2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental toxicant and endocrine disrupting compound with reproductive and developmental effects in humans and model organisms, including zebrafish. Our previous microarray and histological studies found defects in spermatogenesis and fertility of zebrafish in response to acute developmental TCDD exposure. These effects are apparent following exposure during reproductive development, modeling fetal basis of adult-onset disease. Some outcomes of these previous studies (reduced fertility, changes in sex ratio, transcriptomic alterations) are also transgenerational - persisting to unexposed generations - through the male germline. We hypothesized that DNA methylation could be a possible mechanism for these reproductive effects and performed whole genome bisulfite sequencing (WGBS), which identifies whole genome DNA methylation status at the base pair level, on testes of adult zebrafish exposed to TCDD (two separate hour-long exposures to 50 pg/mL TCDD at 3 and 7 weeks post fertilization). In response to TCDD exposure, multiple genes were differentially methylated; many of which are involved in reproductive processes or epigenetic modifications, suggesting a role of DNA methylation in later-life health outcomes. Additionally, several differentially methylated genes corresponded with gene expression changes identified in TCDD-exposed zebrafish testes, indicating a potential link between DNA methylation and gene expression. Ingenuity pathway analysis of WGBS and microarray data revealed genes involved in reproductive processes and development, RNA regulation, the cell cycle, and cellular morphology and development. We conclude that site-specific changes in DNA methylation of adult zebrafish testes occur in response to acute developmental TCDD exposure.

The sex of Chinese tongue sole (Cynoglossus semilaevis) is determined by both genetic sex determination (GSD) and environmental sex determination (ESD), making it an ideal model to study the relationship between sex-determination and temperature. In the present study, transcriptomes of undifferentiated gonads from genetic females and males, as well as differentiated gonads from males, females, and pseudomales under high and normal temperature treatments were generated for comparative transcriptomic analysis. A mean of 68.24 M high-quality clean reads was obtained for each library. Differentially expressed genes (DEGs) between different sexes and environmental treatments were identified, revealing that the heat shock protein gene family was involved in the high temperature induced sex reversal. The Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were enriched in pseudomale and genetic female comparison included neuroactive ligand-receptor interaction, cortisol synthesis and secretion, and steroid hormone biosynthesis. Furthermore, weighted gene co-expression network analyses were conducted on all samples, and two modules were positive correlated with pseudomale under high temperature. An illustrated protein-protein interaction map of the module identified a hub gene, hsc70. These findings provide insights into the genetic network that is involved in sex determination and sexual differentiation, and improve our understanding of genes involved in sex reversal under high temperature.

Schistosomes are the only platyhelminths that have evolved separate sexes, and they exhibit a unique reproductive biology because the female's sexual maturation depends on a constant pairing contact with the male. In the female, pairing leads to gonad differentiation, which is associated with substantial morphological changes, and controls among others the expression of gonad-associated genes. In the male, no morphological changes have been observed after pairing, although first data indicated an effect of pairing on gene transcription. Comprehensive transcriptomic approaches have revealed an unexpected high number of genes that are differentially transcribed in the male after pairing. Their identities suggest roles for the male that are not restricted to feeding and enhanced muscular power to transport paired female and, as assumed before, to induce its sexual maturation by one "magic" factor. Instead, a more complex picture emerges in which both partners live in a reciprocal sender-recipient relationship that not only affects the gonads of both genders but may also involve tactile stimuli, transforming growth factor β signaling, nutritional parts, and neuronal processes, including neuropeptides and G protein-coupled receptor signaling. This review provides a summary of transcriptomics including an overview of genes expressed in a pairing-dependent manner in schistosome males. This may stimulate further research in understanding the role of the male as the recipient of the female's signals upon pairing, the male's "capacitation," and its subsequent competence as a sender of information. The latter process finally transforms a sexually immature, autonomous female without completely developed gonads into a sexually mature, partially non-autonomous female with fully differentiated gonads and enormous egg production capacity.

In the search for new strategies to fight schistosomiasis, the unique reproductive biology of Schistosoma mansoni has come into the focus of research. The development of the gonads and the ability of egg production are fundamental not only for continuing the life cycle but also for pathogenicity. Previous studies of schistosome biology demonstrated an influence of pairing on gonad development of the female and on gene expression profiles in both genders. Due to the limited access to specific tissues, however, most of these studies were done at the level of whole worms neglecting individual tissues that may be targets of pairing-dependent processes. Recently, we established a protocol allowing the isolation of testes and ovaries from adult S. mansoni. Here, we describe tissue-specific qRT-PCR analyses comparing transcript levels of selected genes on the basis of RNA from gonads and whole worms. Gene expression in ovary and testes was in some cases found to be significantly influenced by pairing, which was not traceable in whole worms. Among the candidate genes identified as regulated by pairing in gonads were the frizzled homolog SmFz1 and the two fibroblast growth factor receptor homologs SmFGFR-A and SmFGFR-B. First functional characterizations were done, including comparative qRT-PCR analyses, in situ-localization experiments, heterologous expression in Xenopus oocytes (SmFGFR-A/B), and inhibitor studies using the Fz/Dvl-pathway inhibitor 3289-8625, or BIBF1120 blocking FGFR-signaling. Besides confirming gonad localization and receptor functions, inhibitor-induced phenotypes were observed in vitro such as decreased egg production as well as drastic effects on gonad differentiation, morphology, embryogenesis, and survival of adult worms. In summary, these results emphasise the usefulness of tissue-specific qRT-PCRs for selection of candidate genes with important roles in reproduction, allowing subsequent studies to determine their suitability as drug targets.

Macrobrachium rosenbergii (M. rosenbergii), as a species of common prawn, is a delicacy that is consumed all over the world. By interacting with the target gene 3'-untranslated region (3'-UTR), microRNAs (miRNAs) regulate its expression and ultimately participate in the regulation of reproductive development. However, research focusing on miRNA regulation during gonadal development in M. rosenbergii received very little attention. To explore the association between miRNA and reproduction, we performed RNA sequencing (RNA-seq) on brain and gonad organs in male and female M. rosenbergii. A total of 494 miRNAs were obtained in RNA-seq, including 31 and 59 differentially expressed (DE) miRNAs in the brain and gonads, respectively. Furthermore, 9 DE miRNAs were randomly selected from the brain and gonads, and qRT-PCR was conducted to validate the results of RNA-seq. Interestingly, dpu-miR-133 was found to be substantially expressed in the male brain and testis but poorly expressed in the female brain, ovary, and other organs. Analysis of dpu-miR-133 by Targetscan and MiRanda predicted to target 5-HT1. Furthermore, the dual-luciferase reporter assay manifested that dpu-miR-133 can combine with 5-HT1. Overall, our research work provides basic data for further study on the miRNA-mediated regulation of brain, gonad, and reproductive development of study M. rosenbergii.

Supernumerary B chromosomes are dispensable elements found in several groups of eukaryotes, and their impacts in host organisms are not clear. The cichlid fish Astatotilapia latifasciata presents one or two large metacentric B chromosomes. These elements affect the transcription of several classes of RNAs. Here, we evaluated the epigenetic DNA modification status of B chromosomes using immunocytogenetics and assessed the impact of B chromosome presence on the global contents of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) and the molecular mechanisms underlying these variations. We found that the B chromosome of A. latifasciata has an active pattern of DNA epimarks, and its presence promotes the loss of 5mC in gonads of females with B chromosome (FB+) and promotes the loss of 5hmC in the muscle of males with the B element (MB+). Based on the transcriptional quantification of DNA modification genes (dnmt, tet, and tdg) and their candidate regulators (idh genes, microRNAs, and long non-coding RNAs) and on RNA-protein interaction prediction, we suggest the occurrence of passive demethylation in gonads of FB+ and 5hmC loss by Tet inhibition or by 5hmC oxidation in MB+ muscle. We suggest that these results can also explain the previously reported variations in the transcription levels of several classes of RNA depending on B chromosome presence. The DNA modifications detected here are also influenced by sex. Although the correlation between B chromosomes and sex has been previously reported, it remains unexplained. The B chromosome of A. latifasciata seems to be active and impacts cell physiology in a very complex way, including at the epigenetic level.

Global microarray gene expression analyses previously demonstrated differences in female and male embryos during neurodevelopment. In particular, before sexual maturation of the gonads, the differences seem to concentrate on the expression of genes encoded on the X- and Y-chromosomes. To investigate genome-wide differences in expression during this early developmental window, we combined high-resolution RNA sequencing with qPCR to analyze brain samples from human embryos during the first trimester of development. Our analysis was tailored for maximum sensitivity to discover Y-chromosome gene expression, but at the same time, it was underpowered to detect X-inactivation escapees. Using this approach, we found that 5 out of 13 expressed gametolog pairs showed unbalanced gene dosage, and as a consequence, a male-biased expression. In addition, we found six novel non-annotated long non-coding RNAs on the Y-chromosome with conserved expression patterns in newborn chimpanzee. The tissue specific and time-restricted expression of these long non-coding RNAs strongly suggests important functions during central nervous system development in human males.

Sertoli cells are main players in the male gonads development and their study may shed light on 46,XY disorders of sex development (DSD). Mature primary Sertoli cells are incapable of proliferating in prolonged in vitro cultures and the available Sertoli cell models have several limitations since they derive from mouse or human cancer tissues. We differentiated human fibroblasts (HFs)-derived induced pluripotent stem cells into Sertoli-like cells (SLC) and, in order to characterize this new Sertoli cell model, we performed gene expression analyses by NextGeneration Sequencing techniques. This approach revealed that our putative SLC have reduced expression of pluripotency markers and expressed Sertoli cell markers such as SRY-Related HMG-Box 9 (SOX9), vimentin (VIM), and claudin-11 (CLDN-11). More in detail, the transcriptional profile analysis suggested that these cells are in an early stage of Sertoli cells maturation. Harnessing the power of induced pluripotent stem cells, we were able to generate SLC that show genetic and functional similarities to human Sertoli cells (HSerCs). SLC could become an excellent source of patient-specific Sertoli cells that could be of paramount benefit for both basic research and personalized medicine in sex development and reproductive medicine.

Sex-biased gene expression is the mode through which sex dimorphism arises from a nearly identical genome, especially in organisms without genetic sex determination. Teleost fishes show great variations in the way the sex phenotype forms. Among them, Sparidae, that might be considered as a model family displays a remarkable diversity of reproductive modes. In this study, we sequenced and analyzed the sex-biased transcriptome in gonads and brain (the tissues with the most profound role in sexual development and reproduction) of two sparids with different reproductive modes: the gonochoristic common dentex, Dentex dentex, and the protandrous hermaphrodite gilthead seabream, Sparus aurata. Through comparative analysis with other protogynous and rudimentary protandrous sparid transcriptomes already available, we put forward common male and female-specific genes and pathways that are probably implicated in sex-maintenance in this fish family. Our results contribute to the understanding of the complex processes behind the establishment of the functional sex, especially in hermaphrodite species and set the groundwork for future experiments by providing a gene toolkit that can improve efforts to control phenotypic sex in finfish in the ever-increasingly important field of aquaculture.

Sex-specific markers play an important role in revealing sex-determination mechanism. Sea urchin (Mesocentrotus nudus) is an economically important mariculture species in several Asian countries and its gonads are the sole edible parts for people. However, growth rate and immunocompetence differ by sex in this species, sex-specific markers have not been identified, and the sex-determination mechanism of sea urchin remains undetermined. In this study, type IIB endonuclease restriction-site associated DNA sequencing (2b-RAD-seq) and a genome survey of M. nudus were performed, and three female-specific markers and three female heterogametic single nucleotide polymorphism (SNP) loci were identified. We validated these sex-specific markers via PCR amplification in a large number of individuals, including wild and artificially bred populations. Several open reading frames (ORFs) were predicted, although there are no potential genes known for sex determination and sex differentiation within the scaffold in which the sex-specific markers are located. Importantly, the female-specific sequences and female heterozygous SNP loci indicate that a female heterogametic and male homogametic ZW/ZZ sex-determination system should exist in M. nudus. The results provide a solid basis for revealing the sex-determination mechanism of this species, and open up new possibilities for developing sex-control breeding in sea urchin.

As an economically important fish, Opsariichthys bidens has obvious sexual dimorphism and strong reproductive capacity, but no epigenetics study can well explain its phenotypic variations. In recent years, many microRNAs involved in the regulation of reproductive development have been explored. In this study, the small RNA libraries of O. bidens on the testis and ovary were constructed and sequenced. A total of 295 known miRNAs were obtained and 100 novel miRNAs were predicted. By comparing testis and ovary libraries, 115 differentially expressed (DE) miRNAs were selected, of which 53 were up-regulated and 62 were down-regulated. A total of 64 GO items (padj < 0.01) and 206 KEGG pathways (padj < 0.01) were enriched in the target gene of miRNA. After that, the expression levels of nine DE miRNAs, including let-7a, miR-146b, miR-18c, miR-202-5p, miR-135c, miR-9-5p, miR-34c-3p, miR-460-5p and miR-338 were verified by qRT-PCR. Furthermore, bidirectional prediction of DE miRNAs and sex-related genes was carried out and the targeting correlation between miR-9-5p and nanos1 was verified by Dual-Luciferase reporter assay. Our findings identified the differentially expressed miRNA and paved the way to new possibilities for the follow-up study on the mechanism of miRNA-mRNA interaction in the gonads of O. bidens.

Testicular teratomas and teratocarcinomas are the most common testicular germ cell tumors in early childhood and young men, and they are frequently found unilaterally in the left testis. In 129/SvJ mice carrying a heterozygous copy of the potent modifier of tumor incidence Ter, a point mutation in the dead-end homolog one gene (Dnd1 Ter/+), ∼70% of the unilateral teratomas arise in the left testis. We previously showed that in mice, left/right differences in vascular architecture are associated with reduced hemoglobin saturation and increased levels of the hypoxia inducible factor-1 alpha (HIF-1α) in the left compared to the right testis. To test the hypothesis that systemic reduction of oxygen availability in Dnd1 Ter/+ mice would lead to an increased incidence of bilateral tumors, we placed pregnant females from 129/SvJ Dnd1 Ter/+ intercross matings in a hypobaric chamber for 12-h intervals. Our results show that in 129/SvJ Dnd1 Ter/+ male gonads, the incidence of bilateral teratoma increased from 3.3% to 64% when fetuses were exposed to acute low oxygen conditions for 12-h between E13.8 and E14.3. The increase in tumor incidence correlated with the maintenance of high expression of pluripotency genes Oct4, Sox2 and Nanog, elevated activity of the Nodal signaling pathway, and suppression of germ cell mitotic arrest. We propose that the combination of heterozygosity for the Ter mutation and hypoxia causes a delay in male germ cell differentiation that promotes teratoma initiation.

Sex reversal induced by temperature change is a common feature in fish. Usually, the sex ratio shift occurs when temperature deviates too much from normal during embryogenesis or sex differentiation stages. Despite decades of work, the mechanism of how temperature functions during early development and sex reversal remains mysterious. In this study, we used Chinese tongue sole as a model to identify features from gonad transcriptomic and epigenetic mechanisms involved in temperature induced masculinization. Some of genetic females reversed to pseudomales after high temperature treatment which caused the sex ratio imbalance. RNA-seq data showed that the expression profiles of females and males were significantly different, and set of genes showed sexually dimorphic expression. The general transcriptomic feature of pesudomales was similar with males, but the genes involved in spermatogenesis and energy metabolism were differentially expressed. In gonads, the methylation level of cyp19a1a promoter was higher in females than in males and pseudomales. Furthermore, high-temperature treatment increased the cyp19a1a promoter methylation levels of females. We observed a significant negative correlation between methylation levels and expression of cyp19ala. In vitro study showed that CpG within the cAMP response element (CRE) of the cyp19a1a promoter was hypermethylated, and DNA methylation decreased the basal and forskolin-induced activities of cyp19a1a promoter. These results suggested that epigenetic change, i.e., DNA methylation, which regulate the expression of cyp19a1a might be the mechanism for the temperature induced masculinization in tongue sole. It may be a common mechanism in teleost that can be induced sex reversal by temperature.

As part of our Asian seabass genome project, we are generating an inventory of repeat elements in the genome and transcriptome. The karyotype showed a diploid number of 2n = 24 chromosomes with a variable number of B-chromosomes. The transcriptome and genome of Asian seabass were searched for repetitive elements with experimental and bioinformatics tools. Six different types of repeats constituting 8-14% of the genome were characterized. Repetitive elements were clustered in the pericentromeric heterochromatin of all chromosomes, but some of them were preferentially accumulated in pretelomeric and pericentromeric regions of several chromosomes pairs and have chromosomes specific arrangement. From the dispersed class of fish-specific non-LTR retrotransposon elements Rex1 and MAUI-like repeats were analyzed. They were wide-spread both in the genome and transcriptome, accumulated on the pericentromeric and peritelomeric areas of all chromosomes. Every analyzed repeat was represented in the Asian seabass transcriptome, some showed differential expression between the gonads. The other group of repeats analyzed belongs to the rRNA multigene family. FISH signal for 5S rDNA was located on a single pair of chromosomes, whereas that for 18S rDNA was found on two pairs. A BAC-derived contig containing rDNA was sequenced and assembled into a scaffold containing incomplete fragments of 18S rDNA. Their assembly and chromosomal position revealed that this part of Asian seabass genome is extremely rich in repeats containing evolutionarily conserved and novel sequences. In summary, transcriptome assemblies and cDNA data are suitable for the identification of repetitive DNA from unknown genomes and for comparative investigation of conserved elements between teleosts and other vertebrates.

17α-Methyltestosterone (MT) is a synthetic androgen. The objective of this study was to explore the effects of exogenous MT on the growth and gonadal development of female rare minnow Gobiocypris rarus. Female G. rarus groups were exposed to 25-100 ng/L of MT for 7 days. After exposure for 7 days, the total weight and body length were significantly decreased in the 50-ng/L MT groups. The major oocytes in the ovaries of the control group were vitellogenic oocytes (Voc) and cortical alveolus stage oocytes (Coc). In the MT exposure groups, some fish had mature ovaries with a relatively lower proportion of mature oocytes, and the diameter of the perinucleolar oocytes (Poc) was decreased compared with those of the control group. Ovarian VTG, FSH, LH, 11-KT, E2, and T were significantly increased after exposure to 50 ng/L of MT for 7 days. Unigenes (73,449), 24 known mature microRNAs (miRNAs), and 897 novel miRNAs in the gonads of G. rarus were found using high-throughput sequencing. Six mature miRNAs (miR-19, miR-183, miR-203, miR-204, miR-205, and miR-96) as well as six differentially expressed genes (fabp3, mfap4, abca1, foxo3, tgfb1, and zfp36l1) that may be associated with ovarian development and innate immune response were assayed using qPCR. Furthermore, the miR-183 cluster and miR-203 were differentially expressed in MT-exposed ovaries of the different G. rarus groups. This study provides some information about the role of miRNA-mRNA pairs in the regulation of ovarian development and innate immune system, which will facilitate future studies of the miRNA-RNA-associated regulation of teleost reproduction.

Transgenic technology and selective breeding have great potential for the genetic breeding in both edible fish and ornamental fish. The development of infertility control technologies in transgenic fish and farmed fish is the critical issue to prevent the gene flow with wild relatives. In this study, we report the genome editing of the dead end (dnd1) gene in the zebrafish model, using the CRISPR/Cas9 technology to achieve a loss-of-function mutation in both wild-type zebrafish and transgenic fluorescent zebrafish to develop complete infertility control technology of farmed fish and transgenic fish. We effectively performed targeted mutagenesis in the dnd1 gene of zebrafish with a single gRNA, which resulted in a small deletion (-7 bp) or insertion (+41 bp) in exon 2, leading to a null mutation. Heterozygotes and homozygotes of dnd1-knockout zebrafish were both selected by genotyping in the F 1 and F 2 generations. Based on a comparison of histological sections of the gonads between wild-type, heterozygous, and homozygous dnd1 zebrafish mutants, the dnd1 homozygous mutation (aa) resulted in the loss of germ cells. Still, there was no difference between the wild-type (AA) and dnd1 heterozygous (Aa) zebrafish. The homozygous dnd1 mutants of adult zebrafish and transgenic fluorescent zebrafish became all male, which had normal courtship behavior to induce wild-type female zebrafish spawning. However, they both had no sperm to fertilize the spawned eggs from wild-type females. Thus, all the unfertilized eggs died within 10 h. The targeted mutagenesis of the dnd1 gene using the CRISPR/Cas9 technology is stably heritable by crossing of fertile heterozygous mutants to obtain sterile homozygous mutants. It can be applied in the infertility control of transgenic fluorescent fish and genetically improved farmed fish by selective breeding to promote ecologically responsible aquaculture.

Chromosomal inversions are structural rearrangements that frequently provide genomic substrate for phenotypic diversity. In the ruff Philomachus pugnax, three distinct male reproductive morphs (Independents, Satellites and Faeders) are genetically determined by a 4.5 Mb autosomal inversion. Here we test how this stable inversion polymorphism affects gene expression in males during the lekking season. Gene expression may be altered through disruptions at the breakpoints and the accumulation of mutations due to suppressed recombination. We used quantitative PCR to measure expression of 11 candidate inversion genes across three different tissues (liver, adrenal glands and gonads) and tested for allelic imbalance in four inversion genes across 12 males of all three morphs (8 Independents, 2 Satellites, 2 Faeders). We quantified transcripts of CENPN, an essential gene disrupted by the inversion at the proximal breakpoint, at different exons distributed near and across the breakpoint region. Consistent with dosage dependent gene expression for the breakpoint gene CENPN, we found that expression in Independents was broadly similar for transcripts segments from inside and outside the inversion regions, whereas for Satellites and Faeders, transcript segments outside of the inversion showed at least twofold higher expression than those spanning over the breakpoint. Within the inversion, observed expression differences for inversion males across all four genes with allele-specific primers were consistent with allelic imbalance. We further analyzed gonadal expression of two inversion genes, HSD17B2 and SDR42E1, along with 12 non-inversion genes related to steroid metabolism and signaling in 25 males (13 Independents, 7 Satellites, 5 Faeders). Although we did not find clear morph differentiation for many individual genes, all three morphs could be separated based on gene expression differences when using linear discriminant analysis (LDA), regardless of genomic location (i.e., inside or outside of the inversion). This was robust to the removal of genes with the highest loadings. Pairwise correlations in the expression of genes showed significant correlations for 9-18 pairs of genes within morphs. However, between morphs, we only found a single association between genes SDR42E1 and AROM for Independents and Satellites. Our results suggest complex and wide-ranging changes in gene expression caused by structural variants.

Flathead gray mullet (Mugil cephalus) is a cosmopolitan mugilid species popular in fishery and aquaculture with an economic preference for all-female population. However, it displays neither sexual dimorphisms nor heteromorphic sex chromosomes. We have previously presented a microsatellite-based linkage map for this species locating a single sex determination region (SDR) on linkage group 9 (LG9) with evidence for XX/XY sex determination (SD) mechanism. In this work, we refine the critical SDR on LG9, and propose positional- and functional- candidate genes for SD. To elucidate the genetic mechanism of SD, we assembled and compared male and female genomic sequences of 19 syntenic genes within the putative SDR on mullet's LG9, based on orthology to tilapia's LG8 (tLG8) physical map. A total of 25 sequence-based markers in 12 genes were developed. For all markers, we observed association with sex in at least one of the two analyzed M. cephalus full-sib families, but not in the wild-type population. Recombination events were inferred within families thus setting the SDR boundaries to a region orthologous to ∼0.9 Mbp with 27 genes on tLG8. As the sexual phenotype is evident only in adults, larvae were assigned into two putative sex-groups according to their paternal haplotypes, following a model of XY/XX SD-system. A total of 107 sex-biased differentially expressed genes in larvae were observed, of which 51 were mapped to tLG8 (48% enrichment), as compared to 5% in random control. Furthermore, 23 of the 107 genes displayed sex-specific expression; and 22 of these genes were positioned to tLG8, indicating 96% enrichment. Of the 27 SDR genes, BCCIP, DHX32A, DOCK1, and FSHR (GTH-RI) are suggested as positional and functional gene candidates for SD.

No abstract available