Nano-graphene oxide particles induce inheritable anomalies through altered gene expressions involved in oocyte maturation.

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.

Differentiation Potential of Cultured Extracellular DEAD-Box Helicase 4+ Oogonial Stem Cells from Adult Human Ovaries into Somatic Lineages.

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.

Aqueous extract of large cardamom inhibits vascular damage, oxidative stress, and metabolic changes in fructose-fed hypertensive rats.

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.

Sex-Dependent Bioaccumulation of Nano Zinc Oxide and Its Adverse Effects on Sexual Behavior and Reproduction in Japanese Medaka.

This study investigated the adverse effects of 200 nm zinc oxide particles (nZnO) on sexual behavior and reproduction in Japanese medaka in comparison with ZnSO4 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 ZnSO4. 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 ZnSO4 in males and by nZnO but not ZnSO4 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 ZnSO4. Importantly, the female sex hormone, 17ß-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 ZnSO4.

Sex determination, gonadal sex differentiation, and plasticity in vertebrate species.

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 Alter Proliferation and Meiosis of Germ Cells Only in Genetic Females of Japanese Medaka during Early Embryonic Development.

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.

Long-term drug delivery using implantable electrospun woven polymeric nanotextiles.

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.

Mechanistic understanding of Phenyllactic acid mediated inhibition of quorum sensing and biofilm development in Pseudomonas aeruginosa.

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 (C4-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.

Theranostic Iron Oxide/Gold Ion Nanoprobes for MR Imaging and Noninvasive RF Hyperthermia.

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]Cl2, 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.

Effects of N-Nitrosodiethylamine, a Potent Carcinogen, on Sexual Development, Gametogenesis, and Oocyte Maturation.

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.

Influence of surface passivation of 2-Methoxyestradiol loaded PLGA nanoparticles on cellular interactions, pharmacokinetics and tumour accumulation.

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.

Sequential release of epigallocatechin gallate and paclitaxel from PLGA-casein core/shell nanoparticles sensitizes drug-resistant breast cancer cells.

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.

Sexually dimorphic expression of the sex chromosome-linked genes cntfa and pdlim3a in the medaka brain.

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.

Ol4E-T, a eukaryotic translation initiation factor 4E-binding protein of medaka fish (Oryzias latipes), can interact with nanos3 and vasa in vitro.

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.

Exposure to diethylstilbestrol during embryonic and larval stages of medaka fish (Oryzias latipes) leads to sex reversal in genetic males and reduced gonad weight in genetic females.

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.

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.

A relaxin-like peptide purified from radial nerves induces oocyte maturation and ovulation in the starfish, Asterina pectinifera.

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 change in the Gobiid fish is mediated through rapid switching of gonadotropin receptors from ovarian to testicular portion or vice versa.

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.

Sexual dimorphic expression of genes in gonads during early differentiation of a teleost fish, the Nile tilapia Oreochromis niloticus.

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.