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The inheritable impact of exposure to graphene oxide nanoparticles (GO NPs) on vertebrate germline during critical windows of gamete development remain undetermined to date. Here, we analyzed the transgenerational effects of exposure to nano-graphene oxide particles (nGO) synthesized in house with lateral dimensions 300-600 nm and surface charge of -36.8 mV on different developmental stages of germ cells (GCs): (1) during GCs undergoing early development and differentiation, and (2) during GCs undergoing gametogenesis and maturation in adulthood. Biocompatibility analyses in Japanese medaka embryos showed lethality above 1 µg/ml and also an aberrant increase in germ cell count of both males and females at doses below the lethal dose. However, no lethality or anomalies were evident in adults up to 45 µg/ml. Long term exposure of embryos and adults for 21 days resulted in reduced fecundity. This effect was transmitted to subsequent generations, F1 and F2. Importantly, the inheritable effects of nGO in adults were pronounced at a high dose of 10 µg/ml, while 1 µg/ml showed no impact on the germline indicating lower doses used in this study to be safe. Further, expressions of selected genes that adversely affected oocyte maturation were enhanced in F1 and F2 individuals. Interestingly, the inheritance patterns differed corresponding to the stage at which the fish received the exposure.

The transdifferentiation potential of human oogonial stem cells (hOSCs) isolated using the antibody against extracellular DEAD-Box Helicase 4 (ecDDX4) remains undetermined. Hence, this study isolated OSCs from ovarian cortical pieces of premenopausal women using ecDDX4 antibody by magnetic activated cell sorting and expanded these cells under embryonic stem cell (ESC)-like culture conditions to inves-tigate their transdifferentiation potential. The number of ecDDX4+ cells obtained was variable in each isolation. When cultured on inactivated mouse embryonic fibroblast feeder layer with human leukemia inhibitory factor (hLIF) and basic fibroblast growth factor (bFGF) in Minimum Essential Medium, the hOSCs aggregated, forming ESC-like colonies. The average size of these cells was around 10 μm. hOSCs in culture were positive for alkaline phosphatase and further formed embryoid bodies (EBs) when grown on low attachment plates containing Essential 6 Medium without hLIF and bFGF. Subsequently, EBs differentiated into 3 germ layers, which were confirmed by staining with beta-III tubulin (TUJ1) for ectoderm, alpha-fetoprotein (AFP) for endoderm, and smooth muscle actin (SMA) for mesoderm. Further, using appropriate induction media, the EBs derived from ecDDX4+ hOSCs were differentiated into somatic lineages such as adipocytes, osteoblasts, cardiomyocytes, and neuronal precursor-like cells, which were confirmed by immunofluorescence using antibodies against specific markers for each cell type. This study corroborated the previous findings that ovaries of adult women possess germ cell progenitors that can be isolated using ecDDX4, and these cells can be manipulated as pluripotent stem cells by culturing them under ESC-like culture conditions akin to their male counterparts, the spermatogonial stem cells. Further, these cells could differentiate into somatic lineages under specific signalling environments., (© 2021 S. Karger AG, Basel.)

This study investigated the adverse effects of 200 nm zinc oxide particles (nZnO) on sexual behavior and reproduction in Japanese medaka in comparison with ZnSO 4 and correlated the consequences with the bioaccumulation pattern of the particles in associated organs. nZnO exposure impaired sexual and territorial behaviors and affected fertility by altering sperm viability and motility in males through reactive oxygen species (ROS) induction. Conversely, none of these effects other than behavior loss was seen in males exposed to ZnSO 4 . nZnO exposure to females induced ROS in ovaries, causing follicular growth arrest, atresia, and subfertility. Further, sex-steroid levels were altered by both nZnO and ZnSO 4 in males and by nZnO but not ZnSO 4 in females. Biodistribution studies revealed the deposition of nZnO as particulate matter in the brain, gills, gut, kidney, and ovary. Particle accumulation in the brain was sex specific, as the particles were found in the brain of males but not that of females. A similar trend was seen for zinc levels in males and females exposed to ZnSO 4 . Importantly, the female sex hormone, 17β-estradiol was found to prevent nZnO accumulation in the female brain, emphasizing the need for biodistribution profiling of nanoparticle-based drug delivery vehicles separately in males and females before they are commercialized. This study has demonstrated that the toxic effects of nZnO on the reproductive system were mainly caused by ROS induction, while zinc ions were predominantly responsible for the adverse impact of ZnSO 4 .

Purpose: Since metabolic abnormalities such as elevated glucose level and imbalanced lipid profiles increase the risk for hypertension and cause endothelial dysfunction, we evaluated the effect of aqueous extract of large cardamom (AELC) on fructose-induced metabolic hypertension and oxidative stress., Methods: The male Sprague-Dawley rats were divided into 6 groups with 5 rats in each group, and each group was fed with 10% fructose in drinking water for 8 weeks. Starting from week 5, animals were treated with 50, 100, and 200 mg/kg/day AELC or Losartan (10 mg/kg/day). Systolic, diastolic, and mean arterial blood pressure was measured once in every seven days using the tail-cuff method. Vascular function, plasma nitric oxide (NO), glucose, lipid profiles, serum biochemical, and anti-oxidant parameters were also evaluated., Results: Rats fed with fructose showed higher blood pressure, serum cholesterol, and triglyceride levels, but decreased in the AELC or Losartan treatment group. Treatments with AELC prevented exaggerated plasma glucose and oxidative stress and restored the nitric oxide level in fructose-fed rats. Besides, it also reduced vascular proliferation and improved the relaxation response of acetylcholine in the aorta pre-contracted with phenylephrine., Conclusion: In summary, the obtained results suggest that AELC can prevent and reverse the high blood pressure induced by fructose, probably by restoring nitric oxide level and by improving altered metabolic parameters.

A diverse array of sex determination ( SD ) mechanisms, encompassing environmental to genetic, have been found to exist among vertebrates, covering a spectrum from fixed SD mechanisms (mammals) to functional sex change in fishes (sequential hermaphroditic fishes). A major landmark in vertebrate SD was the discovery of the SRY gene in 1990. Since that time, many attempts to clone an SRY ortholog from nonmammalian vertebrates remained unsuccessful, until 2002, when DMY/dmrt1by was discovered as the SD gene of a small fish, medaka. Surprisingly, however, DMY/dmrt1by was found in only 2 species among more than 20 species of medaka, suggesting a large diversity of SD genes among vertebrates. Considerable progress has been made over the last 3 decades, such that it is now possible to formulate reasonable paradigms of how SD and gonadal sex differentiation may work in some model vertebrate species. This review outlines our current understanding of vertebrate SD and gonadal sex differentiation, with a focus on the molecular and cellular mechanisms involved. An impressive number of genes and factors have been discovered that play important roles in testicular and ovarian differentiation. An antagonism between the male and female pathway genes exists in gonads during both sex differentiation and, surprisingly, even as adults, suggesting that, in addition to sex-changing fishes, gonochoristic vertebrates including mice maintain some degree of gonadal sexual plasticity into adulthood. Importantly, a review of various SD mechanisms among vertebrates suggests that this is the ideal biological event that can make us understand the evolutionary conundrums underlying speciation and species diversity.

Graphene quantum dots (GQDs) have emerged as promising biolabeling agents owing to their stable innate fluorescence, photostability, and biocompatibility as opposed to semiconductor quantum dots. While several studies reported GQDs to be cytocompatible, their potential for reproductive toxicity, particularly to germ cells that can cause transgenerational toxicity, remains unexplored. Here we report the intrinsic toxicity of 2-3 nm sized GQDs synthesized from glucose by a novel bottom-up green chemistry technique on germ cell proliferation and meiosis during early embryonic development. In vitro cell viability studies with a normal ovarian cell line, Chinese Hamster Ovarian cells (CHO), and a primary cell type, Human Umbilical Vein Endothelial Cells (HUVEC), portrayed good cytocompatibility even up to a high concentration of 800 μg/mL. When embryos of Japanese medaka were exposed to GQDs, no developmental toxicity was observed up to 250 μg/mL, beyond which hatchability and survival were affected adversely. In contrast, toxicity to germ cells in developing gonads was apparent in genetically female (XX) embryos exposed to much lower doses (50, 75, and 100 μg/mL), at which in vitro cytotoxicity and embryo hatchability and survival were unaffected. A drastic decline in germ cell number and meiosis was observed at these doses in XX embryos implying anomalies in sexual differentiation of the gametes. Conversely, germ cells of genetically male embryos exposed to GQDs were unaltered. Significantly high levels of reactive oxygen species (ROS) were detected in the XX larvae exposed to GQDs; however, there was no DNA damage, suggesting ROS to be responsible for the adverse effects observed.

A woven nanotextile implant was developed and optimized for long-term continuous drug delivery for potential oncological applications. Electrospun polydioxanone (PDS) nanoyarns, which are twisted bundles of PDS nanofibres, were loaded with paclitaxel (PTX) and woven into nanotextiles of different packing densities. A mechanistic modeling of in vitro drug release proved that a combination of diffusion and matrix degradation controlled the slow PTX-release from a nanoyarn, emphasizing the role of nanostructure in modulating release kinetics. Woven nanotextiles, through variations in its packing density and thereby architecture, demonstrated tuneable PTX-release. In vivo PTX-release, pharmacokinetics and biodistribution were evaluated in healthy BALB/c mice by suturing the nanotextile to peritoneal wall. The slow and metronomic PTX-release for 60 days from the loosely woven implant was extremely effective in enhancing its residence in peritoneum, in contrast to intraperitoneal injections. Such an implantable matrix offers a novel platform for therapy of solid tumors over prolonged durations., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Pseudomonas aeruginosa depends on its quorum sensing (QS) system for its virulence factors' production and biofilm formation. Biofilms of P. aeruginosa on the surface of indwelling catheters are often resistant to antibiotic therapy. Alternative approaches that employ QS inhibitors alone or in combination with antibiotics are being developed to tackle P. aeruginosa infections. Here, we have studied the mechanism of action of 3-Phenyllactic acid (PLA), a QS inhibitory compound produced by Lactobacillus species, against P. aeruginosa PAO1. Our study revealed that PLA inhibited the expression of virulence factors such as pyocyanin, protease, and rhamnolipids that are involved in the biofilm formation of P. aeruginosa PAO1. Swarming motility, another important criterion for biofilm formation of P. aeruginosa PAO1, was also inhibited by PLA. Gene expression, mass spectrometric, functional complementation assays, and in silico data indicated that the quorum quenching and biofilm inhibitory activities of PLA are attributed to its ability to interact with P. aeruginosa QS receptors. PLA antagonistically binds to QS receptors RhlR and PqsR with a higher affinity than its cognate ligands N-butyryl-L-homoserine lactone (C 4 -HSL) and 2-heptyl-3,4-dihydroxyquinoline (PQS; Pseudomonas quinolone signal). Using an in vivo intraperitoneal catheter-associated medaka fish infection model, we proved that PLA inhibited the initial attachment of P. aeruginosa PAO1 on implanted catheter tubes. Our in vitro and in vivo results revealed the potential of PLA as anti-biofilm compound against P. aeruginosa.

This work focuses on the development of a nanoparticulate system that can be used for magnetic resonance (MR) imaging and E-field noninvasive radiofrequency (RF) hyperthermia. For this purpose, an amine-functional gold ion complex (GIC), [Au(III)(diethylenetriamine)Cl]Cl 2 , which generates heat upon RF exposure, was conjugated to carboxyl-functional poly(acrylic acid)-capped iron-oxide nanoparticles (IO-PAA NPs) to form IO-GIC NPs of size ∼100 nm. The multimodal superparamagnetic IO-GIC NPs produced T2-contrast on MR imaging and unlike IO-PAA NPs generated heat on RF exposure. The RF heating response of IO-GIC NPs was found to be dependent on the RF power, exposure period, and particle concentration. IO-GIC NPs at a concentration of 2.5 mg/mL showed a high heating response (δT) of ∼40 °C when exposed to 100 W RF power for 1 min. In vitro cytotoxicity measurements on NIH-3T3 fibroblast cells and 4T1 cancer cells showed that IO-GIC NPs are cytocompatible at high NP concentrations for up to 72 h. Upon in vitro RF exposure (100 W, 1 min), a high thermal response leads to cell death of 4T1 cancer cells incubated with IO-GIC NPs (1 mg/mL). Hematoxylin and eosin imaging of rat liver tissues injected with 100 μL of 2.5 mg/mL IO-GIC NPs and exposed to low RF power of 20 W for 10 min showed significant loss of tissue morphology at the site of injection, as against RF-exposed or nanoparticle-injected controls. In vivo MR imaging and noninvasive RF exposure of 4T1-tumor-bearing mice after IO-GIC NP administration showed T2 contrast enhancement and a localized generation of high temperatures in tumors, leading to tumor tissue damage. Furthermore, the administration of IO-GIC NPs followed by RF exposure showed no adverse acute toxicity effects in vivo. Thus, IO-GIC NPs show good promise as a theranostic agent for magnetic resonance imaging and noninvasive RF hyperthermia for cancer.

In the present work, 2-Methoxyestradiol [2ME2] loaded PLGA nanoparticles [NPs] were stabilized with Casein or poly(ethylene glycol) [PEG] and evaluated for its cellular interactions, pharmacokinetics and tumour accumulation. Surface stabilized PLGA nanoparticles prepared through a modified emulsion route possessed similar size, surface charge, drug loading and release characteristics. Particle-cell interactions as well as the anti-angiogenesis activity were similar for both nanoformulations in vitro. However, in vivo pharmacokinetics and tumour accumulation of the drug were substantially improved for the PEGylated nanoformulation. Reduced protein binding was observed for PEG stabilized PLGA NPs. Thus, it was demonstrated that nanoencapsulation of 2-ME2 within PEGylated PLGA nanocarrier could improve its half-life and plasma concentration and thereby increase the tumour accumulation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

N-Nitrosodiethylamine (DEN), a well-known hepatocarcinogen, is found in certain food products as such or as a metabolic byproduct. This study investigated the effects of DEN on sexual development, gametogenesis, and oocyte maturation in Japanese medaka (Oryzias latipes). DEN reduced the germ cell number dose-dependently during early stages of sexual differentiation in XX larvae, resulting in underdeveloped ovaries in adulthood at low doses. This effect was sex-specific as no such changes were seen in XY larvae. Furthermore, XX and XY larvae that were exposed at a low dose during early life showed a significant reduction in body weight in adulthood. Gonads in sexually immature adult medaka males and females exposed to DEN were in advanced stages in comparison to that of the controls. Gonado-somatic indices were significantly high in treated males and females. DEN induced oocyte maturation in vitro, which was inhibited by cordycepin, demonstrating that it stimulated oocyte maturation through polyadenylation of cyclin B mRNA as in the case of the endogenous maturation-inducing hormone. Altogether, our results have proven that DEN could disrupt or mimic the signaling pathways involved in germ cell development, proliferation, and maturation., (© 2017 S. Karger AG, Basel.)

Nanomedicines consisting of combinations of cytotoxic drugs and molecular targeted therapeutics which inhibit specific downstream signals are evolving as a novel paradigm for breast cancer therapy. This research addresses one such combination of Paclitaxel (Ptx), having several adversities related to the activation of NF-κB pathway, with Epigallocatechin gallate (EGCG), a multiple signaling inhibitor, encapsulated within a targeted core/shell PLGA-Casein nanoparticle. The sequential release of EGCG followed by Ptx from this core/shell nanocarrier sensitized Ptx resistant MDA-MB-231 cells to Ptx, induced their apoptosis, inhibited NF-κB activation and downregulated the key genes associated with angiogenesis, tumor metastasis and survival. More importantly, Ptx-induced expression of P-glycoprotein was repressed by the nanocombination both at the protein and gene levels. This combination also offered significant cytotoxic response on breast cancer primary cells, indicating its translational value., From the Clinical Editor: Breast cancer is the most common cancer in women worldwide. As well as surgery, chemotherapy plays a major role in the treatment of breast cancer. The authors investigated in this article the combination use of a chemotherapeutic agent, Paclitaxel (Ptx), and an inhibitor of NF-?B pathway, packaged in a targeted nano-based delivery platform. The positive results provided a new pathway for future clinical use of combination chemotherapy in breast cancer., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In vertebrates, sex differences in the brain have been attributed to differences in gonadal hormone secretion; however, recent evidence in mammals and birds shows that sex chromosome-linked genes, independent of gonadal hormones, also mediate sex differences in the brain. In this study, we searched for genes that were differentially expressed between the sexes in the brain of a teleost fish, medaka (Oryzias latipes), and identified two sex chromosome genes with male-biased expression, cntfa (encoding ciliary neurotrophic factor a) and pdlim3a (encoding PDZ and LIM domain 3 a). These genes were found to be located 3-4 Mb from and on opposite sides of the Y chromosome-specific region containing the sex-determining gene (the medaka X and Y chromosomes are genetically identical, differing only in this region). The male-biased expression of both genes was evident prior to the onset of sexual maturity. Sex-reversed XY females, as well as wild-type XY males, had more pronounced expression of these genes than XX males and XX females, indicating that the Y allele confers higher expression than the X allele for both genes. In addition, their expression was affected to some extent by sex steroid hormones, thereby possibly serving as focal points of the crosstalk between the genetic and hormonal pathways underlying brain sex differences. Given that sex chromosomes of lower vertebrates, including teleost fish, have evolved independently in different genera or species, sex chromosome genes with sexually dimorphic expression in the brain may contribute to genus- or species-specific sex differences in a variety of traits., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Maternal factors have essential roles in the specification and development of germ cells in metazoans. In Drosophila, a number of genes such as oskar, vasa, nanos, and tudor are required for specific steps in pole cell formation and further germline development. Drosophila cup, another maternal factor, is confirmed as a main factor in normal oogenesis, maintenance, and survival of female germ-line stem cells by interaction with Nanos. Through searching for the homolog of Drosophila cup in the medaka, the homolog of eukaryotic translation initiation factor 4E (eIF4E)-transporter, named Ol4E-T, was identified. Reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization revealed that Ol4E-T is maternally deposited in the embryo and Ol4E-T expression is maintained throughout embryogenesis. Ol4E-T is predominantly expressed in the adult gonads. In the testes, Ol4E-T is expressed in the same regions where medaka vasa, named olvas is expressed. In the ovary, expression of Ol4E-T conforms to that of nanos3 and olvas. Ol4E-T harbors a well-conserved eIF4E-binding motif, YTKEELL, by which Ol4E-T interacts with eIF4E in medaka. Additionally, Ol4E-T can interact with medaka Nanos3 and Olvas, as shown by yeast two hybridization. The spatial expression and interactions between Ol4E-T with germ cell markers Olvas and Nanos3 suggest a role for Ol4E-T in germ-line development in medaka., (Copyright © 2012 Wiley Periodicals, Inc.)

Molecular and cellular mechanisms involved in artificially induced ovarian differentiation were analyzed by exposing embryos of medaka (Oryzias latipes) to a potent nonsteroidal estrogen, diethylstilbestrol (DES). Embryos were exposed for short-exposure (SE) [from 0 to 8 d postfertilization (dpf)] and long-exposure (LE) periods (from 0 to 18/28 dpf) to 1 ng/ml of DES, and status of sexual differentiation in somatic and germ cells of these gonads was analyzed at 8, 18, and 28 dpf by histology, cell proliferation assays, TUNEL assay, and in situ hybridization using sex-specific somatic and germ cell markers. Additionally, gonads of exposed fry were examined after withdrawal of DES to see whether effects of DES in exposed fish were reversible or not. DES induced germ cell proliferation and meiosis in XY fry of SE and LE groups. However, SE induced only a partial reduction in expression of gonadal soma-derived factor, the male-dominant somatic cell marker, and was not sufficient to induce ovarian development after withdrawal of DES. On the contrary, LE resulted in complete loss of such male-specific gene expression in somatic cells of XY gonads, and these gonads underwent sustained ovarian development even after withdrawal of DES. Importantly, LE to DES affected germ cell proliferation in XX gonads adversely during early stages of sexual differentiation, leading to reduced gonad weight in adulthood. Interestingly, apoptosis was not the cause for reduction in germ cell number. Taken together, these results indicated that DES exposure has long-lasting effects on the gonadal development in genetic males (sex reversal) and females (reduced gonad weight) of 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., (Copyright 2010 Elsevier B.V. All rights reserved.)

Gonad-stimulating substance (GSS) of starfish is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to the vertebrate luteinizing hormone (LH). Here, we purified GSS of starfish, Asterina pectinifera, from radial nerves and determined its amino acid sequence. The purified GSS was a heterodimer composed of 2 different peptides, A and B chains, with disulfide cross-linkages. Based on its cysteine motif, starfish GSS was classified as a member of the insulin/insulin-like growth factor (IGF)/relaxin superfamily. The cDNA of GSS encodes a preprohormone sequence with a C peptide between the A and B chains. Phylogenetic analyses revealed that starfish GSS was a relaxin-like peptide. Chemically synthesized GSS induced not only oocyte maturation and ovulation in isolated ovarian fragments, but also unique spawning behavior, followed by release of gametes shortly after the injection. Importantly, the action of the synthetic GSS on oocyte maturation and ovulation was mediated through the production of cAMP by isolated ovarian follicle cells, thereby producing the maturation-inducing hormone of this species, 1-methyladenine. In situ hybridization showed the transcription of GSS to occur in the periphery of radial nerves at the side of tube feet. Together, the structure, sequence, and mode of signal transduction strongly suggest that GSS is closely related to the vertebrate relaxin.

Sex-changing fish Trimma okinawae can change its sex back and forth from male to female and then to male serially, depending on the social status in the harem. T. okinawae is well equipped to respond to its social status by possessing both ovarian and testicular tissues even though only one gonad remains active at one time. Here we investigated the involvement of gonadotropins in sex change by determining the changes in gonadotropin receptor (GtHR) gene expression during the onset of sex change from female to male and male to female. The expression of the GtHR was found to be confined to the active gonad of the corresponding sexual phase. During the sex-change from female to male, initially the ovary had high levels of FSHR and LHR, which eventually went up in the testicular tissue if the fish was bigger. Changing of the gonads started with switching of GtHR expression discernible within 8-12 h of the visual cue. Further in vitro culture of the transitional gonads with a supply of exogenous gonadotropin (human chorionic gonadotropin) revealed that the to-be-active gonad acquired the ability to produce the corresponding sex hormone within 1 d of the activation of GtHR. Conversely, the to-be-regressed gonad did not respond to the exogenous gonadotropin. Our findings show that the gonads of successive sex-changing fish possess the intrinsic mechanism to respond to the social cue differentially. Additionally, this location switching of GtHR expression also could substantiate the importance of the hypothalamo-pituitary-gonadotropic axis.

The Nile tilapia, a gonochoristic teleost fish with an XX/XY sex-determining system, provides an excellent model for studying gonadal sex differentiation because genetic all-females and all-males are available. In this study, we used quantitative real-time RT-PCR to determine the precise timing of the gonadal expression of 17 genes thought to be associated with gonadal sex differentiation in vertebrates. Gonads were isolated from all-female and all-male tilapia before (5-15 days after hatching [dah]) and after (25-70 dah) morphological sex differentiation. The transcript of aromatase (cyp19a1a), an enzyme responsible for producing estradiol-17beta, was expressed only in XX gonads at 5 dah, with a marked elevation in expression thereafter. In contrast, mRNA expression of steroid 11beta-hydroxylase (cyp11b2), an enzyme responsible for the synthesis of 11-ketotestosterone (11-KT, a potent androgen in fish), was found in XY gonads from 35 dah only. These results, combined with the presence of transcripts for other steroidogenic enzymes and estrogen receptors in XX gonads at 5-7 dah, are consistent with our earlier suggestion that estradiol-17beta plays a critical role in ovarian differentiation in tilapia, whereas a role for 11-KT in testicular differentiation is questionable. A close relationship between the expression of foxl2, but not nr5a1 (Ad4BP/SF-1), and that of cyp19a1a in XX gonads suggests an important role for Foxl2 in the transcriptional regulation of cyp19a1a. Dmrt1 exhibited a male-specific expression in XY gonads from 6 dah onward, suggesting an important role for Dmrt1 in testicular differentiation. Sox9 and amh (anti-Mullerian hormone) showed a testis-specific expression, being evident only in the later stages of testicular differentiation. It is concluded that the sex-specific expression of foxl2 and cyp19a1a in XX gonads and dmrt1 in XY gonads during early gonadal differentiation (5-6 dah) is critical for undifferentiated gonads to differentiate into either the ovary or testis in the Nile tilapia.

Recently we reported the isolation of two types of cytochrome P450c17s (steroid 17alpha-hydroxylase/C17, 20 lyase) encoded by two different genes, from genomes of teleost fish. In this study, we characterized the expression profile, enzymatic activity and transcriptional regulation of P450c17-I and -II in the medaka ovary. Similar to tilapia, medaka P450c17-I possessed both hydroxylase and lyase activities, while P450c17-II possessed only the hydroxylase activity. In situ hybridization and gene expression profiles during 48h prior to spawning indicated that P450c17-I is responsible for the production of estradiol-17beta during oocyte growth, while P450c17-II for the production of 17alpha, 20beta-dihydroxy-4-pregnen-3-one during oocyte maturation and cortisol production in the head kidney. Luciferase assays and expression profiles of transcription factors as revealed by real time PCR suggested that P450c17-I and -II expression are tightly controlled by Ad4BP/SF-1, Lrh-1, Foxl2, and Dax1 during the 48 h prior to spawning.

Cytochrome P450c17 is the single enzyme that mediates the 17 alpha-hydroxylase and 17, 20 lyase activities during the biosynthesis of steroid hormones in the gonads and adrenal gland. However, the mechanism underlying its dual action continues to be a controversy in the field of steroidogenesis in fish. In an attempt to resolve this issue, we identified a novel type of P450c17 (P450c17-II) by an in silico analysis from the genomes of six fish species. We cloned P450c17-II from tilapia and medaka, and comparison with the conventional P450c17-I revealed that they differ in gene structure and enzymatic activity. Enzymatic assays by thin-layer chromatography revealed that P450c17-II possesses only the 17 alpha-hydroxylase activity without any 17, 20 lyase activity, unlike P450c17-I, which has both these activities. In testis, both P450c17-I and -II express in the interstitial cells. Remarkable differences, revealed by in situ hybridization, in the expression patterns of the P450c17-I and -II in the ovary and head kidney of tilapia during various stages of development strongly suggest that P450c17-I is responsible for the synthesis of estradiol-17beta in the ovary, whereas P450c17-II is required for the production of C21 steroids such as cortisol in the head kidney. More interestingly, a temporally controlled switching is observable in the expression of these two genes during the steroidogenic shift from estradiol-17beta to the C21 steroid, 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (maturation-inducing hormone of fish oocytes) in the fish ovary, revealing a role for P450c17-II in the production of hormones that induce oocyte maturation in fish.

Although the sex-determining gene SRY/Sry has been identified in mammals, homologues and genes that have a similar function have yet to be identified in nonmammalian vertebrates. Recently, DMY (the DM-domain gene on the Y chromosome) was cloned from the sex-determining region on the Y chromosome of the teleost fish medaka (Oryzias latipes). DMY has been shown to be required for the normal development of male individuals. In this study, we show that a 117-kb genomic DNA fragment that carries DMY is able to induce testis differentiation and subsequent male development in XX (genetically female) medaka. In addition, overexpression of DMY cDNA under the control of the CMV promoter also caused XX sex reversal. These results demonstrate that DMY is sufficient for male development in medaka and suggest that the functional difference between the X and Y chromosomes in medaka is a single gene. Our data indicate that DMY is an additional sex-determining gene in vertebrates.

Increasing evidence suggests the crucial role of estrogen in ovarian differentiation of nonmammalian vertebrates including fish. The present study has investigated the plausible role of Foxl2 in ovarian differentiation through transcriptional regulation of aromatase gene, using monosex fry of tilapia. Foxl2 expression is sexually dimorphic, like Cyp19a1, colocalizing with Cyp19a1 and Ad4BP/SF-1 in the stromal cells and interstitial cells in gonads of normal XX and sex-reversed XY fish, before the occurrence of morphological sex differentiation. Under in vitro conditions, Foxl2 binds to the sequence ACAAATA in the promoter region of the Cyp19a1 gene directly through its forkhead domain and activates the transcription of Cyp19a1 with its C terminus. Foxl2 can also interact through the forkhead domain with the ligand-binding domain of Ad4BP/SF-1 to form a heterodimer and enhance the Ad4BP/SF-1 mediated Cyp19a1 transcription. Disruption of endogenous Foxl2 in XX tilapia by overexpression of its dominant negative mutant (M3) induces varying degrees of testicular development with occasional sex reversal from ovary to testis. Such fish display reduced expression of Cyp19a1 as well as a drop in the serum levels of 17beta-estradiol and 11-ketotestosterone. Although the XY fish with wild-type tilapia Foxl2 (tFoxl2) overexpression never exhibited a complete sex reversal, there were significant structural changes, such as tissue degeneration, somatic cell proliferation, and induction of aromatase, with increased serum levels of 17beta-estradiol and 11-ketotestosterone. Altogether, these results suggest that Foxl2 plays a decisive role in the ovarian differentiation of the Nile tilapia by regulating aromatase expression and possibly the entire steroidogenic pathway.

DMY is the second vertebrate sex-determining gene identified from the fish, Oryzias latipes. In this study, we used two different ways of sex reversal, DMY knock-down and estradiol-17beta (E2) treatment, to determine the possible function of DMY during early gonadal sex differentiation in XY medaka. Our findings revealed that the mitotic and meiotic activities of the germ cells in the 0 day after hatching (dah) DMY knock-down XY larvae were identical to those of the normal XX larvae, suggesting the microenvironment of these XY gonads to be similar to that of the normal XX gonad, where DMY is naturally absent. Conversely, E2 treatment failed to initiate mitosis in the XY gonad, possibly due to an active DMY, even though it could initiate meiosis. Present study is the first to prove that the germ cells in the XY gonad can resume the mitotic activity, if DMY was knocked down.