Embryo development after mitochondrial supplementation from induced pluripotent stem cells

Purpose

The purpose of this study was to evaluate the effects of mitochondrial supplementation (MS) on early embryonic development and to assess the safety of MS treatments using induced pluripotent stem cells (iPSCs) as the mitochondrial donor.

Methods

In this study, we evaluated the effect of MS on early embryonic development using induced pluripotent stem cells (iPSCs) as the donor. Mouse zygotes were injected with either mitochondria from iPSCs or a vehicle solution. Several parameters were evaluated, including the rates of blastocyst formation and implantation, the weight of E13.5 embryos and placentas, the distribution of the donor mitochondrial DNA (mtDNA), and the pattern of methylation in the differentially methylated regions (DMRs) of the H19 and Snrpn genes.

Results

We found that neither the rates of blastocyst formation and implantation nor the weights of E13.5 embryos and placentas were significantly different between the MS and control groups. Additionally, the mtDNA from the iPSC donors could be detected in the muscle tissue of four fetuses and all placentas in the MS group. Finally, the methylation patterns of H19 and Snrpn DMRs remained unchanged by MS.

Conclusions

iPSC-derived mtDNA was directly involved in the process of embryonic development after MS. No adverse effects were seen when using iPSCs as a mitochondrial donor, but it remains to be seen whether this method can improve embryonic development, especially in older mice.

     

Preparation of monoclonal antibodies against pseudorabies virus glycoprotein gC by adenovirus immunization alone or as a boost following DNA priming

The objective of the present study was to demonstrate the usefulness of recombinant adenoviral vector in the generation of monoclonal antibodies (MAb) against natural epitopes of proteins using the glycoprotein gC of pseudorabies virus (PRV) as the target antigen. The recombinant adenovirus expressing the glycoprotein gC (Ad-gC) was constructed according to the AdMax method. Three immunization protocols consisting of various combinations of intramuscular injection of Ad-gC and a plasmid DNA expressing gC (pcDNA-gC) were conducted in BALB/c mice at 2-week intervals. The two groups with the highest antibody levels (Ad-gC/Ad-gC and pcDNA-gC/pcDNA-gC/Ad-gC) were selected for fusion following a final protein boost. Nine MAbs against the glycoprotein gC of PRV were subsequently developed and characterized to be isotypes of IgG1, IgG2a, and IgG2b with ascitic titers ranging from 1:2 x 10(5) to 1:5 x 10(6). Immunofluorescence assay (IFA) and Western blotting analysis confirmed that these MAbs could recognize linear epitopes on the glycoprotein gC of PRV. Our results provide a new strategy for preparation of specific MAb against viral protein.     

The analysis of organization and diversity mechanism in goat immunoglobulin light chain gene loci

The genomic organization of goat immunoglobulin light chains (Igλ and Igκ) loci were annotated based on the goat genome database. The goat Igλ chain located on chromosome 17 contains at least 35 V_λ gene fragments (seven potential functional genes, one ORF and 27 pseudogenes), two J_λ-C_λ clusters arranged in a V_λ(35)-J_λ2-C_λ1-J_λ1-C_λ2 pattern, with another C_λ3 on scaffold. The Igκ locus included 11 Vκ (five potential functional genes, two ORFs and four pseudogene fragments), three J_κ genes and a single C_κ gene ordered in V_κ(35)-J_κ(3)-C_κ pattern on chromosome 11. By analyzing the clonies of Igλ and Igκ, we further found V_λ2 (26.23 %) & V_λ3 (73.11 %), V_κ2 (52.07 %) & V_κ4 (46.15 %) were predominately used in the expression of λ and κ chains respectively. λ chain showed more abundance in connective diversity than κ chain. Besides, somatic hypermutation with higher frequency in both immunoglobulin light chains was the major mechanism for the goat repertoire diversity. These results demonstrated goat immunoglobulin light chain variable region genome loci and repertoire diversity.     

Perfluorooctane sulfonate-induced testicular toxicity and differential testicular expression of estrogen receptor in male mice

Perfluorooctane sulfonate (PFOS, CAS#1763-23-1) causes male reproductive toxicities, but the underlying mechanisms are still unclear. In this study, 0, 0.5 and 10 mg/kg/day PFOS were given by oral gavage to adult mice for 5 weeks. In the 10 mg/kg group, serum testosterone levels decreased significantly. Sperm counts declined which might be associated with the decreased proliferation and increased apoptosis of germ cells. In relation to increased apoptosis, bax, cleaved caspase-9 and cleaved caspase-3 levels elevated significantly, indicating that PFOS induced germ cell apoptosis by activating the mitochondrial pathway. In addition, the increase in levels of testicular estrogen receptor (ER) β was observed in both 0.5 and 10 mg/kg group, whereas a decrease in ERα expression was only observed in 10 mg/kg group. These results suggested that the alterations in testicular ERs expression, together with decreased proliferation and increased apoptosis of germ cells, might be involved in PFOS-induced testicular toxicity.     

Involvement of the neurotensin receptor 1 in the behavioral effects of two neurotensin agonists,NT-2 and NT69L: Lack of hypothermic,antinociceptive and antipsychotic actions in receptor knockout mice

Neurotensin (NT) is a neuropeptide implicated in the pathophysiology of schizophrenia and in mediating the efficacy of antipsychotic drugs. NT is also involved in the regulation of body temperature and pain sensitivity. Using neurotensin receptor 1 (NTR1) knockout (KO) and wild-type (WT) mice, these studies evaluated the involvement of NTR1 in the behavioral responses produced by peripheral administration of NT agonists (NT-2 and NT69L). Animals were characterized in paradigms designed to assess hypothermia, antinociception, and antipsychotic-like effects. Under basal conditions, there were no phenotypic differences between NTR1 KO and WT mice. In WT mice, both NTR1 agonists decreased core body temperature (active doses in mg/kg, i.p., for NT-2 and NT69L, respectively: 1 and 3), increased tail withdrawal latencies (1 and 3), produced decreased spontaneous climbing (0.1, 0.3, 1 and 1, 3, 10) and reversed apomorphine-induced climbing (0.3, 1 and 1, 3). In contrast, none of the effects of either agonist were present in KO mice. These results suggest that NTR1: (1) does not play a major role in the control of basal thermoregulation, nociception or psychomotor stimulation in mice (barring possible developmental plasticity), (2) does mediate these behavioral responses to NT agonists, and (3) may play a role in the potential antipsychotic effects of these agonists.