Loss of Function of Vasoactive-intestinal Peptide Alters Sex Ratio and Reduces Male Reproductive Fitness in Zebrafish.

Vasoactive-intestinal peptide (Vip) is a pleiotropic peptide with a wide range of distribution and functions. Zebrafish possess 2 isoforms of Vip (a and b), in which Vipa is most homologous to the mammalian form. In female zebrafish, Vipa can stimulate LH secretion from the pituitary but is not essential for female reproduction, as vipa-/- females display normal reproduction.

A Thermal-Stable Protein Nanoparticle That Stimulates Long Lasting Humoral Immune Response.

A thermally stable vaccine platform is considered the missing piece of vaccine technology. In this article, we reported the creation of a novel protein nanoparticle and assessed its ability to withstand extended high temperature incubation while stimulating a long-lasting humoral immune response. This protein nanoparticle was assembled from a fusion protein composed of an amphipathic helical peptide derived from the M2 protein of the H5N1 influenza virus (AH3) and a superfolder green fluorescent protein (sfGFP).

Advantages, Factors, Obstacles, Potential Solutions, and Recent Advances of Fish Germ Cell Transplantation for Aquaculture-A Practical Review.

Germ cell transplantation technology enables surrogate offspring production in fish. This technology has been expected to mitigate reproductive barriers, such as long generation time, limited fecundity, and complex broodstock management, enhancing seed production and productivity in aquaculture. Many studies of germ cell transplantation in various fish species have been reported over a few decades.

Gnrh2 maintains reproduction in fasting zebrafish through dynamic neuronal projection changes and regulation of gonadotropin synthesis, oogenesis, and reproductive behaviors.

Restricted food intake, either from lack of food sources or endogenous fasting, during reproductive periods is a widespread phenomenon across the animal kingdom. Considering previous studies show the canonical upstream regulator of reproduction in vertebrates, the hypothalamic Gonadotropin-releasing hormone (Gnrh), is inhibited in some fasting animals, we sought to understand the neuroendocrine control of reproduction in fasted states. Here, we explore the roles of the midbrain neuropeptide, Gnrh2, in inducing reproduction via its pituitary prevalence, gonadotropin synthesis, gametogenesis, and reproductive outputs in the zebrafish model undergoing different feeding regimes.

Knockout of the Gnrh genes in zebrafish: effects on reproduction and potential compensation by reproductive and feeding-related neuropeptides.

Gonadotropin-releasing hormone (GNRH) is known as a pivotal upstream regulator of reproduction in vertebrates. However, reproduction is not compromised in the hypophysiotropic Gnrh3 knockout line in zebrafish (gnrh3-/-). In order to determine if Gnrh2, the only other Gnrh isoform in zebrafish brains, is compensating for the loss of Gnrh3, we generated a double Gnrh knockout zebrafish line.

The gonadotropin-inhibitory hormone (Lpxrfa) system's regulation of reproduction in the brain-pituitary axis of the zebrafish (Danio rerio).

Gonadotropin-inhibitory hormone (GNIH) was discovered in quail with the ability to reduce gonadotropin expression/secretion in the pituitary. There have been few studies on GNIH orthologs in teleosts (LPXRFamide (Lpxrfa) peptides), which have provided inconsistent results. Therefore, the goal of this study was to determine the roles and modes of action by which Lpxrfa exerts its functions in the brain-pituitary axis of zebrafish (Danio rerio).

Neurokinin B regulates reproduction via inhibition of kisspeptin in a teleost, the striped bass.

Kisspeptin and neurokinin B (NKB) are neuropeptides co-expressed in the mammalian hypothalamus and coordinately control GnRH signaling. We have found that Nkb and kisspeptin neurons are distinct in the teleost, striped bass (STB) and capitalized on this phenomenon to study the mode of action of Nkb and its related neuropeptide-F (Nkf), both of which are encoded by the gene. brain slices and administration studies revealed that Nkb/f consistently downregulated , whereas antagonist (AntD) administration restored this effect.

Seasonal expression of arginine vasotocin mRNA and its correlations to gonadal steroidogenic enzymes and sexually dimorphic coloration during sex reversal in the gilthead seabream (Sparus aurata).

Arginine vasotocin is a hormone produced in the hypothalamus of teleost fish that has been shown to regulate gonad development and sexual behavior. To study the role of arginine vasotocin in the gonadal cycle of the hermaphrodite gilthead seabream, Sparus aurata, we cloned the seabream arginine vasotocin (avt) complementary DNA (cDNA). We investigated the expression of brain avt throughout the gonad cycle using real-time quantitative PCR and compared its expression levels to the expression levels of two key gonadal steroidogenic enzymes, cyp19a1a and cyp11b2.

Targeted Mutagenesis of the Hypophysiotropic Gnrh3 in Zebrafish (Danio rerio) Reveals No Effects on Reproductive Performance.

Gnrh is the major neuropeptide regulator of vertebrate reproduction, triggering a cascade of events in the pituitary-gonadal axis that result in reproductive competence. Previous research in mice and humans has demonstrated that Gnrh/GNRH null mutations result in hypogonadotropic hypogonadism and infertility. The goal of this study was to eliminate gnrh3 (the hypophysiotropic Gnrh form) function in zebrafish (Danio rerio) to determine how ontogeny and reproductive performance are affected, as well as factors downstream of Gnrh3 along the reproductive axis.

Production of reproductively sterile fish by a non-transgenic gene silencing technology.

We developed a novel bath-immersion technology to produce large numbers of infertile fish. As seafood consumption shifts from fishery harvests towards artificially propagated species, optimization of aquaculture practices will be necessary to maximize food production and minimize ecological impact. Farming infertile fish is the most effective genetic-containment strategy to support the development of environmentally-responsible aquaculture.

Kisspeptin Antagonists Reveal Kisspeptin 1 and Kisspeptin 2 Differential Regulation of Reproduction in the Teleost, Morone saxatilis.

The importance of kisspeptin in regulating vertebrate reproduction has been well established, but the exact mechanism continues to unfold. Unlike mammals, many lower vertebrates possess a dual kisspeptin system, Kiss1 and Kiss2. To decipher the roles of the kisspeptins in fish, we identified two potential kisspeptin antagonists, pep 234 and pep 359, by screening analogs for their ability to inactivate striped bass Kiss1 and Kiss2 receptors expressed in COS7 cells.

Production of reproductively sterile fish: A mini-review of germ cell elimination technologies.

As seafood consumption shifts from fisheries harvests to artificially propagated aquatic species, the increase of aquaculture activities poses a biological threat to our environment. Selectively bred, non-native and (eventually) genetically engineered farmed fish may escape from aquaculture operations, propagate and/or interbreed with wild stocks and subsequently alter the genetic makeup of populations in the environment. Thus, an effective strategy for bio-containment of farmed fish is critically needed.

Identification of promoter elements responsible for gonad-specific expression of zebrafish Deadend and its application to ovarian germ cell derivation.

We discovered that a 150-bp region of zebrafish deadend (dnd) spanning the translation start codon, exon 1 and part of intron 1 is required to direct heterologous neomycin-resistance gene (neo) expression specifically in the gonad, similar to endogenous dnd. Using an 8.3-kb dnd promoter that contains this 150-bp region, we generated Tg(dnd:neo-dnd) transgenic zebrafish in which the expression of Neo was detected specifically in ovarian germ cells.

Dorsomorphin promotes survival and germline competence of zebrafish spermatogonial stem cells in culture.

Zebrafish spermatogonial cell cultures were established from Tg(piwil1:neo);Tg(piwil1:DsRed) transgenic fish using a zebrafish ovarian feeder cell line (OFC3) that was engineered to express zebrafish Lif, Fgf2 and Gdnf. Primary cultures, initiated from testes, were treated with G418 to eliminate the somatic cells and select for the piwil1:neo expressing spermatogonia. Addition of dorsomorphin, a Bmp type I receptor inhibitor, prolonged spermatogonial stem cell (SSC) survival in culture and enhanced germline transmission of the SSCs following transplantation into recipient larvae.

Inducible Sterilization of Zebrafish by Disruption of Primordial Germ Cell Migration.

During zebrafish development, a gradient of stromal-derived factor 1a (Sdf1a) provides the directional cue that guides the migration of the primordial germ cells (PGCs) to the gonadal tissue. Here we describe a method to produce large numbers of infertile fish by inducing ubiquitous expression of Sdf1a in zebrafish embryos resulting in disruption of the normal PGC migration pattern. A transgenic line of zebrafish, Tg(hsp70:sdf1a-nanos3, EGFP), was generated that expresses Sdf1a under the control of the heat-shock protein 70 (hsp70) promoter and nanos3 3?UTR.

Production of zebrafish offspring from cultured female germline stem cells.

Zebrafish female germline stem cell (FGSC) cultures were generated from a transgenic line of fish that expresses Neo and DsRed under the control of the germ cell specific promoter, ziwi [Tg(ziwi:neo);Tg(ziwi:DsRed)]. Homogeneous FGSC cultures were established by G418 selection and continued to express ziwi for more than 6 weeks along with the germ cell markers nanos3, dnd, dazl and vasa. A key component of the cell culture system was the use of a feeder cell line that was initiated from ovaries of a transgenic line of fish [Tg(gsdf:neo)] that expresses Neo controlled by the zebrafish gonadal soma derived factor (gsdf) promoter.

GnRH isoforms expression in relation to the gonadal cycle and to dominance rank in the gilthead seabream, Sparus aurata.

The manner in which behavior influences the gonadotropin-releasing hormone (GnRH) axis in hermaphroditic fishes is not understood. The Gilthead seabream, Sparus aurata, is a protandrous hermaphrodite with a complex gonadal cycle consisting of a quiescent, pre-spawning, spawning, and post-spawning stage. On two separate experiments, I used real-time quantitative PCR to measure the mRNA expression of three GnRH isoforms in homogenized seabream whole-brain extracts.

Effects of specific and prolonged expression of zebrafish growth factors, Fgf2 and Lif in primordial germ cells in vivo.

Primordial germ cells (PGCs), specified early in development, proliferate and migrate to the developing gonad before sexual differentiation occurs in the embryo and eventually give rise to spermatogonia or oogonia. In this study, we discovered that nanos3 3'UTR, a common method used to label PGCs, not only directed PGC-specific expression of DsRed but also prolonged this expression up to 26 days post fertilization (dpf) when DsRed-nanos3 3'UTR hybrid mRNAs were introduced into 1- to 2-cell-stage embryos. As such, we employed this knowledge to express zebrafish leukemia inhibitory factor (Lif), basic fibroblast growth factor (Fgf2) and bone morphogenetic protein 4 (Bmp4) in the PGCs and evaluate their effects on PGC development in vivo for over a period of 3 weeks.

Zebrafish germline chimeras produced by transplantation of ovarian germ cells into sterile host larvae.

High frequency production of zebrafish germline chimeras was achieved by transplanting ovarian germ cells into sterile Danio hybrid recipients. Ovarian germ cells were obtained from 3-mo-old adult Tg(vasa:DsRed2-vasa);Tg(bactin:EGFP) double transgenic zebrafish by discontinuous Percoll gradient centrifugation. An average of 755 ± 108 DsRed-positive germ cells was recovered from each female.

Zebrafish primordial germ cell cultures derived from vasa::RFP transgenic embryos.

Although embryonic germ (EG) cell-mediated gene transfer has been successful in the mouse for more than a decade, this approach is limited in other species due to the difficulty of isolating the small numbers of progenitors of germ cell lineage (PGCs) from early-stage embryos and the lack of information on the in vitro culture requirements of the cells. In this study, methods were established for the culture of PGCs obtained from zebrafish embryos. Transgenic embryos that express the red fluorescent protein (RFP) under the control of the PGC-specific vasa promoter were used, making it possible to isolate pure populations of PGCs by fluorescence-activated cell sorting (FACS) and to optimize the culture conditions by counting the number of fluorescent PGC colonies produced in different media.

Molecular biology of ovarian aromatase in sex reversal: complementary DNA and 5'-flanking region isolation and differential expression of ovarian aromatase in the gilthead seabream (Sparus aurata).

To elucidate the involvement of aromatase in sex reversal, the gilthead seabream ovarian P450 aromatase (cyp19a1a) cDNA and its 5'-flanking region were isolated and characterized. Northern blot analysis revealed that only one cyp19a1a transcript (2.0 kb) is expressed in the ovary.

Novel expression of gonadotropin subunit genes in oocytes of the gilthead seabream (Sparus aurata).

It is widely believed that FSH and LH, which are known to play key roles in controlling the production of functional oocytes in vertebrates, are synthesized and secreted exclusively by the anterior pituitary. Here we present evidence for the novel expression of FSHbeta, LHbeta, and the common glycoprotein-alpha (Cgalpha) in the gilthead seabream ovary. Using in situ hybridization and immunocytochemistry, FSHbeta was detected in primary-growth and secondary-growth-I oocytes, LHbeta was found in secondary-growth oocytes, and Cgalpha was observed in both primary and secondary-growth oocytes.

Developmental expression of three forms of gonadotropin-releasing hormone and ontogeny of the hypothalamic-pituitary-gonadal axis in gilthead seabream (Sparus aurata).

To address the complexity of the origin of the GnRH system in perciforms, we investigated the ontogenic expression of three GnRHs in gilthead seabream. Using in situ hybridization, chicken (c) GnRH-II mRNA-expressing cells were detected in the hindbrain at 1.5 days postfertilization (DPF) and in the midbrain at 2 DPF and thereafter; the hindbrain signals became undetectable after 10 DPF.

Percutaneous absorption of inorganic lead compounds.

The objective of this study was to determine percutaneous absorption of lead compounds, including lead sulfate, lead oxide, lead powder, and lead stearate. The lead content on the skin surface of 10 lead-battery workers was measured by the method of skin stripping, and urinary lead content of rats was measured with epicutaneous application of four lead compounds: lead sulfate, lead oxide, lead powder, and lead stearate. There were significant amounts of lead on the 9th and 10th skin strippings of the dorsal hand and the back of lead workers.