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.     

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.     

Sex identification of morphologically-undifferentiated Siberian sturgeon with statistical analysis of gene expression patterns

The purpose of this study was to detail a simple strategy for sexing morphologically-undifferentiated fish using statistical analysis of gene expression patterns characterized by quantitative PCR. This approach is especially relevant for species without known genomic sex markers. The method was developed for early identification of female Siberian sturgeon as part of a genomics study. That study documented activation of the enzyme 17ß-hydroxy-steroid-dehydrogenase (hsd17b1) in future ovarian tissue at 3 months of age, concurrent with a small forkhead box L2 (foxl2) peak and emerging cytochrome P450, family 19, subfamily A (cyp19a1) expression. Major cyp19a1 and foxl2 peaks occurred in presumptive female gonads at 5–6 months. This pattern suggested a genetic relay mediating estrogen production throughout differentiation, possibly to maintain gonadal femininity. Genes involved in stem cell proliferation (lim homeobox 2 (lhx2)) and somatic-germ cell interaction maintenance (iroquois homeobox 5 (irx5) and iroquois homeobox 3 (irx3)) were also expressed during molecular differentiation, at 5–6 months. The roles of lhx2, irx3, and irx5 in fish sex differentiation should be confirmed using other methodologies. These results indicate that estrogens are crucial for ovarian differentiation in basal non-teleost fish, consistent with well-established patterns in teleosts, with hsd17b1 as one of the earliest biomarkers of gonadal development.     

黄河鲤性别决定时间和相关基因表达研究

鱼类性别决定和分化机制极为复杂,通过性腺组织切片鉴定得出黄河鲤从未分化性腺发育为Ⅱ期精巢、卵巢的时间为受精后第40天到第80天。选取一些可能参与黄河鲤性别决定分化相关的基因（amh、ar、cyp19a、cyp19b、dax1、dmrt1、er、foxl2、nobox、sox9a、sox9b、zp2）进行实时荧光定量PCR分析各个基因在受精后40d、45d、50d、55d、65d和80d的表达情况。结果显示性别决定相关基因在50d都有高表达,推测45-50 d为性别决定的关键时间。ar、amh、dax1、dmrt1、sox9a、sox9b六个基因在80d雄性表达量升高,且雄性明显高于雌性,推测这些基因参与精巢分化发育过程。cyp19a、cyp19b、foxl2、nobox、zp2五个基因在80d雌性表达升高,且高于雄性,推测其可能参与卵巢分化发育。     

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.     

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.     

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.