Estrogen Signaling Inhibits the Expression of anti-Müllerian hormone (amh) and gonadal-soma-derived factor (gsdf) during the Critical Time of Sexual Fate Determination in Zebrafish.

The mechanism of fish gonadal sex differentiation is complex and regulated by multiple factors. It has been widely known that proper steroidogenesis in Leydig cells and sex-related genes in Sertoli cells play important roles in gonadal sex differentiation. In teleosts, the precise interaction of these signals during the sexual fate determination remains elusive, especially their effect on the bi-potential gonad during the critical stage of sexual fate determination. Recently, all-testis phenotypes have been observed in the cyp17a1-deficient zebrafish and common carp, as well as in cyp19a1a-deficient zebrafish. By mating cyp17a1-deficient fish with transgenic zebrafish Tg(piwil1:EGFP-nanos3UTR), germ cells in the gonads were labelled with enhanced green fluorescent protein (EGFP). We classified the cyp17a1-deficient zebrafish and their control siblings into primordial germ cell (PGC)-rich and -less groups according to the fluorescence area of the EGFP labelling. Intriguingly, the EGFP-labelled bi-potential gonads in cyp17a1+/+ fish from the PGC-rich group were significantly larger than those of the cyp17a1-/- fish at 23 days post-fertilization (dpf). Based on the transcriptome analysis, we observed that the cyp17a1-deficient fish of the PGC-rich group displayed a significantly upregulated expression of amh and gsdf compared to that of control fish. Likewise, the upregulated expressions of amh and gsdf were observed in cyp19a1a-deficient fish as examined at 23 dpf. This upregulation of amh and gsdf could be repressed by treatment with an exogenous supplement of estradiol. Moreover, tamoxifen, an effective antagonist of both estrogen receptor α and ß (ERα and Erß), upregulates the expression of amh and gsdf in wild-type (WT) fish. Using the cyp17a1- and cyp19a1a-deficient zebrafish, we provide evidence to show that the upregulated expression of amh and gsdf due to the compromised estrogen signaling probably determines their sexual fate towards testis differentiation. Collectively, our data suggest that estrogen signaling inhibits the expression of amh and gsdf during the critical time of sexual fate determination, which may broaden the scope of sex steroid hormones in regulating gonadal sex differentiation in fish.

A Potential Role for the Gsdf-eEF1α Complex in Inhibiting Germ Cell Proliferation: A Protein-Interaction Analysis in Medaka (Oryzias latipes) From a Proteomics Perspective.

Gonadal soma-derived factor (gsdf) has been demonstrated to be essential for testicular differentiation in medaka (Oryzias latipes). To understand the protein dynamics of Gsdf in spermatogenesis regulation, we used a His-tag "pull-down" assay coupled with shotgun LC-MS/MS to identify a group of potential interacting partners for Gsdf, which included cytoplasmic dynein light chain 2, eukaryotic polypeptide elongation factor 1 alpha (eEF1α), and actin filaments in the mature medaka testis. As for the interaction with transforming growth factor ß-dynein being critical for spermatogonial division in Drosophila melanogaster, the physical interactions of Gsdf-dynein and Gsdf-eEF1α were identified through a yeast 2-hybrid screening of an adult testis cDNA library using Gsdf as bait, which were verified by a paired yeast 2-hybrid assay. Coimmunoprecipitation of Gsdf and eEF1α was defined in adult testes as supporting the requirement of a Gsdf and eEF1α interaction in testis development. Proteomics analysis (data are available via ProteomeXchange with identifier PXD022153) and ultrastructural observations showed that Gsdf deficiency activated eEF1α-mediated protein synthesis and ribosomal biogenesis, which in turn led to the differentiation of undifferentiated germ cells. Thus, our results provide a framework and new insight into the coordination of a Gsdf (transforming growth factor ß) and eEF1α complex in the basic processes of germ cell proliferation, transcriptional and translational control of sexual RNA, which may be fundamentally conserved across the phyla during sexual differentiation.

Diagnostic Accuracy of Caries and Periapical Lesions on a Monitor with and without DICOM-GSDF Calibration Under Different Ambient Light Conditions.

To evaluate the diagnostic accuracy of caries and periapical lesions on a monitor with and without DICOM part 14: grayscale standard display function (DICOM-GSDF) calibration under different ambient light conditions. Forty digital bitewing radiographs were selected, with or without radiographic images of carious lesions and forty digital periapical radiographs with or without periapical lesions were selected from archives of the Radiology Department at the University Hospital of the Federal University of Sergipe. The gold standard radiographic images were determined through consensus between two radiologists with more than 15 and 30 years of experience. The selected radiographs were evaluated on a LG LED monitor with and without DICOM-GSDF calibration under different ambient light conditions: Lx1 (low ambient lighting), Lx2 (moderate ambient lighting) and Lx3 (high ambient lighting). Kappa (Kw) values determined that evaluator 1 showed almost perfect agreement for all devices, while evaluator 2 presented a substantial agreement for all devices. Monitors with and without DICOM-GSDF calibration have similar accuracy values. The three ambient light conditions analyzed have similar accuracy and can be used for caries lesions diagnosis (p > 0.05); however, the best diagnostic accuracy of periapical lesions was found in Lx 2. The displays with and without DICOM-GSDF calibration studied in this research have similar accuracy and can be used to evaluate digital radiographs without changing the diagnostic capacity. The different ambient lighting conditions did not influence the evaluation of caries lesions. The best diagnostic accuracy of periapical lesions was found in moderate ambient lighting.

Establishment of transgenic zebrafish (Danio rerio) models expressing fluorescence proteins in the oocytes and somatic supporting cells.

The interaction between gonadal somatic support cells and germ cells plays a crucial role in gonadal development. In fish, the process involves various local growth factors such as growth differentiation factor 9 (Gdf9) and gonadal soma-derived factor (Gsdf), which are both members of the transforming growth factor-ß (TGF-ß) superfamily. Gdf9, an oocyte-secreted factor, is a potent regulator of folliculogenesis in both mammals and fish. By contrast, Gsdf is expressed by the gonadal somatic cells (i.e., Sertoli cells in the testis and granulosa cells in the ovary) that support germ cell development. In this study, we established two transgenic zebrafish models, and demonstrated that the 2.7-kb proximal promoter region of gdf9 drove mCherry expression specifically in the oocytes, whereas the 2.1-kb proximal promoter region of gsdf drove enhanced green fluorescent protein (eGFP) expression in the Sertoli cells and granulosa cells. These proximal promoters contained sufficient information to respectively mimic the spatiotemporal expression patterns of endogenous gdf9 and gsdf in zebrafish. In the Tg(gdf9:mCherry) fish, mCherry was weakly expressed in the oocytes at primary growth stage but strongly expressed in those entering the secondary growth phase. In the Tg(gsdf:eGFP) fish, eGFP-positive Sertoli cells were distributed around spermatogenic cysts in the testis, whereas eGFP-positive granulosa cells were located at the outer side of the follicle layer in the ovary. The eGFP-positive Sertoli cells and granulosa cells seemed to have originated from the dorsal epithelium of the gonads. These Tg(gdf9:mCherry) and Tg(gsdf:eGFP) zebrafish models are suitable for studying gonadal development and function especially on the interaction between germ cells and supporting somatic cells.

Transcriptomic analysis of dead end knockout testis reveals germ cell and gonadal somatic factors in Atlantic salmon.

BACKGROUND: Sustainability challenges are currently hampering an increase in salmon production. Using sterile salmon can solve problems with precocious puberty and genetic introgression from farmed escapees to wild populations. Recently sterile salmon was produced by knocking out the germ cell-specific dead end (dnd). Several approaches may be applied to inhibit Dnd function, including gene knockout, knockdown or immunization. Since it is challenging to develop a successful treatment against a gene product already existing in the body, alternative targets are being explored. Germ cells are surrounded by, and dependent on, gonadal somatic cells. Targeting genes essential for the survival of gonadal somatic cells may be good alternative targets for sterility treatments. Our aim was to identify and characterize novel germ cell and gonadal somatic factors in Atlantic salmon. RESULTS: We have for the first time analysed RNA-sequencing data from germ cell-free (GCF)/dnd knockout and wild type (WT) salmon testis and searched for genes preferentially expressed in either germ cells or gonadal somatic cells. To exclude genes with extra-gonadal expression, our dataset was merged with available multi-tissue transcriptome data. We identified 389 gonad specific genes, of which 194 were preferentially expressed within germ cells, and 11 were confined to gonadal somatic cells. Interestingly, 5 of the 11 gonadal somatic transcripts represented genes encoding secreted TGF-ß factors; gsdf, inha, nodal and two bmp6-like genes, all representative vaccine targets. Of these, gsdf and inha had the highest transcript levels. Expression of gsdf and inha was further confirmed to be gonad specific, and their spatial expression was restricted to granulosa and Sertoli cells of the ovary and testis, respectively. Finally, we show that inha expression increases with puberty in both ovary and testis tissue, while gsdf expression does not change or decreases during puberty in ovary and testis tissue, respectively. CONCLUSIONS: This study contributes with transcriptome data on salmon testis tissue with and without germ cells. We provide a list of novel and known germ cell- and gonad somatic specific transcripts, and show that the expression of two highly active gonadal somatic secreted TGF-ß factors, gsdf and inha, are located within granulosa and Sertoli cells.

Bisphenol A induces a shift in sex differentiation gene expression with testis-ova or sex reversal in Japanese medaka (Oryzias latipes).

Bisphenol A (BPA), a very important raw material in the plastics industry, is an endocrine-disrupting chemical in teleost fish. Although BPA induces testis-ova and sex reversal in teleost fish species, the molecular mechanism remains unclear. We evaluated the effects of BPA (measured concentrations: 45, 92, 326, 1030 and 3406 µg/L) on Japanese medaka (Oryzias latipes) using OECD TG234 (2011, Fish Sexual Development Test, OECD Guidelines for the Testing of Chemicals, Section 2). BPA at 1030 and 3406 µg/L induced testis-ova and sex reversal with female-type secondary sexual characteristics in XY males at 30 and 60 days posthatching (dph). Then we examined the BPA effect on the expression of sex differentiation genes related to the testis-ova and sex reversal in XY medaka. BPA exposure (1030 and 3406 µg/L) suppressed gsdf mRNA expression and increased cyp19a1a mRNA expression in XY individuals at stage 38 and 30 dph, although foxl2 mRNA expression showed no change. Interestingly, the concentration of BPA that suppressed gsdf mRNA expression at the larval stage was consistent with that needed to induce testis-ova and sex reversal. These results suggest that the gsdf gene at the embryonic stage can be used as a useful biomarker for predicting the impact of estrogenic endocrine-disrupting chemicals on sexual differentiation in Japanese medaka.

High temperature increases the gsdf expression in masculinization of genetically female Japanese flounder (Paralichthys olivaceus).

In teleosts, sex is plastic and is influenced by environmental factors. Elevated temperatures have masculinizing effects on the phenotypic sex of certain sensitive species. In this study, we reared genetic XX Japanese flounder at a high temperature (27.5 ±â€¯0.5 °C) and obtained a population of sex-reversal XX males (male ratio, 95.24%). We comparatively analyzed the dynamic characteristics of germ cells and gsdf (gonadal soma-derived factor) expression during sexual differentiation for the experimental (27.5 ±â€¯0.5 °C) and control (18 °C ±â€¯0.5 °C) groups. The results revealed that the germ cell proliferation inhibited and gsdf expression up-regulated in the experimental group, and the gsdf mRNA and proteins expressed in somatic cells that had direct contact with germline stem cells (with Nanos 2 protein expression) including spermatogonia and oogonia by ISH (in situ hybridization) and IHC (immunohistochemistry). In addition, we also overexpressed the gsdf in XX flounders, and the germ cell number of XX flounders bearing gsdf gene significantly decreased and sometimes disappeared completely, which was consistent with the results from high-temperature induction. Therefore, based on all the results, we speculated that the high expression of gsdf might inhibit germ cell proliferation during sex differentiation, and eventually cause sex reversal in the high-temperature induced masculinization of XX Japanese flounder.

Loss of Gsdf leads to a dysregulation of Igf2bp3-mediated oocyte development in medaka.

Gonadal soma-derived factor (Gsdf) is a unique TGF-ß factor essential for both ovarian and testicular development in Hd-rR medaka (Oryzias latipes). However, the downstream genes regulated by Gsdf signaling remain unknown. Using a high-throughput proteomic approach, we identified a significant increase in the expression of the RNA-binding protein Igf2bp3 in gsdf-deficient ovaries. We verified this difference in transcription and protein expression against normal gonads using real-time PCR quantification and Western blotting. The genomic structure of igf2bp3 and the syntenic flanking segments are highly conserved across fish and mammals. igf2bp3 expression was correlated with oocyte development, which is consistent with the expression of the igf2bp3 ortholog Vg1-RBP/Vera in Xenopus. In contrast to the normal ovary, cysts of H3K27me3- and Igf2bp3-positive germ cells were dramatically increased in the one-month-old gsdf-deficient ovary, indicating that the gsdf depletion led to a dysregulation of Igf2bp3-mediated oocyte development. Our results provide novel insights into the Gsdf-Igf2bp3 signaling mechanisms that underlie the fundamental process of gametogenesis; these mechanisms may be well conserved across phyla.

Identification of gonadal soma-derived factor involvement in Monopterus albus (protogynous rice field eel) sex change.

We studied molecular events and potential mechanisms underlying the process of female-to-male sex transformation in the rice field eel (Monopterus albus), a protogynous hermaphrodite fish in which the gonad is initially a female ovary and transforms into male testes. We cloned and identified a novel gonadal soma derived factor (GSDF), which encodes a member of the transforming growth factor-beta superfamily. gsdf expression was measured in gonads of female, intersex and male with reverse transcription-PCR and gsdf's role in sex transformation was studied with qPCR, histological analysis and dual-color in situ hybridization assays and compared to other sex-related genes. gsdf was correlated to Sertoli cell differentiation, indicating involvement in testicular differentiation and sex transformation from female to male in this species. A unique expression pattern reveals a potential role of gsdf essential for the sex transformation of rice field eels.

Mutation of Gonadal soma-derived factor induces medaka XY gonads to undergo ovarian development.

Gonochoristic species have a bipotential gonad that develops into a testis or an ovary. In species whose sex is determined by a genetic factor, the expression of a sex-determining gene is the first cue that directs the development of a bipotential gonad. Subsequent expression of downstream genes induces the gonad to develop into a testis or an ovary. The TGF-ß family member Gonadal soma-derived factor (Gsdf) is thought to be an important gene for gonadal development in teleost fish, and it is expressed at higher levels in the testis than in the ovary from early to mature stages. However, there is little functional information about the gene. In this study, we targeted the Gsdf coding region in the medaka fish Oryzias latipes using transcription activator-like effector nucleases (TALENs) and studied the phenotypes of the Gsdf mutant medaka. Although normal and heterozygous XY gonads developed into a testis, all XY gonads with a homozygous mutation in Gsdf developed into an ovary at early developmental stages. However, two-thirds of Gsdf mutant XY gonads developed into testes in the adult stages. These results demonstrate that although a gonad can develop into a complete testis in the absence of Gsdf, Gsdf function is critical for directing the bipotential gonad at early developmental stages. Therefore, Gsdf is an endogenous inducer of testicular development similar to a master sex-determining gene.

Effect of diisobutyl adipate on the expression of biomarker genes that respond to endocrine disruption and on gonadal sexual differentiation in Japanese medaka (Oryzias latipes).

Phthalate and non-phthalate plasticizers are used in polymer materials, such as plastic and rubber. It has recently been found that diisobutyl adipate (DIBA), which is considered an environmentally safe non-phthalate plasticizer, potentially acts as a thyroid disruptor in fish. Here, we investigated the sexual hormone effects of DIBA based on the expression levels of genes that respond to endocrine disruption and sexual hormone activity in the livers and gonads, and on gonadal sexual differentiation in Japanese medaka. Compared with the control group, the mRNA expression of chgH, vtg1, vtg2, and esr1 was significantly suppressed in the livers of DIBA exposed XX individuals. Furthermore, the mRNA expression of gsdf was significantly upregulated and downregulated in the gonads of XX and XY individuals, respectively. The mRNA expressions of esr1 and esr2b were significantly suppressed by DIBA exposure in the gonads of both XX and XY individuals. These observations suggest that DIBA has potential androgenic activity in Japanese medaka. However, normal testes and ovaries were observed in respective XY and XX medaka after DIBA exposure; therefore, these results suggest that DIBA may have weak androgenic activity.

Impact of acetyl tributyl citrate on gonadal sex differentiation and expression of biomarker genes for endocrine disruption in Japanese medaka.

Plasticizers are broadly classified as phthalate or nonphthalate. Recently, acetyl tributyl citrate (ATBC), an environmentally friendly nonphthalate plasticizer, was revealed to have the ability to disrupt thyroid hormone activity in fish species. Therefore, we aimed to assess whether ATBC exhibits any sex hormone (i.e., androgenic or estrogenic) activities. First, we examined the effects of ATBC on gonadal sex differentiation. Subsequently, we analyzed the different expression of biomarker genes that respond to endocrine-disrupting chemicals (EDCs) with sexual hormone activity in the liver. We observed normal testes and ovaries after both XX and XY medakas were exposed to ATBC, indicating that ATBC is not an EDCs with strong sex hormone activity and that it does not induce intersex (testis-to-ova or ovo-to-testis) or sex changes in Japanese medaka. The vitellogenin 1 (vtg1) and vitellogenin 2 (vtg2) mRNA expression levels in the liver of XX medakas were significantly reduced compared with those in the control group, whereas the expression levels of these genes in the liver of XY medakas remained unchanged. Finally, we examined the changes in the expression of biomarker genes that respond to EDCs with sex hormone activity in the gonads. The expression levels of biomarker genes did not differ significantly from that of the control group, although the expression levels of gsdf mRNA tended to increase while that of aromatase mRNA tended to decrease in the ovary of XX medakas following ATBC exposure. Conversely, the expression levels of gsdf and aromatase mRNAs in the testis of XY medakas remained unchanged. These results suggest that ATBC does not exhibit estrogenic activity, although it may have weak androgenic activity or no sexual hormone activity.

Sex-specific meiosis responses to Gsdf in medaka (Oryzias latipes).

The meiotic entry of undifferentiated germ cells is sexually specific and strictly regulated by the testicular or ovarian environment. Germline stem cells with a set of abnormal sex chromosomes and associated autosomes undergo defective meiotic processes and are eventually eliminated by yet to be defined post-transcriptional modifications. Herein, we report the role of gsdf, a member of BMP/TGFß family uniquely found in teleost, in the regulation of meiotic entry in medaka (Oryzias latipes) via analyses of gametogenesis in gsdf-deficient XX and XY gonads in comparison with their wild-type siblings. Several differentially expressed genes, including the FKB506-binding protein 7 (fkbp7), were significantly upregulated in pubertal gsdf-deficient gonads. The increase in alternative pre-mRNA isoforms of meiotic synaptonemal complex gene sycp3 was visualized using Integrative Genomics Viewer and confirmed by real-time qPCR. Nevertheless, immunofluorescence analysis showed that Sycp3 protein products reduced significantly in gsdf-deficient XY oocytes. Transmission electron microscope observations showed that normal synchronous cysts were replaced by asynchronous cysts in gsdf-deficient testis. Breeding experiments showed that the sex ratio deviation of gsdf-/- XY gametes in a non-Mendelian manner might be due to the non-segregation of XY chromosomes. Taken together, our results suggest that gsdf plays a role in the proper execution of cytoplasmic and nuclear events through receptor Smad phosphorylation and Sycp3 dephosphorylation to coordinate medaka gametogenesis, including sex-specific mitotic divisions and meiotic recombination.

Influence of Bisphenol A and 17ß-Trenbolone Exposure in Oryzias Congeners.

Japanese medaka is specified as a model fish in the test guidelines of the Organisation for Economic Co-operation and Development. Recently, populations of Japanese medaka in Japan were divided into two species, the northern Oryzias sakaizumii and the southern O. latipes. Previously, we reported that induction concentrations for sex reversal by exposure to 17α-methyltestosterone differed significantly between these two species, indicating that they respond differently to endocrine-disrupting chemica. In the present study, we examined the effects of exposure to two more endocrine-disrupting chemicals (bisphenol A and 17ß-trenbolone) in O. sakaizumii, and compared the results with those previously reported for O. latipes. Exposure to both bisphenol A and 17ß-trenbolone induced testis-ova formation or sex reversal in O. sakaizumii. Exposure to 17ß-trenbolone also increased expression of gonadal soma-derived factor (gsdf). Least-observed-effect concentrations for gonadal sex differentiation and gsdf expression were lower for O. latipes than for O. sakaizumii after exposure to bisphenol A, and were lower for O. sakaizumii than for O. latipes after exposure to 17ß-trenbolone. These results demonstrate that O. sakaizumii and O. latipes respond differently to androgenic and estrogenic endocrine-disrupting chemicals. Environ Toxicol Chem 2023;42:673-678. © 2022 SETAC.

Evolution, Expression, and Function of Gonadal Somatic Cell-Derived Factor.

Fish gonads develop in very diverse ways different from mammalian gonads. This diversity is contributed by species-specific factors. Gonadal somatic cell-derived factor (Gsdf) is one such factor. The gsdf gene exists mostly in teleosts and is absent in many tetrapods, probably as a result of two gene losses during evolution. The gsdf transcript is expressed mainly in gonadal somatic cells, including Sertoli cell in testis and granulosa cells in ovary; however, these gonadal somatic cells can surround many types of germ cells at different developmental stages depending on the fish species. The function of gsdf is also variable. It is involved in germ cell proliferation, testicular formation, ovarian development and even male sex determination. Here, we summarize the common and diverse expression, regulation and functions of gsdf among different fish species with aspect of evolution.

Involvement of Transforming Growth Factor Beta Family Genes in Gonadal Differentiation in Japanese Eel, Anguilla japonica, According to Sex-Related Gene Expressions.

The gonochoristic feature with environmental sex determination that occurs during the yellow stage in the eel provides an interesting model to investigate the mechanisms of gonadal development. We previously studied various sex-related genes during gonadal sex differentiation in Japanese eels. In the present study, the members of transforming growth factor beta (TGF-ß) superfamily were investigated. Transcript levels of anti-Müllerian hormone, its receptor, gonadal soma-derived factor (amh, amhr2, and gsdf, respectively) measured by real-time polymerase chain reaction (qPCR) showed a strong sexual dimorphism. Transcripts were dominantly expressed in the testis, and their levels significantly increased with testicular differentiation. In contrast, the expressions of amh, amhr2, and gsdf transcripts were low in the ovary of E2-feminized female eels. In situ hybridization detected gsdf (but not amh) transcript signals in undifferentiated gonads. amh and gsdf signals were localized to Sertoli cells and had increased significantly with testicular differentiation. Weak gsdf and no amh signals were detected in early ovaries of E2-feminized female eels. Transcript levels of amh and gsdf (not amhr2) decreased during human chorionic gonadotropin (HCG)-induced spermatogenesis in males. This study suggests that amh, amhr2, and especially gsdf might be involved in the gene pathway regulating testicular differentiation of Japanese eels.

Effects of synthetic sex steroid hormone exposures on gonadal sex differentiation and dynamics of a male-related gene, Gonadal soma-derived factor (Gsdf) and an estrogen up-regulated gene, Choriogenine-H (ChgH) gene expression in the euryhaline Javafish medaka, Oryzias javanicus, based on genetic sexes.

To clarify the basal aspects of sexual development in Javafish medaka, Oryzias javanicus (ZZ/ZW), a model marine species for ecotoxicity testing, we examined the details of gonadal sex differentiation and exogenous sex hormone-dependent sex reversals using genetic sex-linked DNA markers. Sex differences in germ cell numbers were observed at 5 days post hatching (dph), in which there was a significant increase in the germ cells of ZW. In ZW, diplotene oocytes and the ovarian cavity appeared at approximately 10, and 30 dph, respectively. In ZZ, spermatogonial proliferation was observed at approximately 20 dph. A ZZ-dominant expression of Gonadal soma-derived factor (Gsdf) mRNA was detected before hatching. The exposure of embryos to 17α-ethinylestradiol (EE2; 0.1, 1, 10 ng/mL) did not cause sex reversals in most cases. However, EE2 exposures led to significant Choriogenin-H (ChgH) mRNA expression, an estrogen up-regulated gene, in all fry; these exposures did not suppress Gsdf expression in ZZ fry. The exposure of embryos to 17α-methyltestosterone (MT; 0.1, 1, 10 ng/mL) caused sex reversals but only at low frequencies in ZW and ZZ fish. Although the 10 ng/mL MT exposure was accompanied by induction of significant Gsdf expression in ZW fry, induction of ChgH expression was also observed in several fry. Together, the present study indicates for the first time that male-dominant sexual dimorphic expression of Gsdf precedes the first morphological sex difference, i.e., the sex difference in germ cell number, and results strongly suggest that exogenous sex hormone-dependent sex reversal is not induced easily in O. javanicus.

Genotoxic biomarkers and histological changes in marine medaka (Oryzias melastigma) exposed to 17α-ethynylestradiol and 17ß-trenbolone.

Endocrine-disrupting pollutants in marine environments have aroused great concern for their adverse effects on the reproduction of marine organisms. This study aimed to seek promising biomarkers for estrogenic/androgenic chemicals. First, two possible male-specific genes, SRY-box containing gene 9a2 (sox9a2) and gonadal soma-derived factor (gsdf), were cloned from marine medaka (Oryzias melastigma). Then the responses of sox9a2, gsdf, choriogenin (chgH and chgL), vitellogenin (vtg1 and vtg2), and cytochrome P450 aromatase (cyp19a and cyp19b) were investigated after exposure to 17α-ethynylestradiol (EE2) and 17ß-trenbolone (TB) at 2, 10, and 50 ng/L. The results showed that gsdf was specifically expressed in the testes and easily induced in the ovaries after TB exposure, indicating that gsdf was a potential biomarker of environmental androgens. ChgL was a useful biomarker of weak estrogen pollution for its high sensitivity to low levels of EE2. In addition, both EE2 and TB exposure damaged gonadal structures and inhibited gonadal development.

Impact of calibration methods for color medical displays on interpreting brain SPECT images.

PURPOSE: Color images are visualized on medical monitors that are adjusted by a grayscale standard display function (GSDF) or γ2.2. Although the GSDF is visually displayed as a linear graded grayscale, it does not specify how color medical images should be presented. On the other hand, the usual gamma setting for color images is γ2.2, but it has not been standardized. The color standard display function (CSDF) has recently been proposed as a standardized gamma setting for color medical monitors. However, the influence of various gamma settings on image characteristics should be determined. The present study aimed to identify differences in color-scale characteristics on nuclear medicine images displayed on medical monitors adjusted by CSDF, GSDF, and γ2.2. METHODS: Transverse normal (n = 1) and abnormal (n = 5) brain perfusion images were generated using a mathematical digital phantom. Transverse phantom and clinical brain images are shown using the clinically applied eight-color scale. Five nuclear medicine experts visually assessed phantom and clinical images using a defect severity scale that ranged from zero (no defect) to four (defect). Receiver operating characteristic curves were created and areas under the curves (AUCs) were analyzed. Defect scores for the clinical study were evaluated in the nine segments on basal ganglia slice, and defect scores were summed for each patient. RESULTS: The average defect score for color A significantly differed in multiple comparison tests, but not in post hoc tests. The ranges of AUC for CSDF, GSDF, and γ2.2 were 0.86-0.94, 0.82-0.94, and 0.88-0.97, respectively. The AUCs of CSDF in all color scales did not significantly differ from other gamma settings. The summed defect scores of CSDF were similar to those of other gamma settings. CONCLUSION: Nuclear medicine images were equally valid when adjusted by CSDF even at various gamma settings. Nuclear medicine images can be evaluated equally using any gamma setting. Nonetheless, the color gamma setting for medical monitors should be standardized.

Expression and transcriptional regulation of gsdf in spotted scat (Scatophagus argus).

Gonadal soma-derived factor (Gsdf) is critical for testicular differentiation and early germ cell development in teleosts. The spotted scat (Scatophagus argus), with a stable XX-XY sex-determination system and the candidate sex determination gene dmrt1, provides a good model for understanding the mechanism of sex determination and differentiation in teleosts. In this study, we analyzed spotted scat gsdf tissue distribution and gene expression patterns in gonads, as well as further analysis of transcriptional regulation. Tissue distribution analysis showed that gsdf was only expressed in testis and ovary. Real-time PCR showed that both gsdf and dmrt1 were expressed significantly higher in testes at different phases (phase III, IV and V) compared to ovaries at phase II, III and IV, while gsdf was expressed significantly higher in phase II ovaries than those of phase III and IV. Western blot analysis also showed that Gsdf was more highly expressed in the testis than ovary. Immunohistochemistry analysis showed that Gsdf was expressed in Sertoli cells surrounding spermatogonia in the testis, while it was expressed in the somatic cells surrounding the oogonia of the ovary. Approximately 2.7 kb of the 5' upstream region of gsdf was cloned from the spotted scat genomic DNA and in silico promoter analysis revealed the putative transcription factor binding sites of Dmrt1 and Sf1. The luciferase reporter assay, using the human embryonic kidney cells, demonstrated that Dmrt1 activated gsdf expression in a dose-dependent manner in the presence of Sf1 in spotted scat. These results suggest that Gsdf could play a role in regulating the development of spermatogonia and oogonia, and also participate in male sex differentiation by acting as a downstream gene of Dmrt1 in spotted scat.