The single-cell chromatin landscape in gonadal cell lineage specification.

Gonad development includes sex determination and divergent maturation of the testes and ovaries. Recent advances in measuring gene expression in single cells are providing new insights into this complex process. However, the underlying epigenetic regulatory mechanisms remain unclear. Here, we profiled chromatin accessibility in mouse gonadal cells of both sexes from embryonic day 11.5 to 14.5 using single-cell assay for transposase accessible chromatin by sequencing (scATAC-seq). Our results showed that individual cell types can be inferred by the chromatin landscape, and that cells can be temporally ordered along developmental trajectories. Integrative analysis of transcriptomic and chromatin-accessibility maps identified multiple putative regulatory elements proximal to key gonadal genes Nr5a1, Sox9 and Wt1. We also uncover cell type-specific regulatory factors underlying cell type specification. Overall, our results provide a better understanding of the epigenetic landscape associated with the progressive restriction of cell fates in the gonad.

Understanding the genetic sex-determining mechanism in Hyla eximia treefrog inferred from H-Y antigen.

Genetic sex-determining mechanisms have been extensively elucidated in mammals; however, the sex chromosomes, sex-determining genes, and gene regulatory networks involved in sex differentiation remain poorly understood in amphibians. In this study, we investigated the sex-determining mechanism in the Hyla eximia treefrog based on karyotypic analysis and identification of H-Y antigen, a sex-linked peptide that is present in the gonads of the heterogametic sex (XY or ZW) in all vertebrates. Results show a diploid chromosome number 2n = 24 with homomorphic sex chromosomes. The heterogametic sex, ZW-female, were hypothesized based on H-Y antigen mRNA expression in female gonads (24,ZZ/24,ZW). The treefrog H-Y peptide exhibited a high percentage of identity with other vertebrate sequences uploaded to GenBank database. To obtain gene expression profiles, we also obtained the coding sequence of the housekeeping Actb gene. High H-Y antigen expression levels were further confirmed in ovaries using real-time polymerase chain reaction (RT-PCR) during non-breeding season, we noted a decrease in the expression of the H-Y antigen during breeding season. This study provides evidence that sex hormones might suppress H-Y antigen expression in the gonads of heterogametic females 24,ZW during the breeding season. These findings suggest that H-Y gene expression is a well-suited model for studying heterogametic sex by comparing the male and female gonads.

Complete male-to-female sex reversal in XY mice lacking the miR-17~92 cluster.

Mammalian sex determination is controlled by antagonistic gene cascades operating in embryonic undifferentiated gonads. The expression of the Y-linked gene SRY is sufficient to trigger the testicular pathway, whereas its absence in XX embryos leads to ovarian differentiation. Yet, the potential involvement of non-coding regulation in this process remains unclear. Here we show that the deletion of a single microRNA cluster, miR-17~92, induces complete primary male-to-female sex reversal in XY mice. Sry expression is delayed in XY knockout gonads, which develop as ovaries. Sertoli cell differentiation is reduced, delayed and unable to sustain testicular development. Pre-supporting cells in mutant gonads undergo a transient state of sex ambiguity which is subsequently resolved towards the ovarian fate. The miR-17~92 predicted target genes are upregulated, affecting the fine regulation of gene networks controlling gonad development. Thus, microRNAs emerge as key components for mammalian sex determination, controlling Sry expression timing and Sertoli cell differentiation.

Efficacy of gonadal shielding in dose reduction for female pelvic radiography.

This study aimed to evaluate the dose reduction using gonad shielding (GS) during pelvic imaging. Three types of pelvic images (radiography, magnetic resonance and computed tomography) were fused to elucidate the three-dimensional relationship between the position of ovaries and GS. To estimate the dose received by the ovaries, the off-axis dose at any given depth was measured under two different imaging conditions using thermoluminescence dosemeters and a polymethyl methacrylate phantom. The mean ovarian depth was 8.4 cm. The mean estimated ovarian dose without an additional filter was 0.36 mGy without GS and 0.14 mGy with GS. The mean estimated ovarian dose with an additional filter was 0.24 mGy without GS and 0.10 mGy with GS. The efficacy of ovarian dose reduction should be evaluated based on the achieved ovarian dose, considering the ovarian depth and use of additional filtration, rather than the ovarian protection rate of GS.

Differential expression of microRNAs in diapause and non-diapause gonads of Aspongopus chinensis Dallas (Hemiptera: Dinidoridae): implications for reproductive control.

Aspongopus chinensis Dallas, 1851 (Hemiptera: Dinidoridae), an edible and medicinal insect, usually found in China and Southeast Asia, offers substantial potential for various applications. The reproductive cycle of this particular insect occurs annually because of reproductive diapause, leading to inadequate utilization of available natural resources. Despite its considerable ecological importance, the precise mechanisms underlying diapause in A. chinensis are not yet well understood. In this study, we conducted an analysis of comparing the microRNA (miRNA) regulation in the diapause and non-diapause gonads of A. chinensis and identified 303 differentially expressed miRNAs, among which, compared with the diapause group, 76 miRNAs were upregulated and 227 miRNAs downregulated. The results, regarding the Enrichment analysis of miRNA-targeted genes, showed their involvement in several essential biological processes, such as lipid anabolism, energy metabolism, and gonadal growth. Interestingly, we observed that the ATP-binding cassette pathway is the only enriched pathway, demonstrating the capability of these targeted miRNAs to regulate the reproductive diapause of A. chinensis through the above essential pathway. The current study provided the role of gonadal miRNA expression in the control of reproductive diapause in A. chinensis, the specific regulatory mechanism behind this event remained unknown and needed more investigation.

Chronic exposure to gestodene impairs reproductive system in adult female zebrafish (Daniarerio).

Gestodene (GES) is widely used in human therapy and animal husbandry and is frequently detected in aquatic environments. Although GES adversely affects aquatic organisms at trace levels, its effects on the reproductive biology of fish remain inconclusive. In this study, female zebrafish (Danio rerio) were exposed to environmentally relevant levels of GES for the evaluation of the effects of GES on the reproductive system by using endpoints including gene expression, plasma steroid concentrations, histological and morphological analyses, copulatory behavior, and reproductive output. Adult female zebrafish exposed to environmentally relevant concentrations of GES (4.0, 40.2, and 372.7 ng/L) for 60 d demonstrated stagnant ovarian oocyte development, evidenced by an increase in the percentage of perinuclear and atretic oocytes and a decrease in the percentage of late vitellogenic oocytes. GES-exposed females were less attractive to males and had lower copulatory intimacy than females in control. Consequently, spawning (44.3-49.2 %) and egg fertilization rates (27.9-32.0 %) were decreased. The decreased survival of fertilized eggs and hatching rates were accompanied by increased malformations. These negative effects were associated with abnormal transcriptional levels of gonadal steroid hormones, which were regulated by genes (Hsd17ß3, Hsd11ß2, Hsd20ß, Cyp19a1a, and Cyp11b). Overall, our findings suggest that GES impairs the reproductive system of zebrafish, which may threaten population stability.

The expression profiles of cyp19a1, sf-1, esrs and gths in the brain-pituitary during gonadal sex differentiation in juvenile Japanese eels.

Eels are gonochoristic species whose gonadal differentiation initiates at the yellow eel stage and is influenced by environmental factors. We revealed some sex-related genes were sex dimorphically expressed in gonads during gonadal sex differentiation of Japanese eel (Anguilla japonica); however, the expression of sex-related genes in the brain-pituitary during gonadal sex differentiation in eels is still unclear. This study aimed to investigate the sex-related gene expressions in the brain-pituitary and tried to clarify their roles in the brain and gonads during gonadal sex differentiation. Based on our previous histological study, the control eels developed as males, and estradiol-17ß (E2) was used for feminization. Our results showed that during testicular differentiation, the brain cyp19a1 transcripts and aromatase proteins were increased significantly; moreover, the cyp19a1, sf-1, foxl2s, and esrs (except gperb) transcripts in the midbrain/pituitary also were increased significantly. Forebrain gnrh1 transcripts increased slightly during gonadal differentiation of both sexes, but the gnrhr1b and gnrhr2 transcripts in the midbrain/pituitary were stable during gonadal differentiation. The expression levels of gths and gh in the midbrain/pituitary were significantly increased during testicular differentiation and were much higher in males than in E2-feminized females. These results implied that endogenous estrogens might play essential roles in the brain/pituitary during testicular differentiation, sf-1, foxl2s, and esrs may have roles in cyp19a1 regulation in the midbrain/pituitary of Japanese eels. For the GnRH-GTH axis, gths, especially fshb, may be regulated by esrs and involved in regulating testicular differentiation and development in Japanese eels.

Gonadal transcriptome analysis of genes related to sex differentiation and sex development in the Pomacea canaliculata.

As an invasive alien animal, Pomacea canaliculata poses a great danger to the ecology and human beings. Recently, there has been a gradual shift towards bio-friendly control. Based on the development of RNA interference and CRISPR technology as molecular regulatory techniques for pest control, it was determined if the knockout of genes related to sex differentiation in P. canaliculata could induce sterility, thereby helping in population control. However, the knowledge of sex differentiation- and development-related genes in P. canaliculata is currently lacking. Here, transcriptomic approaches were used to study the genes expressed in the two genders of P. canaliculata at various developmental stages. Gonad transcriptomes of immature or mature males and females were compared, revealing 12,063 genes with sex-specific expression, of which 6066 were male- and 5997 were female-specific. Among the latter, 581 and 235 genes were up-regulated in immature and mature females, respectively. The sex-specific expressed genes identified included GnRHR2 and TSSK3 in males and ZAR1 and WNT4 in females. Of the genes, six were involved in reproduction: CCNBLIP1, MND1, DMC1, DLC1, MRE11, and E(sev)2B. Compared to immature snail gonads, the expression of HSP90 and CDK1 was markedly reduced in gonadal. It was hypothesized that the two were associated with the development of females. These findings provided new insights into crucial genetic information on sex differentiation and development in P. canaliculata. Additionally, some candidate genes were explored, which can contribute to future studies on controlling P. canaliculata using molecular regulatory techniques.

Parthenogenetic Stick Insects Exhibit Signatures of Preservation in the Molecular Architecture of Male Reproduction.

After the loss of a trait, theory predicts that the molecular machinery underlying its phenotypic expression should decay. Yet, empirical evidence is contrasting. Here, we test the hypotheses that (i) the molecular ground plan of a lost trait could persist due to pleiotropic effects on other traits and (ii) that gene co-expression network architecture could constrain individual gene expression. Our testing ground has been the Bacillus stick insect species complex, which contains close relatives that are either bisexual or parthenogenetic. After the identification of genes expressed in male reproductive tissues in a bisexual species, we investigated their gene co-expression network structure in two parthenogenetic species. We found that gene co-expression within the male gonads was partially preserved in parthenogens. Furthermore, parthenogens did not show relaxed selection on genes upregulated in male gonads in the bisexual species. As these genes were mostly expressed in female gonads, this preservation could be driven by pleiotropic interactions and an ongoing role in female reproduction. Connectivity within the network also played a key role, with highly connected-and more pleiotropic-genes within male gonad also having a gonad-biased expression in parthenogens. Our findings provide novel insight into the mechanisms which could underlie the production of rare males in parthenogenetic lineages; more generally, they provide an example of the cryptic persistence of a lost trait molecular architecture, driven by gene pleiotropy on other traits and within their co-expression network.

The gonadal niche safeguards human fetal germline cell development following maternal SARS-CoV-2 infection.

During pregnancy, germline development is vital for maintaining the continuation of species. Recent studies have shown increased pregnancy risks in COVID-19 patients at the perinatal stage. However, the potential consequence of infection for reproductive quality in developing fetuses remains unclear. Here, we analyze the transcriptome and DNA methylome of the fetal germline following maternal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We find that infection at early gestational age, a critical period of human primordial germ cell specification and epigenetic reprogramming, trivially affects fetal germ cell (FGC) development. Additionally, FGC-niche communications are not compromised by maternal infection. Strikingly, both general and SARS-CoV-2-specific immune pathways are greatly activated in gonadal niche cells to protect FGCs from maternal infection. Notably, there occurs an "in advance" development tendency in FGCs after maternal infection. Our study provides insights into the impacts of maternal SARS-CoV-2 infection on fetal germline development and serves as potential clinical guidance for future pandemics.

Exploration of cell-cell interactions and the notch signaling pathway in the gonadal niche of Crassostrea gigas.

The Notch signaling pathway plays a pivotal role in governing cell fate determinations within the gonadal niche. This study provides an extensive elucidation of the male and female gonadal niches within Crassostrea gigas. Examination via transmission electron microscopy revealed the presence of desmosome-like connection not only between germ cells and niche cells but also among adjacent niche cells within the oyster gonad. Transcriptomic analysis identified several putative Notch pathway components, including CgJAG1, CgNOTCH1, CgSuh, and CgHey1. Phylogenetic analysis indicated a close evolutionary relationship between CgJAG1, CgNOTCH1, and CgHey1 and Notch members present in Drosophila. Expression profiling results indicated a notable abundance of CgHey1 in the gonads, while CgJAG1 and CgNOTCH1 displayed distinct expression patterns associated with sexual dimorphism. In situ hybridization findings corroborated the predominant expression of CgJAG1 in male niche cells, while CgNOTCH1 was expressed in both male and female germ cells, as well as female niche cells. These findings demonstrate the important role of the Notch signaling pathway in the gonadal niche of oysters.

Gonadal sex and chromosome complement influence the gut microbiome in a mouse model of allergic airway inflammation.

Evidence abounds that gut microbiome components are associated with sex disparities in the immune system. However, it remains unclear whether the observed sex disparity in asthma incidence is associated with sex-dependent differences in immune-modulating gut microbiota, and/or its influence on allergic airway inflammatory processes. Using a mouse model of house dust mite (HDM)-induced allergic inflammation and the four core genotypes (FCGs) model, we have previously reported sex differences in lung inflammatory phenotypes. Here, we investigated associations of gut microbiomes with these phenotypes by challenging FCG mice [mouse with female sex chromosome and male gonad (XXM), mouse with female sex chromosome and female gonad (XXF), mouse with male sex chromosome and male gonad (XYM), and mouse with male sex chromosome and female gonad (XYF); n = 7/group] with HDM (25 µg) or PBS intranasally for 5 wk and collecting fecal samples. We extracted fecal DNA and analyzed the 16S microbiome via Targeted Metagenomic Sequencing. We compared α and ß diversity across genotypes and assessed the Firmicutes/Bacteroidetes (F/B) ratio. When comparing baseline and after exposure for the FCG, we found that the gut F/B ratio was only increased in the XXM genotype. We also found that α diversity was significantly increased in all FCG mice upon HDM challenge, with the highest increase in the XXF, and the lowest in the XXM genotypes. Similarly, ß diversity of the microbial community was also affected by challenge in a gonad- and chromosome-dependent manner. In summary, our results indicated that HDM treatment, gonads, and sex chromosomes significantly influence the gut microbial community composition. We concluded that allergic lung inflammation may be affected by the gut microbiome in a sex-dependent manner involving both hormonal and genetic influences.NEW & NOTEWORTHY Recently, the gut microbiome and its role in chronic respiratory disease have been the subject of extensive research and the establishment of its involvement in immune functions. Using the FCG mouse model, our findings revealed the influence of gonads and sex chromosomes on the microbial community structure before and after exposure to HDM. Our data provide a potential new avenue to better understand mediators of sex disparities associated with allergic airway inflammation.

Reproductive toxic effects of chronic exposure to bisphenol A and its analogues in marine medaka (Oryzias melastigma).

As awareness of BPA's health risks has increased, many countries and regions have implemented strict controls on its use. Consequently, bisphenol analogues like BPF and BPAF are being increasingly used as substitutes. However, these compounds are also becoming increasingly prevalent in the environment due to production, use and disposal processes. The oceans act as a repository for various pollutants, and recent studies have revealed the extensive presence of bisphenols (BPs, including BPA, BPF, BPAF, etc.) in the marine environment, posing numerous health hazards to marine wildlife. Nevertheless, the reproductive toxicity of these chemicals on marine fish is not comprehensively comprehended yet. Thus, the histological features of the gonads and the gene expression profiles of HPG (Hypothalamic-Pituitary-Gonadal) axis-related genes in marine medaka (Oryzias melastigma) were studied after exposure to single and combined BPs for 70 days. The effects of each exposure group on spawning, embryo fertilization, and hatching in marine medaka were also assessed. Furthermore, the impacts of each exposure group on the genes related to methylation in the F2 and F3 generations were consistently investigated. BPs exposure was found to cause follicular atresia, irregular oocytes, and empty follicles in the ovary; but no significant lesions in the testis were observed. The expression of several HPG axis genes, including cyp19b, 17ßhsd, 3ßhsd, and fshr, resulted in significant changes compared to the control group. The quantity of eggs laid and fertilization rate decreased in all groups treated with BPs, with the BPAF-treated group showing a notable reduction in the number of eggs laid. Additionally, the hatching rate showed a more significant decline in the BPF-treated group. The analysis of methylated genes in the offspring of bisphenol-treated groups revealed significant changes in the expression of genes including amh, dnmt1, dnmt3ab, mbd2, and mecp2, indicating a potential transgenerational impact of bisphenols on phenotype through epigenetic modifications. Overall, the potential detrimental impact of bisphenol on the reproduction of marine medaka emphasizes the need for caution in considering the use of BPAF and BPF as substitutes.

Is the use of gonad protection protectors necessary during infants chest radiography?

INTRODUCTION AND OBJECTIVES: To compare gonad doses with and without a gonad protector and to optimize the use of gonadal protectors in infants thorax radiography. MATERIALS AND METHODS: Two pediatric anthropomorphic phantoms are used: an X-ray system for KXO-50SS/DRX-3724HD, and a digital radiography system for CALNEO Smart C12, with and without a gonad protector during infants thorax radiography. A real time skin dosimeter is placed on the X-ray system, and a real time skin dosimeter is inserted on the front side of the mammary gland, the front and back sides of the true pelvis level, and on the ovaries and testes. The X-ray system is irradiated 15 times using phantoms with and without a gonad protector. The measured entrance patient doses values of for the real time skin dosimeter are compared for each phantom, with and without the gonad protector. RESULTS: The medium of measured entrance patient doses values for front side dose of the true pelvis level with and without the protector are 10.00 and 5.00 µGy at newborn, and 10.00 and 0.00µGy at one year, respectively. The medium of measured entrance patient doses values for the back side dose of the true pelvis level with and without the protector are 0.00 and 0.00 µGy at both newborn one year, respectively. The measured entrance patient doses cannot be detected in the ovaries and testes with or without the protector. No significant differences are observed in the measured entrance patient doses values for the front and back side doses of the pelvis, ovaries, and testes at newborn and one year, with and without the protector (p>0.05). CONCLUSIONS: No significant difference was observed in gonad dose measurements with and without the gonad protector during infants chest radiography. We believe that gonadal protector wearing is not necessary.

Valorisation of sea urchin (Paracentrotus lividus) gonads through canning.

Fresh sea urchin (Paracentrotus lividus) gonads are a delicacy with short seasonal availability, very often heterogeneous in size and intrinsic characteristics. This study aimed to valorise this resource through the preparation of canned products (with/without Porphyra spp.) and evaluate their physicochemical and sensory quality (3-12 months). Canning contributed to a decrease in protein, K and most carotenoids contents; and a concentration of lipids, ash, Na and Se levels. A simulated 12-month ageing led to decrease the protein and ß-carotene contents; and the Na and lutein levels concentration. The macroalgae addition resulted in an orange, darker and less soft product, with higher carbohydrates, Na, Se and carotenoids contents. A 25 g-dose contributes to significant daily intakes of protein (8-9%), EPA+DHA (47-53%), I (35-62%) and Se (30-47%). The products were commercially stable/sterile and had good sensory acceptance. Overall, canning constitutes a strategy to provide a nutritionally balanced product available all year-round.

¿Es necesario utilizar protectores de gónadas durante la realización de radiografías de tórax en los lactantes? / Is the use of gonad protection protectors necessary during infants chest radiography?

Introducción y objetivos: Comparar las dosis de radiación en las gónadas con y sin protector gonadal y optimizar el uso de estos protectores al realizar radiografías de tórax a lactantes. Materiales y métodos: Se utilizan 2 maniquíes antropomórficos pediátricos, un sistema de rayos X KXO-50SS/DRX-3724HD, y un sistema de radiografía digital CALNEO Smart C12, con y sin protector de gónadas durante la realización de radiografías de tórax. Se coloca un dosímetro cutáneo en tiempo real en el sistema de rayos X y se inserta un dosímetro cutáneo en tiempo real en la cara anterior de la glándula mamaria, en la cara anterior y posterior de la pelvis verdadera, y en los ovarios y testículos. El sistema de rayos X se irradia 15 veces con maniquíes, con y sin el protector de gónadas. Se comparan los valores de las dosis de entrada del paciente medidos por el dosímetro cutáneo en tiempo real para cada maniquí, con y sin el protector de gónadas. Resultados: Los valores medios de las dosis a la entrada del paciente medidos para la cara anterior a nivel de la pelvis verdadera, con y sin el protector, son 10,00 y 5,00μGy en el recién nacido, y 10,00 y 0,00μGy al año, respectivamente. Los valores medios de las dosis a la entrada del paciente medidos para la cara posterior a nivel de la pelvis verdadera con y sin el protector son de 0,00 y 0,00μGy tanto en el recién nacido como al año, respectivamente. Las dosis a la entrada del paciente medidas no se pueden detectar en los ovarios y los testículos ni con el protector ni sin él. No se observan diferencias significativas en los valores de las dosis a la entrada del paciente medidas en la cara anterior y posterior de la pelvis, los ovarios y los testículos en el recién nacido y al año, con y sin el protector (p>0,05).(AU)

Introduction and objectives: To compare gonad doses with and without a gonad protector and to optimize the use of gonadal protectors in infants thorax radiography. Materials and methods: Two pediatric anthropomorphic phantoms are used: an X-ray system for KXO-50SS/DRX-3724HD, and a digital radiography system for CALNEO Smart C12, with and without a gonad protector during infants thorax radiography. A real time skin dosimeter is placed on the X-ray system, and a real time skin dosimeter is inserted on the front side of the mammary gland, the front and back sides of the true pelvis level, and on the ovaries and testes. The X-ray system is irradiated 15 times using phantoms with and without a gonad protector. The measured entrance patient doses values of for the real time skin dosimeter are compared for each phantom, with and without the gonad protector. Results: The medium of measured entrance patient doses values for front side dose of the true pelvis level with and without the protector are 10.00 and 5.00μGy at newborn, and 10.00 and 0.00μGy at one year, respectively. The medium of measured entrance patient doses values for the back side dose of the true pelvis level with and without the protector are 0.00 and 0.00μGy at both newborn one year, respectively. The measured entrance patient doses cannot be detected in the ovaries and testes with or without the protector. No significant differences are observed in the measured entrance patient doses values for the front and back side doses of the pelvis, ovaries, and testes at newborn and one year, with and without the protector (p>0.05). Conclusions: No significant difference was observed in gonad dose measurements with and without the gonad protector during infants chest radiography. We believe that gonadal protector wearing is not necessary.(AU)

Gonads under stress: A systematic review and meta-analysis on the effects of acute psychosocial stress on gonadal steroids secretion in humans.

Animal research has shown that the hypothalamus-pituitary-gonadal (HPG) axis is inhibited by (chronic and/or severe) stress, which can lead to impaired fertility and reproductive functioning, presumably caused by the inhibition of gonadal steroid secretion and in interactions with glucocorticoids. However, what has not been clarified is how acute psychosocial stress modulates gonadal steroid secretion in humans. Here we summarize the experimental research on the acute effects of stress on the secretion of gonadal steroids in humans. A systematic literature search revealed 21 studies (with N=881 individuals) measuring testosterone, progesterone or estradiol in response to a standardized acute laboratory stressor in healthy humans. Both our literature review and quantitative meta-analysis suggest that in humans, acute stress stimulates rather than inhibits HPG axis activity, although there is a considerable heterogeneity in the reported methods and results. Increased gonadal steroids in response to acute stress contrasts with many animal studies reporting the opposite pattern, at least regarding severe and/or chronic stressors. We discuss methodological issues and challenges for future research and hope to stimulate experimental studies within this area. A better understanding of these mechanisms is needed, and may have important implications for health and disease, as well as the modulation of various behaviors by acute stressors.

Biological patterns of reproduction of the Brazilian sardine Sardinella brasiliensis in the purse seine fishery of Southwest Atlantic Ocean: A long-term assessment.

Recent estimates of the size at first maturity (L50) of Sardinella brasiliensis showed contradictory results with a decreasing in the fish stock biomass encompassed by increasing values of L50. The methodological approach used hereby allowed to separate sardines classified in the virginal maturity stage from those categorized in the recovery stage, and ready for one next spawning event. This study evaluated the hypothesis of the existence of separated stocks experiencing distinct environmental conditions and fishing pressures which may have altered L50 estimates using a robust dataset based on biological samples collected along the entire species distribution area in the southeast-south Brazilian coast [Rio de Janeiro (RJ), São Paulo (SP), Paraná (PR), Santa Catarina (SC) and Rio Grande do Sul (RS)] between 2000 and 2018. A reclassification of the gonadal maturity stages provided a more realistic estimate of L50. Combining biological, reproductive, fishing data and the mean temperature of the catch (MTC), the leave-one-out classification correctly re-assigned individuals with an overall accuracy of 85% [100% (RJ), 45% (SP), 99% (PR), 99% (SC) and 82% (RS)]. The connectivity between the local populations of S. brasiliensis off RJ (23°S) and the southern populations is limited, contrasting to spatial structured semi-discrete population-units found between SP and RS (24°S-30°S). The northern extreme population-unit (RJ, 22°S-23°S) showed an expressive reduction of L50, and a negative correlation was detected between the increasing MTC values and the abundance of early maturing individuals and recruits of the species. Stock specific L50 estimates seemed to act as indicators of long term environmental fluctuations.

Dmrt1 is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia.

Sex is determined by multiple factors derived from somatic and germ cells in vertebrates. We have identified amhy, dmrt1, gsdf as male and foxl2, foxl3, cyp19a1a as female sex determination pathway genes in Nile tilapia. However, the relationship among these genes is largely unclear. Here, we found that the gonads of dmrt1;cyp19a1a double mutants developed as ovaries or underdeveloped testes with no germ cells irrespective of their genetic sex. In addition, the gonads of dmrt1;cyp19a1a;cyp19a1b triple mutants still developed as ovaries. The gonads of foxl3;cyp19a1a double mutants developed as testes, while the gonads of dmrt1;cyp19a1a;foxl3 triple mutants eventually developed as ovaries. In contrast, the gonads of amhy;cyp19a1a, gsdf;cyp19a1a, amhy;foxl2, gsdf;foxl2 double and amhy;cyp19a1a;cyp19a1b, gsdf;cyp19a1a;cyp19a1b triple mutants developed as testes with spermatogenesis via up-regulation of dmrt1 in both somatic and germ cells. The gonads of amhy;foxl3 and gsdf;foxl3 double mutants developed as ovaries but with germ cells in spermatogenesis due to up-regulation of dmrt1. Taking the respective ovary and underdeveloped testis of dmrt1;foxl3 and dmrt1;foxl2 double mutants reported previously into consideration, we demonstrated that once dmrt1 mutated, the gonad could not be rescued to functional testis by mutating any female pathway gene. The sex reversal caused by mutation of male pathway genes other than dmrt1, including its upstream amhy and downstream gsdf, could be rescued by mutating female pathway gene. Overall, our data suggested that dmrt1 is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia.