In vivo induction of male sexual behavior in zebrafish by adding agents to water.

Successful fertilization in fish mating occurs when egg maturation in the ovary of the female, ovulation, sperm maturation in the testis of the male, and reproductive behaviors in both sexes are triggered in synchrony. The male sexual behavior of fish is induced by hormones and pheromones. In a previous study, we demonstrated that externally applied hormones added to the water can induce oocyte maturation and ovulation in female zebrafish. Here, we attempted to establish a similar method to induce the sexual behavior of male zebrafish. The male sex steroid testosterone (Tes) triggered sexual behavior within several hours in vivo when administered directly into the surrounding water. A selective agonist for membrane progesterone receptor (mPR), Org OD-02 (Org), also induced sexual behavior. Through trials of various combinations of compounds, we found that the most effective conditions were achieved by treatment with a mixture of testosterone (Tes) and Org. The effect of treatment was evaluated by the number of fertilized eggs obtained by pairing with females with induced ovulation in vivo. The period necessary for the induction of male sexual behavior was evaluated by time course experiments. The success rate of mating and the number of fertilized eggs reached the maximum level at 3-4 hours of treatment. The duration of hormonal treatment was confirmed by counting the number of hooking occurrences, which is the final cue to induce spawning by females. In summary, we have established a method to induce male sexual behavior in zebrafish in vivo. The method can be used to obtain fertilized eggs in zebrafish by simply adding agents into the water.

Pax7 is involved in leucophore formation in goldfish and gene knockout improves the transparency of transparent goldfish.

Lines with few or no pigment cells have been established in fishes, and these lines are useful for bioimaging. The transparent goldfish (tra) line previously established by N-ethyl-N-nitrosourea (ENU) mutagenesis is also suitable for such experiments. However, in the case of tra, leucophores form in the adult fish, making it difficult to observe the organs inside body from outside the body. In this study, we attempted to create a knockout line of the pax7a and pax7b genes, which are thought to be involved in the formation of leucophores, to further improve the transparency of tra strain.Mutations were introduced by microinjection of the CRISPR/Cas9 mixture into single-cell embryos, mutant individuals were found in F0, and the next generation was generated to confirm the mutation patterns. As a result, multiple mutation patterns, including knockout, were obtained. The same pattern of knockout F1 with pax7a and pax7b mutations was crossed to generate a homozygous knockout in F2.In the resulting pax7b-/- (tra) fish but not in pax7a-/- (tra) fish, the number of leucophores was reduced compared to that in tra, and the transparency of the body was improved. It was suggested that pax7b plays an important role in leucophore formation in goldfish. The established transparent pax7b-/- (tra) goldfish line will be a useful model for bioimaging of the body interior.

Real-Time Observation of Germinal Vesicle Migration During Oocyte Meiotic Cell Division Using Ovarian Fluorescent Transgenic Zebrafish.

The transgenic (TG) zebrafish allows researchers to bio-image specific biological phenomena in cells and tissues in vivo. We established TG lines to monitor changes in the ovaries of live fish. The original TG line with ovarian fluorescence was occasionally established. Although the cDNA integrated into the line was constructed for the expression of enhanced green fluorescent protein (EGFP) driven by the medaka ß-actin promoter, the expression of EGFP is restricted to the oocytes and gills in adult fish. Furthermore, we found that germinal vesicles (GVs) in oocytes of the established line can be observed by relatively strong fluorescence around the GV. In this study, we tried to capture the dynamic processes of germinal vesicle breakdown (GVBD) during meiotic cell division using the GV fluorescent oocytes. As a result, GV migration and GVBD could be monitored in real time. We also succeeded in observing actin filaments involved in the migration of GV to the animal pole. This strain can be used for education in the process of oocyte meiotic cell division.

Establishment of a graphene quantum dot (GQD) based steroid binding assay for the nuclear progesterone receptor (pgr).

Previously, we established a homogeneous assay for membrane progesterone receptor alpha (mPRα) ligands by conjugating semiconductor nanoparticles known as graphene quantum dots (GQDs) to mPRα. When mixed with a progesterone-BSA-fluorescein isothiocyanate conjugate (P4-BSA-FITC), fluorescence occurred by fluorescence resonance energy transfer (FRET) but was reduced by the ligand-receptor binding activity. The established way showed ligand specificity as mPRα protein. In this study, we tried to establish the same way for nuclear progesterone receptor (Pgr). The ligand-binding domain (LBD) of zebrafish Pgr (zPgrLBD) was expressed as a fusion protein with glutathione S-transferase (GST) (GST-zPgrLBD). The recombinant protein was then purified and coupled with GQDs to produce GQD-conjugated GST-zPgrLBD (GQD-GST-zPgrLBD). When mixed with a P4-BSA-FITC and activated by 370 nm light, fluorescence at 520 nm appeared by FRET mechanism. Fluorescence at 520 nm was reduced by adding free progesterone to the reaction mixture. Reduction of fluorescence was induced by zPgr ligands but not by steroids or chemicals that do not interact with zPgr. The results showed the formation of a complex of GQD-GST-zPgrLBD and P4-BSA-FITC with ligand-receptor binding. The binding of the compounds was further confirmed by a radiolabeled steroid binding assay. A homogenous ligand-binding assay for nuclear progesterone receptor has been established.

Establishment of a steroid binding assay for goldfish membrane progesterone receptor (mPR) by coupling with graphene quantum dots (GQDs).

A homogeneous assay was developed to evaluate ligands that target the membrane progesterone receptor alpha (mPRα) of goldfish. This was achieved by employing graphene quantum dots (GQDs), a type of semiconductor nanoparticle conjugated to the goldfish mPRα. When progesterone-BSA-fluorescein isothiocyanate (P4-BSA-FITC) was combined with the other agents, fluorescence was observed through Förster resonance energy transfer (FRET). However, this fluorescence was quenched by binding between the ligand and receptor. This established method demonstrated the ligand selectivity of the mPRα protein. Then, the methylotrophic yeast Pichia pastoris was used to express the goldfish mPRα (GmPRα) protein. The recombinant purified GmPRα protein was coupled with graphene quantum dots (GQDs) to generate GQD-conjugated goldfish mPRα (GQD-GmPRα). Fluorescence at a wavelength of 520 nm was observed through FRET upon the combination of P4-BSA-FITC and subsequent activation by ultraviolet (UV) light. Adding free P4 to the reaction mixture resulted in a decrease in fluorescence intensity at a wavelength of 520 nm. The fluorescence was reduced by the administration of GmPRα ligands but not by steroids that do not interact with GmPRα. The findings indicated that the interaction between the ligand and receptor led to the formation of a complex involving GQD-GmPRα and P4-BSA-FITC. The interaction between the compounds and GQD-GmPRα was additionally validated by a binding experiment that employed the radiolabeled natural ligand [3H]-17α,20ß-dihydroxy-4-pregnen-3-one. We established a ligand-binding assay for the fish membrane progesterone receptor that is applicable for screening compounds.

Evidence of binding between diethylstilbestrol (DES) and the goldfish (Carassius auratus) membrane progesterone receptor α.

BACKGROUND: In a previous study, diethylstilbestrol (DES) was shown to induce oocyte maturation in fish. In the present study, the interaction of DES on goldfish membrane progesterone receptor α (GmPRα) was investigated using a competitive binding assay with radiolabeled steroids. The results indicate that DES exerts its effects on membrane progesterone receptor alpha (mPRα) and induces oocyte maturation through nongenomic steroid mechanisms. This study provides empirical data that demonstrate the binding between DES and GmPRα. METHODS: Binding of DES to GmPRα was achieved by using radiolabeled DES and recombinant GmPRα expressed in culture cells or purified GmPRα proteins that coupled to graphene quantum dots (GQDs). Additionally, the competitive binding of fluorescently labeled progesterone to GmPRα-expressing cells was evaluated. RESULTS: Although significant nonspecific binding of radiolabeled DES to the cell membrane that expresses GmPRα has been observed, specific binding of DES to GmPRα has been successfully identified in the presence of digitonin. Furthermore, the specific binding of DES to GmPRα was confirmed by a binding assay using GQD-GmPRα. The radiolabeled DES was shown to bind to GQD-GmPRα. Additionally, the competition for the binding of fluorescently labeled progesterone to GmPRα-expressing cells was achieved with the DES. CONCLUSIONS: The results of the experiments revealed that DES binds to GmPRα. Thus, it can be concluded that DES induces goldfish oocyte maturation by binding to GmPRα.

Zebrafish prss59.1 is involved in chorion development.

The prss59.1 gene was identified as one of 11 genes that were highly upregulated during the induction of ovulation in zebrafish by using an in vivo ovulation assay. Previously, we conducted biochemical characterization of Prss59.1 and revealed it to be a trypsin-like proteolytic enzyme. In this study, we established a prss59.1 gene knockout strain using the CRISPR/Cas9 system. Phenotypic analysis of prss59.1 knockout fish showed that prss59.1 is associated with chorion elevation, a prominent event in egg activation during fertilization. The chorions of heterozygous and homozygous prss59.1 mutant zebrafish were smaller than those of the wild type. The results suggested that Prss59.1 is necessary for chorion expansion. The homozygous prss59.1 mutant strain, with a small chorion, showed an extremely low survival rate. Fiber-supported knob-like structures (KS) on the chorion showed an abnormal structure in prss59.1 mutants. Prss59.1 was detected in the KS on the chorion. The pores on the chorion were smaller in the prss59.1 mutants than in the wild type. Transmission electron microscopy (TEM) observations of the cross sections of the chorions showed abnormalities in the chorion structure in prss59.1 mutants. These results demonstrated that Prss59.1 is involved in chorion elevation and in proper formation of the chorion, which is necessary for embryo development.

The Mc4r gene is responsible for the development of experimentally induced testicular teratomas.

Teratomas in mice, composed of different tissue types, are derived from primordial germ cells in the fetal gonads. Previously, we identified a locus responsible for experimental testicular teratoma (ETT) formation on chromosome 18, referred to as ett1. The strongest candidate sequence in the ett1 locus was found to be a missense mutation in the melanocortin 4 receptor (Mc4r), Mc4rG25S. We established a strain with a point mutation in the Mc4r gene in the ETT-nonsusceptible LT strain, called LT- Mc4rG25S, by genome editing. Surprisingly, highly developed ovarian teratomas (OTs), rather than testicular teratomas, appeared in the LT-Mc4rG25S strain. The results demonstrated that Mc4r is also one of the genes responsible for OT formation and suggested that missense mutations in Mc4r promote teratoma formation in both sexes. In this study, we performed ETT experiments in different host-graft combinations of the LT-Mc4rG25S and LT strains. Furthermore, the expression of MC4R in germ cells in the testis was demonstrated. Expression of Mc4r in testis was also confirmed by RT-PCR. The results demonstrated that MC4R is expressed in germ cells in the testis and that a point mutation in the Mc4r gene is responsible for ETT formation.

The antagonistic activity of Padina arborescens extracts on mPRα.

The current study attempted to evaluate the antagonistic activity of compounds isolated and purified from the marine algae Padina arborescens during cultivation. The compounds were collected on a filter, concentrated on ODS columns and separated by HPLC. Two peaks that showed competitive progesterone binding activity with membrane progesterone receptor α (mPRα) were purified. Their physiological activity was further uncovered by in vitro and in vivo oocyte maturation and ovulation-inducing assays using zebrafish. The compounds inhibited the induction of oocyte maturation and ovulation. Moreover, the results showed that the compounds have antagonistic activity against mPRα. The purified compounds with antagonistic activity against mPRα would be considered as new pharmaceutical candidate.

Transgenerational effects of bisphenol A on zebrafish reproductive tissues and sperm motility.

In a previous study, we demonstrated the next-generation effects and further transgenerational adverse effects of bisphenol A (BPA) in zebrafish. The adverse effects on reproductive factors, such as gonadal activity, fertility, hatching rate and malformation of embryo caused by the dietary administration on initial generation (F0) male and female zebrafish were continued until third filial (F3) generation. In this study, we examined how much amount of BPA contained in the diet was taken up by the zebrafish. We showed that only about 3.5-6.8% of BPA in the diet was taken into fish body. Also, we confirmed the transgenerational effects caused by 100 times lower amount of BPA than previous study. Even a low amount of BPA (1 µg/g diet) administered to F0 not only caused retraction of the ovaries and testes but also lowered the survival rate and increased the rate of malformation in the offspring. The effects were continued to F3 generation as previously described. Moreover, the sperm motility of the offspring of the BPA-treated ancestral animals was significantly lower, and this adverse effect was continued to F2 generations. These findings demonstrated that BPA at levels comparable to those ingested by humans can cause transgenerational adverse effects on fish reproduction.

Erratum to "Next-generation and further transgenerational effects of bisphenol A on zebrafish reproductive tissues" [Heliyon 4, (9), (September 2018), Article e00788].

[This corrects the article DOI: 10.1016/j.heliyon.2018.e00788.].

Purification and Identification of the 20S Proteasome Complex from Zebrafish.

The proteasome is a large polymeric protease complex responsible for degradation of intracellular proteins and generation of peptides. In this study, we purified a native 20S proteasome protein complex from zebrafish (Danio rerio) from the whole body. The cytosolic fraction of zebrafish hydrolyzed Suc-Leu-Leu-Val-Tyr-MCA (Suc-LLVY-MCA), a well-known substrate for the proteasome, in the presence of sodium dodecyl sulfate. From the cytosolic fraction, the 20S proteasome was purified using five column chromatography steps: DEAE cellulose, Q-Sepharose, Sephacryl S-300 gel, hydroxylapatite, and phenyl Sepharose. Electrophoresis and Western blot analyses showed that zebrafish 20S proteasome subunits have molecular masses ranging from 22 to 33 kDa. The subunit composition of the purified 20S proteasome was identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis after two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separation. Fourteen kinds of 20S subunits were found. As a special characteristic of zebrafish, two proteins of the α1 subunit were identified. In addition, the results suggested that the α8 subunit is in the 20S complex instead of the α4 subunit. In this study, we demonstrated the subunit composition of the 20S proteasome complex present in zebrafish cells.

Establishment of a steroid binding assay for membrane progesterone receptor alpha (PAQR7) by using graphene quantum dots (GQDs).

Currently, semiconductor nanoparticles known as quantum dots (QDs) have attracted interest in various application fields such as those requiring sensing properties, binding assays, and cellular imaging and are the very important in the acceleration of drug discovery due to their unique photophysical properties. Here, we applied graphene quantum dots (GQDs) for the binding assay of membrane progesterone receptor alpha (mPRα), one of the probable membrane receptors that have potential in drug discovery applications. By coupling the amino groups of mPRα with GQDs, we prepared fluorogenic GQD-conjugated mPRα (GQD-mPRα). When mixed with a progesterone-BSA-fluorescein isothiocyanate conjugate (P4-BSA-FITC) to check the ligand receptor binding activity of GQD-mPRα, fluorescence at 520 nm appeared. The fluorescence at 520 nm was reduced by the addition of free progesterone into the reaction mixture. GQD-coupled BSA (GQD-BSA) did not show a reduction in fluorescence at 520 nm. The results demonstrated the formation of a complex of GQD-mPRα and P4-BSA-FITC with ligand receptor binding. We established a ligand binding assay for membrane steroid receptors that is applicable for high-throughput assays.

Biochemical characterization of zebrafish Prss59.1.

Eleven genes, including prss59.1, were selected as candidate ovulation-inducing genes on the basis of microarray analysis and RNA sequencing in our previous study. To address the role of prss59.1, the prss59.1 gene knock-out zebrafish strain is currently being established by genome editing. In this study, for further phenotypic analysis of prss59.1, biochemical characterization of Prss59.1 was conducted using recombinant protein. A C-terminal histidine-tagged version of zebrafish Prss 59.1 was constructed. Although E. coli-produced recombinant Prss59.1 showed almost no activity, peptidase activities appeared after denaturation and renaturation. Zebrafish Prss59.1 showed the highest activity against Lys-MCA. The optimal temperature and pH of the activity toward Lys-MCA were 37 °C and pH 8.0, respectively. The Km value was 0.17 mM. Thus, zebrafish Prss59.1 possesses the closed character of trypsin, as expected from the DNA sequence.

MC4R mutant mice develop ovarian teratomas.

Teratomas in mice, composed of different tissue types, are derived from primordial germ cells (PGCs) in the foetal gonads. The strongest candidate gene in the testicular teratoma locus (Ter) responsible for testicular teratoma formation was identified as mutation in Dnd1, Dnd1R178*. However, the phenotype of mice with a mutated Dnd1 gene was germ cell loss. This suggests that other genes are involved in teratoma formation. Testicular teratomas can also be induced experimentally (experimentally testicular teratomas: ETTs) in 129/Sv mice by transplanting E12.5 foetal testes into adult testes. Previously, we mapped the ett1 locus, which is the locus responsible for ETT formation on chromosome 18. By exome sequence analysis of the 129 and LTXBJ (LT) strains, we identified a missense mutation in the melanocortin 4 receptor (MC4R) gene among 8 genes in the ett1 region. The missense mutation causes a substitution of glycine 25 by serine. Thus, this gene is a candidate for ETT formation. We established the LT-ett1 congenic strain, which introduced the locus responsible for ETT formation genetically into the genomes of a testicular teratoma non-susceptible strain. In this study, we crossed LT-ett1 and a previously established LT-Ter strain to establish the double congenic strain LT-Ter-ett1. Also, we established a strain with a point mutation in the MC4R gene of the LT strain by genome editing, LT-MC4RG25S. Furthermore, double genetically modified strain LT-Ter-MC4RG25S was established to address the relation between Ter and MC4R. Surprisingly, highly developed ovarian teratomas (OTs), instead of testicular teratomas, appeared not only in the LT-Ter-MC4RG25S and LT-MC4RG25S strains but also in the LT-ett1 and LT-Ter-ett1 strains. The incidence of OT formation was high in double genetically modified strains. The results demonstrated that MC4R is one of the genes responsible for OT formation. It was suggested that the effect of the missense mutation in MC4R on teratoma formation was promoted by abnormal germ cell formation by the mutation in DND1.

The Role of DNA Methylation Reprogramming during Sex Determination and Transition in Zebrafish.

DNA methylation is a prevalent epigenetic modification in vertebrates, and it has been shown to be involved the regulation of gene expression and embryo development. However, it remains unclear how DNA methylation regulates sexual development, especially in species without sex chromosomes. To determine this, we utilized zebrafish to investigate DNA methylation reprogramming during juvenile germ cell development and adult female-to-male sex transition. We reveal that primordial germ cells (PGCs) undergo significant DNA methylation reprogramming during germ cell development, and the methylome of PGCs is reset to an oocyte/ovary-like pattern at 9 days post fertilization (9 dpf). When DNA methyltransferase (DNMT) activity in juveniles was blocked after 9 dpf, the zebrafish developed into females. We also show that Tet3 is involved in PGC development. Notably, we find that DNA methylome reprogramming during adult zebrafish sex transition is similar to the reprogramming during the sex differentiation from 9 dpf PGCs to sperm. Furthermore, inhibiting DNMT activity can prevent the female-to-male sex transition, suggesting that methylation reprogramming is required for zebrafish sex transition. In summary, DNA methylation plays important roles in zebrafish germ cell development and sexual plasticity.

Zebrafish stm is involved in the development of otoliths and of the fertilization envelope.

Using an in vivo assay, we selected 11 genes that were highly upregulated during the induction of ovulation in zebrafish using microarray analysis and RNA sequencing. The starmaker gene (stm) was one of these genes. Although stm has been previously reported to be involved in otolith formation during the early development of zebrafish, we detected its expression in eggs and showed that stm was related to fertilization by establishing an stm gene knockout strain using the CRISPR/Cas9 system. Further phenotypic analysis of stm knockout fish was conducted in this study. With a higher nonfertilization rate, the stm mutant strain showed an extremely low survival rate. Otoliths of stm homozygous mutant zebrafish showed abnormal morphology in embryos and adult fish. However, fish did not show any abnormalities in swimming behaviour in either embryos or adults. Stm proteins were detected on the chorion of ovulated eggs before spawning. Fibre-supported knob-like structures on the fertilization envelope (FE) also showed abnormal structures in stm mutants. The Stm protein is necessary for otolith formation, and a lack of Stm causes abnormal otolith formation. The partial defect of otolith formation does not cause defects in swimming behaviour. The Stm protein is expressed in the chorion and is responsible for the formation of fibre-supported knob-like structures on the FE. It was suggested that a lack of Stm caused a lower fertilization rate due to inadequate formation of the FE. LAY SUMMARY: In zebrafish, the protein Starmaker (Stm) was identified as having a role in ovulation. Stm is also known to be required for the formation of ear stones (otoliths) which are needed to keep the body in balance. Zebrafish lacking Stm were produced by genome editing. As expected, Stm-deficient fish formed abnormal otoliths. To investigate the role of Stm in ovulation, fertilization and early development, we tried mating of Stm mutants and observed their juveniles. Although no problem found in ovulation, we found low fertilization rate and abnormal structure of knob-like structure (small pit) on the egg membrane. Survival rate of embryos with abnormal egg membrane was extremely low. It was demonstrated that Stm protein is necessary to form the functional egg membrane to protect embryos from the outside environment.

Pax2a is expressed in oocytes and is responsible for early development and oogenesis in zebrafish.

Eleven genes, including pax2a, were selected as candidate ovulation-inducing genes on the basis of microarray analysis and RNA sequencing in our previous study. The purpose of this study was to investigate the role of the pax2a gene in the ovulation-inducing process. F2 pax2a homozygous mutant zebrafish possessing a deletion of 6 nucleotides were established in this study. However, the deletion included the start codon (ATG) of the pax2a gene, and the Pax2a protein was still detected, which indicated that the deletion caused a shift in the start codon to the next ATG, resulting in a 12-amino acid deletion. F2 pax2a homozygous mutant zebrafish showed ovulation. However, the embryos showed an abnormal oval shape at the epiboly stage that resulted in yolk and tail formation abnormalities and heart edema. The surviving F3 homozygous mutants did not develop ovaries. Pax2a was detected in oocytes and eggs but not after the Prim-22 stage. It is suggested that pax2a is expressed as a maternal gene in oocytes and is necessary for oogenesis and early development.

An agonist for membrane progestin receptor (mPR) induces oocyte maturation and ovulation in zebrafish in vivo.

The maturation and ovulation of fish oocytes are well-characterized biological processes induced by progestins via coordination of nongenomic actions and genomic actions. Previously, we established a procedure that enables the induction of oocyte maturation and ovulation in live zebrafish by simple administration of the natural teleost maturation-inducing hormone 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17,20ß-DHP) into the surrounding water. By this in vivo assay, the potencies of chemicals in inducing or preventing oocyte maturation and ovulation can be evaluated. The potencies of compounds in inducing ovulation of zebrafish oocytes also can be evaluated in vivo with improved in vitro assays. Here, we attempted to evaluate the effect of Org OD 02-0 (Org OD 02), a selective agonist for membrane progestin receptor (mPR), on fish oocyte maturation and ovulation with in vitro and in vivo assays. As reported previously, Org OD 02 triggered oocyte maturation in vitro. The same Org OD 02 triggered oocyte maturation within several hours in vivo. Surprisingly, Org OD 02 even induced ovulation both in in vivo and in vitro. Eggs from Org OD 02-induced ovulation could be fertilized by artificial insemination. The juveniles developed normally. These results indicated that Org OD 02 triggered physiological ovulation in live zebrafish. In summary, we have demonstrated the effect of Org OD 02 on fish oocyte maturation and ovulation in vitro and in vivo. The results suggested that Org OD 02 acted as an agonist not only of mPR but also of nuclear progesterone receptor (nPR).

Rapid Induction of Female-to-Male Sex Change in Adult Zebrafish by Injection of an Aromatase Inhibitor.

Previously, we examined whether aromatase inhibitor (AI) treatment induces a sex change in adult female zebrafish. A 5-month AI treatment regime resulted in the retraction of the ovaries and testis formation. Eight weeks after changing the diet to AI-free food, a large number of normal sperm were obtained. Artificial fertilization using sperm from the sex-changed females was successful. These results demonstrated that sex plasticity remains in the mature ovaries of zebrafish. However, >7 months of treatment was necessary; thus, pairing was unsuccessful. In this study, we tried to induce sex change through the injection of an AI to shorten the time course of sex change. When the AI solution was directly injected into the abdomen of zebrafish, retraction of the ovary was induced within 2 months. The natural mating of sex-changed females with normal females was successful at 3 months. Although the fertilization rate was low, juveniles resulting from these matings developed normally. We succeeded in establishing a method for inducing sex changes in adult zebrafish within 3 months. The procedure will support the study of how sexual plasticity persists in adult zebrafish following sex differentiation and the identification of undifferentiated stem cells.