Cathepsin gene expression in mouse placenta during the latter half of pregnancy

Gene expressions and their interaction are complex and have not been definitely clarified in the placenta. To identify interactions of gene products previously not studied, we applied cDNA subtraction analyses to the placenta between days 12 and 16, days 12 and 14, days 14 and 16 of pregnancy. Among subtracted cDNAs cathepsin M, Q and R in PECs were specifically identified on days 14 and 16 pregnancy. All of these gene expressions exhibited a similar pattern to the mPL-II gene expression determined by northern blot and RT-PCR analyses. By means of in situ hybridization, these mRNAs were localized in the basal and labyrinth zones of the placenta on day 16 of pregnancy. Double staining studies of cathepsin Q or cathepsin R mRNA by in situ hybridization followed by immunohistochemical staining of mPL-II in the same section revealed that signals for cathepsin Q and cathepsin R mRNAs were colocalized in mPL-II immunopositive trophoblast cells in the basal and labyrinth zones of the placenta on day 16 of pregnancy. Possible association of cathepsins with mPL-II may play important roles in placental functions during the latter half of pregnancy in mice.     

Electroejaculation and semen cryopreservation of free-ranging Japanese black bears (Ursus thibetanus japonicus)

The Japanese black bear (Ursus thibetanus japonicus) is endangered for extinction in some areas of Japan, and semen collection and cryopreservation are an important means to preserve genetic resources. The aim of this study was to characterize and cryopreserve semen of free-ranging Japanese black bears. Semen was collected by electroejaculation procedure from 4 free-ranging Japanese black bears at the capture point in the field. Ejaculates containing motile sperm were recovered from all of the animals and ejaculate volume, total sperm count, % motility (percentage of motile spermatozoa), % viability (percentage of spermatozoa that excluded eosin) and % abnormal morphology (range (mean)) were 0.65-2.20 (1.51) ml, 99-1082 (490) x 10(6), 5-100 (31), 42-97 (66) and 20-87 (53), respectively. Three of the 4 ejaculates were diluted with an egg yolk-TRIS-citrate-glucose extender and cryopreserved in liquid nitrogen. Motile spermatozoa were observed after freezing and thawing in all cases. This study showed that electroejaculation would be a useful method for collecting semen from free-ranging Japanese black bears and that at least motile spermatozoa would be obtained by freezing the thus collected electroejaculates.     

Accessory corpora lutea formation in pregnant Hokkaido sika deer (Cervus nippon yesoensis) investigated by examination of ovarian dynamics and steroid hormone concentrations

Generally, sika deer conceive a single fetus, but approximately 80% of pregnant females have two corpora lutea (CLs). The function of the accessory CL (ACL) is unknown; moreover, the process of ACL formation is unclear, and understanding this is necessary to know its role. To elucidate the process of ACL formation, the ovarian dynamics of six adult Hokkaido sika deer females were examined ultrasonographically together with peripheral estradiol-17β and progesterone concentrations. ACLs formed in three females that conceived at the first estrus of the breeding season, but not in those females that conceived at the second estrus. After copulation, postconception ovulation of the dominant follicle of the first wave is induced by an increase in estradiol-17β, which leads to formation of an ACL. A relatively low concentration of progesterone after the first estrus of the breeding season is considered to be responsible for the increase in estradiol-17β after copulation.     

Quadruple or quintuple conversion of hlb, sak, sea (or sep), scn, and chp genes by bacteriophages in non-beta-hemolysin-producing bovine isolates of Staphylococcus aureus

In 13 of 43 non-beta-hemolysin-producing bovine isolates of Staphylococcus aureus, two truncated beta-hemolysin (hlb) genes were demonstrated by PCR and sequencing, and one truncated hlb gene was located beside the integrase (int) gene of phage origin. The staphylokinase (sak) gene was detected in all 13 isolates in which the truncated hlb genes were detected by PCR. Enterotoxin A (sea) and enterotoxin P (sep) genes were also detected in 5 and 2 of the 13 isolates, respectively. Moreover, the scn and chp genes encoding staphylococcal complement inhibitor (SCIN) and chemotaxis inhibitory protein of S. aureus (CHIPS) were detected in 13 and 4 of the 13 isolates, respectively. The bacteriophage induced by mitomycin C treatment was able to lysogenize one beta-hemolysin-producing isolate of S. aureus, and the sak and scn genes were detected from the lysogenized isolate. These results suggest quadruple or quintuple conversion of hlb, sak, sea (or sep), scn, and chp genes by bacteriophages among non-beta-hemolysin-producing bovine isolates of S. aureus.     

Effect of ovarian hormones on maturation of dendritic cells from peripheral blood monocytes in dogs

Previously, we reported that ovarian hormones affect the immune response against E. coli isolated from the dogs affected with pyometra. In order to investigate mechanisms underlying the immune modulation, we examined the effects of ovarian hormones on the generation of dendritic cells (DCs), the most potent antigen presenting cell. DCs were differentiated from peripheral blood monocytes (PBMOs) using a cytokine cocktail. Both estrogen receptor and progesterone receptors were expressed by the PBMOs and immature DCs. When various ovarian hormones were added to the culture for the DC differentiation, progesterone significantly decreased the expression of DC maturation markers, such as CD1a, CD80 and CD86, on mature DCs. Conversely, the addition of estrogen to the cultures increased the expression of CD86, but not other maturation makers. Furthermore, DCs differentiated in the presence of progesterone did not stimulate allogeneic mononuclear cells in PB. Taken together, these results indicate that progesterone diminishes the maturation of DCs, leading to decreased immune responses against invading pathogens.     

Molecular typing and antimicrobial resistance of Salmonella enterica subspecies enterica serovar Choleraesuis isolates from diseased pigs in Japan

Salmonella enterica serovar Choleraesuis (S. Choleraesuis) isolates derived from diseased pigs in Japan during 2001 and 2005 were analyzed for biotype, based on H(2)S production and dulcitol fermentation, pulsed-field gel electrophoresis (PFGE) profile, and antimicrobial resistance profile. S. Choleraesuis biotype Choleraesuis (biotype Choleraesuis) was classified into one genotype, while varietas Kunzendorf (var. Kunzendorf) was classified into two genotypes. The isolates of var. Kunzendorf belonging to one genotype were isolated in a limited area of Japan. Variation in the antimicrobial resistance pattern was observed in isolates of both biotypes Choleraesuis and var. Kunzendorf. We have also shown that the PFGE profile was associated with the biotype and isolation region of each isolate.     

Genetic and enzymatic analyses of metalloprotease (aureolysin) from Staphylococcus aureus isolated from domestic animals.

The gene (aur) encoding the metalloprotease (aureolysin) of Staphylococcus aureus from domestic animals was analyzed by polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (PCR-RFLP) and sequencing. The aur gene was detected in all 74 isolates from cows, pigs and chickens by PCR amplification and was classified into types I and II by PCR-RFLP patterns. The type II aur gene was contained in 36 (94.7%) of 38 protease-positive isolates as judged by skim milk agar plate culture, while type I was contained in 28 (77.8%) of 36 protease-negative isolates. The deduced amino acid sequences of aureolysins from type I and II isolates were almost identical with those of the published data. Subsequently, the two aureolysins were purified from the culture supernatants of type I and II isolates. The molecular weights of purified type I and II aureolysins were both estimated at 34kDa by SDS-polyacrylamide gel electrophoresis. These aureolysins had maximum proteolytic activity at 30-50 degrees C and pH 7.0-8.0. Their activity was inhibited by metal- and zinc-specific inhibitors, such as EDTA, EGTA and 1,10-phenanthroline. Specific activity (activity/protein) of type II aureolysin was two times higher than that of type I. These results indicated that the aur gene is highly conserved with two allelic forms (types I and II) among bovine, porcine and avian isolates of S. aureus.     

Immunohistochemical localization of steroidogenic enzymes in corpus luteum of wild sika deer during early mating season

We analyzed the localization of steroidogenic enzymes (P450 scc, 3 beta HSD, P450 arom and P450 c17) in the corpora lutea of two Hokkaido sika deer (Cervus nippon yesoensis) during the early mating season. Two corpora lutea were found in each female and the timing of formation of the corpora lutea seemed different. P450 scc, and 3 beta HSD, positive luteal cells were found in both corpora lutea. The existence of two functional corpora lutea from the early mating season through pregnancy suggests that progesterone secreted by two or more corpora lutea is necessary for maintenance of pregnancy in sika deer.