Gonadal Gene Research Articles

Identification of estrogen receptor target genes involved in gonadal feminization caused by estrogen in Xenopus laevis

Estrogens and estrogenic endocrine disrupting chemicals can cause gonadal feminization in some vertebrates mainly through estrogen receptor (ER), but the underlying molecular mechanisms are unclear. The present study aimed to identify ER target genes involved in estrogen-caused gonadal feminization in Xenopus laevis. Based on our recent transcriptomic data that 10 nM 17β-estradiol (E2) altered gene transcription in feminizing gonads of male X. laevis at NF stages 48, 50, and 52, we searched estrogen response element (ERE) using the Dragon ERE Finder software in the promoter region of all the E2-regulated genes. As a result, 163 genes containing ERE sequence were identified as predicted ER target genes at NF stage 50 (on the 14th day postfertilization), a crucial stage for gonadal feminization. Then, some of these predicted ER target genes were further investigated, mainly including the genes that were suggested to be involved in E2-caused gonadal feminization and genes being dramatically up or down-regulated by E2. Fifteen genes were demonstrated to be responsive to E2, in turn ER antagonist blocked the E2-regulated transcription. Finally, we identified 10 genes that can bind to ERα by a chromatin immunoprecipitation-qPCR. Taken together, we identified the 10 genes that contain predicted ERE sequences, are responsive to estrogen and ER antagonist, and have ability to bind to ER as ER target genes, including pglyrp2, apoa1, fgb, tdo2, ca6, nags, cpb2, tmprss6, nudc, zwilch. Our results could help to improve the understanding of the molecular mechanisms for gonadal feminization caused by estrogenic endocrine disrupting chemicals in X. laevis, and even in other species.

Transcriptome analysis of gender-biased CYP genes in gonads of the sea cucumber Apostichopus japonicus

Gender differences in physiological characteristics are widespread in animals. Herein, differentially expressed genes (DEGs) in gonads of the sea cucumber Apostichopus japonicus were analysed by transcriptomics, and the results showed that 19,973 genes were commonly expressed in the males and females, 4186 were female-biased, and 2540 were male-biased, 4695 genes were up-regulated in the females and 3436 genes were up-regulated in the males. These DEGs were mainly associated with metabolism, including lipid metabolism, amino acid metabolism, nucleotide metabolism, energy metabolism, and cofactor and vitamin metabolism. 29 Cytochrome P450 (CYP) superfamily genes with gender differential expression were selected, and performed gene identification, phylogenetic, and functional analyses. The results indicated significant roles in multiple metabolic pathways, such as steroid hormone biosynthesis, ovarian steroidogenesis, cortisol synthesis and secretion, arachidonic acid metabolism, linoleic acid metabolism, and retinol metabolism. The findings provide insight into the molecular characteristics of physiological gender differences in sea cucumbers, and will help lay the foundation for the establishment of effective sea cucumber breeding technologies.

Metabolic programming of adipose tissue in female C57Bl 6 mice offspring by maternal high fat feeding and obesity results in greater adaptability to dietary fat content and resistance to high fat diet-induced glucose intolerance compared to male mice

Maternal lifestyle influence may be a factor in the worldwide prevalence of obesity and its complications, including diabetes. Studies investigating the effect of the perinatal maternal environment have produced a range of results, sometimes diametrically opposite. The present study was designed to investigate how obesity and weight gain in pregnant mice affects energy balance, body composition and glucose homeostasis in their offspring, both at a young age on standard diet and when older and fed a high-fat diet. At six weeks of age both male and female offspring from mothers fed a high fat diet had a shorter body length than those from mothers fed standard chow. In contrast to males, female offspring also contained a higher proportion of fat and had elevated circulating leptin and adiponectin. Their gonadal fat pads were heavier and contained larger adipocytes, whereas male offspring had proportionally more smaller adipocytes. Six-week-old female, but not male, offspring had increased gonadal fat gene expression of acetyl CoA carboxylase 1, the rate-limiting step in lipid biosynthesis, and decreased gene expression of carnitine palmitoyl transferase 1, the rate-limiting step in fatty acid oxidation. Maternal high fat diet had no effect on glucose tolerance in six-week-old mice, but this was achieved with higher insulin levels in females. Contrastingly, when the offspring were fed a high fat diet for three months, female, but not male, offspring were leaner than those from mothers fed standard chow. Their gonadal fat depots were lighter and the adipocytes were smaller. Female, but not male, offspring fed high fat diet had decreased gonadal fat gene expression of acetyl CoA carboxylase 1, and increased gene expression of carnitine palmitoyl transferase 1. High fat diet-induced glucose intolerance and elevated plasma insulin concentration were improved in female, but not male, offspring. Plasma leptin and adiponectin remained higher in female offspring on high fat diet with resistin levels being lower. These results suggest that the gonadal fat of female offspring is more adaptable to different levels of dietary fat exposure, increasing storage when levels are low and increasing oxidation when levels are high. This may help female offspring be more resistant to the detrimental effects of high fat diet than male mice.

Gonadotropin inhibitory hormone downregulates steroid hormone secretion and genes expressions in duck granulosa cells.

The mechanisms by which GnIH regulates the steroid synthesis pathway in duck granulosa cells remain poorly understood. In this study, we measured steroid hormone secretion by ELISA and reproduction-associated gene expression by quantitative real-time Polymerase Chain Reaction (qPCR) in duck granulosa cells treated with different concentrations of GnIH (0, 0.1, 1, 10, and 100 ng/mL) for 24 h. The genome-wide expression profiles of GnIH-treated cells (0 and 10 ng/mL) were evaluated by high-throughput RNA sequencing. Compared with untreated cells, the secretion of the steroid hormones E2, E1, P4, and T was downregulated, with that of E1 and P4 reaching statistical significance (P<0.05); in contrast, the secretion of ACV and INH was significantly upregulated (P<0.05) after treatment with 10 and 100 ng/mL GnIH. The expression of encoding steroidogenic proteins and enzymes genes (STAR, CYP11A1, CYP17A1, CYP19A1, and 3-β-HSD) and encoding gonadotropin receptors genes (FSHR, LHR) were significantly declined (P<0.05) in the 10 and 100 ng/mL GnIH treatments. Transcriptome sequencing identified 348 differentially expressed genes (DEGs), including 253 upregulated and 95 downregulated genes. The DEGs were mainly involved in cell growth and death, immune response, and steroid biosynthesis pathways. We identified four novel DEGs (MROH5, LOC113840576, SDR42E1, and LOC113841457) with key roles in the regulation of steroid hormone biosynthesis. Our study revealed changes in gonadal steroid hormone secretion and steroid biosynthesis pathway-related gene expression in duck granulosa cells under the inhibitory effect of GnIH. These data contribute to our understanding of the molecular and genetic mechanisms underlying reproduction in ducks.

Transcriptomic analysis of gene expression in normal goat ovary and intersex goat gonad.

Intersexuality is a congenital reproductive disorder that usually occurs in hornless goats, hindering breeding of goats with hornless traits and the development of the goat industry. In this study, we aimed to identify differentially expressed genes in intersex and normal goat gonads by comparing gene transcription profiles of intersex and normal goat gonads. As intersex goats are genetically based on females, we chose female goats as controls. The goats in the control group and the experimental group were both over one-year old. We evaluated the anatomical characteristics of the reproductive organs of five intersex goats using histopathological methods. The gonads were found to be ovarian and testicular types. RNA-Seq technology was used to identify differentially expressed genes in gonads and normal goat ovary tissues. Transcription analysis results were verified by qPCR. The results showed that 2,748 DEGs were upregulated and 3,327 DEGs were downregulated in intersex ovaries unlike in controls, whereas 2006 DEGs were upregulated and 2032 DEGs were downregulated in the interstitial testes. Many of these genes play important roles in mammalian sex determination and sex differentiation, such as SOX9, WT1, GATA4, DMRT1, DHH, AMH, CYP19A1 and FST. We found that many DEGs are involved in biological developmental regulation by GO and KEGG enrichment analyses, and that most genes associated with the steroid synthesis pathway were downregulated. The DEGs identified in this study may be involved in the regulation of intersex goat sex determination and differentiation, and may increase our understanding of the molecular mechanisms of mammalian sex differentiation.

Expression analysis of DIO2, EYA3, KISS1 and GPR54 genes in year-round estrous and seasonally estrous rams.

The expression characteristics of the hypothalamic–pituitary–gonadal (HPG) axis-related candidate genes, DIO2, EYA3, KISS1 and GPR54, were analyzed in year-round estrous rams (small-tail Han sheep, STH) and seasonally estrous rams (Sunite sheep, SNT) using qPCR. The results were as follows: DIO2 was mainly expressed in pituitary, and KISS1 was specifically expressed in hypothalamus in the two groups. However, EYA3 and GPR54 were widely expressed in the cerebrum, cerebellum, hypothalamus, pituitary, testis, epididymis, vas deferens and adrenal gland tissues in both breeds, with significant differences in the cerebellum, hypothalamus, pituitary, testis and vas deferens tissues. We speculated that DIO2 and KISS1 may have positive roles in different regions in ram year-round estrus. Moreover, the expression patterns of EYA3 and GPR54 suggested that they may regulate the estrous mode of ram via testis and vas deferens. This is the first study to systematically analyze the expression patterns of HPG axis-related genes in rams.

Effects of gonadotropin-releasing hormone analog (GnRHa) immunization on the gonadal transcriptome and proteome of tilapia (Oreochromis niloticus)

Gonadotropin releasing hormone (GnRH) plays an important role in the regulation of vertebrate reproduction. Studies have shown that immunization against GnRHa can induce sexually sterile tilapia. To explore the mechanism behind this, in this study, RNA-seq and data-independent acquisition (DIA) techniques were used to study the transcriptome and proteome of the gonad of tilapia immunized with GnRHa.644 differentially expressed genes (80 upregulated and 564 downregulated) and 1150 differentially expressed proteins (351 upregulated and 799 downregulated) were identified. There were 209 genes with consistent differential expression patterns in the transcriptomic and proteomic analyses, of which 9 were upregulated and 200 downregulated, indicating that the gonad gene expression was inhibited by GnRHa immunization. The downregulated genes were particularly involved in the functions of single-organism process, binding, cellular process, metabolic process and catalytic activity, and associated with the pathways including ECM–receptor interaction, focal adhesion, cardiac muscle contraction and oxidative phosphorylation. The expression of six differentially expressed genes involved in the GnRH signaling pathway was all downregulated. In addition, several important functional genes related to gonadal development after GnRHa immunization were screened.This study confirmed the expression of corresponding genes was affected by GnRHa on the gonad development in tilapia at the molecular level, and laid a foundation for elucidating the mechanism of GnRHa immunization.

Early response to heat stress in Chinese tongue sole (Cynoglossus semilaevis): performance of different sexes, candidate genes and networks

BackgroundTemperature is known to affect living organisms and alter the expression of responsive genes, which affects a series of life processes, such as development, reproduction and metabolism. Several genes and gene families have been involved in high temperature responses, such as heat shock protein (hsp) family, Jumonji family and genes related to cortisol synthesis. Gonad is a vital organ related to the existence of a species. However, the comprehensive understanding of gonadal responses to environmental temperature is limited.ResultsTo explore the effects of environmental temperature on genes and gene networks in gonads, we performed acute heat treatment (48 h) on Chinese tongue sole (Cynoglossus semilaevis). Gonadal transcriptome analysis was conducted on females, pseudomales and males exposed to high (28 °C) and normal (22 °C) temperatures. A total of 1226.24 million clean reads were obtained from 18 libraries. Principal component analysis (PCA) and differentially expressed gene (DEG) analysis revealed different performance of sex responses to heat stress. There were 4565, 790 and 1117 specific genes altered their expression level in females, pseudomales and males, respectively. Of these, genes related to hsp gene family, cortisol synthesis and metabolism and epigenetic regulation were involved in early heat response. Furthermore, a total of 1048 DEGs were shared among females, pesudomales and males, which may represent the inherent difference between high and normal temperatures. Genes, such as eef1akmt3, eef1akmt4, pnmt and hsp family members, were found.ConclusionsOur results depicted for the first time the gonadal gene expression under acute high temperature treatment in Chinese tongue sole. The findings may provide a clue for understanding the responses of genes and networks to environmental temperature.

Chronic exposure to organic oxygen-demanding pollutants at an environmentally realistic concentration affects sperm motility in zebrafish

Wastewater and organic oxygen-demanding pollutants (ODPs) are produced by various factories in China, the United States and other countries. However, whether ODP affects reproductive health remains unclear. To investigate the impact of environmental concentrations of ODP exposure on reproductive health, adult male zebrafish were used to evaluated the effects ODP exposure on the fertility in this study. We found that exposure to ODP reduced the sperm motility of adult male zebrafish. Similarly, the testosterone content of the experimental zebrafish was obviously decreased. Transcription of immune response-related genes, including tumor necrosis factor (tnf)-α, il-1β, and il-8, was upregulated upon exposure to ODP. Mating experiments indicated that the hatching time of the offspring embryos was clearly prolonged upon ODP exposure, but the embryo fertilization rate was not different. These results assumed that exposure to ODP at ambient concentrations visibly affected the sperm motility in adult zebrafish maybe due to the expression of immune response-related genes in the zebrafish male gonads and the release of pro-inflammatory mediators. Therefore, we assumed that the impact of ODP on the reproductive health of aquatic organisms cannot be ignored.

Gonadal transcriptomic analysis and differentially expressed genes between the testes and ovaries in Trachinotus ovatus

The Trachinotus ovatus is a popular aquaculture species in China. There are no obvious morphological differences between male and female fish, even during maturity, prompting research studies on sex-related features of this fish. To examine sex determination- and gonadal development-related genes, we conducted transcriptome analysis of the ovaries and testes of T. ovatus. A total of 345,972,132 high-quality clean reads were obtained from 12 libraries. In addition, 28,137 gonad-expressed unigenes were obtained by mapping the clean reads to the T. ovatus reference genome. A total of 8,237 differentially expressed genes (DEGs) were identified between stage I ovaries and testes, including 3,235 testicular upregulated and 5,002 ovarian upregulated genes. Furthermore, 13,448 DEGs were obtained between stage III ovarian and testicular libraries, including 7,576 testicular upregulated and 5,872 ovarian upregulated genes. The DEGs included some sex-determining genes such as sry, dmrt1, and amh. DEGs between ovarian and testicular libraries were significantly enriched with KEGG pathways that are involved in gonadal development, sex determination, and gonadal function. Then, 10 DEGs (seven testicular upregulated and three ovarian upregulated unigenes) were selected for quantitative real-time PCR analysis. Four genes (zinc-binding protein A33-like, pro-opiomelanocortin-like, leucine-rich repeat and transmembrane domain-containing protein 2-like, and forkhead boxC1) showed a specific testicular expression pattern. The gonadally expressed as well as testicular and ovarian DEGs provide useful information for further research on the reproductive biology of T. ovatus.

Effects of early life stage exposure of largemouth bass to atrazine or a model estrogen (17α-ethinylestradiol)

Endocrine disrupting contaminants are of continuing concern for potentially contributing to reproductive dysfunction in largemouth and smallmouth bass in the Chesapeake Bay watershed (CBW) and elsewhere. Exposures to atrazine (ATR) have been hypothesized to have estrogenic effects on vertebrate endocrine systems. The incidence of intersex in male smallmouth bass from some regions of CBW has been correlated with ATR concentrations in water. Fish early life stages may be particularly vulnerable to ATR exposure in agricultural areas, as a spring influx of pesticides coincides with spawning and early development. Our objectives were to investigate the effects of early life stage exposure to ATR or the model estrogen 17α-ethinylestradiol (EE2) on sexual differentiation and gene expression in gonad tissue. We exposed newly hatched largemouth bass (LMB, Micropterus salmoides) from 7 to 80 days post-spawn to nominal concentrations of 1, 10, or 100 µg ATR/L or 1 or 10 ng EE2/L and monitored histological development and transcriptomic changes in gonad tissue. We observed a nearly 100% female sex ratio in LMB exposed to EE2 at 10 ng/L, presumably due to sex reversal of males. Many gonad genes were differentially expressed between sexes. Multidimensional scaling revealed clustering by gene expression of the 1 ng EE2/L and 100 µg ATR/L-treated male fish. Some pathways responsive to EE2 exposure were not sex-specific. We observed differential expression in male gonad in LMB exposed to EE2 at 1 ng/L of several genes involved in reproductive development and function, including star, cyp11a2, ddx4 (previously vasa), wnt5b, cyp1a and samhd1. Expression of star, cyp11a2 and cyp1a in males was also responsive to ATR exposure. Overall, our results confirm that early development is a sensitive window for estrogenic endocrine disruption in LMB and are consistent with the hypothesis that ATR exposure induces some estrogenic responses in the developing gonad. However, ATR-specific and EE2-specific responses were also observed.

Comparative transcriptome and histomorphology analysis of testis tissues from mulard and Pekin ducks.

Testicular transcriptomes were analyzed to characterize the differentially expressed genes between mulard and Pekin ducks, which will help establish gene expression datasets to assist in further determination of the mechanisms of genetic sterility in mulard ducks. Paraffin sections were made to compare the developmental differences in testis tissue between mulard and Pekin ducks. Comparative transcriptome sequencing of testis tissues was performed, and the expression of candidate genes was verified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). In mulard ducks, spermatogonia and spermatocytes were arranged in a disordered manner, and no mature sperm were observed in the testis tissue. However, different stages of development of sperm were observed in seminiferous tubules in the testis tissue of Pekin ducks. A total of 43.84 Gb of clean reads were assembled into 193 535 UniGenes. Of these, 2131 transcripts exhibited differential expression (false discover rate and fold change ), including 997 upregulated and 1134 downregulated transcripts in mulard ducks as compared to those in Pekin duck testis tissues. Several upregulated genes were related to reproductive functions, including ryanodine receptor 2 (RYR2), calmodulin (CALM), argininosuccinate synthase and delta-1-pyrroline-5-carboxylate synthetase ALDH18A1 (P5CS). Downregulated transcripts included the testis-specific serine/threonine-protein kinase 3, aquaporin-7 (AQP7) and glycerol kinase GlpK (GK). The 10 related transcripts involved in the developmental biological process were identified by GO (Gene Ontology) annotation. The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways indicated that peroxisome proliferator-activated receptors (PPARs) and calcium signaling pathways were significantly () associated with normal testis physiology. The differential expression of select genes implicated in reproductive processes was verified by qRT-PCR, which was consistent with the expression trend of transcriptome sequencing (RNA-seq). Differentially expressed candidate genes RYR2, CALM, P5CS, AQP7 and GK were identified by transcriptional analysis in mulard and Pekin duck testes. These were important for the normal development of the male duck reproductive system. These data provide a framework for the further exploration of the molecular and genetic mechanisms of sterility in mulard ducks. Highlights. The mulard duck is an intergeneric sterile hybrid offspring resulting from mating between Muscovy and Pekin ducks. The transcriptomes of testis tissue from mulard and Pekin ducks were systematically characterized, and differentially expressed genes were screened, in order to gain insights into potential gonad gene expression mechanisms contributing to genetic sterility in mulard ducks.

Insight into the Possible Formation Mechanism of the Intersex Phenotype of Lanzhou Fat-Tailed Sheep Using Whole-Genome Resequencing.

Simple SummaryIndividuals with hermaphroditism are a serious hazard to animal husbandry and production due to their abnormal fertility. The molecular mechanism of sheep intersex formation was unclear. This study was the first to locate the homologous sequence of the goat polled intersex syndrome (PIS) region in sheep and found that the intersex traits of Lanzhou fat-tailed sheep were not caused by the lack of this region. By detecting the selective sweep regions, vital genes associated with androgen biosynthesis and the follicle stimulating hormone response entry were found, including steroid 5 alpha-reductase 2 (SRD5A2), and pro-apoptotic WT1 regulator (PAWR). Additionally, the copy number variations of the four regions on chr9, chr1, chr4, and chr16 may affect the expression of the gonadal development genes, zinc finger protein, FOG family member 2 (ZFPM2), LIM homeobox 8 (LHX8), inner mitochondrial membrane peptidase subunit 2 (IMMP2L) and slit guidance ligand 3 (SLIT3), respectively, and further affect the formation of intersex traits.Intersex, also known as hermaphroditism, is a serious hazard to animal husbandry and production. The mechanism of ovine intersex formation is not clear. Therefore, genome-wide resequencing on the only two intersex and two normal Lanzhou fat-tailed (LFT) sheep, an excellent but endangered Chinese indigenous sheep breed, was performed. Herein, the deletion of homologous sequences of the goat polled intersex syndrome (PIS) region (8787 bp, 247747059–247755846) on chromosome 1 of the LFT sheep was not the cause of the ovine intersex trait. By detecting the selective sweep regions, we found that the genes related to androgen biosynthesis and follicle stimulating hormone response items, such as steroid 5 alpha-reductase 2 (SRD5A2), steroid 5 alpha-reductase 3 (SRD5A3), and pro-apoptotic WT1 regulator (PAWR), may be involved in the formation of intersex traits. Furthermore, the copy number variations of the four regions, chr9: 71660801–71662800, chr1: 50776001–50778000, chr4: 58119201–58121600, and chr16: 778801–780800, may affect the expression of the zinc finger protein, FOG family member 2 (ZFPM2), LIM homeobox 8 (LHX8), inner mitochondrial membrane peptidase subunit 2 (IMMP2L) and slit guidance ligand 3 (SLIT3) genes, respectively, which contribute to the appearance of intersex traits. These results may supply a theoretical basis for the timely detection and elimination of intersex individuals in sheep, which could accelerate the healthy development of animal husbandry.

Chitosan-Based Drug Delivery System: Applications in Fish Biotechnology.

Chitosan is increasingly used for safe nucleic acid delivery in gene therapy studies, due to well-known properties such as bioadhesion, low toxicity, biodegradability and biocompatibility. Furthermore, chitosan derivatization can be easily performed to improve the solubility and stability of chitosan–nucleic acid polyplexes, and enhance efficient target cell drug delivery, cell uptake, intracellular endosomal escape, unpacking and nuclear import of expression plasmids. As in other fields, chitosan is a promising drug delivery vector with great potential for the fish farming industry. This review highlights state-of-the-art assays using chitosan-based methodologies for delivering nucleic acids into cells, and focuses attention on recent advances in chitosan-mediated gene delivery for fish biotechnology applications. The efficiency of chitosan for gene therapy studies in fish biotechnology is discussed in fields such as fish vaccination against bacterial and viral infection, control of gonadal development and gene overexpression and silencing for overcoming metabolic limitations, such as dependence on protein-rich diets and the low glucose tolerance of farmed fish. Finally, challenges and perspectives on the future developments of chitosan-based gene delivery in fish are also discussed.

Bisexual Fertile Triploid Zebrafish (Danio rerio): a Rare Case.

Previous studies have suggested that artificially induced triploid zebrafish are exclusively male-biased. Owing to greatly inhibited gonadal development for the artificially induced triploid fish, they are regarded to be sterile in general. In this article, partially fertile bisexual triploid zebrafish are produced by suppressing extrusion of the second polar body by heat shock. Histological observation confirms that the early gonadal development of these triploid zebrafish is normal. Backcrossing and self-crossing are used to demonstrate that both the female and male triploid zebrafish have partial reproductive ability. Their dynamic of chromosomes during meiosis is revealed from the chromosome preparations of gonads. Examination of the expressed gonadal development-related genes shows some molecular evidence of the normal gonadal development in the triploid zebrafish. Clearly, these fertile bisexual triploid zebrafish can provide a unique system to study sex determination, as well as aneuploidy associated human diseases such as infertility and pregnancy loss.

The Echinodermata PPAR: Functional characterization and exploitation by the model lipid homeostasis regulator tributyltin

The wide ecological relevance of lipid homeostasis modulators in the environment has been increasingly acknowledged. Tributyltin (TBT), for instance, was shown to cause lipid modulation, not only in mammals, but also in fish, molluscs, arthropods and rotifers. In vertebrates, TBT is known to interact with a nuclear receptor heterodimer module, formed by the retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor (PPAR). These modulate the expression of genes involved in lipid homeostasis. In the present work, we isolated for the first time the complete coding region of the Echinodermata (Paracentrotus lividus) gene orthologues of PPAR and RXR and evaluated the ability of a model lipid homeostasis modulator, TBT, to interfere with the lipid metabolism in this species. Our results demonstrate that TBT alters the gonadal fatty acid composition and gene expression patterns: yielding sex-specific responses in fatty acid levels, including the decrease of eicosapentaenoic acid (C20:5 n-3, EPA) in males, and increase of arachidonic acid (20:4n-6, ARA) in females, and upregulation of long-chain acyl-CoA synthetase (acsl), ppar and rxr. Furthermore, an in vitro test using COS-1 cells as host and chimeric receptors with the ligand binding domain (LBD) of P. lividus PPAR and RXR shows that organotins (TBT and TPT (Triphenyltin)) suppressed activity of the heterodimer PPAR/RXR in a concentration-dependent manner. Together, these results suggest that TBT acts as a lipid homeostasis modulator at environmentally relevant concentrations in Echinodermata and highlight a possible conserved mode of action via the PPAR/RXR heterodimer.

Nonspecific expression of fertilization genes in the crown-of-thorns Acanthaster cf. solaris: Unexpected evidence of hermaphroditism in a coral reef predator.

The characterization of gene expression in gametes has advanced our understanding of the molecular basis for ecological variation in reproductive success and the evolution of reproductive isolation. These advances are especially significant for ecologically important keystone predators such as the coral-eating crown-of-thorns sea stars (COTS, Acanthaster) which are the most influential predator species in Indo-Pacific coral reef ecosystems and the focus of intensive management efforts. We used RNA-seq and transcriptome assemblies to characterize the expression of genes in mature COTS gonads. We described the sequence and domain organization of eight genes with sex-specific expression and well known functions in fertilization in other echinoderms. We found unexpected expression of genes in one ovary transcriptome that are characteristic of males and sperm, including genes that encode the sperm-specific guanylate cyclase receptor for an egg pheromone, and the sperm acrosomal protein bindin. In a reassembly of previously published RNA-seq data from COTS testes, we found a complementary pattern: strong expression of four genes that are otherwise well known to encode egg-specific fertilization proteins, including the egg receptor for bindin (EBR1) and the acrosome reaction-inducing substance in the egg coat (ARIS1, ARIS2, ARIS3). We also found histological evidence of both eggs and sperm developing in the same gonad in several COTS individuals from a parallel study. These results suggest the occurrence of hermaphrodites, and the potential for reproductive assurance via self-fertilization. Our findings have implications for management of COTS populations, especially in consideration of the large size and massive fecundity of these sea stars.

Timing of gonadal development and dimorphic expression of sex-related genes in gonads during early sex differentiation in the Yellow River carp

The common carp, a gonochoristic teleost fish with an XX/XY sex-determining system, provides an excellent model for studying cyprinidae gonadal sex differentiation. The present study aims at investigating the detailed process of early gonadal sex differentiation of common carp by combining the expression patterns of sex related genes (cyp19a1a, foxl2, amh, dmrt1, sox9b, sycp3, dmc1) with gonadal histological changes using mono-sex female and male Yellow River carp. H&E staining, immunohistochemistry and immunofluorescence of gonads indicated that the ovaries and testis of common carp were both directly differentiated from undifferentiated gonads. Oogonia and spermatogonia were first observed at 40 dph (days post hatching) and 70 dph in female and male gonads, respectively. Female related genes cyp19a1a and foxl2 and male related genes amh, dmrt1 and sox9b increased significantly in 30–40 dph female, and male gonads, respectively. These suggest that the critical timing of sex determination of common carp takes place between 30 and 40 dph or possibly earlier. Meiotic marker gene dmc1 and sycp3 increased expression significantly in 55 dph female gonads while sycp3 increased expression significantly in 100 dph male gonads. Oocytes in meiosis and spermatocytes were first observed in 55 dph female and 100 dph male gonads, respectively. These indicate that the initiation time of gonadal sex differentiation is 50–55 dph in female common carp and 90–100 dph in male common carp. To our knowledge, this is the first study investigating the detailed process of early gonadal sex differentiation of common carp by combining the expression patterns of sex related genes with gonadal histological changes. We have identified the critical window of sex determination and the initiation times of oogenesis and spermatogenesis in the Yellow River carp. These results provide researchers with valuable information to possibly reveal the mechanisms of sex determination and differentiation of common carp.

Physiological effects of 5α-dihydrotestosterone in male mummichog (Fundulus heteroclitus) are dose and time dependent

Numerous anthropogenic sources, such as pulp mill and sewage treatment effluents, contain androgenic endocrine disrupting compounds that alter the reproductive status of aquatic organisms. The current study injected adult male mummichog (Fundulus heteroclitus) with 0 (control), 1 pg/g, 1 ng/g or 1 μg/g body weight of the model androgen 5α-dihydrotestosterone (DHT) with the intent to induce a period of plasma sex hormone depression, a previously-observed effect of DHT in fish. A suite of gonadal steroidogenic genes were assessed during sex hormone depression and recovery. Fish were sampled 6, 12, 16, 18, 24, 30 and 36 h post-injection, and sections of testis tissue were either snap frozen immediately or incubated for 24 h at 18 °C to determine in vitro gonadal hormone production and then frozen. Plasma testosterone (T) and 11-ketotestosterone (11KT) were depressed beginning 24 h post-injection. At 36 h post-injection plasma T remained depressed while plasma 11KT had recovered. In snap frozen tissue there was a correlation between plasma sex hormone depression and downregulation of key steroidogenic genes including steroidogenic acute regulatory protein (star), cytochrome P450 17a1 (cyp17a1), 3β-hydroxysteroid dehydrogenase (3βhsd), 11β-hydroxysteroid dehydrogenase (11βhsd) and 17β-hydroxysteroid dehydrogenase (17βhsd). Similar to previous studies, 3βhsd was the first and most responsive gene during DHT exposure. Gene responses from in vitro tissue were more variable and included the upregulation of 3βhsd, 11βhsd and star during the period of hormone depression. The differential expression of steroidogenic genes from the in vitro testes compared to the snap frozen tissues may be due to the lack of regulators from the hypothalamo-pituitary-gonadal axis present in whole-animal systems. Due to these findings it is recommended to use snap frozen tissue, not post-incubation tissue from in vitro analysis, for gonadal steroidogenic gene expression to more accurately reflect in vivo responses.

Gonadal disruption after single dose exposure of prothioconazole and prothioconazole-desthio in male lizards (Eremias argus)

Prothioconazole (PTC) is a widely used triazole fungicide with low toxicity, and its desulfurization metabolite, prothioconazole-desthio (PTC-d), is reported to have higher reproductive toxicity to mammals. However, little is known about the reproductive toxicity, much less endocrine disrupting effect, of these two chemicals on reptiles. In this study, we investigated the effects of single dose of PTC/PTC-d (100 mg kg−1 body weight) exposure on the pathomorphism of testes and epididymides, serum sex steroid hormones (testosterone and 17β-estradiol) and transcription of steroidogenic-related genes (STARD, cyp11A, cyp17, cyp19A, 17β-HSD, 3β-HSD, AR and ER-α) in gonads of male lizards (Eremias argus). Although structural disorder existed in PTC-d exposure group, severe gonadal disruption, especially suppression of spermatogenesis was only observed in testis after PTC treatment, which consequently led to the lack of spermatozoa in epididymal ducts. Consistent with this result, T/E2 value in PTC exposure was elevated to a significant higher level compared with control and continually increased over time, while T/E2 value in the PTC-d exposure group slightly increased only at 12 h. These results demonstrated a more serious disruption of PTC on male lizard gonads than PTC-d. In addition, the expression of cyp17 gene was inhibited at 6 h, however, was induced at 12 h, and exhibited negative correlations with STARD, cyp11A and 3β-HSD after PTC exposure at each timepoint. In PTC-d group, the expression of STARD and 3β-HSD were significantly down-regulated, in contrast, cyp11A and cyp17 were up-regulated, and each gene showed consistent changes over time. For 17β-HSD, no significance was observed in both treated groups. This study was the first to compare the gonadal disruption of PTC and PTC-d in male lizards and elucidated that these two chemicals influenced the physiological function of male gonads through differential transcriptional modulation.