Involvement of vascular endothelial growth factor in the formation of the thecal layer and vasculature during follicular development in the ovaries of neonatal female rats

Vascular endothelial growth factor (VEGF) is an important angiogenic factor in the ovary, but the localization of VEGF in the ovary of neonatal animals is poorly understood. A clear understanding of the relationship between the formation of the thecal layer and the cell‐specific expression of the VEGF system during follicular development in the neonatal ovary is still lacking. Immature female Wistar‐Imamichi rats used in this study were killed by decapitation 5, 7, 9 and 11 days after birth, and their ovaries were removed and subjected to histological and immunohistochemical observation. The number of primordial follicles had decreased in the ovaries at day 11 compared with that at day 5. The number of secondary follicles significantly increased with age. In the morphological observation of secondary follicles, we found that the theca layer (70 µm in diameter of follicles) began to form at day 9 and was completely formed at day 11. An endothelial cell marker, CD31, VEGF and Flk‐1 were located in the stromal tissues in the ovaries on each day examined after birth. In particular, in the ovaries at day 9 and day 11, when the secondary follicles appeared, CD31, VEGF and Flk‐1 were expressed in the theca layer. Flt‐1 was expressed in the oocytes of the ovaries at day 5 and day 7, and the sites of its expression changed to stromal and thecal tissues at day 9 and day 11. In conclusion, we provide the first evidence that the theca layer of secondary follicles begin to form at day 9 after birth and that VEGF and Flk‐1 may be able to stimulate the differentiation of stromal‐interstitial cells into thecal cells and the formation of the thecal vasculature in the neonatal rat ovaries, suggesting that the VEGF system may be involved in the formation of the thecal layer and vasculature during folliculogenesis in the neonatal rat.     

PDE4 and PDE5 regulate cyclic nucleotide contents and relaxing effects on carbachol-induced contraction in the bovine abomasum

The effects of various selective phosphodiesterase (PDE) inhibitors on carbachol (CCh)-induced contraction in the bovine abomasum were investigated. Various selective PDE inhibitors, vinpocetine (type 1), erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA, type 2), milrinone (type 3), Ro20-1724 (type 4), vardenafil (type 5), BRL-50481 (type 7) and BAY73-6691 (type 9), inhibited CCh-induced contractions in a concentration-dependent manner. Among the PDE inhibitors, Ro20-1724 and vardenafil induced more relaxation than the other inhibitors based on the data for the IC₅₀ or maximum relaxation. In smooth muscle of the bovine abomasum, we showed the expression of PDE4B, 4C, 4D and 5 by RT-PCR analysis. In the presence of CCh, Ro20-1724 increased the cAMP content, but not the cGMP content. By contrast, vardenafil increased the cGMP content, but not the cAMP content. These results suggest that Ro20-1724-induced relaxation was correlated with cAMP and that vardenafil-induced relaxation was correlated with cGMP in the bovine abomasum. In conclusion, PDE4 and PDE5 are the enzymes involved in regulation of the relaxation associated with cAMP and cGMP, respectively, in the bovine abomasum.     

Changes in the C-reactive protein and 13,14-dihydro-15-keto-prostaglandin F2α concentrations of uterine lavage samples after the administration of povidone-iodine in cows

Changes in the C-reactive protein (CRP) and 13,14-dihydro-15-keto-prostaglandin F_2α (PGFM) concentrations of uterine lavage fluid were examined in cows given an intrauterine povidone-iodine (PI) infusion. The mean polymorphonuclear leukocyte (PMN) ratios (the ratio of PMN to total cells) and CRP concentration of uterine lavage fluid on the day after the treatment were significantly (P<0.05) greater in the PI infusion group (PMN: 53.0 ± 32.7%, CRP: 50.2 ± 32.3 ng/mL) than in the non-treatment control group (PMN: 7.9 ± 21.9%, CRP: 17.2 ± 5.9 ng/mL), whereas there was no significant difference in the mean PGFM concentration between the two groups. The present findings suggest that the uterine CRP level is a useful biomarker of local uterine inflammation in cows.     

Examination of the microbiota of normal cow milk using MinIONTM nanopore sequencing

The aim of this study was to evaluate the microbiota of normal milk in dairy cows and their relationship with host factors, such as the age of the cow (Age), somatic cell counts in milk (SCCs), and days in milk (DIM). We investigated 48 milk samples from 22 cows with no systemic or local clinical signs using MinION^TM nanopore sequencing for a 16S rRNA gene amplicon. Bacterial richness was positively correlated with the DIM (P=0.043), and both the Shannon-Wiener Index and Simpson’s Index, which are metrics of alpha-diversity, were also significantly positively correlated with the SCC (P<0.001). The composition ratios of both Actinobacteria at the phylum level and Kocuria spp. at the genus level in the milk microbiota were significantly correlated with the SCC (P<0.001 and P<0.001, respectively). In the beta-diversity test, the one-way analysis of similarities test showed a significant difference (P=0.0051) between the low- and high-SCC groups. This study clarified that the composition of the normal milk microbiota in this herd was related to the SCC. It also raised the possibility of variations in bacterial genera in the normal milk microbiota between the low- and high-SCC groups. However, to clarify the actual condition of the milk microbiota and to elucidate the relationship with the SCC, it is necessary to perform further analyses taking into account not only the relative abundance, but also the absolute abundance of microbes.     

Removal of sperm tail using trypsin and pre-activation of oocyte facilitates intracytoplasmic sperm injection in mice and rats

We examined various methods to enhance the accessibility of intracytoplasmic sperm injection (ICSI) technology to more users by making the technique easier, more efficient, and practical. First, the methods for artificially removing the mouse sperm tail were evaluated. Trypsin treatment was found to efficiently remove the sperm tails. The resultant sperm cells had a lower oocyte activation capacity; however, the use of activated oocytes resulted in the same fecundity as that of fresh, untreated sperm. Pre-activated oocytes were more resistant to physical damage, showed higher survival rates, and required less time per injection. Testing this method in rats yielded similar results, although the oocyte activation method was different. Remarkably, this method resulted in higher birth rates of rat progeny than with conventional methods of rat ICSI. Our method thereby streamlines mouse and rat ICSI, making it more accessible to laboratories across many disciplines.     

Studies on Endophytic Actinomycetes ( I ) <Emphasis Type="Italic">Streptomyces </Emphasis>sp. Isolated from Rhododendron and Its Antifungal Activity

To survey endophytic actinomycetes as potential biocontrol agents against fungal diseases of rhododendron, young plants of rhododendron were surface-sterilized for use as an isolation source. Nine, six and two isolates, with distinguishing characteristics based on the macroscopic appearance of colonies, were obtained from roots, stems and leaves, respectively, suggesting that various species of actinomycetes grow in the respective organs of this plant as symbionts or parasites. On an agar medium, only isolate R-5 commonly formed a clear growth-inhibition zone against two major fungal pathogens of rhododendron, Phytophthora cinnamomi and Pestalotiopsis sydowiana, indicating that this isolate can produce antifungal material(s). Acetone extracts of a liquid culture of R-5 had a broad antimicrobial spectrum against Gram-positive bacteria, yeast and filamentous fungi. Isolate R-5 was identified as a Streptomyces sp. based on morphological, physiological and chemotaxonomical characteristics. The present results indicate that isolate R-5 is a suitable candidate for the biocontrol of diseases of rhododendron. Received 25 March 2000/ Accepted in revised form 18 May 2000