Direct introduction of gene constructs into the pronucleus-like structure of cloned embryos: a new strategy for the generation of genetically modified pigs

Due to a rising demand of porcine models with complex genetic modifications for biomedical research, the approaches for their generation need to be adapted. In this study we describe the direct introduction of a gene construct into the pronucleus (PN)-like structure of cloned embryos as a novel strategy for the generation of genetically modified pigs, termed “nuclear injection”. To evaluate the reliability of this new strategy, the developmental ability of embryos in vitro and in vivo as well as the integration and expression efficiency of a transgene carrying green fluorescence protein (GFP) were examined. Eighty percent of the cloned pig embryos (633/787) exhibited a PN-like structure, which met the prerequisite to technically perform the new method. GFP fluorescence was observed in about half of the total blastocysts (21/40, 52.5%), which was comparable to classical zygote PN injection (28/41, 68.3%). In total, 478 cloned embryos injected with the GFP construct were transferred into 4 recipients and from one recipient 4 fetuses (day 68) were collected. In one of the fetuses which showed normal development, the integration of the transgene was confirmed by PCR in different tissues and organs from all three primary germ layers and placenta. The integration pattern of the transgene was mosaic (48 out of 84 single-cell colonies established from a kidney were positive for GFP DNA by PCR). Direct GFP fluorescence was observed macro- and microscopically in the fetus. Our novel strategy could be useful particularly for the generation of pigs with complex genetic modifications.     

Retinoblastoma protein promotes uterine epithelial cell cycle arrest and necroptosis for embryo invasion

Retinoblastoma protein (RB) encoded by Rb1 is a prominent inducer of cell cycle arrest (CCA). The hormone progesterone (P₄) promotes CCA in the uterine epithelium and previous studies indicated that P₄ activates RB by reducing the phosphorylated, inactive form of RB. Here, we show that embryo implantation is impaired in uterine‐specific Rb1 knockout mice. We observe persistent cell proliferation of the Rb1‐deficient uterine epithelium until embryo attachment, loss of epithelial necroptosis, and trophoblast phagocytosis, which correlates with subsequent embryo invasion failure, indicating that Rb1‐induced CCA and necroptosis of uterine epithelium are involved in embryo invasion. Pre‐implantation P₄ supplementation is sufficient to restore these defects and embryo invasion. In Rb1‐deficient uterine epithelial cells, TNFα‐primed necroptosis is impaired, which is rescued by the treatment with a CCA inducer thymidine or P₄ through the upregulation of TNF receptor type 2. TNFα is expressed in the luminal epithelium and the embryo at the embryo attachment site. These results provide evidence that uterine Rb1‐induced CCA is involved in TNFα‐primed epithelial necroptosis at the implantation site for successful embryo invasion.     

Excess RNA Editing in Rice Mitochondrial atp9 Transcripts

RNA editing for subunit 9 of ATPase from rice mitochondria wasexamined. C residues at eight positions in the genomic DNA wereconverted to T in the cDNA sequences. Excess RNA editing atnucleotide positions 216 and 254 was identified. The identicalpeptide sequence was deduced from 22 independent cDNA clones. ¹Permanent address: Faculty of Agriculture, Meiji UniversityKawasaki, 214 Japan.     

Three-dimensional reconstructions of haustoria in two parasitic plant species in the Orobanchaceae

Parasitic plants infect other plants by forming haustoria, specialized multicellular organs consisting of several cell types, each of which has unique morphological features and physiological roles associated with parasitism. Understanding the spatial organization of cell types is, therefore, of great importance in elucidating the functions of haustoria. Here, we report a three-dimensional (3-D) reconstruction of haustoria from two Orobanchaceae species, the obligate parasite Striga hermonthica infecting rice (Oryza sativa) and the facultative parasite Phtheirospermum japonicum infecting Arabidopsis (Arabidopsis thaliana). In addition, field-emission scanning electron microscopy observation revealed the presence of various cell types in haustoria. Our images reveal the spatial arrangements of multiple cell types inside haustoria and their interaction with host roots. The 3-D internal structures of haustoria highlight differences between the two parasites, particularly at the xylem connection site with the host. Our study provides cellular and structural insights into haustoria of S. hermonthica and P. japonicum and lays the foundation for understanding haustorium function.

Three-dimensional image reconstruction visualized the spatial organization of cell types in the haustoria of the Orobanchaceae parasitic plants Striga hermonthica and Phtheirospermum japonicum.     

Seasonal and diel changes in cetacean vocalizations monitored by passive acoustic methods in Nemuro Strait adjacent to the Shiretoko World Natural Heritage Site

UNESCO World Natural Heritage sites are established to ensure the long-term conservation of natural areas. Nemuro Strait in northern Japan is adjacent to the Shiretoko World Natural Heritage Site, and attracts various trophic levels of marine species, including marine mammals. Although the coexistence of humans and marine mammals is an important issue in this area, the temporal habitat use of cetaceans in this area is unknown. Here, we document seasonal and diel changes in cetacean vocalizations collected using passive acoustic recording devices during November 2012–March 2014. Killer whale calls occurred in spring and summer, and sperm whale clicks were detected in summer. Pacific white-sided dolphin calls were recorded in summer and late fall. No cetaceans were recorded during the sea ice period in February and March. The dolphin calls and unknown click trains were significantly more frequent at night. In contrast, marginal diel changes in killer whale calls were detected. Our results suggest that the majority of cetaceans utilize Nemuro Strait at night during the ice-free period, and we provide new insights into the habitat use and diversity of marine mammals in the Strait.     

Chloroplast aggregation during the cold-positioning response in the liverwort Marchantia polymorpha

Journal of Plant Research - Under low-light conditions, chloroplasts localize along periclinal cell walls at temperatures near 20 °C, but they localize along anticlinal cell walls near...     

Endogenous cytokines during a lethal infection with Listeria monocytogenes in mice

It has been demonstrated that endogenous cytokines including gamma interferon (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) play protective roles but that IL-4 and IL-10 play detrimental roles in nonlethal Listeria monocytogenes infection in mice. In this paper, we studied the roles of endogenous cytokines in a lethal infection with L. monocytogenes in mice. TNF-alpha and IL-6 titres in the bloodstreams, spleens and livers paralleled bacterial numbers in the organs, and both these cytokines and the bacterial numbers peaked just before the mice died. The high titres of TNF-alpha notably detected in the circulation in lethal infection were different from those in nonlethal infection. The maximum production of IFN-gamma was observed before the peaks of TNF-alpha and IL-6, and IFN-gamma almost disappeared from the bloodstreams and organs just before the mice died. No notable difference of IFN-gamma titres between lethal infection and nonlethal infection in the specimens obtained from mice was observed. IL-10 was also detected in the bloodstreams earlier than the peaks of TNF-alpha and IL-6 during lethal infection, while IL-4 was never detected in the sera. The administration of monoclonal antibodies (mAbs) against TNF-alpha, IFN-gamma, IL-6, IL-4 or IL-10 failed to rescue mice from lethal L. monocytogenes infection, whereas anti-TNF-alpha mAb and anti-IFN-gamma mAb prevented mice from lethality by high-dose endotoxin shock. These results suggest that lethality in L. monocytogenes infection might not be determined solely by these cytokines.