GnRH. Thus, the aim of the present study was to evaluate the anatomical relationship between neurons expressing GnIH and the three GnRH variants by double labelling confocal immunofluorescence in adults of C. dimerus. Our results showed no apparent contacts between GnIH and GnRH1, fiber to fiber interactions between GnIH and GnRH2, and co-localization of GnIH and GnRH3 variant in neurons of the nucleus olfacto-retinalis. In conclusion, whether GnIH regulates the expression or secretion of GnRH1 in this species, an indirect modulation seems more plausible. Moreover, the present results suggest an interaction between GnIH and GnRH2 systems. Finally, new clues were provided to investigate the role of nucleus olfacto-retinalis cells and putative GnIH and GnRH3 interactions in the modulation of the reproductive network in teleost fish.

From molecule to behavior: Brain aromatase (cyp19a1b) characterization, expression analysis and its relation with social status and male agonistic behavior in a Neotropical cichlid fish.

The enzyme aromatase, responsible for the conversion of C19 androgens to C18 estrogens, exists as two paralogue copies in teleost fish: Cyp19a1a mostly expressed in the gonads, referred as gonadal aromatase, and Cyp19a1b, mostly expressed in the brain, accordingly known as brain aromatase. The neural localization of Cyp19a1b is greatly contained within the social behavior network and mesolimbic reward system in fish, suggesting a strong role of estrogen synthesis in the regulation of social behavior. In this work we aimed to analyze the variation in cyp19a1b expression in brain and pituitary of males of a highly social cichlid, Cichlasoma dimerus (locally known as chanchita), and its relation with inter-individual variability in agonistic behavior in a communal social environment. We first characterized chanchita's cyp19a1b mRNA and deduced amino acid sequence, which showed a high degree of conservation when compared to other teleost brain aromatase sequences, and its tissue expression patterns. Within the brain, Cyp19a1b was solely detected at putative radial glial cells of the forebrain, close to the brain ventricles. We then studied the relative expression levels of cyp19a1b by Real Time PCR in the brain and pituitary of males of different social status, territorial vs. non-territorial, and its relationship with an index of agonistic behavior. We found that even though, brain aromatase expression did not differ between types of males, pituitary cyp19a1b expression levels positively correlated with the index of agonistic behavior. This suggests a novel role of the pituitary in the regulation of social behavior by local estrogen synthesis.

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.

A game of two? Gene expression analysis of brain (cyp19a1b) and gonadal (cyp19a1a) aromatase in females of a Neotropical cichlid fish through the parental care period and removal of the offspring.

For many species parental behavior is essential for the survival of the offspring. While the ultimate causes of teleost parental behavior have been widely studied, comparatively little is known about its proximate causes. The aim of this study was to analyze the yet unexplored, potential dual role of brain and gonadal aromatases, the enzymes responsible for the conversion of androgens to estrogens in the brains and gonads of teleosts, respectively, on the different stages of the maternal care period of the biparental cichlid Cichlasoma dimerus, locally known as chanchita. By immunohistochemistry we analyzed the neural distribution of brain aromatase and observed it exclusively within the forebrain, including areas involved in the regulation of parental behavior. We next analyzed the gene expression of brain aromatase in the brain, and gonadal aromatase in the ovary, of female chanchitas through the parental care period. To further characterize the physiological environment associated to maternal care, we also evaluated sex steroid levels (17ß-estradiol, testosterone and 11-ketotestoterone) and ovarian follicle percentage. The onset of parental behavior specifically downregulated sex steroids synthesis and the rate of ovarian maturation, as denoted by a more than 10-fold decrease in steroid levels and delayed detection of mature follicles in females with offspring, compared to females which eggs were removed. Gene expression levels of both aromatases were independent of maternal care at the evaluated time points, even though they varied during the parental care period.

Kisspeptins and their receptors in the brain-pituitary-gonadal axis of Odonthestes bonariensis: Their relationship with gametogenesis along the reproductive cycle.

In vertebrates, the reproduction is controlled by the brain-pituitary-gonadal (BPG) axis and kisspeptin has emerged as a key player of this axis. In this study, we analyzed changes in the expression levels of kiss1, kiss2, and their receptors, kissr2 and kissr3 during gametogenesis in the BPG axis of feral Odontesthes bonariensis. In females, levels of brain kiss1 showed an increase at final maturation (Fm), while kiss2 levels were shown to be high at primary growth (Pg) stage, with no differences in the expression of their receptors. In the pituitary, kiss1 and kiss2 peaked at the cortical alveoli (Ca) stage, and kissr3 at initial vitellogenesis. In parallel, there was an increase of kiss1, kissr2 and kissr3 in the ovary during the Ca stage and both receptors again at Fm stage. In males, the four genes were highly expressed in the brain at the arrested (A) stage. In the pituitary, kiss2 peaked at spermatogonial (SG) and spermatocytary (SC) stages; while kissr3 reached a peak at the spermiogenic stage (SP). In testes, kiss1 and kiss2 significantly increased during the SG and SC stages; meanwhile, kissr2 increased at SG and SC, whereas kissr3 levels were significantly high at SC and SP stages. Taken together these results showed that the kisspeptin system in pejerrey is expressed in the three levels of the BPG axis with different expression profiles during the gonadal cycle. These findings pointed that kisspeptins have different roles in gametogenesis in this species.

Kisspeptin system in pejerrey fish (Odontesthes bonariensis). Characterization and gene expression pattern during early developmental stages.

In vertebrates, kisspeptins and their receptors are known to be related to puberty onset and gonadal maturation, however, there are few studies concerning their role in early development. Here, we characterize the kisspeptin system in the pejerrey, Odontesthes bonariensis, a fish with strong temperature-dependent sex determination. We reconstructed the phylogenetic history of the two ligands (kiss1 and kiss 2) and two receptors (kissr2 and kissr3) in pejerrey in the context of recent classifications of bony fishes, determined their tissue distribution and documented the early expression pattern of these ligands and receptors. Phylogenetic analysis of these gene families clearly resolved the percomorph clade and grouped pejerrey with Beloniformes. Paralogous sets of genes putatively arising from the teleost-specific genome duplication event (3R) were not detected. Kisspeptins and their receptors showed a wide tissue distribution in adult pejerrey, including tissues not related to reproduction. In larvae reared at 24°C, the four kisspeptin elements were expressed in the head from week 1 to week 8 of life, with no differences in transcript levels. Larvae kept at a female-producing temperature (17°C) did not show statistically significant differences in the transcript levels of all analyzed genes during the sex determination/differentiation period; however, in those larvae raised at male producing temperature (29°C), kiss2 levels were increased at week 4 after hatching. These results showed that all members of the kisspeptin system are expressed at this early period, and the increase of kiss2 transcripts at week 4 could be interpreted as it would be related to the differentiation of the brain-pituitary axis in male development.

A multidisciplinary study on social status and the relationship between inter-individual variation in hormone levels and agonistic behavior in a Neotropical cichlid fish.

Social animals with hierarchal dominance systems are particularly susceptible to their social environment. There, interactions with conspecifics and hierarchal position can greatly affect an individual's behavior, physiology and reproductive success. Our experimental model, Cichlasoma dimerus, is a serially-monogamous Neotropical cichlid fish with a hierarchical social system, established and sustained through agonistic interactions. In this work, we aimed to describe C. dimerus social structure and its association with hormonal profiles and testicular cellular composition. We recorded and quantified agonistic interactions from the territorial pair, i.e. the top ranked male and female, and the lowest ranked male of stable social groups. Plasma levels of 11-ketotestosterone (11-KT), testosterone, 17ß-estradiol (E2) and cortisol were measured by ELISA. Results show that territorial pairs cooperatively guarded the territory, but rarely attacked in synchrony. Territorial males had higher testosterone and 11-KT plasma levels than non-territorial males, while E2 and an index of its metabolization from testosterone were higher in non-territorial males. No difference was observed in cortisol levels. Plasma 11-KT and an index of the conversion of testosterone to 11-KT, positively correlated with the frequency of aggressiveness, while E2 showed the opposite pattern. Territorial males had a higher gonadosomatic index than non-territorial males. The quantification of testicular cellular types revealed that the percentage of spermatocytes and spermatids was higher in non-territorial males, while territorial males showed a greater percentage of spermatozoa. Thus, C. dimerus male social position within a stable hierarchy is associated with distinct behaviors, steroid levels and testicular degree of development.

The secretogranin-II derived peptide secretoneurin modulates electric behavior in the weakly pulse type electric fish, Brachyhypopomus gauderio.

Secretoneurin (SN) in the preoptic area and pituitary of mammals and fish has a conserved close association with the vasopressin and oxytocin systems, members of a peptide family that are key in the modulation of sexual and social behaviors. Here we show the presence of SN-immunoreactive cells and projections in the brain of the electric fish, Brachyhypopomus gauderio. Secretoneurin colocalized with vasotocin (AVT) and isotocin in cells and fibers of the preoptic area. In the rostral pars distalis of the pituitary, many cells were both SN and prolactin-positive. In the hindbrain, at the level of the command nucleus of the electric behavior (pacemaker nucleus; PN), some of SN-positive fibers colocalized with AVT. We also explored the potential neuromodulatory role of SN on electric behavior, specifically on the rate of the electric organ discharge (EOD) that signals arousal, dominance and subordinate status. Each EOD is triggered by the command discharge of the PN, ultimately responsible for the basal EOD rate. SN modulated diurnal basal EOD rate in freely swimming fish in a context-dependent manner; determined by the initial value of EOD rate. In brainstem slices, SN partially mimicked the in vivo behavioral effects acting on PN firing rate. Taken together, our results suggest that SN may regulate electric behavior, and that its effect on EOD rate may be explained by direct action of SN at the PN level through either neuroendocrine and/or endocrine mechanisms.

A Y-linked anti-Müllerian hormone duplication takes over a critical role in sex determination.

Gonadal sex determination in vertebrates generally follows a sequence of genetically programmed events. In what is seemingly becoming a pattern, all confirmed or current candidate "master" sex-determining genes reported in this group, e.g., SRY in eutherian mammals, DMY/dmrt1bY in medaka, DM-W in the African clawed frog, and DMRT1 in chicken encode transcription factors. In contrast, here we show that a male-specific, duplicated copy of the anti-Müllerian hormone (amh) is implicated in testicular development of the teleost fish Patagonian pejerrey (Odontesthes hatcheri). The gene, termed amhy because it is found in a single metacentric/submetacentric chromosome of XY individuals, is expressed much earlier than the autosomal amh (6 d after fertilization vs. 12 wk after fertilization) and is localized to presumptive Sertoli cells of XY males during testicular differentiation. Moreover, amhy knockdown in XY embryos resulted in the up-regulation of foxl2 and cyp19a1a mRNAs and the development of ovaries. These results are evidence of a functional amh duplication in vertebrates and suggest that amhy may be the master sex-determining gene in this species. If confirmed, this would be a unique instance of a hormone-related gene, a member of the TGF-ß superfamily, in such a role.

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.