Cloning of brain aromatase gene and expression of brain and ovarian aromatase genes during sexual differentiation in genetic male and female Nile tilapia Oreochromis niloticus

A brain aromatase gene was identified from the Nile tilapia Oreochromis niloticus. The cDNA sequence of this gene differed from that of the ovarian aromatase gene previously reported from this species. Tissue specific expression for both brain and ovarian aromatase genes was examined in the tissues of adult tilapia. Brain aromatase mRNA was expressed in the brain, kidney, eye, ovary, and testis, but not in the liver and spleen. Ovarian aromatase mRNA was expressed in the brain, spleen, ovary, and testis but not in the eye, kidney, and liver. Differential aromatase gene expression between the sexes was investigated in all-male (XY) and all-female (XX) groups of tilapia fry from fertilisation throughout the sexual differentiation period. Semi-quantitative RT-PCR analysis revealed that the initiation of expression of both aromatase genes lay between 3 and 4 dpf (days post fertilisation) in both sexes. The level of brain aromatase mRNA gradually increased throughout the period studied with little difference between the sexes. This contrasted with marked sexual dimorphism of ovarian aromatase mRNA expression. In females, the expression level was maintained or increased gradually throughout ontogeny, while the level in males was dramatically down-regulated between 15 and 27 dpf. Subsequently, the level of ovarian aromatase mRNA expression fluctuated slightly in both sexes, with the expression in females always being higher than in males. These findings clearly suggest that ovarian aromatase plays a decisive role in sexual differentiation in this species and that this is achieved by down-regulation of the expression of this gene in males. Mol. Reprod. Dev. 59: 359-370, 2001.     

Role of aromatase and androgen receptor expression in gonadal sex differentiation of ZW/ZZ-type frogs,Rana rugosa

To elucidate the mechanisms of amphibian gonadal sex differentiation, we examined the expression of aromatase and androgen receptor (AR) mRNAs for days 17-31 after fertilization. The effects of inhibitors and sex steroid hormones were also examined. In ZZ males, expression of AR decreased after day 19, while aromatase expression was low throughout the sampling period. Males treated with 17beta-estradiol (E2) showed increasing aromatase expression after day 21, and formed ovaries. AR antagonist treatment also induced high-level aromatase expression and ovarian differentiation. In males co-treated with an aromatase inhibitor and E2, the undifferentiated gonads developed into testes despite high-level aromatase expression. Males treated with androgen and E2 before and during an estrogen sensitive period, respectively, also formed testes. In ZW females, AR expression persisted at a low-level, while aromatase expression increased after day 18. Short-term treatment with an aromatase inhibitor was ineffective in preventing ovarian differentiation, whereas long-term treatment resulted in testes developing from ovarian structure. Compared with the ZZ males and ZW females, WW females did not exhibit detectable expression of AR, suggesting that the active AR gene(s) itself, or a putative gene regulating AR gene expression, is located on Z chromosomes. From the time lag of aromatase expression between ZW females and ZZ males treated with E2 and the effect of AR antagonist, it was found that in males elevated AR expression suppresses aromatase expression directly or indirectly. Consequently, endogenous androgens, accumulated by blocking estrogen biosynthesis, induced testicular differentiation. The gonadogenesis of males is dependent on sex hormone, whereas that of females has evolved to hormone-independence.     

Steroids, aromatase and sex differentiation of the newt Pleurodeles waltl

In the newt Pleurodeles waltl, genetic sex determination obeys female heterogamety (female ZW, male ZZ). In this species as in most of non-mammalian vertebrates, steroid hormones play a key role in sexual differentiation of gonads. In that context, male to female sex reversal can be obtained by treatment of ZZ larvae with estradiol. Male to female sex reversal has also been observed following treatment of ZZ larvae with testosterone, a phenomenon that was called the "paradoxical effect". Female to male sex reversal occurs when ZW larvae are reared at 32 degrees C during a thermosensitive period (TSP) that takes place from stage 42 to stage 54 of development. Since steroids play an important part in sex differentiation, we focussed our studies on the estrogen-producing enzyme aromatase during normal sex differentiation as well as in experimentally induced sex reversal situations. Our results based on treatment with non-aromatizable androgens, aromatase activity measurements and aromatase expression studies demonstrate that aromatase (i) is differentially active in ZZ and ZW larvae, (ii) is involved in the paradoxical effect and (iii) might be a target of temperature. Thus, the gene encoding aromatase might be one of the master genes in the process leading to the differentiation of the gonad in Pleurodeles waltl.     

Analysis of repetitive DNA sequences in the sex chromosomes of Oreochromis niloticus

In the Nile tilapia, Oreochromis niloticus, sex determination is primarily genetic, with XX females and XY males. While the X and Y chromosomes (the largest pair) cannot be distinguished in mitotic chromosome spreads, analysis of comparative hybridization of X and Y chromosome derived probes (produced, by microdissection and DOP-PCR, from XX and YY genotypes, respectively) to different genotypes (XX, XY and YY) has demonstrated that sequence differences exist between the sex chromosomes. Here we report the characterization of these probes, showing that a significant proportion of the amplified sequences represent various transposable elements. We further demonstrate that concentrations of a number of these individual elements are found on the sex chromosomes and that the distribution of two such elements differs between the X and Y chromosomes. These findings are discussed in relation to sex chromosome differentiation in O. niloticus and to the changes expected during the early stages of sex chromosome evolution.     

Aromatase plays a key role during normal and temperature-induced sex differentiation of tilapia Oreochromis niloticus.

In the tilapia Oreochromis niloticus, sex is determined genetically (GSD), by temperature (TSD) or by temperature/genotype interactions. Functional masculinization can be achieved by applying high rearing temperatures during a critical period of sex differentiation. Estrogens play an important role in female differentiation of non-mammalian vertebrates. The involvement of aromatase, was assessed during the natural (genetic all-females and all-males at 27 degrees C) and temperature-induced sex differentiation of tilapia (genetic all-females at 35 degrees C). Gonads were dissected between 486--702 degree x days. Aromatase gene expression was analyzed by virtual northern and semi-quantitative RT-PCR revealing a strong expression during normal ovarian differentiation concomitant with high levels (465 +/- 137 fg/g) of oestradiol-17 beta (E2-17 beta). This was encountered in gonads after the onset of ovarian differentiation (proliferation of both stromal and germ cells prior to ovarian meiosis). Genetic males exhibited lower levels of aromatase gene expression and E2-17 beta quantities (71 +/- 23 fg/ g). Aromatase enzyme activity in fry heads established a sexual dimorphism in the brain, with high activity in females (377.9 pmol/head/hr) and low activity in males (221.53 pmol/head/hr). Temperature induced the masculinization of genetic females to a different degree in each progeny, but in all cases repression of aromatase expression was encountered. Genetic males at 35 degrees C also exhibited a repression of aromatase expression. Aromatase brain activity decreased by nearly three-fold in the temperature-masculinized females with also a reduction observed in genetic males at 35 degrees C. This suggests that aromatase repression is required in the gonad (and perhaps in the brain) in order to drive differentiation towards testis development. Mol. Reprod. Dev. 59:265-276, 2001.     

Synaptonemal complex analysis in spermatocytes of tilapia, Oreochromis niloticus (Pisces, Cichlidae).

Some adaptations of the synaptonemal complex (SC) whole-mounting technique first used in plants permitted its application to meiotic studies in tilapia, Oreochromis niloticus. Direct observation of the chromosome pairing process and bivalent structure during the meiotic prophase of this fish species by light and electron microscopy permitted the analysis of SCs in autosomes and the possible identification of sex chromosomes. The analysis of SCs in spermatocytes of O. niloticus revealed that all 22 bivalent chromosomes completely paired, except for the occurrence of a size heteromorphism in the terminal region of the largest bivalent associated with the presence of an incompletely paired segment during the synapsis process, which may be the cytological visualization of an XX/XY sex chromosome system in this species.     

两种泥鳅芳香化酶基因的克隆与时空表达

鱼类的性别分化易受发育环境的影响。向性成熟的泥鳅和大鳞副泥鳅个体注射绒毛膜促性腺激素,获得卵子和精子进行人工授精。把胚胎分别置于20℃、25℃和30℃条件下,使其发育。经性腺检查发现随着温度的升高两种泥鳅中雄性个体所占的比例明显升高,获得明显的偏雄比率群体。根据已知细胞色素P450芳香化酶CYP19 b基因序列设计嵌套简并引物用巢式PCR扩增并克隆出了两种泥鳅的CYP19 b的DNA片段。MaCYP19 b片段和Pd-CYP19 b片段分别长1337bp和1473bp。在此基础上用各自的特异引物克隆出两种泥鳅CYP19 b的相应cDNA片段。通过基因组DNA和cDNA序列的比较证明两种泥鳅的CYP19 b基因均包含三个内含子和四个外显子,编码的蛋白质序列长145氨基酸残基。以GAPDH基因为对照,分别对两种泥鳅成体组织和不同发育阶段的胚胎的CYP19 b进行了半定量RT-PCR表达分析,结果表明泥鳅CYP19 b基因只在成体泥鳅卵巢、肾以及原肠胚和神经胚中表达。大鳞副泥鳅CYP19 b基因在成体的脑、卵巢和肾以及神经胚和卵黄吸收期表达。这些结果为揭示细胞色素P450芳香化酶基因与环境性别决定机制的关系奠定了基础。       

The origin and differentiation of the heteromorphic sex chromosomes Z, W, X, and Y in the frog Rana rugosa, inferred from the sequences of a sex-linked gene, ADP/ATP translocase

Sex chromosomes of the Japanese frog Rana rugosa are heteromorphic in the male (XX/XY) or in the female (ZZ/ZW) in two geographic forms, whereas they are still homomorphic in both sexes in two other forms (Hiroshima and Isehara types). To make clear the origin and differentiation mechanisms of the heteromorphic sex chromosomes, we isolated a sex-linked gene, ADP/ATP translocase, and constructed a phylogenetic tree of the genes derived from the sex chromosomes. The tree shows that the Hiroshima gene diverges first, and the rest form two clusters: one includes the Y and Z genes and the other includes the X, W, and Isehara genes. The Hiroshima gene shares more sequence similarity with the Y and Z genes than with the X, W, and Isehara genes. This suggests that the Y and Z sex chromosomes originate from the Hiroshima type, whereas the X and W chromosomes originate from the Isehara-type sex chromosome. Thus, we infer that hybridization between two ancestral forms, with the Hiroshima-type sex chromosome in one and the Isehara-type sex chromosome in the other, was the primary event causing differentiation of the heteromorphic sex chromosomes.      

Treatment with an aromatase inhibitor suppresses high-temperature feminization of genetic male (YY) Nile tilapia

High temperature (36° C) treatment during sexual differentiation caused significant changes in sex ratio in YY male Nile tilapia Oreochromis niloticus fry (64.5% males compared to 100.0% males at 28° C), while dietary treatment with a chemical aromatase inhibitor (AI: Fadrozole™ CGS16949A) during this period suppressed the high temperature feminization (98.9% males). This implies that cytochrome P450 aromatase is mechanistically associated with temperature-dependent sex determination (TSD) in this species. XY male fry did not show significant sex reversal at 36° C. In XX female fry, high temperature treatment resulted in significant masculinization (62.5% males compared with 21.9% males at 28° C), while treatment with AI at either temperature resulted in very high proportions of males (100.0% males at 36° C; 99.0% males at 28° C). These results confirm the importance of aromatase in sexual differentiation in the Nile tilapia below the TSD threshold and suggest that it also plays a role in TSD, at least in the YY genotype.     

Zebrafish sex determination and differentiation: Involvement of FTZ-F1 genes

Sex determination is the process deciding the sex of a developing embryo. This is usually determined genetically; however it is a delicate process, which in many cases can be influenced by environmental factors. The mechanisms controlling zebrafish sex determination and differentiation are not known. To date no sex linked genes have been identified in zebrafish and no sex chromosomes have been identified. However, a number of genes, as presented here, have been linked to the process of sex determination or differentiation in zebrafish. The zebrafish FTZ-F1 genes are of central interest as they are involved in regulating interrenal development and thereby steroid biosynthesis, as well as that they show expression patterns congruent with reproductive tissue differentiation and function. Zebrafish can be sex reversed by exposure to estrogens, suggesting that the estrogen levels are crucial during sex differentiation. The Cyp19 gene product aromatase converts testosterone into 17 beta-estradiol, and when inhibited leads to male to female sex reversal. FTZ-F1 genes are strongly linked to steroid biosynthesis and the regulatory region of Cyp19 contains binding sites for FTZ-F1 genes, further linking FTZ-F1 to this process. The role of FTZ-F1 and other candidates for zebrafish sex determination and differentiation is in focus of this review.     

Temporal difference between testis and ovary determinations with possible involvement of testosterone and aromatase in gonadal differentiation in TSD lacking lizard, Calotes versicolor

In the garden lizard, Calotes versicolor, which lacks identifiable sex chromosomes, incubation temperature also does not have a deterministic effect on the gender. However, the embryos reared at high temperature (33-35 degrees C) have a shorter duration of incubation as well as gonadal differentiation. In contrast, exogenous application of the male hormone testosterone to embryos at ambient temperature (28 degrees C) results in almost all individuals with only testis. Thus the testosterone treatment reverts genic females to males and accelerates the differentiation of testis, a feature similar to the high-temperature treatment. Treatment of eggs with estradiol shows no difference from that seen in the untreated eggs. The present series of experiments was done to establish the "window" of testosterone sensitivity and to understand the interaction between sex hormones and high temperature on gonadal differentiation. The period between day 5 and 15 of embryonic development was the window period of testosterone sensitivity for sex reversal. This period coincided with the formation of the genital ridge and its differentiation into cortex and medulla. Treatment of the 33 degrees C-reared embryos with testosterone resulted in hatchlings of both the sexes, in contrast to only males at the ambient temperature. In contrast, at the same temperature (33 degrees C), all the dihydrotestosterone (nonaromatisable testosterone)-treated embryos hatched into males. However, those given estradiol showed no sex bias regardless of the day of application and the concentration of drug. Eggs were also treated with aromatase inhibitor, CGS 16949 A, at ambient temperature and at 33 degrees C. All the 33 degrees C eggs to which the drug was given on day 25 hatched into males. These results suggest that though high temperature has no direct effect on sex determination in this species, it may have a stimulatory effect on aromatase activity, leading to the conversion of the exogenously applied testosterone into estradiol and permitting ovarian differentiation in the genic females. It also follows from the present report that the pathway of testis formation in Calotes versicolor is triggered much earlier, and irreversibly, than that for the ovary.     

Early origins of the X and Y chromosomes: lessons from tilapia

Differentiated sex chromosome pairs in diverse species display certain common characteristics, normally comprising one largely heterochromatic genetically inactive chromosome and one euchromatic genetically active chromosome (e.g. the mammalian Y and X respectively). It is widely accepted that dimorphic sex chromosomes evolved from homologous pairs of autosomes. Although the exact mechanisms through which the pair diverged are not fully understood, an initial suppression of recombination in the sex-determining region is required by all of the major theories. Here we address the question of the mechanism by which this initial suppression of recombination occurs. Our model postulates that the stochastic, de novo accumulation of heterochromatin in the sex determining region can delay pairing of the sex chromosomes in meiosis, resulting in a decrease in recombination. Data to support this model is presented from the cichlid fish, Oreochromis niloticus. Although such a decrease would in most circumstances be evolutionarily disadvantageous, if the region concerned included the major sex determining gene and other gene(s) with sex-specific functions, then this would be selectively advantageous and could trigger the process(es) which, ultimately, lead to the differentiation of the sex chromosomes.