Affected homologous chromosome pairing and phosphorylation of testis specific histone, H2AX, in male meiosis under FKBP6 deficiency

A gene for FK506 binding protein 6 (Fkbp6) expresses during a specific stage of male and female meiosis. Disruption of the gene influences male reproduction, i.e. arrests spermatogenesis, but not female reproduction. Using the mouse model (targeted disruption), the role of the gene in homologous chromosome pairing has been demonstrated in a previous study. For further understanding the function of Fkbp6 in chromosome synapsis, we evaluated chromosome pairings during male meiosis in the as/as rat, a spontaneous null mutation, and compared them with those of the mouse model. Electron microscopy of the pachytene nuclei unveiled several types of abnormal chromosome pairing in the rat model, as shown in the mouse previously. The frequencies of aberrant pairings in the knockout mice and mutant rats were 42 of 67 nuclei (62.7%) and 20 out of 74 nuclei (27.0%), respectively. In order to clarify the mechanism of male specific infertility in Fkbp6 deficiency, the localization of gammaH2AX, a marker protein of XY chromosome inactivation during male meiosis, was examined. Immunostaining of gammaH2AX unveiled normal localization of the molecule to XY chromosomes (XY body) in both models, showing the independency of FKBP6 in sex chromosome inactivation. Besides the XY body, focal localization of gammaH2AX was observed in accordance with the unsynapsed chromosomes in both types of null animal. These results indicate the fundamental role of Fkbp6 in homologous chromosome synapsis during male meiosis. In conclusion, male specific infertility under Fkbp6 deficiency remains unsolved.     

Endocrine status and development of porcine testicular tissues in host mice

Clarification of the endocrine status of host mice provides us with basic knowledge with which we can manipulate the growth and function of xenografted testicular tissues. We investigated the hormonal profiles of castrated mice grafted with porcine immature testicular tissues from 30 to 210 or more days after grafting (day 0=castration and grafting). The serum follicle stimulating hormone (FSH) concentrations of the host mice declined (P<0.05) from day 60 compared with those of the castrated, ungrafted mice. The serum inhibin and testosterone levels were higher (P<0.05) than those in the castrated, ungrafted mice from days 30 and 90 days, respectively. The inhibin levels further increased (P<0.05) from day 90, during which time the levels were higher (P<0.05) than those in the intact male mice. In the grafts, formation of lumens occurred in the seminiferous cords on day 90 and spermatozoa appeared in the lumens from day 120. However, spermatogenesis in the grafts did not reach the qualitatively normal levels observed in adult boars. The intensity of the immune reaction to inhibin alpha subunits in the Sertoli cells of the grafts decreased with differentiation of the seminiferous tubules. The present findings indicate that a feedback loop was established between the mouse hypothalamo-pituitary axis and the grafted porcine tissues from 60 days post-grafting. The results also indicate that the serum inhibin levels in the host mice remained high even after the appearance of lumens in the seminiferous tubules of the grafted tissues; this is strikingly different to the situation in normal male animals, in which the serum inhibin levels decline at around the time of tubular differentiation. The lack of efferent ducts in the tubules of the grafted tissues probably caused the accumulation of inhibin to be released into the lumens, resulting in high concentrations of circulating inhibin. These high levels of inhibin may directly affect spermatogenic activity and suppress FSH secretion.     

Embryo collection from pigs post‐pseudopregnancy induced by estradiol dipropionate

We aimed to define whether embryo collection carried out after pseudopregnancy was of similar outcome and quality as after artificial abortion. To induce pseudopregnancy, 30 gilts or sows were given 20 mg intramuscular estradiol dipropionate (EDP) 10–11 days after the onset of estrus. Ten additional pigs were inseminated artificially at natural estrus as a control group. Prostaglandin F_2α (PGF_2α) was administered twice with a 24 hr interval beginning 15, 20, or 25 days after EDP‐treatment (n = 10 per group) or between 23 and 39 days after artificial insemination in control pigs. Following this, all pigs were given 1,000 IU equine chorionic gonadotropin and 500 IU human chorionic gonadotropin (hCG) and then inseminated. Embryos were recovered 6 or 7 days after hCG treatment and outcome was recorded. There was no significant difference in the number of normal embryos collected from the pigs with PGF_2α initiated at different time points or from the control group. Embryonic developmental stages 7 days after hCG treatment also did not differ among groups. These results indicate that the use of EDP to induce pseudopregnancy, followed by PGF_2α administration to synchronize estrus for subsequent embryo harvest, is a suitable alternative to the artificial abortion method.     

Molecular changes associated with adaptation of equine influenza H3N8 virus in embryonated chicken eggs

Embryonated chicken eggs (ECEs) are routinely used to isolate equine influenza virus. Propagation of the virus in ECEs results in selection of variants. In the present study, we determined nucleotide sequences of entire coding regions of parent A/equine/Tottori/1/07 (H3N8) and its derivatives that have different passage histories in ECE. After 12 passages, nucleotide sequence analysis predicted 3 amino acid substitutions in hemagglutinin (HA; 2 in HA1 and 1 in HA2). The two amino acid substitutions in HA1 were located in the vicinity of the cell receptor-binding site. Three other amino acid substitutions were predicted in internal proteins, 1 in the M1, 1 in the NP and 1 in the PA. This is the first report showing mutations in the internal protein genes of equine influenza virus associated with adaptation to ECE.     

Use of tween 20 as a substrate for assay of lipase activity in soils

Abstract

A method for assaying the soil lipase activity is described. It involves the titrimetric estimation of the amount of lauric acid released by the lipase activity when the soil is incubated with Tween 20 in the presence of toluene at 30°C for 18 h under agitation. The method is simple and precise and incubation without agitation is also possible. The method has been applied to six different kinds of soils. The lipase activity in the cultivated soils ranged from 22.5 to 75.5 mmol min^?1 g^?1 of dried soil. The K _m value for Tween 20 was 1.8 × 10^?4 m. The optimum pH was approximately 7.5. The hydrolysis of liveen 20 in soil was inhibited by glycerol which was the essential moiety of glyceride. The inhibition by glycerol was found to be competitive. These results indicate that Tween 20 is a potential substrate for the assay of the glyceride hydrolytic activity in soils.     

National-scale estimation of methane emission from paddy fields in Japan: Database construction and upscaling using a process-based biogeochemistry model

We have estimated methane (CH₄) emission from total rice (Oryza sativa L.) paddies in Japan by means of a process-based biogeochemistry model, DeNitrification-DeComposition(DNDC)-Rice, combined with a geographic information system (GIS) database of climate, soil and farming practices. In the GIS database, 2 million ha of rice paddies were divided into 17,408 units according to 136 climate areas, 16 soil types, four classes of drainage rate and two classes of groundwater level, to simulate CH₄ flux from each of the units applying the DNDC-Rice model. As a result, the national-scale CH₄ emission in 1990 was estimated to be 216 Gg carbon (C), 13% lower than a previous inventory estimated by the Tier 2 method. By our Tier 3 approach, a relatively higher CH₄ flux was estimated from eastern regions than from western regions of Japan, presumably due to the differences in climate and water management. Sensitivity analysis and uncertainty assessment indicated that it is important to account for the heterogeneity in soil properties such as field water capacity, iron (Fe) concentration and drainage rate, in order to reduce the uncertainty in regional estimates.     

Source of soil protease based on the splitting sites of a polypeptide

Abstract

Soil protease is an important enzyme in the nitrogen cycle which plays an essential role in the growth of various crops. We have attempted to identify a microbial source of soil protease. Selective soil incubation using antibiotics was suitable for a rough estimation of the groups of microorganisms responsible for the production of soil protease (Hayano and Watanabe 1990; Hayano 1993). Enumeration of proteolytic microorganisms in the field and analysis of their protease-producing ability enabled to evaluate the potential of various soil microorganisms for soil protease production (Watanabe and Hayano 1993a; Watanabe et al. 1994). For the accurate estimation of the soil protease source, the characteristics of the soil protease and microbial protease must be compared directly based on enzymatic properties as indices.