Procedure for bovine ICSI, not sperm freeze-drying, impairs the function of the microtubule-organizing center

This study was designed to investigate whether freeze-dried (FD) bull spermatozoa maintained the function of the microtubule-organizing center (MTOC) after rehydration and intracytoplasmic sperm injection (ICSI). In a preliminary attempt, the cleavage and blastocyst formation rates in FD-ICSI zygotes (36 and 1%, respectively) were found to be considerably lower than those in control ICSI zygotes (67 and 21%, respectively) or in IVF zygotes (78 and 43%, respectively). An alkaline comet assay indicated that the DNA fragmentation index (length of comet tail % DNA liberated) was not significantly different between fresh and FD spermatozoa. In the main experiment, formation of sperm-asters in the FD-ICSI oocytes 7 h postinsemination occurred at a similar rate when compared with the control ICSI oocytes (41 vs. 49%). Among the oocytes exhibiting sperm aster formation, the extent of microtubule network assembly was comparable between the FD-ICSI and control ICSI groups. However, the MTOC of the ICSI oocytes was not as functional as that of IVF oocytes in terms of the aster formation rate (97%) and the fluorescent intensity of the microtubule network (2.0 folds). These results suggest that the freeze-drying process per se had no adverse effect on maintaining the MTOC function in bull spermatozoa.     

Challenging endeavour for preservation of freeze-dried mammalian spermatozoa

Freeze-drying (lyophilization) has been proposed as an alternative method for sperm preservation to overcome the disadvantages of the current cryopreservation method such as the high maintenance cost of frozen stocks, the problems associated with transportation of frozen materials and the potential risk of total loss of the frozen stock. Since freeze-dried spermatozoa after rehydration lose their motility, which is an essential requirement to complete physiological fertilization, a relatively difficult microinsemination technique must be applied to rehydrated spermatozoa. Theoretically, it has been supposed that freeze-dried spermatozoa could maintain their functions and abilities to interact with the oocyte cytoplasm after prolonged storage at refrigerator temperature. However, sufficient yield of transferable blastocysts and production of live offspring derived from freeze-dried sperm samples are still subjects to be challenged and overcome in large domestic species.     

Supplementation of heterologous complement induces anti-Thy-1.1 nephritis in the Mongolian gerbil (Meriones unguiculatus)

Anti-Thy-1.1 nephritis in the rat is a popular experimental model for mesangial proliferative glomerulonephritis (GN). This model is characterized by direct binding of anti-Thy-1.1 antibody with Thy-1.1 antigen expressed on mesangial cells (MCs) of glomeruli in the rat. A single injection of anti-rat thymocyte serum (ARTS) results in GN with proteinuria and extensive mesangiolysis. Development of mesangiolysis and proteinuria are complement-dependent. We previously demonstrated Thy-1.1 antigen, similar to the rat, in thymocytes, brain cells and MCs of the kidney in the Mongolian gerbil (MG). In this study, we attempted to develop a MG nephritis model, but an injection of ARTS did not induce GN. An additional injection of guinea pig serum as a complement after ARTS injection resulted in anti-Thy-1.1 nephritis in MG. Degeneration of MCs and neutrophil infiltration were observed 1 hr after GP serum injection. Mesangiolysis and fibrin exudation occurred 12 hr after the injection and MC proliferation was apparent 7 days after the injection. In the complement-dependent hemolytic test, MG serum could not hemolyze sheep erythrocytes. These results suggested low activity, or depletion of some factors, in complements of MG serum.     

Acoustoelastic birefringence effect in wood II: influence of texture anisotropy on the polarization direction of shear wave in wood

Ultrasonic shear waves were propagated through the breadth direction of a wood beam which was subjected to a bending load such that it was in a plane-stress state. The oscillation direction of the shear waves with respect to the wood beam axis was varied by rotating an ultrasonic sensor, and the relationship between the shear wave velocity and the oscillation direction was examined. The results indicate that when the oscillation direction of the shear wave corresponds to the tangential direction of the wood beam, the shear wave velocity decreases sharply and the relationship between shear wave velocity and rotation angle tends to become discontinuous. When the oscillation of the shear waves occurs in the anisotropic direction of the wood beam instead of in the direction of principal stress, the shear wave velocity exhibits a peak value. In addition, the polarization direction was found to correspond to the direction of anisotropy of the wood beam according to the theory of acoustoelastic birefringence with respect to plane stress. This indicates that when the acoustoelastic birefringence method is applied to stress measurement of wood, it is appropriate to align the oscillation direction of the shear wave with the principal axial direction of anisotropy in order to carry out ultrasonic measurement.     

Attempt at intracytoplasmic sperm injection of in vitro matured oocytes in common minke whales (Balaenoptera acutorostrata) captured during the Kushiro Coast Survey

The present study was conducted during the Kushiro Coast Survey in an attempt to produce common minke whale embryos. In Experiment 1, we attempted to determine the appropriate culture duration (30 or 40 h) for in vitro maturation (IVM) of immature oocytes using the Well of the Well method. In Experiment 2, and intracytoplasmic sperm injection (ICSI) was applied to matured oocytes from prepubertal and adult common minke whales after IVM culture (40 or 48 h), and then their embryonic development was assessed. In Experiment 1, the maturation rate of oocytes cultured for 40 h (30.4%) was significantly higher than that of oocytes cultured for 30 h (6.8%; P<0.01). In Experiment 2, a total of 35 and 46 immature oocytes derived from adult (n=2) and prepubertal (n=6) minke whales, respectively, were cultured for 40 or 48 h. The maturation rate in the oocytes from the adult whales (34.2%) tended to be higher than that of the oocytes from the prepubertal whales (19.6%), but there was no significant difference. Following ICSI, 3 out of the 10 inseminated and cultured oocytes from the adult whales cleaved (2-, 8-, and 16-cell stages); all of these oocytes had been matured for 40 in culture. However, these oocytes did not develop to further stages. Only one of the 6 oocytes derived from the prepubertal whales, IVM cultured for 40 h and inseminated, developed to the 4-cell stage. The present results indicate that a 40 h IVM culture produces significantly higher rates of in vitro maturation than a 30 h IVM culture for common minke whale oocytes. Following ICSI, some oocytes cleaved to the 16-cell stage, but no further development was observed.     

Significance of Mn and Fe for growth of coastal marine diatom <Emphasis Type="Italic">Thalassiosira weissflogii</Emphasis>

The significance of Mn and Fe for the growth of a coastal marine diatom Thalassiosira weissflogii was investigated by performing culture experiments containing macronutrients with either Mn or Fe, or both. Only the addition of both Mn and Fe induced the highest growth rates and maximal cell yields. Maximal growth was maintained in continuous culture media, which were repeatedly prepared by an inoculation of pre-culture and the addition of both Mn and Fe to the control culture medium containing macronutrients. In particular, it was found that the full growth recovery in Mn-sufficient medium (without added Fe) is accomplished by the addition of Fe even after several days’ incubation. On the contrary, there was no sufficient growth recovery by the addition of Mn after a long incubation time in Fe-sufficient medium but without additional Mn. These results suggest that T. weissflogii in Mn-sufficient waters retains the ability for full physiological recovery for a long time, probably resulting from the decrease in the oxidative stress of phytoplankton by the production of antioxidant enzyme Mn superoxide dismutase during a long incubation period.     

Involvement of programmed cell death in suppression of the number of root nodules formed in soybean induced by Bradyrhizobium infection

Legumes establish symbiosis with nitrogen-fixing rhizobia through root nodules to acquire nitrogen. Legumes control nodule number through systemic (autoregulation of nodulation) as well as local regulation. Moreover, plants defend themselves against bacteria and other pathogens through the induction of localized (localized acquired resistance) and systemic (SAR, systemic acquired resistance; ISR, induced systemic resistance) responses. Herein, we show that the number of root nodules is suppressed by programmed cell death (PCD), and is simultaneously controlled by SAR and ISR in soybean (Glycine max [L.] Merr.). The wild-type soybean cultivar Williams 82 showed markedly fewer root nodule primordia and PCD symptoms, including accelerated DNA degradation, enhanced generation of reactive oxygen species (visualized by 3,3′-diaminobenzidine staining), and excessive cell death (detected on staining with trypan blue) compared to the hypernodulation mutant NOD1-3. These results suggest that PCD suppresses the formation of root nodules in wild-type soybean. In addition, microarray and gene ontology analyses showed that essential components of hypersensitive response (HR) or disease resistance, such as resistance (R) genes, mitogen-activated protein kinase cascade, SAR, salicylic acid, jasmonic acid, ethylene, etc., were activated in wild-type plants. These analyses corroborate the above findings, demonstrating that the suppression of root nodule formation by PCD is accompanied by HR, and is simultaneously controlled by SAR and ISR in soybean. These findings provide new insight into the control of nodulation to balance nutritional requirements and energy status in legumes.     

Fine thermal structure of a coronal active region

The determination of the fine thermal structure of the solar corona is fundamental to constraining the coronal heating mechanisms. The Hinode X-ray Telescope collected images of the solar corona in different passbands, thus providing temperature diagnostics through energy ratios. By combining different filters to optimize the signal-to-noise ratio, we observed a coronal active region in five filters, revealing a highly thermally structured corona: very fine structures in the core of the region and on a larger scale further away. We observed continuous thermal distribution along the coronal loops, as well as entangled structures, and variations of thermal structuring along the line of sight.