Characterization of Subcellular Dynamics of Sterol Methyltransferases Clarifies Defective Cell Division in smt2 smt3, a C-24 Ethyl Sterol-Deficient Mutant of Arabidopsis.

An Arabidopsis sterol mutant, smt2 smt3, defective in sterolmethyltransferase2 (SMT2), exhibits severe growth abnormalities. The loss of C-24 ethyl sterols, maintaining the biosynthesis of C-24 methyl sterols and brassinosteroids, suggests specific roles of C-24 ethyl sterols. We characterized the subcellular localizations of fluorescent protein-fused sterol biosynthetic enzymes, such as SMT2-GFP, and found these enzymes in the endoplasmic reticulum during interphase and identified their movement to the division plane during cytokinesis. The mobilization of endoplasmic reticulum-localized SMT2-GFP was independent of the polarized transport of cytokinetic vesicles to the division plane. In smt2 smt3, SMT2-GFP moved to the abnormal division plane, and unclear cell plate ends were surrounded by hazy structures from SMT2-GFP fluorescent signals and unincorporated cellulose debris. Unusual cortical microtubule organization and impaired cytoskeletal function accompanied the failure to determine the cortical division site and division plane formation. These results indicated that both endoplasmic reticulum membrane remodeling and cytokinetic vesicle transport during cytokinesis were impaired, resulting in the defects of cell wall generation. The cell wall integrity was compromised in the daughter cells, preventing the correct determination of the subsequent cell division site. We discuss the possible roles of C-24 ethyl sterols in the interaction between the cytoskeletal network and the plasma membrane.

Improvement of the ayu (Plecoglossus altivelis) draft genome using Hi-C sequencing.

OBJECTIVE: The ayu or sweetfish Plecoglossus altivelis is ray-finned fish that is widely distributed in East Asia. The genome size of ayu was estimated at approximately 420 Mb. Previously, we reported on ayu draft genome assembly by whole-genome shotgun using Illumina short reads and PacBio long reads; however, the assembly was not to chromosome level. Therefore, to improve the draft genome sequence of ayu to chromosome level, we performed in situ Hi-C sequencing as a source of linkage information. RESULTS: The ayu genome assembly yielded 28 large scaffolds that corresponded to the karyotype of ayu (n = 28). The resulting ayu genome assembly has a N50 scaffold length of 17.0 Mb, improved from 4.3 Mb. The high-quality reference genome will be helpful for phylogenetic research on bony fishes and for breeding programs in ayu aquaculture.

Y-specific amh allele, amhy, is the master sex-determining gene in Japanese flounder Paralichthys olivaceus.

Japanese flounder (Paralichthys olivaceus) is an important marine fish species of both fisheries and aquaculture in Northeast Asia. The commercial interest for all-female progenies due to several sex-related traits has prompted basic research on the mechanisms of sex determination in this species. By conducting a linkage analysis of the sex-determining locus, we initially identified 12 microsatellite markers linked to sex in 11 scaffolds, whose localization was restricted to a specific region of linkage group 9. Sequence analysis of this region identified 181 genes based on the UniProt database annotations. Among them, the amh gene was considered a potential candidate for sex determination because this gene is known to have taken over the role of sex determination in many teleosts. An in-depth sequence analysis of both the coding and non-coding regions of amh in XX and XY individuals detected nine SNPs linked with maleness. However, because these substitutions were synonymous, the upstream and downstream regions of amh were also investigated and a male-specific variant with deletions in the promoter region was detected. This truncated Y-specific amh variant was named amhy, and the amh shared by both sexes was named amhx. The association analysis using both females and males of the genotypic sex inferred by the presence/absence of amhy found complete association with phenotypic sex and genotype. Gene expression analysis in larvae derived from a single-pair progeny by quantitative real-time PCR detected amhy transcripts in the larval trunks between 20 and 100 days after hatching only in XY larvae. Localization of amhy by in situ hybridization was detected in presumptive Sertoli cells of XY gonads. Expression of amhx was almost undetectable in both XX and XY genotypes. Loss of Amh function by CRISPR-Cas9 induced male-to-female sex reversal, indicating that this gene was necessary for the masculinization of XY individuals. In conclusion, the complete linkage of amhy with males, its early expression in XY gonads before testicular differentiation, and the induction of sex reversal by loss-of-function mutation support the view that amhy is the sex-determining gene in this species.

Exploration and characterization of chemical stimulators to maximize the wax ester production by Euglena gracilis.

Euglena gracilis, a phototrophic protist, is a valuable biomass producer that is often employed in sustainable development efforts. E. gracilis accumulates wax esters as byproducts during anaerobic ATP production via the reductive tricarboxylic acid cycle, utilizing the storage carbohydrate ß-1,3-glucan paramylon as the carbon source. Here, we report a library screening for chemical stimulators that accelerate both wax ester production and paramylon consumption. Among the 115 compounds tested, we identified nine compounds that increased wax ester production by more than 2.0-fold relative to the solvent control. In the presence of these nine compounds, the paramylon content decreased compared with the control experiment, and the residual paramylon content varied between 7% and 26% of the initial level. The most active compound, 1,4-diaminoanthracene-9,10-dione (OATQ008), stimulated wax ester production up to 2.7-fold within 24 h, and 93% of the cellular paramylon was consumed. In terms of the structural features of the chemical stimulators, we discuss the potential target sites to stimulate wax ester production in mitochondria under anaerobic conditions.

A Y-linked anti-Müllerian hormone type-II receptor is the sex-determining gene in ayu, Plecoglossus altivelis.

Whole-genome duplication and genome compaction are thought to have played important roles in teleost fish evolution. Ayu (or sweetfish), Plecoglossus altivelis, belongs to the superorder Stomiati, order Osmeriformes. Stomiati is phylogenetically classified as sister taxa of Neoteleostei. Thus, ayu holds an important position in the fish tree of life. Although ayu is economically important for the food industry and recreational fishing in Japan, few genomic resources are available for this species. To address this problem, we produced a draft genome sequence of ayu by whole-genome shotgun sequencing and constructed linkage maps using a genotyping-by-sequencing approach. Syntenic analyses of ayu and other teleost fish provided information about chromosomal rearrangements during the divergence of Stomiati, Protacanthopterygii and Neoteleostei. The size of the ayu genome indicates that genome compaction occurred after the divergence of the family Osmeridae. Ayu has an XX/XY sex-determination system for which we identified sex-associated loci by a genome-wide association study by genotyping-by-sequencing and whole-genome resequencing using wild populations. Genome-wide association mapping using wild ayu populations revealed three sex-linked scaffolds (total, 2.03 Mb). Comparison of whole-genome resequencing mapping coverage between males and females identified male-specific regions in sex-linked scaffolds. A duplicate copy of the anti-Müllerian hormone type-II receptor gene (amhr2bY) was found within these male-specific regions, distinct from the autosomal copy of amhr2. Expression of the Y-linked amhr2 gene was male-specific in sox9b-positive somatic cells surrounding germ cells in undifferentiated gonads, whereas autosomal amhr2 transcripts were detected in somatic cells in sexually undifferentiated gonads of both genetic males and females. Loss-of-function mutation for amhr2bY induced male to female sex reversal. Taken together with the known role of Amh and Amhr2 in sex differentiation, these results indicate that the paralog of amhr2 on the ayu Y chromosome determines genetic sex, and the male-specific amh-amhr2 pathway is critical for testicular differentiation in ayu.

RADSex: A computational workflow to study sex determination using restriction site-associated DNA sequencing data.

The study of sex determination and sex chromosome organization in nonmodel species has long been technically challenging, but new sequencing methodologies now enable precise and high-throughput identification of sex-specific genomic sequences. In particular, restriction site-associated DNA sequencing (RAD-Seq) is being extensively applied to explore sex determination systems in many plant and animal species. However, software specifically designed to search for and visualize sex-biased markers using RAD-Seq data is lacking. Here, we present RADSex, a computational analysis workflow designed to study the genetic basis of sex determination using RAD-Seq data. RADSex is simple to use, requires few computational resources, makes no prior assumptions about the type of sex-determination system or structure of the sex locus, and offers convenient visualization through a dedicated R package. To demonstrate the functionality of RADSex, we re-analysed a published data set of Japanese medaka, Oryzias latipes, where we uncovered a previously unknown Y chromosome polymorphism. We then used RADSex to analyse new RAD-Seq data sets from 15 fish species spanning multiple taxonomic orders. We identified the sex determination system and sex-specific markers in six of these species, five of which had no known sex-markers prior to this study. We show that RADSex greatly facilitates the study of sex determination systems in nonmodel species thanks to its speed of analyses, low resource usage, ease of application and visualization options. Furthermore, our analysis of new data sets from 15 species provides new insights on sex determination in fish.

Rhizotaxis Modulation in Arabidopsis Is Induced by Diffusible Compounds Produced during the Cocultivation of Arabidopsis and the Endophytic Fungus Serendipita indica.

Rhizotaxis is established under changing environmental conditions via periodic priming of lateral root (LR) initiation at the root tips and adaptive LR formation along the primary root (PR). In contrast to the adaptable LR formation in response to nutrient availability, there is little information on root development during interactions with beneficial microbes. The Arabidopsis root system is characteristically modified upon colonization by the root endophytic fungus Serendipita indica, accompanied by a marked stimulation of LR formation and the inhibition of PR growth. This root system modification has been attributed to endophyte-derived indole-3-acetic acid (IAA). However, it has yet to be clearly explained how fungal IAA affects the intrinsic LR formation process. In this study, we show that diffusible compounds (chemical signals) other than IAA are present in the coculture medium of Arabidopsis and S. indica and induce auxin-responsive DR5::GUS expression in specific sections within the pericycle layer. The DR5::GUS expression was independent of polar auxin transport and the major IAA biosynthetic pathways, implicating unidentified mechanisms responsible for the auxin response and LR formation. Detailed metabolite analysis revealed the presence of multiple compounds that induce local auxin responses and LR formation. We found that benzoic acid (BA) cooperatively acted with exogenous IAA to generate a local auxin response in the pericycle layer, suggesting that BA is one of the chemical signals involved in adaptable LR formation. Identification and characterization of the chemical signals will contribute to a greater understanding of the molecular mechanisms underlying adaptable root development and to unconventional technologies for sustainable agriculture.

A SNP in a Steroidogenic Enzyme Is Associated with Phenotypic Sex in Seriola Fishes.

Vertebrate sex development consists largely of two processes: "sex determination," the initial bifurcation of sexual identity, and "sex differentiation," which subsequently facilitates maleness or femaleness according to the sex determination signal. Steroid hormones promote multiple types of sexual dimorphism in eutherian mammals and avians [1-3], in which they are indispensable for proper sex differentiation. By contrast, in many poikilothermic vertebrates, steroid hormones have been proposed to be key players in sex determination as well as sex differentiation [4-8]. This hypothesis was introduced more than 50 years ago but has never been rigorously tested due to difficulties in discriminating the roles of steroids in sex determination and differentiation. We found that a missense SNP in the gene encoding the steroidogenic enzyme 17ß-hydroxysteroid dehydrogenase 1 (Hsd17b1) was perfectly associated with ZZ/ZW sex determination in Seriola fishes. Biochemical analyses revealed that a glutamate residue present specifically in Z-type HSD17B1 attenuated interconversion between 17-keto and 17ß-hydroxy steroids relative to the allelic product from the W chromosome, which harbors glycine at that position, by disrupting the hydrogen bond network between the steroid and the enzyme's catalytic residues. Hsd17b1 mRNA is constitutively expressed in undifferentiated and differentiating gonads of both genotypic sexes, whereas W-type mRNA is expressed only in genotypic females. Meanwhile, Cyp19a1 is predominantly expressed in differentiating ovary. We conclude that the combination of Hsd17b1 alleles determines sex by modulating endogenous estrogen levels in Seriola species. These findings strongly support the long-standing hypothesis on steroids in sex determination.

Ovarian aromatase loss-of-function mutant medaka undergo ovary degeneration and partial female-to-male sex reversal after puberty.

Although estrogens have been generally considered to play a critical role in ovarian differentiation in non-mammalian vertebrates, the specific functions of estrogens during ovarian differentiation remain unclear. We isolated two mutants with premature stops in the ovarian aromatase (cyp19a1) gene from an N-ethyl-N-nitrosourea-based gene-driven mutagenesis library of the medaka, Oryzias latipes. In XX mutants, gonads first differentiated into normal ovaries containing many ovarian follicles that failed to accumulate yolk. Subsequently, ovarian tissues underwent extensive degeneration, followed by the appearance of testicular tissues on the dorsal side of ovaries. In the newly formed testicular tissue, strong expression of gsdf was detected in sox9a2-positive somatic cells surrounding germline stem cells suggesting that gsdf plays an important role in testicular differentiation during estrogen-depleted female-to-male sex reversal. We conclude that endogenous estrogens synthesized after fertilization are not essential for early ovarian differentiation but are critical for the maintenance of adult ovaries.

A novel C-type lectin gene is a strong candidate gene for Benedenia disease resistance in Japanese yellowtail, Seriola quinqueradiata.

Little is known about mechanisms of resistance to parasitic diseases in marine finfish. Benedenia disease is caused by infection by the monogenean parasite Benedenia seriolae. Previous quantitative trait locus (QTL) analyses have identified a major QTL associated with resistance to Benedenia disease in linkage group Squ2 of the Japanese yellowtail/amberjack Seriola quinqueradiata. To uncover the bioregulatory mechanism of Benedenia disease resistance, complete Illumina sequencing of BAC clones carrying genomic DNA for the QTL region in linkage group Squ2 was performed to reveal a novel C-type lectin in this region. Expression of the mRNA of this C-type lectin was detected in skin tissue parasitized by B. seriolae. Scanning for single nucleotide polymorphisms (SNPs) uncovered a SNP in the C-type lectin/C-type lectin-like domain that was significantly associated with B. seriolae infection levels. These results strongly suggest that the novel C-type lectin gene controls resistance to Benedenia disease in Japanese yellowtails.

Evolution of Shh endoderm enhancers during morphological transition from ventral lungs to dorsal gas bladder.

Shh signalling plays a crucial role for endoderm development. A Shh endoderm enhancer, MACS1, is well conserved across terrestrial animals with lungs. Here, we first show that eliminating mouse MACS1 causes severe defects in laryngeal development, indicating that MACS1-directed Shh signalling is indispensable for respiratory organogenesis. Extensive phylogenetic analyses revealed that MACS1 emerged prior to the divergence of cartilaginous and bony fishes, and even euteleost fishes have a MACS1 orthologue. Meanwhile, ray-finned fishes evolved a novel conserved non-coding sequence in the neighbouring region. Transgenic assays showed that MACS1 drives reporter expression ventrally in laryngeal epithelium. This activity has been lost in the euteleost lineage, and instead, the conserved non-coding sequence of euteleosts acquired an enhancer activity to elicit dorsal epithelial expression in the posterior pharynx and oesophagus. These results implicate that evolution of these two enhancers is relevant to the morphological transition from ventral lungs to dorsal gas bladder.

Critical Involvement of Environmental Carbon Dioxide Fixation to Drive Wax Ester Fermentation in Euglena.

Accumulation profiles of wax esters in Euglena gracilis Z were studied under several environmental conditions. The highest amount of total wax esters accumulated under hypoxia in the dark, and C28 (myristyl-myristate, C14:0-C14:0) was prevalent among all conditions investigated. The wax ester production was almost completely suppressed under anoxia in the light, and supplying exogenous inorganic carbon sources restored wax ester fermentation, indicating the need for external carbon sources for the wax ester fermentation. 13C-labeling experiments revealed specific isotopic enrichment in the odd-numbered fatty acids derived from wax esters, indicating that the exogenously-supplied CO2 was incorporated into wax esters via the propionyl-CoA pathway through the reverse tricarboxylic acid (TCA) cycle. The addition of 3-mercaptopicolinic acid, a phosphoenolpyruvate carboxykinase (PEPCK) inhibitor, significantly affected the incorporation of 13C into citrate and malate as the biosynthetic intermediates of the odd-numbered fatty acids, suggesting the involvement of PEPCK reaction to drive wax ester fermentation. Additionally, the 13C-enrichment pattern of succinate suggested that the CO2 assimilation might proceed through alternative pathways in addition to the PEPCK reaction. The current results indicate that the mechanisms of anoxic CO2 assimilation are an important target to reinforce wax ester fermentation in Euglena.

Diversification of sterol methyltransferase enzymes in plants and a role for ß-sitosterol in oriented cell plate formation and polarized growth.

Phytosterols are classified into C24-ethylsterols and C24-methylsterols according to the different C24-alkylation levels conferred by two types of sterol methyltransferases (SMTs). The first type of SMT (SMT1) is widely conserved, whereas the second type (SMT2) has diverged in charophytes and land plants. The Arabidopsis smt2 smt3 mutant is defective in the SMT2 step, leading to deficiency in C24-ethylsterols while the C24-methylsterol pathway is unchanged. smt2 smt3 plants exhibit severe dwarfism and abnormal development throughout their life cycle, with irregular cell division followed by collapsed cell files. Preprophase bands are occasionally formed in perpendicular directions in adjacent cells, and abnormal phragmoplasts with mislocalized KNOLLE syntaxin and tubulin are observed. Defects in auxin-dependent processes are exemplified by mislocalizations of the PIN2 auxin efflux carrier due to disrupted cell division and failure to distribute PIN2 asymmetrically after cytokinesis. Although endocytosis of PIN2-GFP from the plasma membrane (PM) is apparently unaffected in smt2 smt3, strong inhibition of the endocytic recycling is associated with a remarkable reduction in the level of PIN2-GFP on the PM. Aberrant localization of the cytoplasmic linker associated protein (CLASP) and microtubules is implicated in the disrupted endocytic recycling in smt2 smt3. Exogenous C24-ethylsterols partially recover lateral root development and auxin distribution in smt2 smt3 roots. These results indicate that C24-ethylsterols play a crucial role in division plane determination, directional auxin transport, and polar growth. It is proposed that the divergence of SMT2 genes together with the ability to produce C24-ethylsterols were critical events to achieve polarized growth in the plant lineage.

Expression of 3ß-hydroxysteroid dehydrogenase (hsd3b), star and ad4bp/sf-1 during gonadal development in medaka (Oryzias latipes).

In most vertebrates, sex steroids play a critical role in gonadal development, maturation of germ cells, and development of secondary sexual characteristics. Sex steroids are synthesized in steroid-producing cells (SPCs) in the testis known as Leydig cells, as well as in thecal and granulosa cells in the ovary. In SPCs, cholesterol is sequentially catalyzed by a set of steroidogenic factors and enzymes in order to produce sex steroids. Therefore, integrated expression of the genes involved in steroidogenesis is critical for the proper production of sex steroids. In the present study, regulatory mechanisms of steroidogenic factors and enzymes were examined. We focused on hsd3b, star and ad4bp/sf-1 as well as the description of temporal and spatial expression of these genes during gonadal development in medaka (Oryzias latipes). During testicular development, hsd3b, star and ad4bp/sf-1 were co-expressed in the interstitial somatic cells subsequent to the formation of the seminiferous tubule precursor, suggesting that ad4bp/sf-1 regulated the transcription of both hsd3b and star. During ovarian development, the expression pattern of hsd3b coincided with that of cyp11a1, but not with that of aromatase. Although ad4bp/sf-1 was mainly expressed in presumptive follicular cells, it was also detected in hsd3b positive interstitial cells in the developing ovary. Contrary to our expectations, the onset of star expression occurred during a later stage of ovarian development than the expression of other steroidogenic enzymes. Thus, the regulation mechanism of star transcription appears to differ from that of the other steroidogenic enzymes in the developing ovary, but not in the developing testis.

Expression of gonadal soma derived factor (GSDF) is spatially and temporally correlated with early testicular differentiation in medaka.

In the teleost fish, medaka (Oryzias latipes), the sex is genetically determined at the time of fertilization. The males are heterogametic with XY chromosome composition, while females are of XX chromosome composition. The male sexual differentiation is initiated in XY embryos of medaka by the sex-determining gene Dmy. In this study, we have cloned the gonadal soma derived factor (Gsdf) from medaka and characterized its expression pattern during the initiation of morphological testicular differentiation. By real-time PCR, an XY-specific up-regulation was detected in the expression levels of Gsdf in the whole embryos of medaka at 6days post fertilization (dpf), coincident with the initiation of testicular differentiation in the XY gonads. Whole mount and section in situ hybridizations reaffirmed that Gsdf was expressed exclusively in primordial gonads of only the genetic males at 6dpf. Conversely, the expression of Gsdf was found to be very weak in the XX gonads during embryogenesis. Importantly, Gsdf and Dmy were found to be co-localized in the same somatic cells in the XY gonads. When the XY embryos were treated with estradiol-17beta, in order to reverse their phenotypic sex, a decline was observed in the expression of Gsdf in these embryos by real-time PCR.

Cloning and expression of medaka cholesterol side chain cleavage cytochrome P450 during gonadal development.

Cholesterol side chain cleavage cytochrome P450 (P450scc, Cyp11a) is responsible for the first step in steroidogenesis, catalyzing the conversion of cholesterol to prognenolone. To investigate the differentiation of steroid-producing cells and the function of sex steroids during gonadal differentiation in the teleost fish, medaka (Oryzias latipes), we isolated the full length cDNA of medaka P450scc and analyzed the expression pattern of P450scc mRNA during gonadal development using in situ hybridization. At hatching, and just after the initiation of morphological sex differentiation, we did not detect any P450scc expression in both sexes. In male gonads, expression of P450scc was detected in the interstitial somatic cells 15 days after hatching following the formation of the seminiferous tubule precursor, and was maintained in the interstitial somatic cells throughout testicular development. In the female gonad, expression of P450scc was initially detected in interstitial somatic cells 5 days after hatching. Subsequently, the expression of P450scc was continuously detected in the interstitial somatic cells of the developing ovary. This expression pattern of P450scc differed from that of female specific steroidogenic enzyme P450arom. Both P450scc and P450arom expressing cells, only P450scc expressing cells, and only P450arom expressing cells were observed. Our results suggest that expression of steroidogenic enzymes is regulated by various mechanisms during ovarian development.

Gonadal sex differentiation and expression of Sox9a2, Dmrt1, and Foxl2 in Oryzias luzonensis.

Oryzias luzonensis is closely related to the medaka, O. latipes. The sex of both species is determined by an XX-XY system. However, the testis determining gene (DMY/Dmrt1bY) found in O. latipes does not exist in O. luzonensis. Instead, a different gene is thought to act as a testis determining gene. In this study, we focused the gonadal sex differentiation process in O. luzonensis under different testis determining gene. First, we observed the gonadal development of O. luzonensis histologically. We then analyzed the expression of Sox9a2/Sox9b, Dmrt1, and Foxl2 during early development. Our results suggest that the sexual differentiation of germ cells in O. luzonensis is initiated later than in O. latipes. However, the timing of the sexual differentiation of the supporting cell linage is similar between the species.

Preferential localization of neural cell recognition molecule NB-2 in developing glutamatergic neurons in the rat auditory brainstem.

NB-2 is a neuronal cell recognition molecule that is preferentially expressed in auditory pathways. Mice deficient in the NB-2 gene exhibit aberrant responses to acoustic stimuli. Here we examined the expression and localization of NB-2 in the auditory brainstem during development in the rat. NB-2 was strongly expressed in the ventral cochlear nucleus (VCN), ventral acoustic stria, lateral and medial superior olivary complex (SOC), superior paraolivary nucleus, medial nucleus of the trapezoid body (MNTB), ventrolateral lemniscus, and central nucleus of the inferior colliculus (CIC). In the VCN and CIC, NB-2 was expressed in the regions that are known to respond to high frequencies. In situ hybridization combined with immunohistochemistry suggested that NB-2 is expressed only in neurons. NB-2 was colocalized with glutamatergic elements in the neuropil and the calyces of Held but not with glycinergic or GABAergic elements. NB-2 expression in the SOC was first detected at embryonic day (E)19, reached a maximum level at postnatal day (P)7, and declined thereafter. Immunolabeling with VGLUT1 and VGLUT2, markers for mature and premature glutamatergic synapses, respectively, in combination with NB-2 immunolabeling revealed that NB-2 is expressed at glutamatergic synapses. Collectively, our findings suggest that NB-2 plays a key role in maturation of glutamatergic synapses in the brainstem during the final stages of auditory development.

Dax1 suppresses P450arom expression in medaka ovarian follicles.

Dax1 is a member of an unusual orphan nuclear receptor family, and is known to regulate P450arom in mammals and is involved in sex differentiation in some vertebrates. To investigate whether Dax1 is involved in the regulation of the steroidogenic pathway for estrogen biosynthesis in medaka ovarian follicles, we isolated Dax1 cDNA from adult medaka ovaries and analyzed its expression pattern in medaka gonads. In adult ovaries, Dax1 mRNA was detected only in postvitellogenic follicles and was not detected in previtellogenic and vitellogenic follicles. In adult testis, Dax1 mRNA was not detected. We compared the expression pattern of Dax1 with that of Foxl2, Ad4BP/Sf-1, P450c17, and P450arom by in situ hybridization using adjacent sections. In contrast to Dax1 expression, these genes were co-expressed in vitellogenic follicles but were not detected in postvitellogenic follicles. Thus, in medaka ovarian follicles, Dax1 did not show any overlapping expression patterns against Foxl2, Ad4BP/Sf-1, P450c17, and P450arom. Moreover, co-transfection experiments demonstrated that Dax1 inhibits Ad4BP/Sf-1- and Foxl2-mediated P450arom expression. On the other hand, during early sex differentiation, Dax1 mRNA was not detected in both males and females. Our results suggest that Dax1 down-regulates Ad4BP/Sf-1- and Foxl2-mediated P450arom expression in medaka ovarian follicles.

Molecular cloning and analysis of gonadal expression of Foxl2 in the medaka, Oryzias latipes.

Foxl2 is a member of the winged helix/forkhead family of transcription factors and is known to be involved in ovarian development in some vertebrates. To address the role of Foxl2 in ovarian differentiation in medaka, we isolated Foxl2 cDNA and analyzed its expression patterns during sex differentiation. Expression of Foxl2 started in somatic cells surrounding germ cells in XX gonads, just after initiation of ovarian differentiation, and was maintained in granulosa cells throughout ovarian development. In the adult ovary, Foxl2 was expressed in previtellogenic and vitellogenic follicles, but expression ceased in postvitellogenic follicles. In contrast, Foxl2 mRNA could not be detected in testes. In addition, Foxl2 and aromatase mRNAs were co-localized in some somatic cells located on the ventral side of developing XX gonads. Our results suggested that Foxl2 was not involved in ovarian determination, but was involved in differentiation of granulosa cells in medaka.