Partial cloning of CYP2C23a genes and hepatic protein expression in eight representative avian species

Large interspecies differences in avian xenobiotic metabolism have been revealed by microsome‐based studies, but specific enzyme isoforms in different bird species have not yet been compared. We have previously shown that CYP2C23 genes are the most induced CYP isoforms in chicken liver. In this study, we collected partial CYP2C23a gene sequences from eight avian species (ostrich, blue‐eared pheasant, snowy owl, great‐horned owl, Chilean flamingo, peregrin falcon, Humboldt penguin, and black‐crowned night heron) selected to cover the whole avian lineage: Paleognathae, Galloanserae, and Neoaves. Genetic analysis showed that CYP2C23 genes of Galloanserae species (chicken and blue‐eared pheasant) had unique characteristics. We found some duplicated genes (CYP2C23a and CYP2C23b) and two missing amino acid residues in Galloanserae compared to the other two lineages. The genes have lower homology than in other avian lineages, which suggests Galloanserae‐specific rapid evolutionary changes. These genetic features suggested that the Galloanserae are not the most representative avian species, considering that the Neoaves comprise more than 95% of birds. Moreover, we succeeded in synthesizing an antipeptide polyclonal antibody against the region of CYP2C23 protein conserved in avians. However, comparative quantitation of CYP2C23 proteins in livers from six species showed that expression levels of these proteins differed no more than fourfold. Further study is needed to clarify the function of avian CYP2C23 proteins.     

Classification of the spermatogenic cycle,seasonal changes of seminiferous tubule morphology and estimation of the breeding season of the large Japanese field mouse (Apodemus speciosus) in Toyama and Aomori prefectures, Japan

The large Japanese field mouse, Apodemus speciosus, is a potential indicator of environmental stress, but this function has not been confirmed by histological studies. Since environmental stress affects the reproductive function of mice, we determined the reproductive characteristics of this species at two locations: Toyama (36°35ʹN, 137°24ʹE) and Aomori (40°35ʹN, 140°57ʹE). Mice were captured during May–November (n=119) and July–November (n=146) at these locations, respectively. We classified the breeding season from the numbers of pregnant females and young, in addition to the spermatogenic cycle and seasonal changes in seminiferous tubule morphology of males. Testicular weight was measured, and seminiferous tubule morphology was examined histologically. Fourteen stages were found in the seminiferous epithelium cycle based on acrosome formation and spermatid head morphology. At both locations, the breeding season peaked from late summer to early autumn and possibly in spring. Spermatogenic activity was classified into 4 periods from June to November: resting around June and October–November; resumptive around July; active around August; and degenerative around September. During the resting period, the seminiferous tubules consisted of Sertoli cells, spermatogonia and spermatocytes. Spermatogenesis began during the resumptive period, and spermatids were observed. During the active period, active spermatogenesis and a broad lumen were observed. During the degenerative period, spermatogenesis ended, and Sertoli cells, spermatogonia, spermatocytes and degenerating exfoliated round spermatids were observed. This study provides scientific information about the testicular histopathological evaluations of the large Japanese field mouse for its use as an index species of environmental pollution.     

Karnovsky’s fixative prevents artifacts appearing as vacuolation derived from tissue processing in kidneys treated with antisense oligonucleotide

Antisense oligonucleotide (ASO) therapies have been identified as a new treatment modality for intractable diseases. In kidneys treated with ASOs, vacuoles, in addition to basophilic granules, are often observed in the proximal tubules. Some reports have described that these vacuoles are likely to be a secondary phenomenon resulting from the extraction of ASOs during tissue processing. In this study, we compared renal morphology after fixation with Karnovsky’s fixative or 4% paraformaldehyde phosphate buffer (PFA) with that of 10% neutral-buffered formaldehyde solution (NBF). Female Sprague-Dawley rats, intravenously treated four times with 50 mg/kg locked nucleic acid containing antisense oligonucleotides (LNA-ASOs) for 1 or 2 weeks, were examined. Microscopically, vacuoles and basophilic granules in the proximal tubules were observed in the kidneys fixed with NBF. Basophilic granules are indicative of the accumulation of ASOs. Moreover, some of the vacuoles also contained faint basophilic granules, suggesting that the vacuoles were relevant to the accumulation of ASOs. Although moderate vacuolation was observed in the proximal tubules, the majority of the vacuolated epithelia were negative for kidney injury molecule-1 on immunohistochemical staining. Vacuoles in the proximal tubules were not observed in samples subjected to Karnovsky’s fixation, although basophilic granules were observed. In samples subjected to PFA fixation, vacuoles and basophilic granules were observed in the proximal tubules, similar to those in samples subjected to NBF fixation. Overall, our findings demonstrated the possibility of overestimation of vacuolation due to artifacts during tissue processing when using conventional NBF fixation. Karnovsky’s fixative is considered a useful alternative for distinguishing artificial vacuoles from true nephrotoxicity.     

Effect of suckling on the reproductive performance and metabolic status of obese Japanese black cattle during the early postpartum period

The aim of our study was to investigate the effects of suckling on reproductive performance and metabolic status of obese (mean body condition score of more than 4.0 on a scale of 1-5) maternal Japanese Black cows during early postpartum period. We used 7 postpartum Japanese Black cattle. Four cows were suckled ad libitum (suckled) until completion of their first artificial insemination (AI), while 3 cows were not suckled at all because they were separated from their calves immediately after parturition (non-suckled). Body weight and plasma concentrations of metabolites and hormones were measured from wk 1 to 9 postpartum. Ovarian activity was detected using plasma progesterone concentration, and all cows received their first AI after application of the Ovsynch protocol at approximately 4 months postpartum. Although body weights of non-suckled cows increased during experimental period (P<0.05), those of suckled cows remained unchanged. Plasma concentrations of glucose of non-suckled cows were higher at wk 2 postpartum (P<0.05) and their levels of non-esterified fatty acid tended to be lower at wk 1 and 2 postpartum compared with suckled cows (P<0.1); however, these differences between groups were not observed with progression of postpartum period. In addition, plasma insulin concentrations of non-suckled cows were higher than those of suckled cows during experimental period (P<0.05). During sampling period (wk 0 to 9 postpartum), onset of normal ovarian cycle was observed in all non-suckled and 2 of 4 suckled cows, and it was delayed in other 2 suckled cows compared with non-suckled cows; however, 3 suckled cows conceived at the first AI after application of the Ovsynch protocol; none of non-suckled cows conceived at this time. Overall, we suggest that suckling seems to reduce increase of body weight after parturition, although it does not improve obesity, and influences conception despite delay in resumption of normal ovarian cyclicity in obese Japanese Black cows.     

Parasexual recombination in Macrophomina pseudophaseolina and vegetative compatibility reactions in M. euphorbiicola

European Journal of Plant Pathology - Charcoal rot is an economically important fungal disease of economically important crops. Several species of the Macrophomina genus, such as M....