Intrauterine air impairs embryonic postimplantation development in mice.

OBJECTIVE: Although most embryologists load air bubbles into the catheter along with embryos during embryo transfer, the effects of these air bubbles on embryo transfer success rate are not clear. STUDY DESIGN: Air bubbles were nonsurgically injected into unilateral uterine horns of mice to demonstrate the negative effects of intrauterine air bubbles on embryonic development. RESULTS: Our data showed that when air bubbles are nonsurgically injected into unilateral uterine horns of pregnant 4days mice the litter size is significantly decreased. Four days after the introduction of air, abnormal decidua and dead conceptuses were detected in the uterine horns receiving the air bubbles. In addition, intrauterine air also significantly impaired murine embryo transfer success rates, and induced an increase in endometrial capillary permeability and decidualization in mice on day 4 of pseudopregnancy. These results strongly indicated that the air bubbles loaded into embryo transfer catheters to bracket the embryo-containing medium may have negative effect on embryonic implantation and development. CONCLUSIONS: Intrauterine air impaired murine embryonic postimplantation development, and this provided some clues for improving embryo transfer techniques in human.

Dry ice is a reliable substrate for the distribution of frozen mouse spermatozoa: A multi-centric study.

Disseminating mouse stocks as frozen materials offers both ethical and logistical advantages over live animal shipment, minimizing the welfare issues and avoiding some of the complex custom regulations that are associated with live animal transportation. Embryo freezing in liquid nitrogen (LN2) at -196 °C has traditionally been the method of choice for archiving mouse lines. However, spermatozoa freezing is emerging as a more convenient alternative due to the application of innovative cryopreservation and recovery protocols. In addition, frozen spermatozoa are less sensitive to post-freezing temperature fluctuations. Here we demonstrated that spermatozoa frozen using standard laboratory protocols can be safely stored in dry ice (-79 °C) for at least seven days. The protocol we report here is robust and has been validated in a multi-centric study involving mouse spermatozoa samples exchanged between five European Mouse Mutant Archive (EMMA) nodes. Furthermore, following shipment on dry ice the spermatozoa can be returned to LN2 for long term storage without any noticeable detrimental effect. This protocol permits frozen spermatozoa to be shared and shipped in dry ice between biorepositories, networks and scientific institutions at low cost, using common courier companies, while avoiding the complexities, risks and hazards associated with using a traditional LN2 dry-shipper.

HCV J6/JFH1 tilts the capability of myeloid-derived dendritic cells to favor the induction of immunosuppression and Th17-related inflammatory cytokines.

PURPOSE: How HCV virus affects the function of dendritic cells (DCs) and their ability to induce CD4+ T cell response remains not fully understood. This study was done to elucidate the impact of HCV on the function of DCs and on DC's capability to induce CD4+ T-cell response. METHODS: Monocyte-derived DCs (MoDCs) were treated with cell-culture HCV (HCVcc). The effects of HCVcc on DC maturation, CD40L-induced DC maturation, and cytokine production and the capacity of DCs to induce Th cytokine production of allogeneic CD4+ T cells were evaluated. RESULTS: HCVcc exposure increased expression of both IL-6 and IL-10 by MoDCs. HCV-exposed MoDCs also selectively facilitated allogeneic CD4+ T cells to further produce Th17-related cytokines interleukin 1 (IL-1), IL-6, and IL-17A. Pretreatment of IL-17A inhibited HCV production in Huh7.5 cells, suggesting that induction of Th17 cells may be beneficial to host anti-HCV immunity. Paradoxically, induction of IL-10 expression and the failure of HCV-exposed MoDCs to facilitate other Th cell development may hinder the anti-viral immunity. CONCLUSIONS: This study highlights both the therapeutic potential of IL-17A in treating HCV infection and the cautious consideration of HCV-induced immunosuppression in DC-based therapy.

Alanine scanning mutagenesis of hepatitis C virus E2 cysteine residues: Insights into E2 biogenesis and antigenicity.

Envelope glycoprotein 2 (E2) of hepatitis C virus contains 18 conserved cysteine (Cys) residues in its ectodomain. By cysteine-alanine mutagenesis and function analysis, six Cys in H77 E2 (C494, C508, C552, C564, C607 and C644) were found to be indispensable for recognition by conformation-dependent mAb H53. Removal of any of these Cys residues did not affect E2 heterodimerization with E1, but notably reduced E1E2 transmembrane transportation. These Cys together with C429 and C503 were required for conformation-dependent mAb H48 recognition. All of the above Cys except C607 were required for H77 and Con1 E2 binding to CD81. None of individual mutation of above Cys affected the ability of E2 to induce neutralizing antibodies in mice. Mouse antibodies mainly recognize E2 linear epitopes and are unrelated to epitopes recognized by human E2 antibodies. The findings provide new insights for understanding the biogenesis of functional HCV envelope proteins and HCV neutralizing immunity.

Conservation of mouse models through embryo freezing.

The ability to interrogate the entire coding sequence of the mouse combined with the tools to manipulate the genome has firmly established the mouse as the model organism of choice for studying the causes of human disease. Consequently, a huge number of novel mouse models are generated each year to support active research programs. However, it is neither ethically justifiable, nor economically viable to maintain mouse colonies on the shelf that are not part of active research programs. This means that novel mouse lines have to be preserved in some way. If this is not done and the line is simply killed off, the genetics will be lost to future generations of scientists. This article describes the current practices used in cryopreservation laboratories to archive and recover mouse embryos frozen using controlled-rate freezing and vitrification techniques.

In vitro fertilization in mice using the MBCD-GSH protocol.

Historically, timed mating of either naturally cycling or superovulated females has been the mainstay of pre-implantation embryo production. However, rising cage costs and the rapid expansion of biomedical research programs has necessitated the development of high-throughput approaches to mouse embryo production. In vitro fertilization (IVF) represents one such versatile tool offering many advantages to busy mouse facilities in terms of efficient use of space and resources. For example, strains can be taken off the shelf, frozen quickly as sperm, and recovered at a later date, small colonies can be rapidly expanded to meet demand, and IVF can be used to rescue strains that fail to breed or where the founder male is ill or has died suddenly. This article describes an IVF protocol currently used by many reproductive technologists to assist mouse biology programs.

Transporting mouse embryos and germplasm as frozen or unfrozen materials.

The 21st century has seen a huge proliferation in the availability of genetically altered mice. The availability of these resources has been accompanied by ever greater opportunities for international collaborations between laboratories involving the exchange of mouse strains. This exchange can involve significant costs in terms of animal welfare and transportation expenses. In an attempt to mitigate some of these costs, the mouse community has developed a battery of techniques that can be used to avoid transporting live mice. Transporting frozen embryos and sperm at liquid nitrogen (LN2 ) temperatures using dry shippers has been common practice for some time. However, current advances in this field have refined transportation procedures and introduced new techniques for disseminating embryos and sperm: for example, shipping frozen sperm on dry ice, exchanging unfrozen epididymides from which sperm can be extracted, and transporting frozen/thawed embryos in isotonic media. This article discusses some of the current practices used by laboratories to transport mouse strains around the world without having to exchange live mice.

Contemporary techniques for freezing mouse spermatozoa.

Each year, thousands of new mouse models are generated around the world to further biomedical research. Unfortunately, the cost of maintaining mouse colonies makes it uneconomical to keep strains on the shelf that are not part of active research programs. Ideally, these retired strains should be archived. If this is not done and the line is simply killed off, the genetics are lost to future generations of scientists. Traditionally, embryo freezing has been used to cryopreserve mice, but this is expensive, time consuming, requires large numbers of donor females, and usually involves invasive superovulation procedures. Sperm freezing circumvents all of these disadvantages and is rapidly becoming the technique of choice for many repositories. This has been made possible through the use of refined cryoprotective agents and the development of improved in vitro fertilization techniques. This article describes two popular sperm freezing techniques employed by mouse repositories to archive spermatozoa using cryoprotective agents supplemented with either L-glutamine or monothioglycerol.

The Arg719 residue at the C-terminal end of the stem region in hepatitis C virus JFH-1 E2 glycoprotein promotes viral infection.

Hepatitis C virus (HCV) envelope glycoprotein E2 is involved in virus assembly and initial entry into host cells. The tertiary organization of the E2 ectodomain is mainly composed of domains I-III, followed by the stem (ST) region and transmembrane (TM) domain. The ST region is critical for reorganizing the envelope glycoproteins during the membrane fusion process. While this region is relatively flexible, the physicochemical properties of its amino acid residues are conserved. Whether and how this physicochemical conservation is required for HCV infection is still unclear. The last residue of the E2 ST region evolved to be either an arginine or lysine among different HCV strains, suggesting that the residues confer different functions during HCV infection. To address this possibility, we constructed an R719K point mutant in the JFH-1 strain (genotype 2a) in the context of the cell-culture derived HCV (HCVcc) system. Compared with wild-type (wt) HCV, the R719K mutant exhibited decreased growth, and its extracellular and intracellular infectivity were also significantly decreased at 48 and 72 h post-electroporation. Correspondingly, less RNA and HCV core protein was observed in the supernatant for the R719K mutant, as well as less efficient RNA replication and protein expression. These findings indicate that the 719th residue of arginine on E2 is critical to promote HCV replication and infection. The data provide new clues for the biochemical function of E2, which is required for efficient HCV assembly and infection.

Overview of new developments in and the future of cryopreservation in the laboratory mouse.

The large-scale mutagenesis programmes underway around the world are generating thousands of novel GA mouse strains that need to be securely archived. In parallel with advances in mutagenesis, the procedures used to cryopreserve mouse stocks are being continually refined in order to keep pace with demand. Moreover, the construction of extensive research infrastructures for systematic phenotyping is fuelling demand for these novel strains of mice and new approaches to the distribution of frozen and unfrozen embryos and gametes are being developed in order to reduce the dependency on the transportation of live mice. This article highlights some contemporary techniques used to archive, rederive, and transport mouse strains around the world.

Three different functional microdomains in the hepatitis C virus hypervariable region 1 (HVR1) mediate entry and immune evasion.

High genetic heterogeneity is an important characteristic of hepatitis C virus (HCV) that contributes to its ability to establish persistent infection. The hypervariable region 1 (HVR1) that includes the first 27 amino acid residues of the E2 envelope glycoprotein is the most variable region within the HCV polyprotein. HVR1 plays a major role in both HCV cell entry and immune evasion, but the respective contribution of specific amino acid residues is still unclear. Our mutagenesis analyses of HCV pseudoparticles and cell culture-derived HCV using the H77 isolate indicate that five residues at positions 14, 15, and 25-27 mediate binding of the E2 protein to the scavenger receptor class B, type I receptor, and any residue herein is indispensable for HCV cell entry. The region spanning positions 16-24 contains the sole neutralizing epitope and is dispensable for HCV entry, but it is involved in heparan binding. More importantly, this region is necessary for the enhancement of HCV entry by high density lipoprotein and interferes with virus neutralization by E2-neutralizing antibodies. Residues at positions 1-13 are also dispensable for HCV entry, but they can affect HCV infectivity by modulating binding of the envelope protein to scavenger receptor class B, type I. Mutations occurring at this site may confer resistance to HVR1 antibodies. These findings further our understanding about the mechanisms of HCV cell entry and the significance of HVR1 variation in HCV immune evasion. They have major implications for the development of HCV entry inhibitors and prophylactic vaccines.

Significance of palmitoylation of CD81 on its association with tetraspanin-enriched microdomains and mediating hepatitis C virus cell entry.

CD81, a co-receptor for hepatitis C virus (HCV), is a member of the tetraspanin superfamily and is heavily palmitoylated in the juxtamembrane cysteine residues. Palmitoylation plays an important role in protein-protein interactions and association with cholesterol-rich domains of membranes. In this study, Huh7 cells expressing wild-type or palmitoylation-defective CD81 were generated to analyze whether palmitoylation of CD81 is involved in HCV cell entry. Our data showed that de-palmitoylation of CD81 dramatically reduced its association with tetraspanin CD151, but did not influence CD81 partition in detergent-resistant membranes. Moreover, de-palmitoylated CD81 decreased the host cell susceptibility to HCV. Notably, CD151-specific antibodies and siRNA inhibited HCV cell entry, and detachment of CD81 with CD151 decreased the lateral movement of virus particle/CD81 complex to areas of cell-cell contact. These results suggest that palmitoylation of CD81 should facilitate HCV entry, at least in part, by regulating the association of CD81 with tetraspanin-enriched microdomains.

Ultrasound enhanced methanol penetration of zebrafish (Danio rerio) embryos measured by permittivity changes using impedance spectroscopy.

Studies on permittivity changes in fish embryos measured by impedance spectroscopy after ultrasound treatment during exposure to cryoprotectant is reported here for the first time. The permittivity changes of zebrafish embryos in cryoprotectant solutions before and after ultrasound treatment were measured using impedance spectroscopy. Zebrafish (Danio rerio) embryos at 50% epiboly stage were exposed to 2 M methanol for 25 min before ultrasound treatment for 5 min at 22 degrees C. Embryos were treated with ultrasound in different frequencies (24 and 48 kHz) and voltages (50, 100, 150 and 175 V) combinations. The results showed a clear increasing trend of permittivity from voltage 50 to 175 V over lower impedance frequency range of 10-10(3) Hz indicating increased methanol penetration into the embryos after ultrasound treatment. The embryo survival was not compromised after ultrasound treatment under conditions used in the present study. The use of impedance spectroscopy technique provides a useful none-invasive tool for detecting changes of cryoprotectant penetration in fish embryos after ultrasound treatment. The technique is especially useful for the selection of the suitable cryoprotectants in embryo cryopreservation and may also allow quantitative measurements in embryo membrane permeability studies.

Cryopreservation of cynomolgus monkey (Macaca fascicularis) spermatozoa in a chemically defined extender.

AIM: To establish a method for cynomolgus monkey sperm cryopreservation in a chemically defined extender. METHODS: Semen samples were collected by electro-ejaculation from four sexually mature male cynomolgus monkeys. The spermatozoa were frozen in straws by liquid nitrogen vapor using egg-yolk-free Tes-Tris mTTE synthetic extender and glycerol as cryoprotectant. The effects of glycerol concentration (1 %, 3 %, 5 %, 10 % and 15 % [v/v]) and its equilibration time (10 min, 30 min, 60 min and 90 min) on post-thaw spermatozoa were examined by sperm motility and sperm head membrane integrity. RESULTS: The post-thaw motility and head membrane integrity of spermatozoa were significantly higher (P0.05) for 5 % glycerol (42.95 +/- 2.55 and 50.39+/- 2.42, respectively) than those of the other groups (1%: 19.19 +/- 3.22 and 24.84 +/- 3.64; 3%: 34.23 +/- 3.43 and 41.37 +/- 3.42; 10%:15.68 +/- 2.36 and 21.39 +/- 3.14; 15%: 7.47 +/- 1.44 and 12.90 +/- 2.18). The parameters for 30 min equilibration(42.95 +/- 2.55 and 50.39 +/- 2.42) were better (P0.05) than those of the other groups (10 min: 31.33 +/- 3.06 and 38.98 +/- 3.31; 60 min: 32.49 +/- 3.86 and 40.01 +/- 4.18; 90 min: 31.16 +/- 3.66 and 38.30 +/- 3.78). Five percent glycerol and 30 min equilibration yielded the highest post-thaw sperm motility and head membrane integrity. CONCLUSION: Cynomolgus monkey spermatozoa can be successfully cryopreserved in a chemically defined extender, which is related to the concentration and the equilibration time of glycerol.