Establishment of a Conditional Transgenic System Using the 2A Peptide in the Female Mouse Germline

Transgenic mice are essential research tools in developmental biology studies. The 2A peptide allows multiple genes to be expressed simultaneously at comparable levels in somatic cells, but there are no reports of it being used successfully in germ cells. We constructed a Cre/loxP-based conditional vector containing the 2A peptide to significantly enhance the expression of a reporter and target gene from a constitutive promoter in oocytes. Mice with a transgene insertion containing the chicken β-actin promoter, floxed EGFP-polyA cassette, mCherry reporter, 2A peptide and target gene DNA methyltransferase 3A2 (Dnmt3a2) were crossed with TNAP- or Vasa-Cre mice to produce offspring, in which mCherry and DNMT3A2 proteins were highly expressed in oocytes upon Cre-mediated removal of EGFP-polyA. This novel transgenic mouse line based on the 2A expression system can serve as a useful tool for examining gene function during oogenesis.     

Sporadic endemicity of zoonotic Paragonimus in raccoon dogs and Japanese badgers from Miyazaki Prefecture,Japan

Paragonimiasis is a zoonotic trematode infection caused by Paragonimus spp. To determine the recent status of Paragonimus infections in wild animals, this study investigated Paragonimus spp. in 39 raccoon dogs and 54 Japanese badgers from March 2019 to January 2021 in Miyazaki Prefecture, and examined metacercariae in freshwater crabs. Triploid P. westermani was found in one raccoon dog (2.6%), and metacercariae were recovered from Eriocheir japonica captured near the infected animal collected. One Japanese badger (1.9%) harbored P. skrjabini miyazakii; this prevalence was lower than the approximately 30% that was reported in the 1970s. Results indicated that zoonotic Paragonimus was sporadically prevalent in wild animals. Further investigation in various animals is awaited to elucidate current wildlife reservoirs for those Paragonimus.     

Effects of testosterone on rat placental development

We investigated the morphological effects of testosterone on placental development in a rat model of polycystic ovarian syndrome (PCOS). Testosterone propionate (TP), which was subcutaneously administered to pregnant rats with 5 mg/animal from gestation day (GD) 14 to GD 18, induced a maternal weight reduction without mortality or clinical signs from GD 19 onwards. A decrease in fetal and placental weight, an increase in intrauterine growth retardation (IUGR) rates, and histological changes in the placenta were observed on GD 21 but not on GD15 or 17. Histopathologically, on GD 21, the trophoblast septa thickened, and the maternal sinusoids were narrowed in the labyrinth zone, resulting in a small placenta. Additionally, the placental weight, thickness, and histological morphology in the labyrinth zone on GD 21 in the TP-treated group were nearly identical to those on GD 17 in the control and TP-treated groups. Therefore, it was assumed that the testosterone-induced small placenta was induced in association with the developmental inhibition of the fetal part of the placentas from GD 17 onwards.     

Combination of serum phosphorylated neurofilament heavy subunit and hyperintensity of intramedullary T2W on magnetic resonance imaging provides better prognostic value of canine thoracolumbar intervertebral disc herniation

The aim of this study was to evaluate the prognostic value of concurrent measurement of serum phosphorylated neurofilament heavy subunit (pNF-H) concentration and intramedullary T2W hyperintensity in paraplegic to paraplegic dogs. Our hypothesis was that concurrent measurement of these would provide a more accurate prediction of functional outcome in dogs with thoracolumbar intervertebral disc herniation (IVDH). A prospective case-control clinical study was designed using 94 dogs with acute onset of thoracolumbar IVDH. The association of serum pNF-H concentration, T2W hyperintensity on sagittal MRI (T2H/L2), deep pain perception and surgical outcome were evaluated with logistic regression analysis after three months for all 94 surgically treated dogs. Sensitivity to predict non-ambulatory outcome was compared among pNF-H and T2H/L2 and combination of both. Logistic regression analysis indicated that serum pNF-H concentration and T2H/L2 were significantly correlated with surgical outcome (P<0.05); however, deep pain perception was not (P=0.41). The results of logistic regression analysis indicated that the odds ratios of unsuccessful long-term outcome were 2.6 for serum pNF-H concentration, 1.9 for T2H/L2 and 2.3 for deep pain sensation. The sensitivity and specificity to predict non-ambulatory outcome for using serum parameter pNF-H>2.6 ng/ml, using T2H/L2 value of>0.84 and using both serum pNF-H and T2H/L2, were 95% and 75.7%, 65% and 86.5%, and 90.0% and 97.5%, respectively. Therefore, combined measurements of serum pNF-H and T2H/L2 might be useful for predicting long-term outcome in dogs with thoracolumbar IVDH.     

Experimental Final Hosts of Metagonimus Hakubaensis (Trematoda: Heterophyidae) and Their Suitability to the Fluke

Seven laboratory mammal and bird species were orally inoculated with 200–1,000 encysted Metagonimus hakubaensis metacercariae that had been isolated from naturally infected lampreys (Lethenteron reissneri) captured in Aomori Prefecture. At 8 and 15 days post-infection, adult flukes were recovered from all of the laboratory animals tested, and therefore, hamster, rat, mouse, dog, cat, chicken and quail were considered as final hosts of M. hakubaensis. Recovery rates of the fluke were higher in dogs and hamsters than in cats, rats, mice, chickens and quails. The flukes recovered from dogs and hamsters showed increased body length and higher fecundity than those recovered from the other hosts. These results indicate that the suitability of dogs and hamsters for M. hakubaensis infection is higher than that of the other laboratory animals.     

A Comparison of the Histological Structure of the Placenta in Experimental Animals

The primary function of the placenta is to act as an interface between the dam and fetus. The anatomic structure of the chorioallantoic placenta in eutherian mammals varies between different animal species. The placental types in eutherian mammals are classified from various standpoints based on the gross shape, the histological structure of the materno-fetal interface, the type of materno-fetal interdigitation, etc. Particularly, the histological structure is generally considered one of the most useful and instructive classifications for functionally describing placental type. In this system, three main types are recognized according to the cell layers comprising the interhemal area: (1) epitheliochorial type (horses, pigs and ruminants), (2) endotheliochorial type (carnivores) and (3) hemochorial type (primates, rodents and rabbits). The number of cell layers in the interhemal area is considered to modify the transfer of nutrients between maternal and fetal blood and is one of the important factors with respect to the difference in placental permeability between animal species. Therefore, in reproductive and developmental toxicity studies, careful attention should be paid to the histological structure of the interhemal area when extrapolating information concerning placental transfer characteristics to different animal species.     

Epigenetic assessment of environmental chemicals detected in maternal peripheral and cord blood samples

Epigenetic alteration is an emerging paradigm underlying the long-term effects of chemicals on gene functions. Various chemicals, including organophosphate insecticides and heavy metals, have been detected in the human fetal environment. Epigenetics by DNA methylation and histone modifications, through dynamic chromatin remodeling, is a mechanism for genome stability and gene functions. To investigate whether such environmental chemicals may cause epigenetic alterations, we studied the effects of selected chemicals on morphological changes in heterochromatin and DNA methylation status in mouse ES cells (ESCs). Twenty-five chemicals, including organophosphate insecticides, heavy metals and their metabolites, were assessed for their effect on the epigenetic status of mouse ESCs by monitoring heterochromatin stained with 4￠,6-diamino-2-phenylindole (DAPI). The cells were surveyed after 48 or 96 h of exposure to the chemicals at the serum concentrations of cord blood. The candidates for epigenetic mutagens were examined for the effect on DNA methylation at genic regions. Of the 25 chemicals, five chemicals (diethyl phosphate (DEP), mercury (Hg), cotinine, selenium (Se) and octachlorodipropyl ether (S-421)) caused alterations in nuclear staining, suggesting that they affected heterochromatin conditions. Hg and Se caused aberrant DNA methylation at gene loci. Furthermore, DEP at 0.1 ppb caused irreversible heterochromatin changes in ESCs, and DEP-, Hg- and S-421-exposed cells also exhibited impaired formation of the embryoid body (EB), which is an in vitro model for early embryos. We established a system for assessment of epigenetic mutagens. We identified environmental chemicals that could have effects on the human fetus epigenetic status.     

Advanced maternal age induces fetal growth restriction through decreased placental inflammatory cytokine expression and immune cell accumulation in mice

Advanced maternal age is a risk factor for female infertility, and placental dysfunction is considered one of the causes of pregnancy complications. We investigated the effects of advanced maternal aging on pregnancy outcomes and placental senescence. Female pregnant mice were separated into three groups: young (3 months old), middle (8–9 months old), and aged (11–13 months old). Although the body weights of young and middle dams gradually increased during pregnancy, the body weight of aged dams only increased slightly. The placental weight and resorption rate were significantly higher, and live fetal weights were reduced in a maternal age-dependent manner. Although mRNA expression of senescence regulatory factors (p16 and p21) increased in the spleen of aged dams, mRNA expression of p16 did not change and that of p21 was reduced in the placenta of aged dams. Using a cytokine array of proteins extracted from placental tissues, the expression of various types of senescence-associated secretory phenotype (SASP) factors was decreased in aged dams compared with young and middle dams. The aged maternal placenta showed reduced immune cell accumulation compared with the young placenta. Our present results suggest that models using pregnant mice older than 8 months are more suitable for verifying older human pregnancies. These findings suggest that general cellular senescence programs may not be included in the placenta and that placental functions, including SASP production and immune cell accumulation, gradually decrease in a maternal age-dependent manner, resulting in a higher rate of pregnancy complications.