Nutrient starvation affects expression of LC3 family at the feto-maternal interface during murine placentation

LC3 − the mammalian homolog of Atg8 − was found as autophagosome membrane binding protein in mammals and widely used as an autophagosomal marker. LC3A, B and C show different expression patterns in each tissue. The aim of this study was to reveal the differences of expression patterns among LC3 families in mouse placenta under normal condition and nutrient starving condition. LC3A and B were highly expressed in decidual cells. LC3A and B were increased in D14 compared with D12 and D16 in mouse placenta, while LC3C was decreased. Starvation induced increase in LC3B expression specifically. Immunohistochemistry showed different expression patterns among LC3A, B and C. LC3A expression in syncytiotrophoblast was vanished by starvation. The results of real time RT-PCR suggested differences between D12 and D16 in autophagic cascade induced by starvation. Taken together, this study suggests that autophagy could play a role in placental invasion system and that nutrient starvation affects LC3B expression.     

DNA methylation analysis on satellite I region in blastocysts obtained from somatic cell cloned cattle

Many observations have been made on cloned embryos and on adult clones by somatic cell nuclear transfer (SCNT), but it is still unclear whether the progeny of cloned animals is presenting normal epigenetic status. Here, in order to accumulate the information for evaluating the normality of cloned cattle, we analyzed the DNA methylation status on satellite I region in blastocysts obtained from cloned cattle. Embryos were produced by artificial insemination (AI) to non‐cloned or cloned dams using semen from non‐cloned or cloned sires. After 7 days of AI, embryos at blastocyst stage were collected by uterine flushing. The DNA methylation levels in embryos obtained by using semen and/or oocytes from cloned cattle were similar to those in in vivo embryos from non‐cloned cattle. In contrast, the DNA methylation levels in SCNT embryos were significantly higher (P < 0.01) than those in in vivo embryos from non‐cloned and cloned cattle, approximately similar to those in somatic cells used as donor cells. Thus, this study provides useful information that epigenetic status may be normal in the progeny of cloned cattle, suggesting the normality of germline cells in cloned cattle.     

Suppurative meningoencephalitis and perineuritis caused by Streptococcus gallolyticus in a Japanese Black calf

A 179-day-old calf, which was weak and stunted, showed neurological signs and was euthanized. Postmortem examination revealed extensive and severe cloudy area in the meninges, and pleural pneumonia. Gram-positive cocci were isolated from systemic organs. Biochemical and 16S rRNA gene sequence analyses identified the isolate as Streptococcus gallolyticus, and its subspecies was suggested to be gallolyticus (SGG). The isolate was classified as a novel sequence type (ST115) by the multilocus sequence typing scheme for SGG and showed susceptibility to penicillin, ampicillin, amoxicillin, florfenicol, sulfamethoxazole-trimethoprim, and chloramphenicol. Histopathologically, suppurative meningoencephalitis and perineuritis were detected. As SGG has been isolated solely from a cow with mastitis in Japan, this is the first SGG infection in a calf with suppurative meningoencephalitis and perineuritis in this country.     

Differentially methylated CpG sites related to fertility in Japanese Black bull spermatozoa: epigenetic biomarker candidates to predict sire conception rate

For semen suppliers, predicting the low fertility of service bull candidates before artificial insemination would help prevent economic loss; however, predicting bull fertility through in vitro assessment of semen is yet to be established. In the present study, we focused on the methylated CpG sites of sperm nuclear DNA and examined methylation levels to screen new biomarkers for predicting bull fertility. In frozen-thawed semen samples collected from Japanese Black bulls, for which the sire conception rate (SCR) was recorded, the methylation level of each CpG site was analyzed using human methylation microarray. According to regression analysis, 143 CpG sites related to SCR were significantly differentially methylated. Whole genome bisulfite sequence data were obtained from three semen samples and the differentially methylated regions (DMRs) that included the target CpG sites selected by human methylation microarray were confirmed. Using combined bisulfite restriction analysis, fertility-related methylation changes were detected in 10 DMRs. With the exception of one DMR, the methylation levels of these DMRs were significantly different between groups with high fertility (> 50%) and low fertility (< 40%). From multiple regression analysis of methylation levels and SCR, three DMRs were selected that could effectively predict bull fertility. We suggest that these fertility-related differences in spermatozoal methylation levels could be new epigenetic biomarkers for predicting bull fertility.     

Growth model for the endangered cyprinid fish Tribolodon nakamurai based on otolith analyses

A growth model for the endangered cyprinid fish Tribolodon nakamurai was derived following otolith analyses of 16 wild and 53 reared specimens. The asteriscus was the most appropriate to measure size among three otolith elements, and its height OH  mm was used as size index of otolith. Standard length L  cm was best back-calculated using the Gompertz model, L  = 70.0·exp[–exp{−0.553 (OH   –  2.73)}]. Translucent zones on the lapilli, analyzed from 5-year-old-reared fish, were regarded as winter slow-growing zones. The ages of 10 wild specimens of 37.0–48.1 cm standard length were calculated as 7–10 years by counting the translucent zones on the lapilli. Age t was best back-calculated using the allometry model, t  = 1.33· OH ^1.37. The growth trajectory of T.   nakamurai followed a slender S curve, three typical growth models, von Bertalanffy, Logistic and Gompertz, and Richards' model, which is a general formula of the above three, being fitted using the maximum likelihood method. The Gompertz model, L_t  = 60.2·exp[–exp{−0.258( t  − 4.68)}], was found by Akaike's information criterion (AIC) to be the statistically most acceptable growth model.     

Isolation and characterization of arylacetamide deacetylase in cynomolgus macaques

Arylacetamide deacetylase (AADAC), a microsomal serine esterase, hydrolyzes drugs, such as flutamide, phenacetin and rifampicin. Because AADAC has not been fully investigated at molecular levels in cynomolgus macaques, the non-human primate species widely used in drug metabolism studies, cynomolgus AADAC cDNA was isolated and characterized. The deduced amino acid sequence, highly homologous (92%) to human AADAC, was more closely clustered with human AADAC than the dog, rat or mouse ortholog in a phylogenetic tree. AADAC was flanked by AADACL2 and SUCNR1 in the cynomolgus and human genomes. Moreover, relatively abundant expression of AADAC mRNA was found in liver and jejunum, the drug-metabolizing organs, in cynomolgus macaques, similar to humans. The results suggest molecular similarities of AADAC between cynomolgus macaques and humans.     

An intracytoplasmic injection of deionized bovine serum albumin immediately after somatic cell nuclear transfer enhances full-term development of cloned mouse embryos

In mouse somatic cell nuclear transfer (SCNT), polyvinylpyrrolidone (PVP) is typically included in the nuclear donor injection medium. However, the cytotoxicity of PVP, which is injected into the cytoplasm of oocytes, has recently become a cause of concern. In the present study, we determined whether bovine serum albumin deionized with an ion-exchange resin treatment (d-BSA) was applicable to the nuclear donor injection medium in SCNT as an alternative to PVP. The results obtained showed that d-BSA introduced into the cytoplasm of an enucleated oocyte together with a donor nucleus significantly enhanced the rate of in vitro development of cloned embryos to the blastocyst stage compared with that of a conventional nuclear injection with PVP in SCNT. We also defined the enhancing effects of d-BSA on the blastocyst formation rate when d-BSA was injected into the cytoplasm of oocytes reconstructed using the fusion method with a hemagglutinating virus of Japan envelope before oocyte activation. Furthermore, immunofluorescence experiments revealed that the injected d-BSA increased the acetylation levels of histone H3 lysine 9 and histone H4 lysine 12 in cloned pronuclear (PN) and 2-cell embryos. The injection of d-BSA before oocyte activation also increased the production of cloned mouse offspring. These results suggested that intracytoplasmic injection of d-BSA into SCNT oocytes before oocyte activation was beneficial for enhancing the in vitro and in vivo development of mouse cloned embryos through epigenetic modifications to nuclear reprogramming.