Epigenetic control of cyp19a1a expression is critical for high temperature induced Nile tilapia masculinization

In fish species with temperature-dependent sex determination (TSD) or genotypic sex determination plus temperature effects (GSD + TE), temperature can either affect sex differentiation or determine the sex. However, it is unknown if epigenetic control of cyp19a1a expression is critical for high temperature induced masculinization in the freshwater fish Nile tilapia. We analyzed the cyp19a1a DNA methylation levels in three age groups and found that they were lower in females than in males. At 8 months of age, males had DNA methylation levels of the cyp19a1a promoter that were almost twice as high as those of females. Exposure to high temperatures increased the cyp19a1a promoter DNA methylation levels from 30.87 ± 4.56% to 48.34 ± 0.92% (P = 0.035) in females and from 50.33 ± 7.38% to 51.66 ± 4.75% in males (P = 0.867). The increases in the cyp19a1a promoter DNA methylation levels were associated with the mRNA expression levels and might play a role in promoting gonadal differentiation in high temperature induced group females toward the male pathway. Western blot analysis revealed that the cyp19a1a protein expression levels in females significantly declined after high temperature treatment; only a slight decline was recorded in male fish. These results reveal that epigenetic control of cyp19a1a mRNA and protein expression is related to the environmental temperature and sex ratios in fish with TSD or GSD + TE.     

Molecular cloning and characterization of amh,dax1 and cyp19a1a genes and their response to 17α-methyltestosterone in Pengze crucian carp

The proteins encoded by amh, dax1 and cyp19a1a play important roles in gonad differentiation. Their functions have been far less studied in teleosts. In this study, the full-length cDNAs of amh, dax1 and cyp19a1a were cloned and characterized in a triploid gynogenic fish, the Pengze crucian carp. Their expression profilings in juvenile development, adult tissues and juveniles exposed to 100 ng/L 17α-methyltestosterone (MT) were investigated. Results showed that their putative proteins shared high identities to their counterparts in cyprinid fish species, respectively. The tissue distribution results indicated that amh and cyp19a1a were predominantly expressed in the ovary and dax1 was dominantly expressed in the liver. Gene profiling in the developmental stages showed that all the three target genes had a consistent highest expression at 48 days post hatching (dph). The period of 48 dph appeared to be a key time during the process of the gonad development of Pengze crucian carp. 100 ng/L MT significantly increased the mRNA expression of amh at 2- and 4-week exposures and enhanced dax1 and cyp19a1a at 6-week exposure. The present study indicated that MT could influence the gonad development in Pengze crucian carp by disturbing sex-differentiation associated gene expression. Furthermore, the present study will be of great significance to broaden the understanding of molecular mechanisms of the physiological processes of reproduction in fish.     

Characterization and function of the T-box 1 gene in Chinese giant salamander Andrias davidianus

We characterized the Andrias davidianus T-box 1 (Tbx1) gene. Tbx1 expression was high in testis and low in other examined tissues. Immunohistochemistry detected tbx1 expression in somatic and germ cells 62 days post-hatching (dph), prior to gonad differentiation. At 210 dph, after gonad differentiation, tbx1 was expressed in spermatogonia and testis somatic cells and in granulosa cells in ovary. Tbx1 expression was up-regulated in ovary after high temperature treatment. In the neomale, tbx1 expression showed a similar profile to normal males, and vice-versa for genetic male. Over-expression of tbx1 in females after injection of TBX1 protein down-regulated the female-biased genes cyp19a and foxl2 and up-regulated the male-biased amh gene. When tbx1 was knocked down by tbx1/siRNA, cyp19a and foxl2 expression was up-regulated, and expression of amh, cyp26a, dmrt1, and wt1 was down-regulated. Results suggest that tbx1 influenced sex-related gene expression and participates in regulation of A. davidianus testis development.     

Natural sex change in mature protogynous orange-spotted grouper (Epinephelus coioides): gonadal restructuring,sex hormone shifts and gene profiles

Sexual patterns of teleosts are extremely diverse and include both gonochorism and hermaphroditism. As a protogynous hermaphroditic fish, all orange-spotted groupers (Epinephelus coioides) develop directly into females, and some individuals change sex to become functional males later in life. This study investigated gonadal restructuring, shifts in sex hormone levels and gene profiles of cultured mature female groupers during the first (main) breeding season of 2019 in Huizhou, China (22° 42′ 02.6″ N, 114° 32′ 10.1″ E). Analysis of gonadal restructuring revealed that females with pre-vitellogenic ovaries underwent vitellogenesis, spawning and regression and then returned to the pre-vitellogenic stage in the late breeding season, at which point some changed sex to become males via the intersex gonad stage. A significant decrease in the level of serum 17β-estradiol (E2) was observed during ovary regression but not during sex change, whereas serum 11-ketotestosterone (11-KT) concentrations increased significantly during sex change with the highest concentration in newly developed males. Consistent with serum hormone changes, a significant decrease in cyp19a1a expression was observed during ovary regression but not during sex change, whereas the expression of cyp11c1 and hsd11b2 increased significantly during sex change. Interestingly, hsd11b2 but not cyp11c1 was significantly upregulated from the pre-vitellogenic ovary stage to the early intersex gonad stage. These results suggest that a decrease in serum E2 concentration and downregulation of cyp19a1a expression are not necessary to trigger the female-to-male transformation, whereas increased 11-KT concentration and upregulation of hsd11b2 expression may be key events for the initiation of sex change in the orange-spotted grouper.     

Molecular characterization and functional analysis of cyp11a and cyp11b in black rockfish (Sebastes schlegelii)

The cyp11 includes cyp11a and cyp11b in most mammals and teleosts, encoded cholesterol side chain lyase and 11β-hydroxylase, respectively. It is essential in steroid hormone synthesis. However, studies on the regulation of cyp11 are limited, especially in teleosts. In this study, the molecular characterization and function of cyp11a and cyp11b of black rockfish was investigated. Both of them showed high homology with other teleost counterparts by phylogenetic analysis. The expression of cyp11a and cyp11b exhibited a clear sexually dimorphic pattern, with a higher expression level in testis than that of in ovaries. During the different developmental stages (40 dpf, 80 dpf, 190 dpf, 360 dpf, 720 dpf), the expression of cyp11a was earlier than cyp11b. In situ hybridization results showed that cyp11a and cyp11b were mainly expressed in oogonia and oocytes of the ovary. They were located in spermatogonia and interstitial compartment in the 1.5-year-old gonads, and spermatocytesgonia and the peritubular myoid cell of the testis in the 2.5-year-old gonads. To explore the distinct roles of cyp11a and cyp11b in gonads, oestrogen and androgens were used to stimulate the primary testicular and ovarian cells. The expressions of cyp11a and cyp11b were tested under different dose of 17α-methyltestosterone (17α-MT) and 17β-estradiol (E2). The results showed cyp11a was significantly increased at 10⁻⁶ mol ml^–1 of 17α-MT and 10⁻⁸ mol ml^–1 of E2 in ovary and 10⁻¹⁰ mol ml^–1 of 17α-MT and E2 in testis, while cyp11b was significantly decreased after 17α-MT and E2 treatment. These results indicated that cyp11a and cyp11b were likely to have different functions, and also implied they might play an important roles in the differentiation of gonads and the synthesis of steroids in black rockfish.     

A longitudinal study of epigenetic variation in twins

DNA methylation is a key epigenetic mechanism involved in the developmental regulation of gene expression. Alterations in DNA methylation are established contributors to inter-individual phenotypic variation and have been associated with disease susceptibility. The degree to which changes in loci-specific DNA methylation are under the influence of heritable and environmental factors is largely unknown. In this study, we quantitatively measured DNA methylation across the promoter regions of the dopamine receptor 4 gene (DRD4), the serotonin transporter gene (SLC6A4/SERT) and the X-linked monoamine oxidase A gene (MAOA) using DNA sampled at both ages 5 and 10 years in 46 MZ twin-pairs and 45 DZ twin-pairs (total n=182). Our data suggest that DNA methylation differences are apparent already in early childhood, even between genetically identical individuals, and that individual differences in methylation are not stable over time. Our longitudinal-developmental study suggests that environmental influences are important factors accounting for interindividual DNA methylation differences, and that these influences differ across the genome. The observation of dynamic changes in DNA methylation over time highlights the importance of longitudinal research designs for epigenetic research.     

Simulated microgravity‐induced epigenetic changes in human lymphocytes

Real space flight and modeled microgravity conditions result in changes in the expression of genes that control important cellular functions. However, the mechanisms for microgravity‐induced gene expression changes are not clear. The epigenetic changes of DNA methylation and chromatin histones modifications are known to regulate gene expression. The objectives of this study were to investigate whether simulated microgravity alters (a) the DNA methylation and histone acetylation, and (b) the expression of DNMT1, DNMT3a, DNMT3b, and HDAC1 genes that regulate epigenetic events. To achieve these objectives, human T‐lymphocyte cells were grown in a rotary cell culture system (RCCS) that simulates microgravity, and in parallel under normal gravitational conditions as control. The microgravity‐induced DNA methylation changes were detected by methylation sensitive‐random amplified polymorphic DNA (MS‐RAPD) analysis of genomic DNA. The gene expression was measured by Quantitative Real‐time PCR. The expression of DNMT1, DNMT3a, and DNMT3b was found to be increased at 72 h, and decreased at 7 days in microgravity exposed cells. The MS‐RAPD analysis revealed that simulated microgravity exposure results in DNA hypomethylation and mutational changes. Gene expression analysis revealed microgravity exposure time‐dependent decreased expression of HDAC1. Decreased expression of HDAC1 should result in increased level of acetylated histone H3, however a decreased level of acetylated H3 was observed in microgravity condition, indicating thereby that other HDACs may be involved in regulation of H3 deacetylation. The findings of this study suggest that epigenetic events could be one of the mechanistic bases for microgravity‐induced gene expression changes and associated adverse health effects. J. Cell. Biochem. 111: 123–129, 2010. © 2010 Wiley‐Liss, Inc.     

Genome-wide mapping of DNA methylation in Nile Tilapia

Cytosine DNA methylation is crucial for gene regulation and maintenance of genome stability. However, the detailed nile tilapia methylome remains uncharacterized. In this study, we present the first high-resolution methylome of tilapia gonad generated using methylated DNA immunoprecipitation (MeDIP) and high-throughput sequencing. In the ovary, 265 and 56 methylation peaks were identified in the genebody and promoter region of 145 genes, respectively. In the testis, 293 and 80 methylation peaks were identified in the genebody and promoter region of 144 genes. Furthermore, 8 and 49 genes showed differentially higher and lower promoter-region methylation rates, respectively, in the ovary relative to those of the testis. Quantitative PCR results revealed that the expression level of fibroblast growth factor 16 (fgf16), sialidase-3-like, fibroblast growth factor 20, aromatase (cyp19a), estrogen receptor, and gonadotropin receptor II precursor were negatively correlated to their methylation levels in the ovary and testis. The methylated levels of cyp19a and fgf16 were validated by bisulfite sequencing PCR technology, and the results were consistent with the MeDIP results. Thus, apart from generating the first methylation map, this study produced a candidate gene repository that provides additional options to explore the relationship between DNA methylation and sex differentiation or maintenance.     

Differential expression of foxl2 and cyp19a1a mRNA during gonad developmental stages in great sturgeon Huso huso

This study aimed to determine the sex specificity and expression pattern of foxl2 and cyp19a1a genes in great sturgeon Huso huso gonads during gonadal sex differentiation and development. The results revealed that foxl2 and cyp19a1a mainly expressed in female gonads and during gonad development the foxl2 and cyp19a1a mRNA expression is required for ovarian development.     

Germ line control of female sex determination in zebrafish

A major transition during development of the gonad is commitment from an undifferentiated “bi-potential” state to ovary or testis fate. In mammals, the oogonia of the developing ovary are known to be important for folliculogenesis. An additional role in promoting ovary fate or female sex determination has been suggested, however it remains unclear how the germ line might regulate this process. Here we show that the germ line is required for the ovary versus testis fate choice in zebrafish. When the germ line is absent, the gonad adopts testis fate. These germ line deficient testes have normal somatic structures indicating that the germ line influences fate determination of surrounding somatic tissues. In germ line deficient animals the expression of the ovary specific gene cyp19a1a fails to be maintained whereas the testis genes sox9a and amh remain expressed. Furthermore, we observed decreased levels of the ovary specific genes cyp19a1a and foxL2 in germ line deficient animals prior to morphological sex differentiation of the gonad. We propose that the germ line has a common role in female sex determination in fish and mammals. Additionally, we show that testis specification is sufficient for masculinization of the fish pointing to a direct role of hormone signaling from the gonad in directing sex differentiation of non-gonadal tissues.     

Variable histone modifications at the Avy metastable epiallele

The ability of environmental factors to shape health and disease involves epigenetic mechanisms that mediate gene-environment interactions. Metastable epiallele genes are variably expressed in genetically identical individuals due to epigenetic modifications established during early development. DNA methylation within metastable epialleles is stochastic due to probabilistic reprogramming of epigenetic marks during embryogenesis. Maternal nutrition and environment have been shown to affect metastable epiallele methylation patterns and subsequent adult phenotype. Little is known, however, about the role of histone modifications in influencing metastable epiallele expression and phenotypic variation. Utilizing chromatin immunoprecipitation followed by qPCR, we observe variable histone patterns in the 5’ long terminal repeat (LTR) of the murine viable yellow agouti (A^vy) metastable epiallele. This region contains 6 CpG sites, which are variably methylated in isogenic A^vy/a offspring. Yellow mice, which are hypomethylated at the Avy LTR and exhibit constitutive ectopic expression of agouti (a), also display enrichment of H3 and H4 di-acetylation (p=0.08 and 0.09, respectively). Pseudoagouti mice, in which A^vy hypermethylation is thought to silence ectopic expression, exhibit enrichment of H4K20 tri-methylation (p=0.01). No differences are observed for H3K4 tri-methylation (p=0.7), a modification often enriched in the promoter of active genes. These results show for the first time the presence of variable histone modifications at a metastable epiallele, indicating that DNA methylation acts in concert with histone modifications to affect inter-individual variation of metastable epiallele expression. Therefore, the potential for environmental factors to influence histone modifications, in addition to DNA methylation, should be addressed in environmental epigenomic studies.     

Expression and characterization of the cyp19a gene and its responses to estradiol/letrozole exposure in Chinese soft‐shelled turtle (Pelodiscus sinensis)

Cytochrome P450 aromatase (CYP19) catalyzes the conversion of androgens to estrogens and is critical in sex differentiation. CYP19 exists as the ovarian type and brain type. Herein, we cloned the full‐length ovarian cyp19a gene from the Chinese soft‐shelled turtle, Pelodiscus sinensis (pscyp19a). We determined the distribution of pscyp19a in adult tissue and evaluated its expression during embryonic development, following treatment with 17β‐estradiol (E2) or letrozole (LE). The pscyp19a complementary DNA is 2,285 bp in length and comprises a 1,512 bp open reading frame that encodes a protein of 503 AA. The nucleotide sequence and amino acid of pscyp19a shared significant identity with other vertebrate sequences. Expression of pscyp19a was high in the ovary (p < 0.01), and exhibited modest expression in the female brain and intestine. Expression of pscyp19a displayed significant differences between sexes during early embryo development stages; expression increased gradually during embryonic development in females, but the opposite trend was observed in males. Female embryos treated with different concentrations of E2 and LE displayed altered pscyp19a expression compared with untreated individuals, and E2 clearly induced pscyp19a expression. These results indicate that pscyp19a gene plays important roles in early developmental stages in Chinese soft‐shelled turtle, and may assist future studies on sex differentiation and sex control in this and similar species.     

Gestational low protein diet in the rat mediates Igf2 gene expression in male offspring via altered hepatic DNA methylation

Biological responses to environmental stress, including nutrient limitation are mediated in part by epigenetic modifications including DNA methylation. Insulin-like growth factor II (Igf2) and H19 are subject to epigenetic modifications leading to genomic imprinting. The present study was designed to test the effect of maternal low protein diet on the Igf2/H19 locus in offspring. Pregnant Sprague-Dawley rats were fed diets containing 180 g/kg casein (control) or 90 g/kg (LP) casein with either 1 mg/kg (LP) or 3 mg/kg folic acid (LPF). LP diet increased Igf2 and H19 gene expression in the liver of day 0 male offspring and the addition of folic acid reduced the mRNA level in LPF rats to that of the control group. DNA methylation in Imprinting Control Region (ICR) of Igf2/H19 locus increased significantly following maternal LP diet but rats fed the LPF diet did not exhibit the hypermethylation. The Differential Methylation Region 2 (DMR2) did not show any change in methylation in either LP or LPF rats. The expression of Dnmt1 and Dnmt3a, the members of DNA methyltransferase family, and methyl CpG-binding domain 2 (Mbd2) was significantly increased following the maternal LP diet but did not differ between the control and LPF group. There is a strong correlation between methylation of ICR with the expression of Igf2 and H19. These results suggested that maternal exposure to a low protein diet and folic acid during gestation alters gene expression of Igf2 and H19 in the liver by regulating the DNA methylation of these genes. The DNA methyltransferase machinery may be involved into the programming of imprinted genes through the imprinted control region.     

Genome‐wide promoter methylation analysis identifies epigenetic silencing of MAPK13 in primary cutaneous melanoma

The involvement of epigenetic alterations in the pathogenesis of melanoma is increasingly recognized. Here, we performed genome‐wide DNA methylation analysis of primary cutaneous melanoma and benign melanocytic nevus interrogating 14 495 genes using BeadChip technology. This genome‐wide view of promoter methylation in primary cutaneous melanoma revealed an array of recurrent DNA methylation alterations with potential diagnostic applications. Among 106 frequently hypermethylated genes, there were many novel methylation targets and tumor suppressor genes. Highly recurrent methylation of the HOXA9, MAPK13, CDH11, PLEKHG6, PPP1R3C, and CLDN11 genes was established. Promoter methylation of MAPK13, encoding p38δ, was present in 67% of primary and 85% of metastatic melanomas. Restoration of MAPK13 expression in melanoma cells exhibiting epigenetic silencing of this gene reduced proliferation, indicative of tumor suppressive functions. This study demonstrates that DNA methylation alterations are widespread in melanoma and suggests that epigenetic silencing of MAPK13 contributes to melanoma progression.