Leydig cell hyperplasia in an ENU-induced mutant mouse with germ cell depletion

Repro22 is an N-ethyl-N-nitrosourea (ENU)-induced mutation in mice showing depletion of both male and female germ cells. In the present study, we investigated the male phenotypes of the mutant mouse at the adult stage. The repro22/repro22 homozygous mice showed reduced body weights as well as markedly reduced testis weights. Histological examination of the testes at 4 and 10 months of age showed no germ cells in the seminiferous tubules of the affected testis while a number of Sertoli cells were observed in the tubules. In addition to the germ cell depletion, the testes of the affected mouse contained expanded intertubular spaces that were filled by Leydig cell-like interstitial cells. These interstitial cells were confirmed to be Leydig cells by immunohistochmical staining using anti-3beta-HSD antibody. The estimated number of Leydig cells in the affected testes at 10 months of age increased approximately 2 fold compared with those of normal testes. Furthermore, the plasma testosterone levels of the affected mice at 10 months of age were significantly higher than those of the normal mice. These findings indicated that the repro22/repro22 mouse developed hyperplasia of Leydig cells that was presumably caused by the absence of germ cells in the seminiferous tubules.     

Association of swine vertnin (VRTN) gene with production traits in Duroc pigs improved using a closed nucleus breeding system

Vertnin (VRTN) is involved in the variation of vertebral number in pigs and it is located on Sus scrofa chromosome 7. Vertebral number is related to body size in pigs, and many reports have suggested presence of an association between body length (BL) and meat production traits. Therefore, we analyzed the relationship between the VRTN genotype and the production and body composition traits in purebred Duroc pigs. Intramuscular fat content (IMF) in the Longissimus muscle was significantly associated with the VRTN genotype. The mean IMF of individuals with the wild‐type genotype (Wt/Wt) (5.22%) was greater than that of individuals with the Wt/Q (4.99%) and Q/Q genotypes (4.79%). In addition, a best linear unbiased predictor of multiple traits animal model showed that the Wt allele had a positive effect on the IMF breeding value. No associations were observed between the VRTN genotype and other production traits. The VRTN genotype was related to BL. The Q/Q genotype individuals (100.0 cm) were longer than individuals with the Wt/Q (99.5 cm) and Wt/Wt genotypes (98.9 cm). These results suggest that in addition to the maintenance of an appropriate backfat thickness value, VRTN has the potential to act as a genetic marker of IMF.     

The histopathogenesis of paralytic rabies in six-week-old C57BL/6J mice following inoculation of the CVS-11 strain into the right triceps surae muscle

A fatal encephalomyelitis was developed after intracerebral and hind limb inoculation of in 6-week-old C57BL/6J mice by the inoculation of fixed rabies virus (CVS-11 strain), intracerebrally and into hind. After the intracerebral inoculation, virus antigens were detected in the cerebral cortex and hippocampus at 2 days postinoculation (PI), and later spread centrifugally to thalamus, brain stem, cerebellum, spinal cord and spinal ganglia. At 4 days PI, severe apoptosis and DNA fragmentation were observed in the hippocampus and cerebral cortex. All mice infected intracerebrally were dead without limb paralysis at from 10 to 11 days PI. In contrast, mice infected with virus intramuscularly were persistently observed virus antigens in the myocytes at the site of inoculation from 2 days PI. At 4 days PI, the antigens were demonstrated in the spinal dorsal root ganglia, spinal cord and muscle spindles without their detection in the cerebrum and hippocampus. There were no apoptosis in the spinal cord and dorsal root ganglia, however hind limb paralysis was found in all infected mice. Hind limb paralysis was progressed to quadriparalysis, and mice were dead from 11 to 13 days PI. From 4 days PI, necrosis of neuron was observed in the the spinal and dorsal ganglia with infiltration of lymphocyte. This study suggested that the necrosis of spinal neurons was more important to cause the paralysis of hind limb rather than the severe cerebral infection and apoptosis in C57BL/6J mice infected with CVS-11 strain. The virus primarily replicated in the muscles was ascended the spinal cord via afferent fibers and retrogradely invaded the cerebrum, and with subsequent spread to muscle spindles.     

Stem productivity in relation to nitrogen concentration and carbon isotopic composition (δ13C) in leaves of hinoki cypress (Chamaecyparis obtusa Endlicher) plantations in Shikoku district,Japan

The stem productivity of the hinoki cypress (Chamaecyparis obtusa Endlicher) in relation to plant nitrogen status and water-use efficiency was investigated in the Okuono (OKU) and Karakawa (KRK) areas in Shikoku district, Japan, where abundant precipitation occurs. The nitrogen concentration and carbon isotopic composition (δ¹³C) in the leaves were used as indexes of plant nitrogen status and water-use efficiency, respectively. The leaf nitrogen concentration increased with decreasing soil carbon/nitrogen (C/N) ratio and with increasing soil pH. There was a marginally significant negative correlation between leaf δ¹³C and soil water content in the KRK area, but leaf δ¹³C in the OKU area did not correlate with the soil water condition, and increased on the upper slope. The results suggest that hinoki trees in the KRK area have higher water-use efficiency (high leaf δ¹³C) under lower soil water conditions. In the OKU area, meanwhile, leaf δ¹³C in the upper slope was higher due to adaptation to adverse conditions. When 12 plots in two areas were included, the mean height and stem increments increased with increasing leaf nitrogen concentration and with decreasing leaf δ¹³C. These findings suggest that nitrogen acquisition is a primary factor for stem productivity in the areas concerned but the productivity of some forests is restricted by the soil water condition or other conditions, as indicated by the high value of leaf δ¹³C. The measurement of nitrogen concentration and δ¹³C in leaves can provide us with valuable insights into the relative importance of nitrogen, water and other conditions on stem productivity in the two areas.     

Successful long term culture of immature porcine sertoli cells in the reconstructed testicular cell cord

The ultimate goal of this study was to establish an in vitro system to produce sperms. To pursue this goal, immature porcine testicular cells were cultured in stereostructural form and cultured testicular cord was investigated morphologically. At 4 weeks of age, the seminiferous tubules of the porcine testes consisted of undifferentiated germ cells (gonocytes and undifferentiated spermatogonia) and immature Sertoli cells. The interstitial tissue was largely occupied by Leydig cells. The testes were enzymatically digested, and the dispersed cells were encapsulated with alginate either immediately or after freeze-thawing. The resulting testicular cell cords were cultured for up to 10 weeks. After 2 weeks of culture, Sertoli cells, which were identified by their inhibin-positive reaction in immunohistochemistry, and Leydig cells, which were identified by their morphological characteristics, were observed in the cords. Neither undifferentiated nor differentiated types of germ cells were detected. The number of cells in the cords progressively decreased during the culture period. In order to discover the fate of the Sertoli cells, the level of inhibin in the spent media was determined. Inhibin in the media was at a detectable level after 2 days of culture. The levels increased and peaked at 2 weeks. When frozen-thawed testicular cells were applied to the culture, the peak level was maintained for over 8 weeks, in contrast to the gradual decrease of inhibin level when fresh cells were cultured. These results indicate that the culture conditions can sustain the survival of Sertoli cells. Further improvement is required for proliferation and differentiation of germ cells.     

Synthetic microRNA‐205 exhibited tumour suppression in spontaneous canine malignant melanoma by intratumoral injection

MicroRNAs (miRNA) are small, noncoding RNA molecules consisting of 18 to 25 nucleotides. Malignant melanomas (MMs) are one of the most common malignancies in both dogs and humans. We previously reported that chemically modified synthetic miRNA‐205 (miR‐205BP/S3) inhibits melanoma growth in vitro and in vivo. The present study aimed to evaluate the efficacy of intratumoral administration of synthetic miR‐205 for spontaneous CMMs and to evaluate its potential as systemic therapy. Ten dogs with various stages of MM were treated with miR‐205BP/S3 injected into tumours. Adverse effects (AEs) were assessed in accordance with the Veterinary Cooperative Oncology Group‐Common Terminology Criteria for Adverse Events (VCOG‐CTCAE) v1.1 guidelines. Five cases attained complete remission (CR), three attained stable disease (SD), and two cases displayed characteristics of progressive disease (PD). In all cases, no changes were observed in the blood parameters upon miRNA administration, and miR‐205BP/S3 administration did not yield any side effects. The present results suggest that intratumoral administration of miR‐205BP/S3 is a potentially applicable treatment for canine melanoma.