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Overview of germline specification and sexual differentiation. (A) The zebrafish germline is specified by inheritance of maternal germ plasm (yellow) which can be seen at the animal pole and in select cleavage furrows of the early embryo (cartoon is animal pole view of 4-cell stage). (B) After specification, PGCs proliferate and migrate to the gonad anlage. (C) If PGC numbers are limiting then the developing fish will begin differentiation as a male. If sufficient PGCs are present, then a bi-potential ovary develops and meiosis I and progression of oogenesis commences through diplotene arrest at the end of prophase I. If adequate stage Ib oocytes are produced, then the somatic gonad and oocyte differentiate further but if there are insufficient stage Ib oocytes, male promoting factors increase in abundance and the juvenile ovary is replaced by a male gonad. Even after the ovary matures, sustained communication between the germline and the somatic gonad cells are required to maintain the expression of pro-female factors and female fate and to prevent oocyte death and transdifferentiation as a male
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Zebrafish have emerged as a major model organism to study vertebrate reproduction due to their high fecundity and external development of eggs and embryos. The mechanisms through which zebrafish determine their sex have come under extensive investigation, as they lack a definite sex-determining chromosome and appear to have a highly complex method...
Citations
... Whether all germ cell in these cysts continue along the oocyte trajectory is unclear because at this stage, the gonad has yet to adopt an ovary or testis fate. D. rerio lacks sexually dimorphic chromosomes (such as X and Y) and instead rely on a complex interaction between genetic and environmental factors to determine male or female fate (reviewed in ref. 158). In other model systems, including the fruit fly and mouse, only a fraction of cyst cells develops into oocytes, whereas the rest become nurse cells that feed their contents into the growing oocyte and then die 151 . ...
The germline provides the genetic and non-genetic information that passes from one generation to the next. Given this important role in species propagation, egg and sperm precursors, called primordial germ cells (PGCs), are one of the first cell types specified during embryogenesis. In fact, PGCs form well before the bipotential somatic gonad is specified. This common feature of germline development necessitates that PGCs migrate through many tissues to reach the somatic gonad. During their journey, PGCs must respond to select environmental cues while ignoring others in a dynamically developing embryo. The complex multi-tissue, combinatorial nature of PGC migration is an excellent model for understanding how cells navigate complex environments in vivo. Here, we discuss recent findings on the migratory path, the somatic cells that shepherd PGCs, the guidance cues somatic cells provide, and the PGC response to these cues to reach the gonad and establish the germline pool for future generations. We end by discussing the fate of wayward PGCs that fail to reach the gonad in diverse species. Collectively, this field is poised to yield important insights into emerging reproductive technologies.
... The M-AB strain can be maintained by regular care and thus will be in demand in the zebrafish research community. Sex is determined by the sex chromosome in mice, rats and medakas, whereas it is determined by both genetic and environmental factors in zebrafish 28 . Despite enormous efforts in sex determination studies, genetic factors that determine sex in zebrafish have not been unveiled. ...
Inbred strains of organisms are genetically highly uniform and thus useful for life science research. We have previously reported the ongoing generation of the zebrafish IM strain from the India (IND) strain through full sib-pair mating for 16 generations. However, the IM fish laid a small number of offspring and had a short lifespan, implying the need for discreet care in breeding. Here, we report the subsequent establishment of IM strain as well as the generation of a new inbred zebrafish strain, Mishima-AB (M-AB). M-AB was derived from the *AB strain by full sib-pair mating for over 20 generations, which fulfills the general criterion for the establishment of an inbred strain. In contrast to the IM case, maintenance of the M-AB strain by sib-pair mating required almost no special handling. Genome sequencing of IM individuals from the 47th generation and M-AB individuals from the 27th generation revealed that SNP-based genomic heterogeneity across whole-genome nucleotides was 0.008% and 0.011%, respectively. These percentages were much lower than those of the parental IND (0.197%) and *AB (0.086%) strains. These results indicate that the genomes of these inbred strains were highly homogenous. We also demonstrated the successful microinjection of antisense morpholinos, CRISPR/Cas9, and foreign genes into M-AB embryos at the 1-cell stage. Overall, we report the establishment of a zebrafish inbred strain, M-AB, which is capable of regular breeding and genetic manipulation. This strain will be useful for the analysis of genetically susceptible phenotypes such as behaviors, microbiome features and drug susceptibility.
... A second batch of experiments was performed with the rbfox1 sa15940 fish segregating them by genotype and sex. Sex was phenotypically determined as it cannot be genotyped in zebrafish [24,25]. In all instances, all fish were genotyped, sized-matched and 13 individuals were randomly selected per condition. ...
RBFOX1 is a highly pleiotropic gene that contributes to several psychiatric and neurodevelopmental disorders. Both rare and common variants in RBFOX1 have been associated with several psychiatric conditions, but the mechanisms underlying the pleiotropic effects of RBFOX1 are not yet understood. Here we found that, in zebrafish, rbfox1 is expressed in spinal cord, mid- and hindbrain during developmental stages. In adults, expression is restricted to specific areas of the brain, including telencephalic and diencephalic regions with an important role in receiving and processing sensory information and in directing behaviour. To investigate the contribution of rbfox1 to behaviour, we used rbfox1sa15940, a zebrafish mutant line with TL background. We found that rbfox1sa15940 mutants present hyperactivity, thigmotaxis, decreased freezing behaviour and altered social behaviour. We repeated these behavioural tests in a second rbfox1 mutant line with a different genetic background (TU), rbfox1del19, and found that rbfox1 deficiency affects behaviour similarly in this line, although there were some differences. rbfox1del19 mutants present similar thigmotaxis, but stronger alterations in social behaviour and lower levels of hyperactivity than rbfox1sa15940 fish. Taken together, these results suggest that mutations in rbfox1 lead to multiple behavioural changes in zebrafish that might be modulated by environmental, epigenetic and genetic background effects, and that resemble phenotypic alterations present in Rbfox1-deficient mice and in patients with different psychiatric conditions. Our study, thus, highlights the evolutionary conservation of rbfox1 function in behaviour and paves the way to further investigate the mechanisms underlying rbfox1 pleiotropy on the onset of neurodevelopmental and psychiatric disorders.
... Although the triggers for sex determination vary among species, ranging from genetic to environmental factors [2], the downstream genes and signaling pathways involved in sex differentiation are conserved, and their expression patterns show sexual dimorphism. For instance, dmrt1, gsdf, amh, and sox9 are highly expressed in testes, while foxl2 and cyp19a1a are predominantly expressed in ovaries [3][4][5]. In addition to ovary and testis development, sex differentiation is accompanied by a range of sexual dimorphisms, such as the growth rate, color, and size [6]. ...
... Integrating the analysis of ATAC-seq and RNA-seq revealed that some genes are associated with both relatively increased and decreased peaks simultaneously and that open chromatin regions could either improve or repress gene expression, which contributed to forming complex regulatory networks. Just like some well-known genes related to sex differentiation, dmrt1 can stimulate the expression of sox9a and repress the expression of foxl2, whereas foxl2 can inhibit dmrt1 and sox9a [3,4]. ...
Epigenetic modifications are critical in precisely regulating gene expression. The common carp (Cyprinus carpio) is an economically important fish species, and females exhibit faster growth rates than males. However, the studies related to epigenetic modifications in the common carp gonads are limited. In this study, we conducted the Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) and Bisulfite sequencing (BS-seq) to explore the roles of epigenetic modifications in the common carp gonads. We identified 84,207 more accessible regions and 77,922 less accessible regions in ovaries compared to testes, and some sex-biased genes showed differential chromatin accessibility in their promoter regions, such as sox9a and zp3. Motif enrichment analysis showed that transcription factors (TFs) associated with embryonic development and cell proliferation were heavily enriched in ovaries, and the TFs Foxl2 and SF1 were only identified in ovaries. We also analyzed the possible regulations between chromatin accessibility and gene expression. By BS-seq, we identified 2087 promoter differentially methylated genes (promoter-DMGs) and 5264 gene body differentially methylated genes (genebody-DMGs) in CG contexts. These genebody-DMGs were significantly enriched in the Wnt signaling pathway, TGF-beta signaling pathway, and GnRH signaling pathway, indicating that methylation in gene body regions could play an essential role in sex maintenance, just like methylation in promoter regions. Combined with transcriptomes, we revealed that the expression of dmrtb1-like, spag6, and fels was negatively correlated with their methylation levels in promoter regions. Our study on the epigenetic modifications of gonads contributes to elucidating the molecular mechanism of sex differentiation and sex maintenance in the common carp.
... The zebrafish dmrt1 mutant develops mainly as a fertile female. 55,56 The dmrt1 functions in male sex determination and testis development, 57 and the mutant dmrt1 developed sterile males with testicular dysgenesis. 56 The decreased transcriptions of dmrt1 in the ovaries of F0 females also supported the female bias in zebrafish. ...
Imidacloprid (IMI) and thiamethoxam (THM) are ubiquitous in aquatic ecosystems. Their negative effects on parental fish are investigated while intergenerational effects at environmentally relevant concentrations remain unclear. In this study, F0 zebrafish exposed to IMI and THM (0, 50, and 500 ng L-1) for 144 days post-fertilization (dpf) was allowed to spawn with two modes (internal mating and cross-mating), resulting in four types of F1 generations to investigate the intergenerational effects. IMI and THM affected F0 zebrafish fecundity, gonadal development, sex hormone and VTG levels, with accumulations found in F0 muscles and ovaries. In F1 generation, paternal or maternal exposure to IMI and THM also influenced sex hormones levels and elevated the heart rate and spontaneous movement rate. LncRNA-mRNA network analysis revealed that cell cycle and oocyte meiosis-related pathways in IMI groups and steroid biosynthesis related pathways in THM groups were significantly enriched in F1 offspring. Similar transcriptional alterations of dmrt1, insl3, cdc20, ccnb1, dnd1, ddx4, cox4i1l, and cox5b2 were observed in gonads of F0 and F1 generations. The findings indicated that prolonged paternal or maternal exposure to IMI and THM could severely cause intergenerational toxicity, resulting in developmental toxicity and endocrine-disrupting effects in zebrafish offspring.
... In most animals, gonadal sex is determined by signals from the somatic cells, such as mice [5]. Unlike in most mammals, zebrafish gonadal sex differentiation is mainly under the control of multiple genes and the number of germ cells [6][7][8]. Although the complete loss of germ cells lead to all-male phenotype along with sterility; interestingly, we and our collaborators found that those sterile males could behave like wild-type males to induce the female oviposition [9,10]. ...
Background:
Sexually dimorphic mating behaviors differ between sexes and involve gonadal hormones and possibly
sexually dimorphic gene expression in the brain. However, the associations among the brain, gonad, and sexual
behavior in teleosts are still unclear. Here, we utilized germ cells-free tdrd12 knockout (KO) zebrafish, and steroid synthesis
enzyme cyp17a1-deficient zebrafish to investigate the differences and interplays in the brain–gonad–behavior
axis, and the molecular control of brain dimorphism and male mating behaviors.
Methods:
Tdrd12+/−; cyp17a1+/− double heterozygous parents were crossed to obtain tdrd12−/−; cyp17a1+/+ (tdrd12
KO), tdrd12+/+; cyp17a1−/− (cyp17a1 KO), and tdrd12−/−; cyp17a1−/− (double KO) homozygous progenies. Comparative
analysis of mating behaviors were evaluated using Viewpoint zebrafish tracking software and sexual traits were
thoroughly characterized based on anatomical and histological experiments in these KOs and wild types. The steroid
hormone levels (testosterone, 11-ketotestosterone and 17β-estradiol) in the brains, gonads, and serum were measured
using ELISA kits. To achieve a higher resolution view of the differences in region-specific expression patterns
of the brain, the brains of these KOs, and control male and female fish were dissected into three regions: the forebrain,
midbrain, and hindbrain for transcriptomic analysis.
Results:
Qualitative analysis of mating behaviors demonstrated that tdrd12−/− fish behaved in the same manner
as wild-type males to trigger oviposition behavior, while cyp17a1−/− and double knockout (KO) fish did not exhibit
these behaviors. Based on the observation of sex characteristics, mating behaviors and hormone levels in these
mutants, we found that the maintenance of secondary sex characteristics and male mating behavior did not depend
on the presence of germ cells; rather, they depended mainly on the 11-ketotestosterone and testosterone levels
secreted into the brain–gonad regulatory axis. RNA-seq analysis of different brain regions revealed that the brain transcript
profile of tdrd12−/− fish was similar to that of wild-type males, especially in the forebrain and midbrain. However,
the brain transcript profiles of cyp17a1−/− and double KO fish were distinct from those of wild-type males and were
partially biased towards the expression pattern of the female brain. Our results revealed important candidate genes
... Consequently, we hypothesized that dietary crude oil exposure during development affects sex differentiation and thus skews the sex ratio in adulthood in the zebrafish (Danio rerio). We exposed zebrafish to crude oil through diet (<0.2, 6.5, 11.4, and 17.5 mg/kg food) from 20 to 35 dpfthe period of sex differentiation in this species (Aharon and Marlow, 2021;Kossack and Draper, 2019;Rodríguez-Marí et al., 2010) -and determined the number of females and males at 90 dpf. To better comprehend if dietary crude oil exposure during sex determination compromises other aspects of phenotype, we also assessed multiple fitness-related phenotypic traits (i.e., body mass and length, condition factor, heart rate, oxygen consumption, and the fish capacity to tolerate hypoxia). ...
... Our experiments were designed to maximize the effects of PAH exposure by timing it to occur during the sensitive period for sex differentiation in zebrafish (Fig. 1). Thus, dietary exposure to crude oil was performed in larvae from 20 to 35 dpf, corresponding to the period for sex differentiation (Aharon and Marlow, 2021;Rodríguez-Marí et al., 2010). Fish at 19 dpf and 27 dpf were exposed to their treatment diet (10 mg) twice a day. ...
... Asterisks in panel A indicate statistically significant differences in sampling days as per post hoc analyses. differentiation is largely diverse and plastic in fishes (Aharon and Marlow, 2021;Baroiller et al., 2009a;Capel, 2017;Devlin and Nagahama, 2002;Liew et al., 2012;Luckenbach and Yamamoto, 2018). Male-skewed sex ratios, intersexual and female-skewed sex ratios can result from the exposure to environmental stressors (i.e., temperature, (Baroiller et al., 2009a)). ...
Dietary crude oil exposure has detrimental morpho-physiological effects in fishes, including endocrine disruption. However, little is known about how it influences sex differentiation and its potential for skewing sex ratios of populations. Appropriate sex ratio is important for maintaining effective population size and structure. Deviations of these ratios can compromise population growth and maintenance and may induce changes in a species' evolutionary trajectory. We assessed the potential of dietary exposure to crude oil (6.5, 11.4, and 17.5 mg/kg food) to alter sex differentiation in the zebrafish (Danio rerio) (20-35 days post fertilization (dpf)) and subsequently skew the adult (90 dpf) sex ratio. Multiple health- and fitness-related phenotypic traits (i.e., body mass and length, condition factor, heart rate, oxygen consumption, and their capacity to cope with hypoxia) were also assessed to better understand the effects of dietary crude oil exposure. We showed that dietary exposure to crude oil during the process of sex differentiation skewed sex ratio towards males (up to 0.34:1 female to male ratio in the highest oil concentration). Remarkably, this effect occurred independently of affecting physiological variables and female gonad characteristics, thus highlighting just how subtle the effects of dietary crude oil exposure can be. Our results suggest that, although fish were in an apparently healthy state during experimentation, sex ratio was still impacted, potentially compromising the resilience of the population. Therefore, considering how complex chemical mixtures affect organisms at several levels (molecular-individual) in experimental designs is warranted to better understand the implications of the exposures and the hazards that populations face in the wild.
... Hence, as previously demonstrated [20], ovarian development is promoted by abundance of PGCs also at early developmental stages. Moreover, some proteins, such as Dead end and Nanos3, are involved in the control of PGC survival and migration, and their silencing results in allmale development [21]. ...
Background:
AMBRA1 is an intrinsically disordered protein, working as a scaffold molecule to coordinate, by protein-protein interaction, many cellular processes, including autophagy, mitophagy, apoptosis and cell cycle progression. The zebrafish genome contains two ambra1 paralogous genes (a and b), both involved in development and expressed at high levels in the gonads. Characterization of the zebrafish paralogous genes mutant lines generated by CRISPR/Cas9 approach showed that ambra1b knockout leads to an all-male population.
Results:
We demonstrated that the silencing of the ambra1b gene determines a reduction of primordial germ cells (PGCs), a condition that, in the zebrafish, leads to the development of all-male progeny. PGC reduction was confirmed by knockdown experiments and rescued by injection of ambra1b and human AMBRA1 mRNAs, but not ambra1a mRNA. Moreover, PGC loss was not rescued by injection with human AMBRA1 mRNA mutated in the CUL4-DDB1 binding region, thus suggesting that interaction with this complex is involved in PGC protection from loss. Results from zebrafish embryos injected with murine Stat3 mRNA and stat3 morpholino suggest that Ambra1b could indirectly regulate this protein through CUL4-DDB1 interaction. According to this, Ambra1+/- mice showed a reduced Stat3 expression in the ovary together with a low number of antral follicles and an increase of atretic follicles, indicating a function of Ambra1 in the ovary of mammals as well. Moreover, in agreement with the high expression of these genes in the testis and ovary, we found significant impairment of the reproductive process and pathological alterations, including tumors, mainly limited to the gonads.
Conclusions:
By exploiting ambra1a and ambra1b knockout zebrafish lines, we prove the sub-functionalization between the two paralogous zebrafish genes and uncover a novel function of Ambra1 in the protection from excessive PGC loss, which seems to require binding with the CUL4-DDB1 complex. Both genes seem to play a role in the regulation of reproductive physiology.
... Sex determination mechanisms are an intriguing research topic in evolutionary biology. Generally, the sex determination factors influence gonad differentiation by controlling downstream gene expression (Aharon, Marlow, 2021). Vertebrates, especially mammals and birds, possess a conserved X/Y or W/Z sex determination system with highly differentiated sex chromosomes containing specific sex determination genes. ...
Sex determination is a hot topic in evolutionary biology. The mechanism of sex determination is complex and diverse in shellfish, while no sex chromosomes have been found in mollusks. The Pacific abalone (Haliotis discus hannai) is one of the most economically important cultured shellfish species in China, and its sex determination mechanism is still unclear. Here, we identified the sex-determination region (SDR) of Pacific abalone and developed five stable sex-specific markers through genome-wide association studies (GWAS) based on re-sequencing data from 803 abalones. These markers can be used for sex discrimination in polyploid and sexually immature individuals in abalone breeding projects. In addition, haplotype analysis and quantitative expression of candidate sex-related genes showed that RhoGAPp190, Skp1, Cul2, and Otulin were potentially involved in the sex determination of Pacific abalone by the ubiquitin-mediated proteolysis pathway. Our study could help to better understand the molecular mechanisms that underpin sex determination and reproductive regulation in mollusks.
... In most animals, gonadal sex is determined by signals from the somatic cells, such as mice [4]; however, there is no evidence on whether the complete loss of germ cells could affect the formation of SSCs and sexual behaviors. Zebrafish gonadal sex differentiation is mainly under the control of multiple genes and the number of germ cells [5][6][7]. Interestingly, we and our collaborators found that some of the sex-reversed sterile males behaved like wide-type males to induce the female oviposition [8,9]. Therefore, how gonadal germ cells and somatic cells affect the formation of SSCs and male-typical mating behaviors warrant deep investigation. ...
Background: Sexually dimorphic mating behaviors differ between sexes and involve gonadal hormones and possibly sexually dimorphic gene expression in the brain. However, the associations among the brain, gonad, and sexual behavior in teleosts are still unclear. Here, we utilized germ cells-free tdrd12 knockout (KO) zebrafish, and steroid synthesis enzyme cyp17a1-deficient zebrafish to investigate the differences and interactions in the brain-gonad-behavior axis, and the molecular control of brain dimorphism and male mating behaviors.
Methods: Tdrd12+/-; cyp17a1+/- double heterozygous parents were crossed to obtain tdrd12-/-;cyp17a1+/+ (tdrd12 KO), tdrd12+/+;cyp17a1-/- (cyp17a1 KO), and tdrd12-/-;cyp17a1-/- (double KO) homozygous progenies. Comparative analysis of mating behaviors were evaluated by using Viewpoint zebrafish tracking software and sexual traits were thoroughly characterized based on anatomical and histological experiments in these KOs and wide-types. The steroid hormone levels (testosterone, 11-ketotestosterone and estrogen) in the brains, gonads, and serum were measured using ELISA kits. To achieve a higher-resolution view of the differences in region-specific expression patterns of the brain, the brains of these KOs, and control male and female fish were dissected into three regions: the forebrain, midbrain, and hindbrain for transcriptomic analysis.
Results: Qualitative analysis of mating behaviors demonstrated that tdrd12-/- fish behaved in the same manner as wild-type males to trigger oviposition behavior, while cyp17a1-/- and double knockout (KO) fish did not exhibit these behaviors. Based on the observation of sex characteristics, mating behaviors and hormone levels in these mutants, we found that the maintenance of secondary sex characteristics and male mating behavior did not depend on the presence of germ cells; rather, they depended mainly on the 11-ketotestosterone and testosterone levels secreted into the brain-gonad-regulatory axis. RNA-seq analysis of different brain regions revealed that the brain transcript profile of tdrd12-/- fish was similar to that of wild-type males, especially in the forebrain and midbrain. However, the brain transcript profiles of cyp17a1-/- and double KO fish were distinct from those of wild-type males and were partially biased towards the expression pattern of the female brain. Our results revealed key genes and signaling pathways, such as synaptic signaling/neurotransmission, MAPK signaling, and steroid hormone pathways, that shape brain dimorphism and modulate male mating behavior in zebrafish.
Conclusions: Our results provide comprehensive analyses and new insights regarding the endogenous interactions in the brain-gonad-behavior axis. Moreover, this study revealed the key genes and neural signaling pathways of different brain regions that are involved in modulating brain dimorphism and male mating behavior in zebrafish, which would significantly light up the understanding the neuroendocrine and molecular mechanisms modulating brain dimorphism and male mating behavior in zebrafish and other teleost fish.
