Expression Of Dmrt1 Research Articles

Biochemical, histological, and molecular investigation of fish exposed to a brewery effluent

BackgroundBreweries' industrial processes produce much effluent, which, if improperly managed, could contaminate the aquatic bodies that receive them. The effect of brewery effluent on fish health was investigated to assess the extent of pollution on the fish population. Samples of Clarias gariepinus L were exposed to varying concentrations of brewery effluent. After exposure, key biochemical indicators of oxidative stress response were analyzed. Messenger RNA expressions of genes associated with growth, reproduction, oxidative stress, and toxicity were quantified. The histopathological architecture of the liver was also examined.ResultsExposure of fish to the brewery effluent increased conjugated diene and malondialdehyde levels, while the activity of antioxidant enzymes (catalase and SOD) significantly (p < 0.05) decreased relative to control fish. The effluent increased the mRNA expression of growth hormones (GHs), CAMKIIg, MEL1C, IL-1β, CYP11A, HSP70, and DMRT1, while decreasing the expression of HSD17B and FOXL2 when compared to control fish (p < 0.05). The histological study revealed liver degeneration from the normal architectural structure with simple steatosis and structurally enlarged central vein coupled with Mallory bodies.ConclusionThe results indicate that effluent discharge from the brewery industry can harm fish population by altering antioxidant enzymes and expression of growth hormones. Hence, prior discharge into water bodies, industrial effluents should be effectively treated to meet standards and guidelines stipulated by environmental regulatory bodies.

Dmrt1 Is Responsible for Androgen-Induced Masculinization in Nile Tilapia.

17α-Methyltestosterone (MT) is a widely used androgen for all-male fish production in aquaculture. However, the molecular mechanism underlying MT-induced masculinization remains unclear. In this study, we aim to identify the key gene responsible for MT-induced masculinization using the Nile tilapia (Oreochromis niloticus) amhy, dmrt1, and gsdf mutants, which exhibit male-to-female sex reversal. Nile tilapia fry from these three mutant lines were treated with 50 μg/g MT from 5 to 30 days after hatching (dah). The results showed that amhy and gsdf mutants, but not dmrt1 mutants, were masculinized by the MT treatment. Gonadal transcriptome analysis revealed that genes involved in steroidogenesis and germ cell development in MT-treated dmrt1 mutants exhibited a similar expression pattern to that of the wild type (WT) XX. In addition, the dmrt1 mutants cannot be masculinized by co-treatment with MT and the aromatase inhibitor fadrozole. The MT treatment completely blocked early steroidogenic enzyme (Star2, Cyp17a2, and Cyp19a1a) expression independent of amhy, gsdf, and dmrt1. A luciferase analysis showed that MT directly suppressed basal and Sf-1-activated cyp19a1a promoter activity through ara and arb in cultured HEK293 cells. Furthermore, MT treatment inhibited germ cell proliferation in amhy and gsdf mutants but not in dmrt1 mutants. Consistently, dmrt1 expression was induced in MT-treated WT XX, -amhy, and -gsdf mutants. Taken together, these results suggest that dmrt1 is indispensable for MT-induced masculinization in Nile tilapia and that MT functions by inhibiting early steroid synthesis and activating dmrt1 to promote testis development.

Effects of 17α-methyltestosterone and letrozole on growth and gonadal development in largemouth bass (Micropterus salmodies).

In order to optimize the parameters for reversing masculinization and establish the techniques for sex induction of pseudo-males and creation of all-female fry in largemouth bass (Micropterus salmodies, LMB), 15-day-old LMB (1.00 ± 0.10cm in length, 0.10 ± 0.01g in weight) were fed a diet supplemented with either 17α-methyltestosterone (MT) or letrozole (LE) and their combination. The experimental groups were M20 (20mg/kg MT), L20 (20mg/kg LE) and M10L10 (10mg/kg MT and 10mg/kg LE). The control group, named C, was not feed MT or LE. After 60 days, exogenous hormone in the diets was stopped and the effects of MT and LE on growth, male ratio, and gonadal development in LMB were evaluated. At 12-month-old, blood and gonadal tissue samples were collected to measure sex steroid hormones levels, analyze expression levels of dmrt1 and cyp19a1a genes, as well as examine the gonads morphology. The results showed no significant differences in growth between the experimental groups and the control group after a 60-day feeding period with the formulated diet (p > 0.05). The sex reversal ratio of M20, L20, M10L10 were 95.00%, 80.00%, 76.47%, respectively. The gonadal tissue sections showed that the gonadal structure of masculinized fish morphologic resembled that of control male fish. At 12-month-old, the sex reversal ratio in M20, L20, M10L10 and C groups were 100%, 86.67%, 73.33% and 50.00%, respectively. The testicular of pseudo-male fish in the M20 group exhibited well-developed morphology similarities to that of the control group males. However, the testes of pseudo-male fish in the L20 and M10L10 groups were smaller size Estradiol (E2) levels in the experimental groups was significantly lower than those in the control group females (p < 0.05), while testosterone (T) levels were significantly higher than that of the control group (p < 0.05). Compared to the female fish in the control group, pseudo-male fish from all experimental groups showed significantly upregulated expression of dmrt1 (p < 0.05), and significantly downregulated expression of cyp19a1a (p < 0.05). Pseudo-males selected from group M20 exhibited a significantly higher proportion of female offspring (92.00%) compared to the control group (46.50%). In summary, 20mg/kg MT was the optimal inducing concentration.

Thyroid Hormone Regulates Postnatal Development of the Rete Testis in Mice.

Thyroid hormone regulates the rate of testis maturation in mammals. Manipulations of thyroid hormone levels in neonatal animals affect various aspects of testis biology. However, there were no studies examining the effects of thyroid hormone on the rete testis (RT). Here, we used animal models of neonatal hyperthyroidism (injections of triiodothyronine, or T3) and hypothyroidism (goitrogen PTU treatment) and found that higher levels of thyroid hormone accelerate RT development, while lower levels of thyroid hormone delay it. T3 and PTU treatments influence RT size, proliferation of RT cells, and expression of DMRT1 and androgen receptor in the RT. T3 supplementation accelerates RT development in an organ testicular culture, which indicates the local action of thyroid hormone. Additionally, it was found that follicle stimulated hormone could be involved in the regulation of both RT proliferation and RT size. The fact that RT cells in a cell culture do not respond to T3 suggests indirect action of thyroid hormone on the RT in vivo or the loss of the responsiveness to the hormone in vitro.

MiR-34 negatively regulates the expression of Dmrt and related genes in the testis of mud crab Scylla paramamosain

The mud crab (Scylla paramamosain) is a commercially significant marine decapod crustacean. Due to its obvious sexual dimorphism, the mechanism of sex differentiation and gonadal development has attracted significant research interest. The Dmrt (double-sex and mab-3 related transcription factor) genes are vital in animal gonadal development and sex differentiation. In the present study, miR-34 was predicted to target the 3′ end of Dmrt-1, idmrt-2, Dmrt-3, Dsx and Dmrt-like genes by prediction software, and the interactions between miR-34 and these Dmrt genes were validated by in vivo and in vitro experiments. Dual luciferase assay results indicated that miR-34 mimics/inhibitors co-transfected with plasmid vectors with 3′ end of Dmrt-1, idmrt-2, Dmrt-3, Dsx and Dmrt-like, respectively, led to a significant decrease/increase of fluorescence activity in HEK293T cells. In vivo experiments showed that injection of agomir-34 significantly inhibited Dmrt-1, idmrt-2, Dsx and Dmrt-like expression, while injection of antagomir-34 caused the opposite result. However, Dmrt-3 expression was not affected by injection of miR-34 reagents. Meanwhile, the expression of spermatogenesis and testicular development-related molecular marker genes (IAG, foxl2 and vasa) in mud crabs was significantly changed after injecting the miR-34 reagent in vivo. Furthermore, the result of immunoblotting proved that the expression level of Dmrt-like protein can be regulated by miR-34. These results imply that miR-34 is indirectly involved in sex differentiation and testicular development of S. paramamosain by regulating Dmrt-1, idmrt-2, Dsx and Dmrt-like genes.

Differential expression and genetic polymorphism of DMRT1 and FOXL2 genes in the gonads of Kadaknath chicken

In chicken, the left ovary is functional and the right ovary becomes rudimentary in the later stage of the embryonic development. The expression and genetic polymorphism of the DMRT1 and FOXL2 genes are assessed in order to understand the distinct developing process of gonads in male and female embryonic gonads. In the current study, 19th day Kadaknath chicken gonadal tissue samples were collected from the embryos and RT-PCR analysis and DNA sequencing was done. According to the expression studies, the left ovaries relative expression of FOXL2 was 2.14 and 12.65 factor greater than that of the right ovaries and testes, while the right testis relative expression of DMRT1 was 3.78 and 3.83 fold higher than that of the left and right ovaries. DNA sequence analysis in the left ovary identified a nonsynonymous alteration (c.289C&gt;G) involving histidine to aspartic acid at the 289th position in the FOXL2 coding region. The 51st position of exon 6 of DMRT1 has a G→A transition SNP that is not part of the coding sequence (CDS). Males showed higher levels of the DMRT1 gene than females, while females expressed higher levels of FOXL2. It was found that the DMRT1 and FOXL2 genes are polymorphic. The genetic variability in the gonads and the differences in DMRT1 and FOXL2 gene expression allow learning more about the critical mechanisms underlying chicken gonadal development.

Roles of estrogen receptors during sexual reversal in Pelodiscus sinensis.

The Chinese soft-shelled turtle, Pelodiscus sinensis, exhibits distinct sexual dimorphism, with the males growing faster and larger than the females. During breeding, all-male offspring can be obtained using 17β-estradiol (E2). However, the molecular mechanisms underlying E2-induced sexual reversal have not yet been elucidated. Previous studies have investigated the molecular sequence and expression characteristics of estrogen receptors (ERs). In this study, primary liver cells and embryos of P. sinensis were treated with ER agonists or inhibitors. Cell incubation experiments revealed that nuclear ERs (nERs) were the main pathway for the transmission of estrogen signals. Our results showed that ERα agonist (ERα-ag) upregulated the expression of Rspo1, whereas ERα inhibitor (ERα-Inh) downregulated its expression. The expression of Dmrt1 was enhanced after ERα-Inh + G-ag treatment, indicating that the regulation of male genes may not act through a single estrogen receptor, but a combination of ERs. In embryos, only the ERα-ag remarkably promoted the expression levels of Rspo1, Wnt4, and β-catenin, whereas the ERα-Inh had a suppressive effect. Additionally, Dmrt1, Amh, and Sox9 expression levels were downregulated after ERβ inhibitor (ERβ-Inh) treatment. GPER agonist (G-ag) has a significant promotion effect on Rspo1, Wnt4, and β-catenin, while the inhibitor G-Inh does not affect male-related genes. Overall, these results suggest that ERs play different roles during sexual reversal in P. sinensis and ERα may be the main carrier of estrogen-induced sexual reversal in P. sinensis. Further studies need to be performed to analyze the mechanism of ER action.

Overexpression of Kdm6b induces testicular differentiation in a temperature-dependent sex determination system.

In reptiles, such as the red-eared slider turtle ( Trachemys scripta elegans), gonadal sex determination is highly dependent on the environmental temperature during embryonic stages. This complex process, which leads to differentiation into either testes or ovaries, is governed by the finely tuned expression of upstream genes, notably the testis-promoting gene Dmrt1 and the ovary-promoting gene Foxl2. Recent studies have identified epigenetic regulation as a crucial factor in testis development, with the H3K27me3 demethylase KDM6B being essential for Dmrt1 expression in T. s. elegans. However, whether KDM6B alone can induce testicular differentiation remains unclear. In this study, we found that overexpression of Kdm6b in T. s. elegans embryos induced the male development pathway, accompanied by a rapid increase in the gonadal expression of Dmrt1 at 31°C, a temperature typically resulting in female development. Notably, this sex reversal could be entirely rescued by Dmrt1 knockdown. These findings demonstrate that Kdm6b is sufficient for commitment to the male pathway, underscoring its role as a critical epigenetic regulator in the sex determination of the red-eared slider turtle.

Effects of exogenous steroid hormones on growth, body color, and gonadal development in the Opsariichthys bidens.

To investigate the effects of exogenous steroid hormones on growth, body color, and gonadal development in the Opsariichthys bidens (O. bidens), synthetic methyltestosterone (MT) and 17β-estradiol (E2) were used for 28 days' treatment of 4-month-old O. bidens before the breeding season. Our results suggested that MT had a significant growth-promoting effect (P < 0.05), whereas E2 played an inhibitory role. On the body surface, the females in the MT group showed gray stripes, and the fish in other groups showed no obvious stripes. The males with MT treatment displayed brighter blue-green stripes compared to the CK and E2 groups. The histological analysis showed that the MT significantly promoted testes development in males, blocked oocyte development, and caused massive apoptosis in females, whereas the E2 group promoted ovarian development and inhibited testes development. Based on qRT-PCR analysis, in females, the expression of igf-1, dmrt1, and cyp19a1a genes revealed that E2 treatment resulted in down-regulation of igf-1 expression and up-regulation of cyp19a1a expression. In males, igf-1 and dmrt1 were significantly up-regulated after MT treatment, and E2 treatment led to down-regulation of igf-1. Therefore, this study demonstrates that MT and E2 play an important role in reversing the morphological sex characteristics of females and males.

Alternative splicing of histone demethylase Kdm6bb mediates temperature-induced sex reversal in the Nile tilapia

Sex determination in many fish species is remarkably plastic and temperature sensitive. Nile tilapia display a genetic sex-determination system (XX/XY). However, high-temperature treatment during critical thermosensitive periods can induce XX females into XXm pseudo-males, and this phenomenon is termed temperature-induced sex reversal (TISR). To investigate the molecular mechanism of TISR in Nile tilapia, we performed Iso-seq analysis and found a dramatic effect of high temperature on gene alternative splicing (AS). Kdm6bb histone demethylase showed a novel AS at intron 5 that generates Kdm6bb_tv1 transcripts without intron 5 and Kdm6bb_tv2 with intron 5. Kdm6bb_tv1 encodes a full-length protein while Kdm6bb_tv2 encodes a truncated protein. Expression analysis revealed that intron 5 splicing of Kdm6bb is male and gonad biased at larval stage, and only gonad biased at adult stage. High-temperature treatment induced intron 5 splicing in the gonads of XX and XY fish, resulting in increased Kdm6bb_tv1 expression. To directly test the role of Kdm6bb_tv1 in Nile tilapia TISR, we knocked out expression of Kdm6bb_tv1. However, Kdm6bb_tv1-/- homozygous mutants showed embryonic lethality. Overexpression of Kdm6bb_tv1, but not Kdm6bb_tv2, induced sex reversal of XX females into pseudo-males. Overexpression of Kdm6bb_tv1, as with high-temperature treatment, modified the promotor region of Gsdf and Dmrt1 by demethylating the trimethylated lysine 27 of histone 3 (H3K27me3), thereby increasing expression. Collectively, these studies demonstrate that AS of Kdm6bb intron 5 increases the expression of Kdm6bb_tv1, which acts as a direct link between high temperature and activation of Gsdf and Dmrt1 expression, leading to male sex determination.

Production of sterile mono-sex triploid yellow drum (Nibea albiflora): genotypic females and sex-reversed phenotypic males with emphasis on utilization as surrogate broodstock.

Production of sterile mono-sex fish is of great significance for sustainable aquaculture as well as germ cell transplantation. In this study, we aimed to produce mono-sex triploid yellow drum, including genotypic females (XXX female) and sex-reversed phenotypic males (XXX male). Firstly, the mono-female triploids were produced through cold-shock treatment on eggs fertilized with sperm from neo-males. Then, the mono-male triploids were produced by the sex reversal of mono-female triploids with oral administration of letrozole (LZ). We comparatively investigated the growth and gonadal development in the mono-sex triploids. The results showed that the triploids displayed similar growth performance to their diploids throughout their first year, but had impaired gonadosomatic index and gametogenesis. No mature gametes were produced in the triploids during their first spawning season. Meanwhile, we analyzed the process of gametogenesis in the both sex of triploids. Ultrastructure of gametogenesis showed that the germ cells arrested at abnormal metaphase 1 in females, while males had irregular meiotic divisions, variable-sized spermatid and degenerated cells. The expression levels of meiosis-related genes (i.e., sycp3 and rec8) confirmed the abnormal meiosis in the triploids. Furthermore, the gonadal development was also determined by the expression patterns of vasa, dmrt1 and cyp19a1a. Abnormal expression of vasa mRNA and protein were detected in triploids. High cyp19a1a expression levels suggested the sex steroid hormones production might be at least partially functional in triploid females. In addition, high dmrt1 expression levels confirmed the masculinization and testicular development of sex-reversed triploid males by LZ. Our findings provide an efficient protocol to produce sterile mono-sex triploid yellow drum and provide new insights into the mechanism of gonadal sterility of triploid fish.

DMRT1 is a testis-determining gene in rabbits and is also essential for female fertility

DMRT1 is the testis-determining factor in several species of vertebrates, but its involvement in mammalian testes differentiation, where SRY is the testis-determining gene, remains ambiguous. So far, DMRT1 loss-of-function has been described in two mammalian species and induces different phenotypes: Disorders of Sex Development (46, XY DSD) in men and male infertility in mice. We thus abolished DMRT1 expression by CRISPR/Cas9 in a third species of mammal, the rabbit. First, we observed that gonads from XY DMRT1−/− rabbit fetuses differentiated like ovaries, highlighting that DMRT1 is involved in testis determination. In addition to SRY, DMRT1 is required in the supporting cells to increase the expression of the SOX9 gene, which heads the testicular genetic cascade. Second, we highlighted another function of DMRT1 in the germline since XX and XY DMRT1−/− ovaries did not undergo meiosis and folliculogenesis. XX DMRT1−/− adult females were sterile, showing that DMRT1 is also crucial for female fertility. To conclude, these phenotypes indicate an evolutionary continuum between non-mammalian vertebrates such as birds and non-rodent mammals. Furthermore, our data support the potential involvement of DMRT1 mutations in different human pathologies, such as 46, XY DSD as well as male and female infertility.

DMRT1 is a testis-determining gene in rabbits and is also essential for female fertility.

DMRT1 is the testis-determining factor in several species of vertebrates, but its involvement in mammalian testes differentiation, where SRY is the testis-determining gene, remains ambiguous. So far, DMRT1 loss-of-function has been described in two mammalian species and induces different phenotypes: Disorders of Sex Development (46, XY DSD) in men and male infertility in mice. We thus abolished DMRT1 expression by CRISPR/Cas9 in a third species of mammal, the rabbit. First, we observed that gonads from XY DMRT1-/- rabbit fetuses differentiated like ovaries, highlighting that DMRT1 is involved in testis determination. In addition to SRY, DMRT1 is required in the supporting cells to increase the expression of the SOX9 gene, which heads the testicular genetic cascade. Second, we highlighted another function of DMRT1 in the germline since XX and XY DMRT1-/- ovaries did not undergo meiosis and folliculogenesis. XX DMRT1-/- adult females were sterile, showing that DMRT1 is also crucial for female fertility. To conclude, these phenotypes indicate an evolutionary continuum between non-mammalian vertebrates such as birds and non-rodent mammals. Furthermore, our data support the potential involvement of DMRT1 mutations in different human pathologies, such as 46, XY DSD as well as male and female infertility.

Experiencing short heat waves early in development changes thermal responsiveness of turtle embryos to later heat waves.

Although physiological responses to the thermal environment are most frequently investigated using constant temperatures, the incorporation of thermal variability can allow for a more accurate prediction of how thermally sensitive species respond to a rapidly changing climate. In species with temperature-dependent sex determination (TSD), developmental responses to incubation temperature are mediated by several genes involved in gonadal differentiation. Kdm6b and Dmrt1 respond to cool incubation temperatures and are associated with testis development, while Foxl2 and Cyp19A1 respond to warm incubation temperatures and are associated with ovary development. Using fluctuating incubation temperatures, we designed two studies, one investigating how conflicting thermal cues affect the timing of commitment to gonadal development, and another investigating the rapid molecular responses to conflicting thermal cues in the red-eared slider turtle (Trachemys scripta). Using gene expression as a proxy of timing of commitment to gonadal fate, results from the first study show that exposure to high amounts of conflicting thermal cues during development delays commitment to gonadal fate. Results from the second study show that Kdm6b splice variants exhibit differential responses to early heat wave exposure, but rapidly (within 2 days) recover to pre-exposure levels after the heat wave. Despite changes in the expression of Kdm6b splice variants, there was no effect on Dmrt1 expression. Collectively, these findings demonstrate how short exposures to heat early in development can change how embryos respond to heat later in development.

DMRT1 regulates human germline commitment

Germline commitment following primordial germ cell (PGC) specification during early human development establishes an epigenetic programme and competence for gametogenesis. Here we follow the progression of nascent PGC-like cells derived from human embryonic stem cells in vitro. We show that switching from BMP signalling for PGC specification to Activin A and retinoic acid resulted in DMRT1 and CDH5 expression, the indicators of migratory PGCs in vivo. Moreover, the induction of DMRT1 and SOX17 in PGC-like cells promoted epigenetic resetting with striking global enrichment of 5-hydroxymethylcytosine and locus-specific loss of 5-methylcytosine at DMRT1 binding sites and the expression of DAZL representing DNA methylation-sensitive genes, a hallmark of the germline commitment programme. We provide insight into the unique role of DMRT1 in germline development for advances in human germ cell biology and in vitro gametogenesis.

Improving effects of telmisartan on spermatogenic disorder induced by fractionated low-dose irradiation in mice.

Male infertility is a hot problem worldwide, but there are few treatments, especially male infertility caused by irradiation is difficult to treat. The aim of this study was to investigate and evaluate novel drugs for the treatment of male infertility caused by irradiation. we randomly divided 18 male BALB/c mice into 3 groups: control, irradiated, and telmisartan. Both irradiated and telmisartan group completed whole-body 0.5Gy five times irradiation, and the telmisartan group received intraperitoneal injection of telmisartan (1.2mg/kg) daily on the next day after irradiation, and all groups were sampled on day 25 after irradiation. Sperm motility results show that total sperm motility of irradiated group was significantly lower compared with control group, and testicular HE results showed that testis in irradiated group were severely damaged. Compared with irradiated group, the total sperm motility, sperm concentration, testicular index, Johnsen score, and the seminiferous tubule layer numbers were higher in telmisartan group (P < 0.05). The immunohistochemical staining showed γ-H2AX expression is higher in telmisartan group compared with irradiated group. And the relative mRNA expression of PLZF, GFRA1, STRA8, DMRT1, SPO11, SYCP2, OVOL2, CCNA1, TJP3, RUNX2, TXNDC2 TNP1, and PRM3 in telmisartan group was all significantly higher than irradiated group (P < 0.05). In conclusion, in vivo experiments confirmed that telmisartan ameliorated the spermatogenic disorder in mice caused by fractionated low-dose irradiation via promoting spermatogenesis.

Effect ofhigh temperatures onsex ratio anddifferential expression analysis (RNA-seq) ofsex-determining genes inOreochromis niloticus from different river basins inBenin.

The high temperature sex reversal process leading to functional phenotypic masculinization during development has been widely described in Nile tilapia (Oreochromis n iloticus) under laboratory or aquaculture conditions and in the wild. In this study, we selected five wild populations of O. niloticus from different river basins in Benin and produced twenty full-sib families of mixed-sex (XY and XX) by natural reproduction. Progenies were exposed to room temperature or high (36.5°C) temperatures between 10 and 30 days post-fertilization (dpf). In control groups, we observed sex ratios from 40% to 60% males as expected, except for 3 families from the Gobé region which showed a bias towards males. High temperature treatment significantly increased male rates in each family up to 88%. Transcriptome analysis was performed by RNA-sequencing (RNA-seq) on brains and gonads from control and treated batches of six families at 15 dpf and 40 dpf. Analysis of differentially expressed genes, differentially spliced genes, and correlations with sex reversal was performed. In 40 dpf gonads, genes involved in sex determination such as dmrt1, cyp11c1, amh, cyp19a1b, ara, and dax1 were upregulated. In 15 dpf brains, a negative correlation was found between the expression of cyp19a1b and the reversal rate, while at 40 dpf a negative correlation was found between the expression of foxl2, cyp11c1, and sf1 and positive correlation was found between dmrt1 expression and reversal rate. Ontology analysis of the genes affected by high temperatures revealed that male sex differentiation processes, primary male sexual characteristics, autophagy, and cilium organization were affected. Based on these results, we conclude that sex reversal by high temperature treatment leads to similar modifications of the transcriptomes in the gonads and brains in offspring of different natural populations of Nile tilapia, which thus may activate a common cascade of reactions inducing sex reversal in progenies.

Insights into Early Ontogenesis of Salmo salar: RNA Extraction, Housekeeping Gene Validation and Transcriptional Expression of Important Primordial Germ Cell and Sex-Determination Genes

The challenge in extracting high-quality RNA impedes the investigation of the transcriptome of developing salmonid embryos. Furthermore, the mRNA expression pattern of important PGC and SD genes during the initial embryonic development of Salmo salar is yet to be studied. So, in the present study, we aimed to isolate high-quality RNA from eggs and developing embryos to check vasa, dnd1, nanos3a, sdf1, gsdf, amh, cyp19a, dmrt1 and foxl2 expression by qPCR. Additionally, four HKGs (GAPDH, UB2L3, eEf1a and β-actin) were validated to select the best internal control for qPCR. High-quality RNA was extracted, which was confirmed by spectrophotometer, agarose gel electrophoresis and Agilent TapeStation analysis. UB2L3 was chosen as a reference gene because it exhibited lower intra- and inter-sample variation. vasa transcripts were expressed in all the developmental stages, while dnd1 was expressed only up to 40 d°C. Nanos3a was expressed in later stages and remained at its peak for a shorter period, while sdf1 showed an irregular pattern of mRNA expression. The mRNA expression levels of SD genes were observed to be upregulated during the later stages of development, prior to hatching. This study presents a straightforward methodology for isolating high-quality RNA from salmon eggs, and the resulting transcript profiles of significant PGC and SD genes in S. salar could aid in improving our comprehension of reproductive development in this commercially important species.

Expression pattern analysis of anti-Mullerian hormone in testis development of pearlscale angelfish (Centropyge vrolikii).

In vertebrates, anti-Mullerian hormone (Amh) secreted by Sertoli cells (SC) performs a pivotal function in male sex differentiation. Compared with that of higher vertebrates, the expression pattern of Amh is more diversified in fish. In this study, the full-length complementary DNA (cDNA) of Amh in Centropyge vrolikii (Cv-Amh) was cloned and analysed, which was 2,470 bp, including a 238 bp 5'UTR, a 1,602 bp ORF and a 633 bp 3'UTR; the similarity of Amh between Cv-Amh and other fish is relatively high. The quantitative real-time PCR (qRT-PCR) results of healthy tissues and gonads at sex reversal stages in C. vrolikii showed that the expression level of Amh in the testis was significantly higher than that in other tissues (P < 0.05). Amh was weakly expressed in the vitellogenic stage ovary and perinucleolus stage ovary, but its expression significantly increased in the gonads at the hermaphroditic stage, and finally reached the highest in the pure testis after sexual reversal. The results of in situ hybridization indicated that the positive signal of Amh was strongly concentrated in SCs of testis. After Amh knockdown in the gonads, the effect on sex-related genes was tested using qRT-PCR. Among these, the expression of Dmrt1, Cyp11a, Hsd11b2, Sox8 and Sox9 significantly decreased, whereas that of Cyp19a, Sox4, Foxl2 and Sox3 increased. These results suggested that Amh could be the pivotal gene in reproductive regulation in C. vrolikii, and the data will contribute to sex-related research of C. vrolikii in the future.

Identification and functional analysis of Dmrt1 gene and the SoxE gene in the sexual development of sea cucumber, Apostichopus japonicus.

Members of the Doublesex and Mab-3-related transcription factor (Dmrt) gene family handle various vital functions in several biological processes, including sex determination/differentiation and gonad development. Dmrt1 and Sox9 (SoxE in invertebrates) exhibit a very conserved interaction function during testis formation in vertebrates. However, the dynamic expression pattern and functional roles of the Dmrt gene family and SoxE have not yet been identified in any echinoderm species. Herein, five members of the Dmrt gene family (Dmrt1, 2, 3a, 3b and 5) and the ancestor SoxE gene were identified from the genome of Apostichopus japonicus. Expression studies of Dmrt family genes and SoxE in different tissues of adult males and females revealed different expression patterns of each gene. Transcription of Dmrt2, Dmrt3a and Dmrt3b was higher expressed in the tube feet and coelomocytes instead of in gonadal tissues. The expression of Dmrt1 was found to be sustained throughout spermatogenesis. Knocking-down of Dmrt1 by means of RNA interference (RNAi) led to the downregulation of SoxE and upregulation of the ovarian regulator foxl2 in the testes. This indicates that Dmrt1 may be a positive regulator of SoxE and may play a role in the development of the testes in the sea cucumber. The expression level of SoxE was higher in the ovaries than in the testes, and knocking down of SoxE by RNAi reduced SoxE and Dmrt1 expression but conversely increased the expression of foxl2 in the testes. In summary, this study indicates that Dmrt1 and SoxE are indispensable for testicular differentiation, and SoxE might play a functional role during ovary differentiation in the sea cucumber.