Loss of gdnfa disrupts spermiogenesis and male courtship behavior in zebrafish.

Spermatogenesis is essential for establishment and maintenance of reproduction in male vertebrates. Spermatogenesis, which is mainly regulated by the combined action of hormones, growth factors, and epigenetic factors, is highly conserved. Glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor-ß superfamily. In this study, global gdnfa knockout and Tg (gdnfa: mcherry) transgenic zebrafish lines were generated. Loss of gdnfa resulted in disorganized testes, decreased gonadosomatic index, and low percentage of mature spermatozoa. In the Tg (gdnfa: mcherry) zebrafish line, we found that gdnfa was expressed in Leydig cells. The mutation in gdnfa significantly decreased Leydig cell marker gene expression and androgen secretion in Leydig cells. In addition, courtship behavior was disrupted in the male mutants. We present in vivo data showing that global knockout of gdnfa disrupts spermiogenesis and male courtship behavior in zebrafish. The first viable vertebrate model with a global gdnfa knockout may be valuable for studying the role of GDNF in animal reproduction.

A novel electrochemical sensor based on N, S co-doped liquorice carbon/functionalized MWCNTs nanocomposites for simultaneous detection of licochalcone A and liquiritin.

In this study, nanoporous C was prepared from the roots, stems and leaves of liquorice and modified via element doping. Then, the nitrogen and sulfur co-doped liquorice carbon (N, S-LC) and functional multiwall carbon nanotubes (f-MWCNTs) were dispersed using an ultrasonic dispersion technology, and a binary nanocomposite was prepared. N, S-LC plays a key role in the formation and electrochemical efficiency of binary nanocomposites, and electrochemical impedance spectroscopy indicates that the binary complexes formed by the hybridisation of N, S-LC and f-MWCNTs can improve the electron transfer ability of an electrode. The electrochemical behaviour of Licochalcone A (LicA) and Liquiritin (LQ) on GCE modified by N, S-LC/f-MWCNTs binary nanocomposite was investigated via differential pulse voltammetry (DPV) and cyclic voltammetry (CV). The interpeak potential difference between LicA and LQ were 0.48 V. The calibration curves for LicA and LQ were obtained in the range of 0.4-70.0 µM and 0.1-150.0 µM, respectively, and the detection limits are 33 and 25 nM (S/N=3), respectively. The modified electrode has been successfully applied to the simultaneous determination of LicA and LQ in licorice with satisfactory recoveries as confirmed by HPLC method.

Overexpression of growth hormone improved hepatic glucose catabolism and relieved liver lipid deposition in common carp (Cyprinus carpio L.) fed a high-starch diet.

Growth hormone (GH) is important for regulating insulin secretion and carbohydrate metabolism, and its role in mammalian models of diabetes is relatively worked out. Although some fish species were used as models for diabetes research, the effects of GH on insulin and glucose catabolism and anabolism in these models remain to be clarified. In this study, we investigated the effect of GH on insulin and glucose catabolism and anabolism in an omnivorous fish using GH transgenic (T) common carp that consistently overexpressed GH and wild-type (WT) common carp. We compared the intestinal morphology, and digestive and absorptive capacity of fish fed commercial feed. We also analyzed the growth performance, insulin level, glucose catabolism and anabolism, lipid deposition, and lipid catabolism and anabolism in T carp and WT carp fed diets containing either 30% or 40% starch. In the intestine of T carp, α-amylase activity was enhanced, the number of goblet cells and intestinal villi surface area was increased, and the expression level of glucose transport protein-related genes (glut2 and sglt1) was upregulated when compared to these indicators in WT carp. When fed either a normal or high-starch diet, the growth performance of T carp was better than that of WT carp. Compared with WT carp, serum insulin was increased and glucose was decreased, hepatic expression level of igf-1 and glycolysis-related genes was increased, and the activity level of a hepatic enzyme related to glycolysis was enhanced in T carp. When fed with a high-starch diet, the serum alanine aminotransferase activity, hepatic lipid content, and malondialdehyde content were significantly lower in T carp than in WT carp. These results indicated that overexpression of GH (1) enhanced carbohydrate digestion and absorption in the carp intestine, (2) did not induce insulin resistance and improved glucose catabolism and utilization in carp, and (3) relieved liver lipid deposition. Our data might provide new insights into potential ways to improve glucose utilization in fish and diabetes treatments.

Improved liver lipid catabolism and utilization in growth hormone transgenic common carp (Cyprinus carpio L.) through enhanced lipolytic and fatty acid ß-oxidation pathways.

Growth hormone (GH) transgenic common carp (Cyprinus carpio L.) show desirable aquaculture traits. Their specific growth rate (SGR) and feed efficiency (FE) are approximately 12% and 17% higher than the wild-type (WT) common carp, respectively. However, the mechanisms of lipid catabolism (lipolysis and fatty acid ß-oxidation) and utilization in GH transgenic common carp are still unclear. In this study, we firstly compared the lipid metabolism of GH transgenic (initial weight 3.72 ± 0.32 g) and WT (initial weight 3.30 ± 0.28 g) common carp fed with a normal fat level diet (6% lipid, 33% protein) for two months, then compared the growth performance of GH transgenic (initial weight 3.65 ± 0.33 g) and WT (initial weight 3.27 ± 0.26 g) common carp fed with different fat levels diets (6% lipid and 12% lipid, 33% protein) for two months. We found that the lipid content in serum, liver and whole body was significantly reduced in GH transgenic common carp, the hepatic activities of the lipolytic enzymes hormone-sensitive lipase and adipose triglyceride lipase were enhanced, and the hepatic expression level of hormone-sensitive lipase was upregulated. In addition, the mitochondrion numbers were increased, and the expression level of carnitine palmitoyltransferase-1a and carnitine palmitoyltransferase-1b was upregulated in the liver of GH transgenic common carp. GH transgenic common carp showed higher weight gain and SGR than that in WT carp when fed with a normal-fat diet as they did when fed with a high-fat diet, and GH transgenic common carp showed higher FE than that in WT carp when fed with a high-fat diet. These results suggested that the lipid catabolism and utilization was improved in the GH transgenic common carp liver through enhanced lipolytic and fatty acid ß-oxidation pathways. Our study provides new insights into improving lipid utilization in some aquaculture fish species.

Isolation and Characterization of Germline Stem Cells in Protogynous Hermaphroditic Monopterus albus.

Germline stem cells (GSCs) are a group of unique adult stem cells in gonads that act as important transmitters for genetic information. Donor GSCs have been used to produce offspring by transplantation in fisheries. In this study, we successfully isolated and enriched GSCs from the ovary, ovotestis, and testis of Monopterus albus, one of the most important breeding freshwater fishes in China. Transcriptome comparison assay suggests that a distinct molecular signature exists in each type of GSC, and that different signaling activities are required for the maintenance of distinct GSCs. Functional analysis shows that fGSCs can successfully colonize and contribute to the germline cell lineage of a host zebrafish gonad after transplantation. Finally, we describe a simple feeder-free method for the isolation and enrichment of GSCs that can contribute to the germline cell lineage of zebrafish embryos and generate the germline chimeras after transplantation.

Sinhcaf-dependent histone deacetylation is essential for primordial germ cell specification.

Primordial germ cells (PGCs) are the progenitor cells that give rise to sperm and eggs. Sinhcaf is a recently identified subunit of the Sin3 histone deacetylase complex (SIN3A-HDAC). Here, we provide evidence that Sinhcaf-dependent histone deacetylation is essential for germ plasm aggregation and primordial germ cell specification. Specifically, maternal-zygotic sinhcaf zebrafish mutants exhibit germ plasm aggregation defects, decreased PGC abundance and male-biased sex ratio, which can be rescued by re-expressing sinhcaf. Overexpression of sinhcaf results in excess PGCs and a female-biased sex ratio. Sinhcaf binds to the promoter region of kif26ab. Loss of sinhcaf epigenetically switches off kif26ab expression by increasing histone 3 acetylation in the promoter region. Injection of kif26ab mRNA could partially rescue the germ plasm aggregation defects in sinhcaf mutant embryos. Taken together, we demonstrate a role of Sinhcaf in germ plasm aggregation and PGC specialization that is mediated by regulating the histone acetylation status of the kif26ab promoter to activate its transcription. Our findings provide novel insights into the function and regulatory mechanisms of Sinhcaf-mediated histone deacetylation in PGC specification.

Complete Depletion of Primordial Germ Cells Results in Masculinization of Monopterus albus, a Protogynous Hermaphroditic Fish.

Primordial germ cells (PGCs) play an important role in sexual fate determination and gonadal development in gonochoristic fish, such as zebrafish and medaka. However, little is known about the function of PGCs in hermaphroditic fish. Rice field eel (Monopterus albus), a protogynous hermaphroditic fish, is an economically valuable aquaculture species. We eliminated PGCs in rice field eels during embryogenesis via morpholino-mediated knockdown dead end (dnd). The PGCs-depleted gonads developed into testis-like structures with Sertoli cells and Leydig cells. The gene expression pattern of 15-month-old PGCs-depleted gonads showed that male-biased genes, dmrt1, sox9a, gsdf, and amh, were significantly higher than that of the WT, whereas female-biased genes, foxl2 and cyp19a1a, were significantly decreased. These results indicate that PGCs are essential for ovarian differentiation in rice field eel, and PGCs-depleted gonads develop into sterile males without undergoing the female and intersex stages. Our study is the first to identify the role of PGCs in sex differentiation in rice field eel, a protogynous hermaphrodite teleost. And it is of great significance in rice field eel for discovering the underlying mechanism of sex differentiation and establishing sex control technology.

Dynamics Analysis and Design for a Bidirectional Super-Ring-Shaped Neural Network With n Neurons and Multiple Delays.

Recently, the dynamics of delayed neural networks has always incurred the widespread concern of scholars. However, they are mostly confined to some simplified neural networks, which are only made up of a small amount of neurons. The main cause is that it is difficult to decompose and analyze generally high-dimensional characteristic matrices. In this article, for the first time, we can solve the computing issues of high-dimensional eigenmatrix by employing the formula of Coates flow graph, and the dynamics is considered for a bidirectional neural network with super-ring structure and multiple delays. Under certain circumstances, the characteristic equation of the linearized network can be transformed into the equation with integration element. By analyzing the equation, we find that the self-feedback coefficient and the delays have significant effects on the stability and Hopf bifurcation of the network. Then, we achieve some sufficient conditions of the stability and Hopf bifurcation on the network. Furthermore, the obtained conclusions are applied to design a standardized high-dimensional network with bidirectional ring structure, and the scale of the standardized high-dimensional network can be easily extended or reduced. Afterward, we propose some designing schemes to expand and reduce the dimension of the standardized high-dimensional network. Finally, the results of theories are coincident with that of experiments.

Associations Between Oral Health Status, Perceived Stress, and Neuropsychiatric Symptoms Among Community Individuals With Alzheimer's Disease: A Mediation Analysis.

Community individuals with Alzheimer's disease (AD) experience oral disease alongside neuropsychiatric symptoms (NPS) with disease progression. Despite growing evidence for the link between oral health and cognitive status, few studies have investigated the associations between oral health and NPS, especially based on individuals' experience of AD. The primary aim of this study was to examine (a) the difference in oral health-related stressors among individuals with AD, mild cognitive impairment (MCI), and subjective cognitive decline (SCD); and (b) the associations of these stressors with NPS under the framework of the stress process model (SPM). A cross-sectional study was conducted among individuals diagnosed with AD (n = 35), MCI (n = 36) or SCD (n = 35), matched for age, sex education, and body mass index (BMI). Multiple regression and mediation model analyses were performed to explore predictors and their relationships with NPS based on the SPM. Data collection comprised four sections: (a) individual context; (b) oral health-related stressors, including dental caries, periodontal status, oral hygiene, the geriatric oral health assessment index (GOHAI), oral salivary microbiota, pro-inflammatory cytokines, and oral health behavior; (c) subjective stressors (i.e., perceived stress [PS]); and (d) NPS. Decayed, missing, and filled teeth (DMFT), missing teeth (MT), loss of attachment (LoA), plaque index (PLI), PS, oral health behavior, GOHAI, pro-inflammatory cytokines, and salivary bacterial composition were significantly different among the three groups; these parameters were poorer in the AD group than SCD and/or MCI group. LoA, PLI, PS, and pain or discomfort in the GOHAI were directly associated with NPS. PLI, LoA, and psychosocial function in the GOHAI indirectly affected NPS, and this relationship was mediated by PS. Individuals with AD reported greater oral health-related stressors. This study identifies direct and indirect associations linking oral health-related stressors and PS with NPS in individuals with AD. Our findings suggest that targeted dental care and oral-related stressor control may be valuable for managing NPS.

Dynamic Gene Expression and Alternative Splicing Events Demonstrate Co-Regulation of Testicular Differentiation and Maturation by the Brain and Gonad in Common Carp.

The common carp (Cyprinus carpio) accounts for approximately 10% of the annual freshwater aquaculture production and is an ideal model to study cyprinidae reproduction. Female common carp grow faster than the males; therefore, related research presents an opportunity with high application value. Although we have a detailed understanding of common carp's early gonadal differentiation process, information about genome-wide gene expression, regulation, and underlying molecular mechanisms during this process remain limited. Here, time-course data comprising six key stages during testicular differentiation and maturation were investigated to further understand the molecular mechanisms underlying the testicular development in cyprinid species. After integrating these time-series data sets, common carp genome, including 98,345 novel transcripts and 3,071 novel genes were re-annotated and precisely updated. Gene co-expression network analysis revealed that the ubiquitin-mediated proteolysis pathway was essential for metabolism during testicular differentiation in the endocrine system of C. carpio. Functional enrichment analyses indicated that genes mainly related to amino acid metabolism and steroid hormone synthesis were relatively highly expressed at the testicular undifferentiation stages, whereas genes associated with cell cycle and meiosis were expressed from the beginning of testicular differentiation until maturation. The dynamics of alternative splicing events demonstrated that exon skipping accounted for majority of the alternative splicing events in the testis and the brain during gonad development. Notably, several potential male-specific genes (fanci and sox30) and brain-specific genes (oxt, gad2, and tac1, etc.) were identified. Importantly, we traversed beyond the level of transcription to test for stage- and gonad-specific alternative splicing patterns between the brain and testis. This study is the first to describe a comprehensive landscape of alternative splicing events and gene expression patterns during gonadogenesis in common carp. This work is extremely valuable to elucidate the mechanisms underlying gonadal differentiation in Cyprinidae as well as other fish species.

Multiple Developmental Defects in sox11a Mutant Zebrafish with Features of Coffin-Siris Syndrome.

A previous study suggested that human Coffin-Siris syndrome is related to the mutation of SOX11. Since the homozygous SOX11 mutant mice died soon after birth, no suitable model was available for the study of the pathogenic mechanism of Coffin-Siris syndrome. To solve this problem, we generated two viable homozygous zebrafish mutants, sox11am/m and sox11bm/m . We found that the sox11am/m mutant possessed Coffin-Siris syndrome features. The sox11am/m mutants exhibited growth deficiency from 3.3 hpf embryos to adulthood. Furthermore, the sox11am/m mutant also displayed microcephaly, narrow pupillary distance, achondroplasia, and bone deformity in adults. Growth deficiency could be rescued by the injection of sox11a mRNA at the one-cell stage. In addition, the expression levels of genes related to cartilage and bone were downregulated in the sox11am/m mutant, indicating that sox11a mainly affected the growth and development of zebrafish by regulating the expression of genes related to skeletal development. Our results indicate that sox11am/m mutant zebrafish offered a potential model system to help with the search for pathogenic mechanisms of human Coffin-Siris syndrome.

Secretoneurin is a secretogranin-2 derived hormonal peptide in vertebrate neuroendocrine systems.

Secretogranin-2 (SCG2) is a large precursor protein that is processed into several potentially bioactive peptides, with the 30-43 amino acid central domain called secretoneurin (SN) being clearly evolutionary conserved in vertebrates. Secretoneurin exerts a diverse array of biological functions including regulating nervous, endocrine, and immune systems in part due to its wide tissue distribution. Expressed in some neuroendocrine neurons and pituitary cells, SN is a stimulator of the synthesis and release of luteinizing hormone from both goldfish pituitary cells and the mouse LßT2 cell line. Neuroendocrine, paracrine and autocrine signaling pathways for the stimulation of luteinizing hormone release indicate hormone-like activities to regulate reproduction. Mutation of the scg2a and scg2b genes using TALENs in zebrafish reduces sexual behavior, ovulation, oviposition, and fertility. A single injection of the SNa peptide enhanced reproductive outcomes in scg2a/scg2b double mutant zebrafish. Evidence in goldfish suggests a new role for SN to stimulate food intake by actions on other feeding-related neuropeptides. Expression and regulation of the Scg2a precursor mRNA in goldfish gut also supports a role in feeding. In rodent models, SN has trophic-like properties promoting both neuroprotection and neuronal plasticity and has chemoattractant properties that regulate neuroinflammation. Data obtained from several cellular models suggest that SN binds to and activates a G-protein coupled receptor (GPCR), but a bona fide SN receptor protein needs to be identified. Other signaling pathways for SN have been reported which provides alternatives to the GPCR hypothesis. These include AMP-activated protein kinase (AMPK), extracellular signal-regulated kinases (ERK), mitogen-activated protein kinase (MAPK)and calcium/calmodulin-dependent protein kinase II in cardiomyocytes, phosphatidylinositol 3-kinase (PI3K) and Akt/Protein Kinase B (AKT, and MAPK in endothelial cells and Janus kinase 2/signal transducer and activator of transcription protein (JAK2-STAT) signaling in neurons. Some studies in cardiac cells provide evidence for cellular internalization of SN by an unknown mechanism. Many of the biological functions of SN remain to be fully characterized, which could lead to new and exciting applications.

Kisspeptin2 regulates hormone expression in female zebrafish (Danio rerio) pituitary.

Kisspeptin2 is a neuropeptide widely found in the brain and multiple peripheral tissues in the zebrafish. The pituitary is the center of synthesis and secretes various endocrine hormones. However, Kiss2 innervation in the zebrafish pituitary is unknown. In this study, the organization of Kiss2 cells and structures in the zebrafish pituitary by promoter-driving mCherry-labeling Kiss2 neurons were investigated. Kiss2 neurons in the hypothalamus do not project into the pituitary. Kiss2 cells are found in the female pituitary. Unidentified Kiss2 cells and extensions are located in the proximal pars distalis (PPD), similar to the distribution of Gnrh3 fibers. Kiss2 structures reside alongside Gnrh3 fibers. No Kiss2 structures are found in the male pituitary. The transcriptional expression of the kisspeptin receptor kiss1rb is detected in both female and male pituitaries. In situ hybridization shows that kiss1rb-positive cells are located in the PPD and pars intermedia (PI). In vitro Kiss2-10 treatment stimulates Akt and Erk phosphorylation and significantly induces lhß, fshß, and prl1 mRNA expression in the female pituitary. The results in this study suggest that Kiss2 and Kiss1rb may form an independent paracrine or autocrine system in the female zebrafish pituitary. Kiss2 and Kiss1rb signaling regulates the expression of pituitary hormones.

Targeted mutation of secretogranin-2 disrupts sexual behavior and reproduction in zebrafish.

The luteinizing hormone surge is essential for fertility as it triggers ovulation in females and sperm release in males. We previously reported that secretoneurin-a, a neuropeptide derived from the processing of secretogranin-2a (Scg2a), stimulates luteinizing hormone release, suggesting a role in reproduction. Here we provide evidence that mutation of the scg2a and scg2b genes using TALENs in zebrafish reduces sexual behavior, ovulation, oviposition, and fertility. Large-scale spawning within-line crossings (n = 82 to 101) were conducted. Wild-type (WT) males paired with WT females successfully spawned in 62% of the breeding trials. Spawning success was reduced to 37% (P = 0.006), 44% (P = 0.0169), and 6% (P < 0.0001) for scg2a-/- , scg2b-/- , and scg2a-/-;scg2b-/- mutants, respectively. Comprehensive video analysis indicates that scg2a-/-;scg2b-/- mutation reduces all male courtship behaviors. Spawning success was 47% in saline-injected WT controls compared to 11% in saline-injected scg2a-/-;scg2b-/- double mutants. For these mutants, spawning success increased 3-fold following a single intraperitoneal (i.p.) injection of synthetic secretoneurin-a (P = 0.0403) and increased 3.5-fold with injection of human chorionic gonadotropin (hCG). Embryonic survival at 24 h remained on average lower in scg2a-/-;scg2b-/- fish compared to WT injected with secretoneurin-a (P < 0.001). Significant reductions in the expression of gonadotropin-releasing hormone 3 in the hypothalamus, and luteinizing hormone beta and glycoprotein alpha subunits in the pituitary provide evidence for disrupted hypothalamo-pituitary function in scg2a and scg2b mutant fish. Our results indicate that secretogranin-2 is required for optimal reproductive function and support the hypothesis that secretoneurin is a reproductive hormone.

A novel fluorescent probe based on naphthalimide for imaging nitroreductase (NTR) in bacteria and cells.

A nitroreductase (NTR) responsive fluorescent probe, Na-NO2, comprising p-nitrobenzyl as the unique recognition group and 1,8-naphthalimide as fluorophore, was synthesized. Na-NO2 showed remarkable fluorescence "turn-on" signal in the presence of NTR under DMSO/H2O (1:19, v/v) buffered with PBS (pH = 7) solution in the presence of NADH (300 µM). Furthermore, the probe has a low detection limit down to 3.4 ng/mL and it is very sensitive towards the NTR in Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), normal and tumor cells such as HL-7702, HepG-2 and MCF-7.

Gnrh3 Regulates PGC Proliferation and Sex Differentiation in Developing Zebrafish.

Gonadotropin-releasing hormone (Gnrh) plays important roles in reproduction by stimulating luteinizing hormone release, and subsequently ovulation and sperm release, ultimately controlling reproduction in many species. Here we report on a new role for this decapeptide. Surprisingly, Gnrh3-null zebrafish generated by CRISPR/Cas9 exhibited a male-biased sex ratio. After the dome stage, the number of primordial germ cells (PGCs) in gnrh3-/- fish was lower than that in wild-type, an effect that was partially rescued by gnrh3 overexpression. A terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) analysis revealed no detectable apoptosis of PGCs in gnrh3-/- embryos. Proliferating PGCs could be detected in wild-type embryos, while there was no detectable signal in gnrh3-/- embryos. Compared with wild type, the phosphorylation of AKT was not significantly different in gnrh3-/- embryos, but the phosphorylation of ERK1/2 decreased significantly. Treatment with a Gnrh analog (Alarelin) induced ERK1/2 phosphorylation and increased PGC numbers in both wild-type and gnrh3-/- embryos, and this was blocked by the MEK inhibitor PD0325901. The relative expression of sox9a, amh, and cyp11b were significantly upregulated, while cyp19a1a was significantly downregulated at 18 days post-fertilization in gnrh3-/- zebrafish. Taken together, these results indicate that Gnrh3 plays an important role in early sex differentiation by regulating the proliferation of PGCs through a MAPK-dependent path.

GSH-responsive anti-mitotic cell penetrating peptide-linked podophyllotoxin conjugate for improving water solubility and targeted synergistic drug delivery.

Podophyllotoxin (PPT) is a chemotherapeutic agent which has shown significant anti-cancer effects through inhibiting microtubule assembly. However, because of the poor water solubility and obvious side effects, PPT cannot be used in clinical cancer therapy. In order to solve these problems, a novel glutathione-responsive PPT conjugate has been synthesized in which PPT was linked to an anti-mitotic cell penetrating peptide (PRA) via a disulfide linkage. In particular, the as-prepared PPT-PRA conjugate can self-assemble into vesicle in water, furthermore, another anti-cancer drug (doxorubicin was chosen as an example) can be loaded in the vesicle for synergistic drug delivery. For better cancer cells targeting, the vesicle was then modified with folic acid (FA). The results indicated that the as-prepared FA modified drug-loaded vesicle not only could overcome the poor water solubility and side effects of PPT but also exhibited targeted toxicity and synergistic therapeutic effect.

Secretogranin-II plays a critical role in zebrafish neurovascular modeling.

Secretoneurin (SN) is a neuropeptide derived from specific proteolytic processing of the precursor secretogranin II (SgII). In zebrafish and other teleosts, there are two paralogs named sgIIa and sgIIb. Our results showed that neurons expressing sgIIb were aligned with central arteries in the hindbrain, demonstrating a close neurovascular association. Both sgIIb-/- and sgIIa-/-/sgIIb-/- mutant embryos were defective in hindbrain central artery development due to impairment of migration and proliferation of central artery cells. Further study revealed that sgIIb is non-cell autonomous and required for central artery development. Hindbrain arterial and venous network identities were not affected in sgIIb-/- mutant embryos, and the mRNA levels of Notch and VEGF pathway-related genes were not altered. However, the activation of MAPK and PI3K/AKT pathways was inhibited in sgIIb-/- mutant embryos. Reactivation of MAPK or PI3K/AKT in endothelial cells could partially rescue the central artery developmental defects in the sgIIb mutants. This study provides the first in vivo evidence that sgIIb plays a critical role in neurovascular modeling of the hindbrain. Targeting the SgII system may, therefore, represent a new avenue for the treatment of vascular defects in the central nervous system.

Growth Hormone Overexpression Disrupts Reproductive Status Through Actions on Leptin.

Growth and reproduction are closely related. Growth hormone (GH)-transgenic common carp exhibit accelerated growth and delayed reproductive development, which provides an amenable model to study hormone cross talk between the growth and reproductive axes. We analyzed the energy status and reproductive development in GH-transgenic common carp by using multi-tissue RNA sequencing, real-time-PCR, Western blotting, ELISA, immunofluorescence, and in vitro incubation. The expression of gys (glycogen synthase) and igfbp1 (insulin-like growth factor binding protein) as well as blood glucose concentrations are lower in GH-transgenic carp. Agrp1 (agouti-related protein 1) and sla (somatolactin a), which are related to appetite and lipid catabolism, are significantly higher in GH-transgenic carp. Low glucose content and increased appetite indicate disrupted metabolic and energy deprivation status in GH-transgenic carp. Meanwhile, the expression of genes, such as gnrhr2 (gonadotropin-releasing hormone receptor 2), gthα (gonadotropin hormone, alpha polypeptide), fshß (follicle stimulating hormone, beta polypeptide), lhß [luteinizing hormone, beta polypeptide] in the pituitary, cyp19a1a (aromatase A) in the gonad, and cyp19a1b (aromatase B) in the hypothalamus, are decreased in GH-transgenic carp. In contrast, pituitary gnih (gonadotropin inhibitory hormone), drd1 (dopamine receptor D1), drd3 (dopamine receptor D3), and drd4 (dopamine receptor D4) exhibit increased expression, which were associated with the retarded reproductive development. Leptin receptor mRNA was detected by fluorescence in situ hybridization in the pituitary including the pars intermedia and proximal pars distalis, suggesting a direct effect of leptin on LH. Recombinant carp Leptin protein was shown to stimulate pituitary gthα, fshß, lhß expression, and ovarian germinal vesicle breakdown in vitro. In addition to neuroendocrine factors, we suggest that reduced hepatic leptin signaling to the pituitary might be part of the response to overexpression of GH and the resulting delay in puberty onset.

Fam60al as a novel factor involved in reprogramming of somatic cell nuclear transfer in zebrafish (Danio rerio).

The main reason for abnormal development of cloned animals or embryos, and inefficient animal cloning, is a poor understanding of the reprogramming mechanism. To better comprehend reprogramming and subsequent generation of pluripotent stem cells, we must investigate factors related to reprogramming of somatic cells as nuclear donors. As we know, fam60al (family with sequence similarity 60, member A, like) is a coding gene only found in zebrafish and frog (Xenopus laevis) among vertebrates. However, until now, its functions have remained unknown. Here, we generated a zebrafish fam60al-/- mutant line using transcription activator-like effector nucleases (TALENs), and found that both nanog and klf4b expression significantly decreased while myca expression significantly increased in fam60al-/- mutant embryos. Concurrently, we also uncovered that in developmentally arrested embryos of somatic cell nuclear transfer, nanog, klf4b and myca expression was down-regulated, accompanying a decrease of fam60al expression. Interestingly, we identified a long noncoding RNA (lncRNA) of fam60al, named fam60al-AS, which negatively regulated fam60al by forming double-stranded RNA (dsRNA). RNase protection assay and real-time PCR confirmed these findings. Taken together, these results suggest that fam60al is a novel factor related to the reprogramming of somatic cell nuclear transfer in zebrafish, which is regulated by its reverse lncRNA.