Molecular mechanisms of sex determination and gonadal sex differentiation in fish

We have used various genetic and molecular approaches to investigate the mechanisms of sex determination and gonadal sex differentiation in fish. DMY was identified as the sex-determining gene of medaka. In tilapia, endogenous estrogens act as natural inducers of ovarian differentiation, while DMRT1 may be important for testicular differentiation. The roles of these regulators in sex determination and gonadal sex differentiation were ascertained using a gene or hormonal blockade strategy.     

斑节对虾Sox基因HMG盒的PCR扩增及SSCP分析

SRY基因是人类及哺乳动物睾丸决定因子的最佳候选基因,HMG盒是SRY基因编码蛋白质的唯一功能区,在性别决定中极其重要且高度保守,许多进化程度上明显不同的物种中都能检测出SRY基因的HMG盒,即Sox基因。斑节对虾是一种重要经济虾类,其性别决定机制研究薄弱,至今未找到性染色体。本研究根据人的SRY基因的HMG盒保守区的核苷酸序列,设计了1对兼并引物,通过PCR扩增出斑节对虾的Sox基因,并对扩增产物进行SSCP分析。结果表明,斑节对虾雌雄个体均存在Sox基因,从雌雄个体中均扩增出约350bp和220bp两条基因片段,推测其中350bp片段含内含子,SSCP结果显示斑节对虾内存在Sox基因家族的不同成员,且雌雄存在差异最后我们讨论了对虾性别决定可能的机制。     

GnRH gene products downregulate their neighboring genes encoding protein tyrosine phosphatases

The decapeptide gonadotropin-releasing hormone (GnRH) has multiple functions, including the regulation of gonadotropin secretion, reproductive behavior, and cell proliferation. In this paper, we have found that in the medaka (Oryzias latipes) genome, gonadotropin-releasing hormone (GnRH) genes are adjacent to type IV protein tyrosine phosphatase (PTP) genes encoding PTPα and PTPε, which are known to regulate cellular activity via interacting with voltage-gated potassium channel. We have subsequently demonstrated using an in vitro medaka whole-brain culture system that GnRH downregulates the PTPα and PTPε gene expression. Inhibition of intracellular type IV PTP signaling, which probably results in the modulation of cellular activity, may account for multiplicity of GnRH function.     

Differences in sex reversion and growth between normal and neomale stock in half-smooth tongue sole,Cynoglossus semilaevis

The present study evaluated the difference in the sex ratio (the genotypic and phenotypic female ratios) and the growth parameters between normal male offspring and neomale offspring under tank culture conditions. The phenotypic female ratio of normal male offspring (20.46–63.79 %) in four families and three stocks was remarkably higher than that of neomale offspring (5.21–10.53 %) in three neomale families and three neomale stocks (p < 0.01), while there was no significant difference between normal male offspring (30.41–70.60 %) and neomale offspring (29.7–70.83 %) in the genotypic female ratio (p > 0.05). The sex reversion ratio (SRR) was analyzed based on the genotypic and phenotypic female ratios. In neomale offspring, the SRR ranged from 84.25 to 92.53 %, while in normal offspring the SRR ranged from 9.65 to 34.60 %. There was a significant difference between normal male offspring and neomale offspring in the SRR (p < 0.01). The weight and length were measured at the ages of 300, 600, and 720 days. The growth rate was analyzed by the statistics, and there was a significant higher growth rate in the normal family than the neomale family (p < 0.05). The results suggest that the slower growth in the neomale offspring is responsible for the high SRR.     

Estrogen-induced and suppressed gene expression during XY sex reversal in a teleost fish, tilapia

To identify the down-stream gene products of estrogen during ovarian differentiation, we performed subtractive hybridization using mRNA derived from normal and estrogen treated XY gonads. The clones obtained after subtractive hybridization screening were re-screened by reverse-Northern analysis. Finally seven up-regulated and three down-regulated gene products were obtained.     

Analysis of plasmids encoding the tyrosine decarboxylase gene in Tetragenococcus halophilus isolated from fish sauce

In order to elucidate mechanisms of tyramine accumulation during fish sauce production, two tyramine-producing bacterial strains, referred to as TyrA and TyrB, were isolated from fish sauce mash accumulating over 141 mg of tyramine per 100 g of sample. Both strains were identified as Tetragenococcus halophilus based on phenotypic characterization and a 16S rRNA gene sequence analysis. Molecular analysis of the tyramine-producing gene in the two strains confirmed the presence of a ~30-kb plasmid encoding a single copy of the pyridoxal phosphate-dependent tyrosine decarboxylase gene (tdcA) along with three other genes related to tyramine synthesis (tdc cluster). The complete nucleotide sequences of plasmids extracted from the two strains indicated that both plasmids were almost identical, except for a 1.6-kb transposon sequence in the plasmid from the strain TyrB. Both plasmids had a replication region, a plasmid maintenance region, and two putative mobile genetic elements located upstream and downstream of the tdc cluster. This structure was identical to that of tetragenococcal plasmids encoding histidine decarboxylase (hdcA), which were sequenced previously. These results suggest a common origin for plasmids encoding hdcA and tdcA. In addition, the genes for both these biogenic amines are distributed among tetragenococcal species via this plasmid.     

中华鳖SOX9基因在胚胎期性腺中的表达模式及在性逆转中的表达变化

SOX9基因在脊椎动物性别决定和性腺分化中扮演重要的调控作用。从mRNA和蛋白水平分析中华鳖SOX9基因在不同组织中的差异性表达、在胚胎性腺和成年睾丸中的细胞定位以及在性逆转中的表达变化,研究SOX9基因在中华鳖性别分化中的调控作用。Real-time PCR结果显示,SOX9基因在中华鳖雄性性腺中特异性表达。免疫荧光染色分析显示,SOX9蛋白在雄性18期胚胎性腺中开始表达,随着性腺的发育,SOX9蛋白定位于性腺Sertoli前体细胞细胞核中;而在雌性胚胎性腺并未见其表达。此外,在雌激素诱导的雄性向雌性性逆转胚胎中,SOX9基因显著下调,而在芳香化酶抑制剂诱导的雌性向雄性性逆转胚胎中,SOX9基因表达则显著上升。研究表明,SOX9基因为中华鳖雄性特异性基因,参与雄性性腺的发育过程,可能在中华鳖早期性别分化过程中起调控作用。     

Analysis of plasmids encoding the histidine decarboxylase gene in Tetragenococcus muriaticus isolated from Japanese fermented seafoods

In order to analyze the genes related to histamine production in halophilic lactic acid bacteria, 16 strains of histamine-producing bacteria were isolated from three fermented seafoods produced in the Hokuriku region of Japan. Phenotypic and 16S rRNA gene sequence analyses identified all of the strains as those of Tetragenococcus muriaticus. Pyruvoyl-dependent histidine decarboxylase gene (hdcA) was determined from all strains using the PCR method with an hdcA-specific detection primer set. Genetic analyses (Southern blot and restriction fragment length polymorphism analysis) of hdcA and genes related to histamine production (the hdc cluster) confirmed that all of the strains harbored 21–23 kbp plasmids encoding a single copy of hdcA. The four representative strains were selected based on isolation source and genetic analysis, and subsequently full sequences of plasmids harbored in these strains were determined. hdc cluster sequences from the plasmids showed very high similarity (>99 %) to known hdc clusters of T. halophilus, Lactobacillus hilgardii 0006, and other lactic acid bacteria. The structures of the plasmids, the replication region, the hdc cluster, and the plasmid maintenance system were conserved between the plasmids present in new isolates and the T. halophilus strains. These results indicate that plasmids encoding hdcA are widely distributed among T. halophilus and T. muriaticus and function in both species.     

Transactivational property of chemicals via medaka glucocorticoid receptor 1b using a stable reporter gene assay

The transactivational property of natural and synthetic chemicals via medaka GR1b was investigated after development of a stable cell line for the reporter gene assay. In our study, cortisol was the most potent agonist among the natural corticoids assayed for potency [EC₅₀ (concentration of agonist provoking a response halfway between the baseline and maximum response) 68 nM] and efficacy. Three artificial corticosteroids, namely, dexamethasone [EC₅₀ 16 nM, relative agonistic activity to cortisol (RAA) 144 %], prednisolone (EC₅₀ 81 nM, RAA 116 %) and clobetasol propionate (EC₅₀ 0.10 nM, RAA 220 %), showed strong agonistic activity and were more potent than the original corticoid, F. All synthetic corticoids used in our study were full agonists. Interestingly, melengestrol acetate, a synthetic progestogen, induced luciferase activity in a dose-dependent manner. Based on its EC₅₀ value and RAA of 29 nM and 57 %, respectively, this molecule was assessed as a partial agonist. None of the other steroids and chemicals assayed in our study induced an agonistic response. In conclusion, we successfully developed a stable reporter gene assay that can be used to assess the transactivational property of glucocorticoid-like chemicals toward medaka GR1b.     

Estimation of the mode of inheritance of thermal tolerance in the guppy <Emphasis Type="Italic">Poecilia reticulata</Emphasis>

High water temperature influences the survival, growth, and maturation of fish. Genetically characterizing thermal tolerance is one of the most important subjects in fish culture. To identify the genetic characterization of thermal tolerance, this characteristic was compared among strains, and among parents and their offspring, in the guppy Poecilia reticulata. In the strain comparison, significant differences in survival rate were observed among the five strains examined, and between females and males. Females exhibited greater tolerance than males in four of five strains examined. In the comparison between parents and their offspring, stronger influence of female parent than of male parent was observed. Offspring obtained from surviving females exhibited greater tolerance than those from dead females. This tendency was typically observed in male offspring. The survival rate in male offspring obtained from dead female parents was lower than that of those from surviving females. The high-temperature tolerance of male parents did not influence this characteristic in offspring as strongly as that of female parents. These results suggest that the major gene or genes, which has a dominant resistant allele and a recessive sensitive allele, are probably passed on by sex-linked inheritance, located on the X chromosome.     

Characterization of a low-density lipoprotein receptor,Lrp13, in Chinese tongue sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>) and medaka (<Emphasis Type="Italic">Oryzias latipes</Emphasis>)

As an important economic marine species cultured in China, Chinese tongue sole (Cynoglossus semilaevis) has interested us due to its sexual dimorphism and ZW/ZZ sex determination system. In a previous study, dmrt1 was identified as a dosage-dependent male-determining gene. In the present study, a female-specific expressed gene, cse0440, initially annotated as lrp1b-like, was identified from chromosome W of C. semilaevis. In view of the differences between cse0440 and lrp1b in terms of expression pattern, a phylogenetic analysis containing 85 LRP proteins was constructed and provided an evidence to re-annotate cse0440 as cseLRP13. In addition, two orthologues of cseLRP13 were separately identified from W and Z chromosomes: cseLRP13-W and cseLRP13-Z. The subsequent multiple sequence alignment and syntenic arrangements of LRP13 in C. semilaevis, Japanese medaka (Oryzias latipes), large yellow croaker (Larimichthys crocea), striped bass (Morone saxatilis), white perch (Morone americana) and Fugu rubripes (Takifugu rubripes) further supported this re-annotation. RT-PCR and in situ hybridization revealed that cselrp13 was exclusively expressed in the oocytes and follicles of ovaries. These results suggested that lrp13 may play important roles in female reproduction. In future, with the advancement of micromanipulation in flatfish, the detailed function of two lrp13 orthologues in C. semilaevis will be elucidated.     

Glycoprotein hormone receptor family in medaka (Oryzias latipes)

Four types of GPHR cDNAs have been cloned from ovary and testis of medaka (Oryzias latipes) and their gene constructions have been determined. Two of them are closely related to known fish receptors for FSH and LH, respectively. Changes in their mRNA levels were examined during the course of oogenesis. FSH receptor mRNA could not be detected from 20 h before ovulation, whereas LH mRNA remained 5 h before ovulation.     

Complete mitochondrial genome sequence of Japanese cockle Fulvia mutica (Cardiidae)

The complete mitochondrial genome (mt-genome) sequence of Fulvia mutica (Veneroida; Cardiidae) was determined. The genome is 19,110 bp in size and contains 42 genes, including the ATP synthase subunit 8 gene (atp8). All genes are on the same strand, as in other marine bivalves. It is extremely different in gene arrangement and size from that of Acanthocardia tuberculata, the only species belonging to Cardiidae with complete genome sequence data. The presence of putative atp8 genes in two additional reported bivalve species, A. tuberculata and Sinonovacula constricta was also inferred by revising their deposited sequence data. It was suggested that atp8 genes of heterodont bivalves could be translated to 37–39 amino acid sequences highly conserved within families, excluding Hiatella arctica with 53 amino acids. The mt-genome of F. mutica also contains two large duplicated regions related to different sequence motifs. One of the regions consists of five nearly identical copies of the 154 bp motif that includes a transfer RNA gene for cysteine. This region exhibited polymorphism in the number of repeats among individuals, suggesting the existence of a variable number of tandem repeats, which was expected to provide valuable information for developing useful genetic markers for phylogenetic study and population genetics.     

Expression of <Emphasis Type="Italic">mep50</Emphasis> in adult and embryos of medaka fish (<Emphasis Type="Italic">Oryzias latipes</Emphasis>)

Protein arginine methylation is important for gene regulation and biological processes. Methylosome protein 50 (Mep50) is identified as a partner of protein arginine methyltransferase 5 (Prmt5), a major enzyme capable of symmetric dimethylation, in mammals and Xenopus. The isolation and characterization of medaka mep50 were reported in this paper. Medaka Mep50 is a homolog of human MEP50 with six WD40 domains. Medaka mep50 was ubiquitously expressed in the adult tissues and had maternal origin with continuous and dynamical expression during embryonic development detected by RT-PCR and in situ hybridization. A strong interaction of medaka Mep50 and Prmt5 was shown by yeast two hybridization. The expression pattern of mep50 is similar to that of prmt5 in medaka. The results suggested that medaka Mep50 could be a partner of Prmt5 and might play major roles in a variety of tissues in medaka.     

The effect of cyclooxygenase (COX) inhibitors on Japanese medaka (Oryzias latipes) reproduction parameters fed with high level of arachidonic acid (20:4 n-6)

Broodstock of mature Japanese medaka (Oryzias latipes Temminck and Schlegel, 1846) (mean weight 0.45 ± 0.03 g; age 6 months) were maintained in glass aquaria (8 males and 8 females in each aquarium). The fish were fed commercial feed (group C) or this feed was supplemented with arachidonic acid ARA (2 % of total fatty acids in feed; group A) and one of the COX inhibitors: resveratrol (group AR) or NS-398 (group ANS) with amount 20 mg kg^?1 BW day^?1 of one of the COX inhibitors. The effect of 2 weeks of feeding experimental diets on reproductive parameters (fecundity, embryo and larval survival, hatching rate, and larvae body weight) was analyzed. Larvae (obtained from experimental dietary treatments) were reared for 2 weeks. The percent of spawning female and fecundity was similar in dietary treatments. COX inhibitors in broodstock diet caused a decrease in embryo survival rate. Eggs of medaka from group ANS and AR revealed the lowest hatchability rate. In contrast, group A revealed a higher hatchability rate at the same time. Larval body weight after hatching was significantly the highest in this group. At the end of the experiment, larvae body weight ranged from 1.62 ± 0.43 mg in group AN to 2.89 ± 0.74 mg in group A. These results show that the COX inhibitors decreased gamete and larval quality regardless of high levels of ARA in broodstock diet.     

Life history traits of leopard coralgrouper Plectropomus leopardus in the Okinawa Islands, southwestern Japan

The life history of the leopard coralgrouper Plectropomus leopardus was examined for the purpose of stock evaluation and to help maintain populations in the Okinawa Islands, southwestern Japan. Age was estimated from cut and burnt otoliths, and gonads were observed histologically to reveal the growth, spawning period and relationships between age and both sexual development and sexual maturation for P. leopardus in waters north of Okinawa Island. The three parameters in the von Bertalanffy growth equation, L _∞ , k, and t ₀, were estimated at 61.2 cm fork length, 0.289, and 0.41, respectively. The oldest individual obtained among the specimens was 18.8 years. The spawning period started in May and lasted until July. During this period, 50 % of females reached maturity at 43.3 cm fork length and at 5 years of age. Due to the sexual transition from female to male, the sex ratio decreased to 50 % at 59.8 cm fork length and at 10.3 years of age.     

Study of sex determination system in ship sturgeon, Acipenser nudiventris using meiotic gynogenesis

The sex ratios and sex determination mechanism of gynogenetic diploids of ship sturgeon, Acipenser nudiventris, have been investigated to verify the possibilities of sex control by chromosome manipulation in this species. Meiotic gynogenesis was induced in a female ship sturgeon using cold shock after egg activation with UV-irradiated sperm of a male Siberian sturgeon. Microsatellite DNA analysis was applied for verification of uniparental inheritance in the gynogenetic diploid group of fish. All the analyzed gynogenetic diploids possessed only maternal genotype in the examined experimental group of fish. In this study, a minimum of two distinctly selected diagnostic loci in the offspring was used to confirm exclusively maternal contribution. Also, these fish were analyzed for sex diagnostic. Histological analysis of gonads from gynogenetic diploids, obtained from one family, showed 73.3 % of females and 27.7 % of males. The observed sex ratio has suggested that the ship sturgeon have a female heterogametic sex determination system. Gynogenesis in this species with female heterogametic sex determination system will have important role in breeding program and reclamation of its natural population to produce both female and male progeny, while this species has been introduced in the red list of IUCN (International Union for Conservation of Nature and Natural Resources).