The Mechanism for Primordial Germ-Cell Migration Is Conserved between Japanese Eel and Zebrafish

Primordial germ cells (PGCs) are segregated and specified from somatic cells during early development. These cells arise elsewhere and have to migrate across the embryo to reach developing gonadal precursors. Several molecules associated with PGC migration (i.e. dead-end, nanos1, and cxcr4) are highly conserved across phylum boundaries. However, since cell migration is a complicated process that is regulated spatially and temporally by multiple adaptors and signal effectors, the process is unlikely to be explained by these known genes only. Indeed, it has been shown that there are variations in PGC migration pattern during development among teleost species. However, it is still unclear whether the actual mechanism of PGC migration is conserved among species. In this study, we studied the migration of PGCs in Japanese eel (Anguilla japonica) embryos and tested the migration mechanism between Japanese eel and zebrafish (Danio rerio) for conservation, by transplanting eel PGCs into zebrafish embryos. The experiments showed that eel PGCs can migrate toward the gonadal region of zebrafish embryos along with endogenous PGCs, even though the migration patterns, behaviors, and settlements of PGCs are somewhat different between these species. Our results demonstrate that the migration mechanism of PGCs during embryonic development is highly conserved between these two distantly related species (belonging to different teleost orders)

Short term non-invasive ventilation post-surgery improves arterial blood-gases in obese subjects compared to supplemental oxygen delivery - a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>In the immediate postoperative period, obese patients are more likely to exhibit hypoxaemia due to atelectasis and impaired respiratory mechanics, changes which can be attenuated by non-invasive ventilation (NIV). The aim of the study was to evaluate the duration of any effects of early initiation of short term pressure support NIV vs. traditional oxygen delivery via venturi mask in obese patients during their stay in the PACU.</p> <p>Methods</p> <p>After ethics committee approval and informed consent, we prospectively studied 60 obese patients (BMI 30-45) undergoing minor peripheral surgery. Half were randomly assigned to receive short term NIV during their PACU stay, while the others received routine treatment (supplemental oxygen via venturi mask). Premedication, general anaesthesia and respiratory settings were standardized. We measured arterial oxygen saturation by pulse oximetry and blood gas analysis on air breathing. Inspiratory and expiratory lung function was measured preoperatively (baseline) and at 10 min, 1 h, 2 h, 6 h and 24 h after extubation, with the patient supine, in a 30 degrees head-up position. The two groups were compared using repeated-measure analysis of variance (ANOVA) and t-test analysis. Statistical significance was considered to be P < 0.05.</p> <p>Results</p> <p>There were no differences at the first assessment. During the PACU stay, pulmonary function in the NIV group was significantly better than in the controls (p < 0.0001). Blood gases and the alveolar to arterial oxygen partial pressure difference were also better (p < 0.03), but with the addition that overall improvements are of questionable clinical relevance. These effects persisted for at least 24 hours after surgery (p < 0.05).</p> <p>Conclusion</p> <p>Early initiation of short term NIV during in the PACU promotes more rapid recovery of postoperative lung function and oxygenation in the obese. The effect lasted 24 hours after discontinuation of NIV. Patient selection is necessary in order to establish clinically relevant improvements.</p> <p>Trial Registration#</p> <p>DRKS00000751; <url>http://www.germanctr.de</url></p

Technical note: Viability and motility of vitrified/thawed primordial germ cell isolated from common carp (Cyprinus carpio) somite embryos

The feasibility of cryopreserving common carp (Cyprinus carpio) primordial germ cells (PGC) by vitrification of whole embryos at the 22 to 28 somite stage was investigated. Green fluorescent protein (GFP)-labeled PGC were cooled rapidly using liquid nitrogen after exposure to a pretreatment solution containing 1.5 M cryoprotectant (ethylene glycol or dimethyl sulfoxide, 30 or 50 min) and a vitrification solution containing 3 M cryoprotectant and 0.5 M sucrose (5, 10, 20, or 30 min). Embryonic cells that were pretreated for 30 min and vitrified for 20 min with ethylene glycol had the greatest rate of survival of embryonic cells (68.6%; P < 0.01), an optimal highest percentage of viable PGC (73.8% to 74.9%; P < 0.05), and no evidence of ice formation after thawing. The vitrified/ thawed PGC were transplanted into blastula-stage embryos from goldfish (Carassius auratus). The PGC maintained their motility and moved to the gonadal ridge of the host embryo. Thus, the combination of vitrification and transplantation to produce germ-line chimeras is a powerful tool for the artificial production of next-generation offspring

Vitrification of primordial germ cells using whole embryos for gene-banking in loach, Misgurnus anguillicaudatus

In gene-banking, primordial germ cells (PGCs), which are embryonic precursor cells of germ cells, are useful for cryopreservation because PGCs have a potential to differentiate into both eggs and sperm via germ-line chimera. Here, we have established vitrification methods for PGCs cryopreservation using 12- to 17-somite stage embryos in loach, Misgurnus anguillicaudatus, which were dechorionated, removed their yolk and injected with green fluorescent protein (GFP) -nos1 3'UTR mRNA to visualize their PGCs. In order to optimize cryopreservation medium for vitrification, the toxicity of cryoprotectants was analyzed. Different concentrations (2, 3, 4, 5 m) of dimethyl sulfoxide (DMSO), methanol (MeOH), ethylene glycol (EG) and propylene glycol (PG) as cryoprotectants were tested. Then, 5 m DMSO showed significantly-high toxicity. Based on this information, combinations called DMP (2 m (14.2% [v/v]) DMSO, 2 m (8.1% [v/v]) MeOH and 2 m (14.4% [v/v]) PG), DP (2 m (14.2% [v/v]) DMSO and 4 m (28.7% [v/v]) PG) and DE (2.1 m (15% [v/v]) DMSO and 2.7 m (15% [v/v]) EG) were evaluated for their toxicities and efficacy of PGCs cryopreservation using two types of equilibration step: direct immersion of cryopreservation media (one-step) and serial exposure to half and full concentration of cryopreservation media (two-step). Viable PGCs were obtained from post-thaw embryos which were cryopreserved by DP and DE with both 1- and 2-step equilibrations. Despite DP showing the highest toxicity, it gave the highest survival rate of embryonic cells after cryopreservation. When PGCs recovered from vitrified embryos were transplanted into host embryos at the blastula stage, the transplanted PGCs were able to migrate to a host genital ridge similarly as endogenous PGCs. It suggests that our methods could be useful to create a germ-line chimera for the production of gametes from PGCs of cryopreserved embryos.Biooriented Technology Research Advancement Institution (BRAIN) of JapanBio-oriented Technology Research Advancement Institution (BRAIN) of Japa

Sexual dimorphism of gonadal structure and gene expression in germ cell-deficient loach, a teleost fish

Germ cell-deficient fish usually develop as phenotypic males. Thus, the presence of germ cells is generally considered to be essential for female gonadal differentiation or the maintenance of ovarian structure. However, little is known of the role of germ cells in the determination of the sexual fate of gonadal somatic cells. We have established an inducible germ cell deficiency system in the loach (Misgurnus anguillicaudatus, Cypriniformes: Cobitidae), a small freshwater fish, using knockdown of the dead end gene with a morpholino antisense oligonucleotide. Interestingly, loach lacking germ cells could develop as either phenotypic males or females, as characterized morphologically by the presence or absence of bony plates in the pectoral fins, respectively. The phenotypic males and females had testicular and ovarian structures, respectively, but lacked germ cells. Gene expression patterns in these male and female germ cell-deficient gonads were essentially the same as those in gonads of normal fish. Our observations indicate that sexually dimorphic gonads can develop in germ cell-deficient loach. In contrast to the situation in other model fish species, the gonadal somatic cells in phenotypic females autonomously differentiated into ovarian tissues and also played a role in the maintenance of gonadal structure. On the basis of our observations, we propose two possible models to explain the role of germ cells in sex determination in fish