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.

CRISPR/dCas9-Mediated DNA Methylation Editing on emx2 in Chinese Tongue Sole (Cynoglossus semilaevis) Testis Cells.

DNA methylation is a key epigenetic mechanism orchestrating gene expression networks in many biological processes. Nonetheless, studying the role of specific gene methylation events in fish faces challenges. In this study, we validate the regulation of DNA methylation on empty spiracles homeobox 2 (emx2) expression with decitabine treatment in Chinese tongue sole testis cells. We used the emx2 gene as the target gene and developed a new DNA methylation editing system by fusing dnmt3a with catalytic dead Cas9 (dCas9) and demonstrated its ability for sequence-specific DNA methylation editing. Results revealed that utilizing dCas9-dnmt3a to target emx2 promoter region led to increased DNA methylation levels and decreased emx2 expression in Chinese tongue sole testis cells. More importantly, the DNA methylation editing significantly suppressed the expression of MYC proto-oncogene, bHLH transcription factor (myc), one target gene of emx2. Furthermore, we assessed the off-target effects of dCas9-dnmt3a and confirmed no significant impact on the predicted off-target gene expression. Taken together, we developed the first DNA methylation editing system in marine species and demonstrated its effective editing ability in Chinese tongue sole cells. This provides a new strategy for both epigenetic research and molecular breeding of marine species.

Photobiomodulation Reduces the Cytokine Storm Syndrome Associated with COVID-19 in the Zebrafish Model.

Although the exact mechanism of the pathogenesis of coronavirus SARS-CoV-2 (COVID-19) is not fully understood, oxidative stress and the release of pro-inflammatory cytokines have been highlighted as playing a vital role in the pathogenesis of the disease. In this sense, alternative treatments are needed to reduce the level of inflammation caused by COVID-19. Therefore, this study aimed to investigate the potential effect of red photobiomodulation (PBM) as an attractive therapy to downregulate the cytokine storm caused by COVID-19 in a zebrafish model. RT-qPCR analyses and protein-protein interaction prediction among SARS-CoV-2 and Danio rerio proteins showed that recombinant Spike protein (rSpike) was responsible for generating systemic inflammatory processes with significantly increased levels of pro-inflammatory (il1b, il6, tnfa, and nfkbiab), oxidative stress (romo1) and energy metabolism (slc2a1a and coa1) mRNA markers, with a pattern similar to those observed in COVID-19 cases in humans. On the other hand, PBM treatment was able to decrease the mRNA levels of these pro-inflammatory and oxidative stress markers compared with rSpike in various tissues, promoting an anti-inflammatory response. Conversely, PBM promotes cellular and tissue repair of injured tissues and significantly increases the survival rate of rSpike-inoculated individuals. Additionally, metabolomics analysis showed that the most-impacted metabolic pathways between PBM and the rSpike treated groups were related to steroid metabolism, immune system, and lipid metabolism. Together, our findings suggest that the inflammatory process is an incisive feature of COVID-19 and red PBM can be used as a novel therapeutic agent for COVID-19 by regulating the inflammatory response. Nevertheless, the need for more clinical trials remains, and there is a significant gap to overcome before clinical trials can commence.

Molecular and Physiological Effects of 17α-methyltestosterone on Sex Differentiation of Black Rockfish, Sebastes schlegelii.

It is widely known that all-female fish production holds economic value for aquaculture. Sebastes schlegelii, a preeminent economic species, exhibits a sex dimorphism, with females surpassing males in growth. In this regard, achieving all-female black rockfish production could significantly enhance breeding profitability. In this study, we utilized the widely used male sex-regulating hormone, 17α-methyltestosterone (MT) at three different concentrations (20, 40, and 60 ppm), to produce pseudomales of S. schlegelii for subsequent all-female offspring breeding. Long-term MT administration severely inhibits the growth of S. schlegelii, while short term had no significant impact. Histological analysis confirmed sex reversal at all MT concentrations; however, both medium and higher MT concentrations impaired testis development. MT also influenced sex steroid hormone levels in pseudomales, suppressing E2 while increasing T and 11-KT levels. In addition, a transcriptome analysis revealed that MT down-regulated ovarian-related genes (cyp19a1a and foxl2) while up-regulating male-related genes (amh) in pseudomales. Furthermore, MT modulated the TGF-ß signaling and steroid hormone biosynthesis pathways, indicating its crucial role in S. schlegelii sex differentiation. Therefore, the current study provides a method for achieving sexual reversal using MT in S. schlegelii and offers an initial insight into the underlying mechanism of sexual reversal in this species.

Transcriptome Analysis Revealed the Early Heat Stress Response in the Brain of Chinese Tongue Sole (Cynoglossus semilaevis).

As a common influencing factor in the environment, temperature greatly influences the fish that live in the water all their life. The essential economic fish Chinese tongue sole (Cynoglossus semilaevis), a benthic fish, will experience both physiological and behavioral changes due to increases in temperature. The brain, as the central hub of fish and a crucial regulatory organ, is particularly sensitive to temperature changes and will be affected. However, previous research has mainly concentrated on the impact of temperature on the gonads of C. semilaevis. Instead, our study examines the brain using transcriptomics to investigate specific genes and pathways that can quickly respond to temperature changes. The fish were subjected to various periods of heat stress (1 h, 2 h, 3 h, and 5 h) before extracting the brain for transcriptome analysis. After conducting transcriptomic analyses, we identified distinct genes and pathways in males and females. The pathways were mainly related to cortisol synthesis and secretion, neuroactive ligand-receptor interactions, TGF beta signaling pathway, and JAK/STAT signaling pathway, while the genes included the HSP family, tshr, c-fos, c-jun, cxcr4, camk2b, and igf2. Our study offers valuable insights into the regulation mechanisms of the brain's response to temperature stress.

Thyroid Hormones Deficiency Impairs Male Germ Cell Development: A Cross Talk Between Hypothalamic-Pituitary-Thyroid, and-Gonadal Axes in Zebrafish.

In vertebrates, thyroid hormones are critical players in controlling different physiological processes such as development, growth, metabolism among others. There is evidence in mammals that thyroid hormones are also an important component of the hormonal system that controls reproduction, although studies in fish remain poorly investigated. Here, we tested this hypothesis by investigating the effects of methimazole-induced hypothyroidism on the testicular function in adult zebrafish. Treatment of fish with methimazole, in vivo, significantly altered zebrafish spermatogenesis by inhibiting cell differentiation and meiosis, as well as decreasing the relative number of spermatozoa. The observed impairment of spermatogenesis by methimazole was correlated with significant changes in transcript levels for several genes implicated in the control of reproduction. Using an in vitro approach, we also demonstrated that in addition to affecting the components of the brain-pituitary-peripheral axis, T3 (triiodothyronine) also exerts direct action on the testis. These results reinforce the hypothesis that thyroid hormones are an essential element of multifactorial control of reproduction and testicular function in zebrafish and possibly other vertebrate species.

Gdnf Acts as a Germ Cell-Derived Growth Factor and Regulates the Zebrafish Germ Stem Cell Niche in Autocrine- and Paracrine-Dependent Manners.

Glial cell line-derived neurotrophic factor (GDNF) and its receptor (GDNF Family Receptor α1-GFRα1) are well known to mediate spermatogonial stem cell (SSC) proliferation and survival in mammalian testes. In nonmammalian species, Gdnf and Gfrα1 orthologs have been found but their functions remain poorly investigated in the testes. Considering this background, this study aimed to understand the roles of the Gdnf-Gfrα1 signaling pathway in zebrafish testes by combining in vivo, in silico and ex vivo approaches. Our analysis showed that zebrafish exhibit two paralogs for Gndf (gdnfa and gdnfb) and its receptor, Gfrα1 (gfrα1a and gfrα1b), in accordance with a teleost-specific third round of whole genome duplication. Expression analysis further revealed that both ligands and receptors were expressed in zebrafish adult testes. Subsequently, we demonstrated that gdnfa is expressed in the germ cells, while Gfrα1a/Gfrα1b was detected in early spermatogonia (mainly in types Aund and Adiff) and Sertoli cells. Functional ex vivo analysis showed that Gdnf promoted the creation of new available niches by stimulating the proliferation of both type Aund spermatogonia and their surrounding Sertoli cells but without changing pou5f3 mRNA levels. Strikingly, Gdnf also inhibited late spermatogonial differentiation, as shown by the decrease in type B spermatogonia and down-regulation of dazl in a co-treatment with Fsh. Altogether, our data revealed that a germ cell-derived factor is involved in maintaining germ cell stemness through the creation of new available niches, supporting the development of spermatogonial cysts and inhibiting late spermatogonial differentiation in autocrine- and paracrine-dependent manners.

Toxicity of spike fragments SARS-CoV-2 S protein for zebrafish: A tool to study its hazardous for human health?

Despite the significant increase in the generation of SARS-CoV-2 contaminated domestic and hospital wastewater, little is known about the ecotoxicological effects of the virus or its structural components in freshwater vertebrates. In this context, this study evaluated the deleterious effects caused by SARS-CoV-2 Spike protein on the health of Danio rerio, zebrafish. We demonstrated, for the first time, that zebrafish injected with fragment 16 to 165 (rSpike), which corresponds to the N-terminal portion of the protein, presented mortalities and adverse effects on liver, kidney, ovary and brain tissues. The conserved genetic homology between zebrafish and humans might be one of the reasons for the intense toxic effects followed inflammatory reaction from the immune system of zebrafish to rSpike which provoked damage to organs in a similar pattern as happen in severe cases of COVID-19 in humans, and, resulted in 78,6% of survival rate in female adults during the first seven days. The application of spike protein in zebrafish was highly toxic that is suitable for future studies to gather valuable information about ecotoxicological impacts, as well as vaccine responses and therapeutic approaches in human medicine. Therefore, besides representing an important tool to assess the harmful effects of SARS-CoV-2 in the aquatic environment, we present the zebrafish as an animal model for translational COVID-19 research.

Molecular characterization and expression analysis of anti-Müllerian hormone in common carp (Cyprinus carpio) adult testes.

Anti-Müllerian hormone (Amh) is a member of the transforming growth factor-ß (Tgf-ß) superfamily required in the regression of Müllerian ducts during gonadal sex differentiation of higher vertebrates. Teleost fish lack Müllerian ducts, but identified Amh orthologs have been shown to exert crucial functions during sex determination and differentiation of several species of teleosts. However, the function of Amh during gametogenesis in adult fish remains poorly investigated. Therefore, to expand present knowledge on the role of Amh in teleosts, the present study aimed to isolate and clone full-length amh cDNA in the common carp, Cyprinus carpio, and examine its expression levels throughout the male reproductive cycle and in response to different hormone treatments of testicular explants. Molecular cloning and characterization showed that the common carp Amh precursor amino acid sequence shared common features to other fish Amh precursors, including a conserved C-terminus (Tgf-ß domain) and a double proteolytic cleavage site (R-X-X-R-X-X-R) upstream to the Tgf-ß domain. Expression analysis showed amh dimorphic expression in the adult gonads with higher expression in the testes than ovaries. In testes, amh mRNA was detected in Sertoli cells contacting different types of germ cells, although the expression was greatest in Sertoli cells associated with type A undifferentiated spermatogonia. Expression analysis during the reproductive cycle showed that amh transcripts were down-regulated during the developing phase, which is characterized by an increased proliferation of type A undifferentiated spermatogonia and Sertoli cells and appearance of spermatocytes (meiosis) in the testes. Furthermore, ex vivo experiments showed that a 7 day exposure to Fsh or estrogens was required to decrease amh mRNA levels in common carp testicular explants. In summary, this study provided information on the molecular characterization and transcript abundance of amh in common carp adult testes. Altogether, these data will be useful for further investigations on sex determination and differentiation in this species, and also to improved strategies for improved carp aquaculture, such as inhibiting precocious maturation of males.

Cortisol Directly Stimulates Spermatogonial Differentiation, Meiosis, and Spermiogenesis in Zebrafish (Danio rerio) Testicular Explants.

Cortisol is the major endocrine factor mediating the inhibitory effects of stress on vertebrate reproduction. It is well known that cortisol affects reproduction by interacting with the hypothalamic-pituitary-gonads axis, leading to downstream inhibitory and stimulatory effects on gonads. However, the mechanisms are not fully understood. In this study, we provide novel data demonstrating the stimulatory effects of cortisol on spermatogenesis using an ex vivo organ culture system. The results revealed that cortisol treatment did not modulate basal androgen production, but it influenced transcript levels of a selected number of genes involved in the zebrafish testicular function ar (androgen receptor), star (steroidogenic acute regulatory), cyp17a1 (17α-hydroxylase/17,20 lyase/17,20 desmolase), cyp11a2 (cytochrome P450, family 11, subfamily A, polypeptide 2), hsd11b2 (11-beta hydroxysteroid dehydrogenase), cyp2k22 (cytochrome P450, family 2, subfamily K, polypeptide 22), fkbp5 (FKBP prolyl isomerase 5), grα (glucocorticoid receptor alpha), and grß (glucocorticoid receptor beta) in a short-term culture. We also showed that cortisol stimulates spermatogonial proliferation and differentiation in an androgen independent manner as well as promoting meiosis and spermiogenesis by increasing the number of spermatozoa in the testes. Moreover, we demonstrated that concomitant treatment with RU 486, a potent glucocorticoid receptor (Gr) antagonist, did not affect the cortisol effects on spermatogonial differentiation but blocked the induced effects on meiosis and spermiogenesis. Supporting the Gr-mediated effects, RU 486 nullified the cortisol-induced expression of sycp3l (synaptonemal complex protein 3), a marker for the meiotic prophase that encodes a component of the synaptonemal complex. This is consistent with in silico analysis that found 10 putative GREs (glucocorticoid response elements) upstream of the zebrafish sycp3l. Finally, we also showed that grα mRNA is expressed in Sertoli and Leydig cells, but also in several types of germ cells, including spermatogonia and spermatocytes. Altogether, this evidence indicates that cortisol exerts paracrine roles in the zebrafish testicular function and spermatogenesis, highlighting its effects on spermatogonial differentiation, meiosis, and spermiogenesis.

Characterization of vasa homolog in a neotropical catfish, Jundiá (Rhamdia quelen): Molecular cloning and expression analysis during embryonic and larval development.

We have characterized the full-length vasa cDNA from Jundiá, Rhamdia quelen (Heptapteridae, Siluriformes). vasa encodes a member of the DEAD-box protein family of ATP-dependent RNA helicases. This protein is highly conserved among different organisms and its role is associated with RNA metabolism. In the majority of the investigated species, vasa is restricted to the germ cell lineage and its expression has been used to study germline development in many organisms, including fish. The deduced R. quelen vasa amino acid sequence displayed high similarity with Vasa protein sequences from other organisms, and did not cluster with PL10 or P68 DEAD-box protein subfamilies. We also reported that there is no other isoform for vasa mRNA in R. quelen gonads. Expression analysis by RT-PCR and qPCR showed vasa transcripts exclusively expressed in the germ cells of R. quelen gonads. R. quelen vasa mRNA was maternally inherited, and was detected in the migrating primordial germ cells (PGCs) until 264 h post-fertilization during embryonic and larval development. This work has characterized for the first time the full-length R. quelen vasa cDNA, and describes its expression patterns during R. quelen embryonic and larval development. Our results will contribute to the basic reproductive biology of this native species, and will support studies using vasa as a germ cell marker in different biotechnological studies, such as germ cell transplantation.

Characterization of Gnrh/Gnih elements in the olfacto-retinal system and ovary during zebrafish ovarian maturation.

Gonadotropin releasing hormone (GnRH) is one of the key players of brain-pituitary-gonad axis, exerting overall control over vertebrate reproduction. In zebrafish, two variants were characterized and named as Gnrh2 and Gnrh3. In this species, Gnrh3, the hypohysiotropic form, is expressed by neurons of the olfactory-retinal system, where it is related with food detection, intra/interspecific recognition, visual acuity and retinal processing modulation. Previous studies have reported the presence of Gnrh receptors in the zebrafish retina, but not yet in the zebrafish olfactory epithelium. The current study analyzed the presence of gnrh2 and gnrh3, their receptors (gnrhr 1,2,3 and 4) and gnih (gonadotropin inhibitory hormone) transcripts, as well as the Gnrh3 protein in the olfactory epithelium (OE), olfactory bulb (OB), retina and ovary during zebrafish ovarian maturation. We found an increase of gnrh receptors transcripts in the OE at the final stages of ovarian maturation. In the OE, Gnrh3 protein was detected in the olfactory receptor neurons cilia and in the olfactory nerve fibers. Interestingly, in the OB, we found an inverse expression pattern between gnih and gnrh3. In the retina, gnrhr4 mRNA was found in the nuclei of amacrine, bipolar, and ganglion cells next to Gnrh3 positive fibers. In the ovary, gnrh3, gnrhr2 and gnrhr4 transcripts were found in perinucleolar oocytes, while gnih in oocytes at the cortical alveolus stage. Our results suggested that Gnrh/Gnih elements are involved in the neuromodulation of the sensorial system particularly at the final stages of maturation, playing also a paracrine role in the ovary.