Synthesis, processing, and subcellular localization of mouse ADAM3 during spermatogenesis and epididymal sperm transport

To elucidate synthesis, processing, and subcellular localization of mouse ADAM3 (cyritestin) during spermatogenesis and epididymal sperm transport, we carried out immunoblotting and immunohistochemical analysis of testicular germ cells, and epididymal and vas deferens sperm, using affinity-purified anti-ADAM3 antibody. ADAM3 was initially synthesized as a 110-kDa precursor in round spermatids, and the precursor was then processed into a 42-kDa mature protein during the sperm transport into and/or once in the epididymis. The mature ADAM3 was localized on the anterior part of capacitated sperm heads and was rapidly removed from the head region during the calcium ionophore A23187-induced acrosome reaction. These results demonstrate that the mature form of ADAM3 is involved in the binding of sperm to the egg zona pellucida, not in the membrane fusion between sperm and egg.     

Effects of high potassium chloride supplementation on growth rate and renal function in mice

Fifty‐four mice were assigned to a control diet group or a KCl diet group to clarify the effects of KCl supplementation on growth rate, water intake and renal function in mice, and 5% of KCl was supplemented in KCl diets for 1–4 weeks. Bodyweights of KCl supplemented mice were significantly lower than those of control mice, and bodyweights of control and KCl supplemented mice at 28 days after treatment were 45.5 and 41.2 g, respectively. Feed intake was not affected by KCl supplementation, but water intake of KCl supplemented mice was significantly higher than that of control mice. Bone weights of KCl supplemented mice at 2 weeks after treatment were significantly lower than those of control mice. Serum urea nitrogen concentration at 4 weeks and serum K and Cl concentrations at 2 weeks were significantly lower in KCl supplemented mice. Histological alteration using hematoxylin–eosin and Sirius red staining was not found in the kidney of each mouse at 1, 2, and 4 weeks after treatment. These results suggest that high KCl supplementation decreases the rate of bodyweight gain and increases water intake in mice.     

Birth of normal offspring from mouse eggs activated by a phospholipase Czeta protein lacking three EF-hand domains

Sperm-specific phospholipase C, PLCzeta, is a candidate for the Ca(2+) oscillation-inducing factor that is introduced into the ooplasm upon sperm-egg fusion. In addition to the 647-residue full-length PLCzeta, s-PLCzeta lacking the N-terminal 110 amino acids is known to be present in the mouse testis. In this study, we attempted to obtain full-term offspring from s-PLCzeta-activated eggs by round spermatid injection. Metaphase II-arrested eggs injected with a high RNA concentration of s-PLCzeta RNA normally developed to blastocysts. When the round spermatid nucleus was injected into telophase II-stage eggs previously activated by s-PLCzeta RNA, three live offspring were successfully obtained by transfer of the developed 4-cell embryos to pseudopregnant mice. These three offspring all grew to be normal adults and reproduced healthy second-generation mice.     

The effects of migration on the immunity of Black-Headed Gulls (Chroicocephalus ridibundus: Laridae)

In order to elucidate the relationship between migration period and immunity related to susceptibility, we conducted research on Black-headed gulls (Chroicocephalus ridibundus). We captured 260 gulls and collected their peripheral blood. Their leukocyte (WBC) count, percentages of heterophils (Het) and lymphocytes (Lym), heterophil and lymphocyte ratio (H/L ratio), and CD4 and CD8α expression levels (CD4 and CD8α, respectively) were quantitatively analyzed over three migration periods (Autumn migration, Wintering, Spring migration). In Adult gulls, WBC counts and CD4 levels significantly increased. Moreover, the Het and H/L ratio decreased from the Autumn migration to Wintering. Conversely, only WBC counts and CD4 levels measurements significantly decreased from Wintering to Spring migration (P<0.05). The tested parameters of the Tokyo-bay population show a greater significant difference than the measurements of immunity of the Mikawa-bay population. This study suggests that the migratory period has a negative effect on an aspect of the immune system. Including the period-difference in the immune systems in the local population, it is necessary to investigate the relationship between the ecology of migratory birds and their immunity.     

Genetic variation and cross-reactivity of Clostridium septicum alpha-toxin

Clostridium septicum alpha-toxin genes were sequenced with the polymerase chain reaction (PCR) products amplified from DNAs of 25 C. septicum strains, and were classified into 10 patterns. Alpha-toxins were purified from the culture supernatant of four C. septicum strains (strains No. 44, Kagoshima 8, Mie and Tokachi) which were specially chosen from patterns of the deduced amino acid sequences. The molecular weights of the alpha-toxins were not different according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. However, the isoelectric points between the alpha-toxins of No. 44 and Tokachi strains differed markedly. Cross-neutralization tests were performed with purified alpha-toxins and antitoxins in mice and in Vero cells. Each antitoxin showed roughly the same titers against the four alpha-toxins in mice and completely identical titers against these in Vero cells. Calves immunized with toxoid prepared from the culture supernatant of No.44 strain were challenged by exposure to spores of Mie strain. The toxoid conferred protection against the challenge in calves. From these results, although genetic variation has been observed within the C. septicum alpha-toxin gene, C. septicum strains toxoid of strain No.44 induces protective immunity against exposure to C. septicum that produce other subtypes of alpha-toxin containing several different amino acid residues.     

QUANTITATIVE CONTRAST-ENHANCED ULTRASONOGRAPHY OF CANINE SPLEEN

Contrast-enhanced ultrasonography has an important role in the detection of tumors in humans. The second-generation contrast agent Sonazoid has the ability of real-time contrast imaging along with parenchymal imaging. The purposes of this study were to determine the effect and duration of Sonazoid on the changes in gray-scale enhancement of canine spleen and to establish an appropriate protocol for contrast-enhanced ultrasonography of canine spleen. Six healthy beagles were injected with an intravenous bolus of Sonazoid. In the spleen parenchyma, the enhancement was maintained up to 30 min after injection. Moreover, for 5–22 s after injection, gray-scale enhancement of splenic arteries afforded arterial imaging. Perfusion of the kidney may be investigated from 3.6 s to 3.5 min after injection of Sonazoid. These results suggest that Sonazoid is applicable to canine spleen parenchymal imaging and that the optimal time for the parenchymal imaging is 7–30 min after injection. The findings of this quantitative study should prove useful in the evaluation of diffuse or focal splenic and renal diseases in dogs.     

Plasma concentration of anti-diuretic hormone and urine volume in response to intraruminal administration of acetate, propinata and butyrate in suckling calves

Acetate, propionate, and butyrate were intraruminally administered to dry feed-fed suckling calves to evaluate their effects on plasma ketone bodies, anti-diuretic hormone (ADH) concentrations, and urine volume. Four male Holstein calves (5–7 weeks old) were given 1.0 L of warm water or 0.5 mole of one of the acids in 1.0 L of warm water. A 4 × 4 Latin square design was adopted for the experiment. The acetate group showed significantly higher plasma acetate concentrations than the other three groups between 0.25 h and 2.0 h after administration ( P  < 0.01). Plasma glucose concentrations did not differ markedly among the groups. The butyrate group showed significantly higher plasma ketone body concentrations than the other three groups until the end of the experiment ( P  < 0.01). Plasma ADH concentrations quickly rose in the butyrate group and remained significantly higher than in the other three groups from 0.25 h to 2.5 h after administration ( P  < 0.05). In accordance with the elevation of plasma ADH levels, the butyrate group showed decreases in urine volume and increases in urine osmolarity ( P  < 0.05). Plasma osmolarity and hematocrit values (Ht) were not different among the groups. These results suggest that the administration of acetate and propionate had little effect on ADH secretion.