Immunolocalization of steroidogenic enzymes in the corpus luteum and placenta of the Japanese Shiba goat

In this study, we performed immunohistochemistry of cholesterol side-chain cleavage cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase (3betaHSD), cytochrome 17alpha-hydroxylase P450 (P450c17), and cytochrome P450 aromatase (P450arom) in the corpus luteum and placenta of Shiba goats. The aim was to clarify the steroidogenic capability of the corpus luteum and placenta of Shiba goats. Ovaries containing corpora lutea were obtained from four adult Shiba goats during the luteal phase (day10; n=2) and pregnancy (90 and 120 days of gestation). Placenta was obtained from one Shiba goat on day 120 of gestation. The sections of the ovaries and placentae were immunostained using the avidin-biotin-peroxidase complex method (ABC) with polyclonal antibodies generated against steroidogenic enzymes of mammalian origin. All luteal cells expressed P450scc, 3betaHSD, P450c17 and P450arom. The distribution of P450scc, 3betaHSD, P450c17 and P450arom were not different during the luteal phase and pregnancy. P450arom showed a weak positive staining in late pregnancy (120 days). In addition, immunoreactions for P450c17 and P450arom were observed in syncytiotrophoblast of the placenta of one Shiba goat. These results indicate that, in Shiba goats, corpus luteum is not only an important source of progesterone but also has the ability to synthesize androgen and estrogen during the luteal phase and pregnancy. Also the placenta has the ability to synthesize androgen and estrogen in late pregnancy.     

Immunolocalization of nerve growth factor (NGF) and its receptors (TrkA and p75LNGFR) in the reproductive organs of Shiba goats

The objective of this study was to determine the immunolocalization of NGF and its receptors (TrkA and p75LNGFR) in the reproductive tract of the Japanese Shiba goats. Five adult goats were used in this study and sections of ovaries, uteri and oviducts were immunostained by the avidin-biotin-peroxidase complex method (ABC). The results showed that NGF and its receptors (TrkA and p75LNGFR) were expressed in granulosa cells, theca cells, interstitial cells and lutein cells in ovaries. Immunoreactions for NGF, TrkA and p75LNGFR were also detectable in epithelial cells and muscle cells of the ampulla and isthmus of the oviduct, and in epithelial cells and uterine glands of the uterus. These results strongly suggest autocrine and paracrine regulation of reproductive function by NGF in the reproductive tract of female Shiba goats.     

Immunogenicity and safety of an attenuated Bordetella bronchiseptica vaccine in pigs

The immunogenicity and safety of an attenuated Bordetella bronchiseptica vaccine for swine atrophic rhinitis (AR) was evaluated in 22 hysterectomy-produced, colostrum-deprived pigs and 18 conventional pigs. None of 8 pigs inoculated at 7 days of age intranasally with greater than or equal to 3 X 10(5) colony-forming units (CFU) of vaccinal strain/pig and 2 of 5 pigs inoculated at 7 days of age intranasally with 3 X 10(4) CFU of the vaccinal strain/pig developed AR after intranasal challenge exposure with a virulent strain at postinoculation week (PIW) 3. The remaining 3 vaccinated pigs and 4 nonvaccinated pigs developed AR. Thirteen pigs were inoculated intranasally with 3 X 10(6) to 3 X 10(9) CFU of the vaccinal strain at 7 days of age. At PIW 12, the pigs were killed and necropsied. None of the pigs had clinical signs of AR and/or pneumonia. Virulence was studied by transmission of vaccinal strain through 3 serial growing passages on the nasal mucosa of a litter of hysterectomy-produced colostrum-deprived pigs. Inoculum (nasal swab samples from 2 pigs 4 days after inoculation with 10(8) CFU of vaccinal strain at 5 days of age) was inoculated into the nasal cavity of 2 nonvaccinated pigs. This procedure was repeated 3 times. After the 1st passage, the vaccinal strain was recovered on postinoculation day 4, but after postinoculation day 4, the vaccinal strain was not recovered until the end of the 3rd passage. Turbinate atrophy or pneumonia was not recognized in these inoculated pigs. The vaccinal strain provided immunogenicity without ill effects.     

Interaction between the helicase domain of the tobacco mosaic virus replicase and a tobacco arginine decarboxylase

In a yeast two-hybrid screening test for tobacco proteins that interact with TMV replicase using the helicase (H) domain as bait, a cDNA clone was selected that encodes a polyamine biosynthetic enzyme, arginine decarboxylase (ADC). In yeast cells, the C-terminal internal region of ADC interacted with the H domain. This observation was confirmed in vitro by far-Western blotting. Inhibition of the binding between the H domain and the IRnHEL (I region and N-terminus of helicase domain) region by ADC using a yeast three-hybrid assay suggested possible interference of the heterodimerization of 126K and 183K by ADC.The nucleotide sequence data of pADCF reported in this study is available in the DDBJ/EMBL/GenBank databases under accession number AB110952     

A simple calculation for obtaining shunt fractions of portosystemic shunts

Since the isotopes can not be utilized for veterinary patients in Japan, the authors developed a simple calculation formula of shunt fraction of portosystemic shunt based on the hepatic circulation model. The shunt fraction can be calculated utilizing only 2 portal pressure measurements of pre-shunt ligation and temporary or permanent shunt ligation. The calculated shunt fraction can obtained pre-ligation and post-ligation either temporally or permanent complete shunt ligation: complete ligation group of PSS (n=59) had 48.2 +/- 16.9% of shunt fractions, whereas the partial ligation group (n=48) had 71.6 +/- 10.7% of shunt fractions.     

Simultaneous observation of the GnRH pulse generator activity and plasma concentrations of metabolites and insulin during fasting and subsequent refeeding periods in Shiba goats

The time course of GnRH pulse generator activity and plasma concentrations of energy substrates and insulin were simultaneously observed in female goats during 4-day fasting and subsequent refeeding in the presence or absence of estrogen for a better understanding of the mechanism of energetic control of gonadotropin secretion in ruminants. The GnRH pulse generator activity was electrophysiologically assessed with the intervals of characteristic increases in multiple-unit activity (MUA volleys) in the mediobasal hypothalamus. In estradiol-treated ovariectomized (OVX+E2) goats, the MUA volley intervals increased as fasting progressed. Plasma concentrations of non-esterified fatty acid and ketone body increased, while those of acetic acid and insulin decreased during fasting. The MUA volley intervals and plasma concentrations of those metabolites and insulin were restored to pre-fasting levels after subsequent refeeding. In ovariectomized (OVX) goats, changes in plasma metabolites and insulin concentrations were similar to those in OVX+E2 goats, but the MUA volley intervals were not altered. The present results demonstrated that fasting suppressed GnRH pulse generator activity in an estrogen-dependent manner. Changes in plasma concentrations of energy substrates and insulin during fasting were associated with the GnRH pulse generator activity in the presence of estrogen, but not in the absence of the steroid in female goats.     

Maternal exposure to low doses of bisphenol a has no effects on development of female reproductive tract and uterine carcinogenesis in Donryu rats

Effects of maternal exposure to low doses of bisphenol A (BPA), including those comparable with human exposure levels, on growth and development of the female reproductive system and uterine carcinogenesis in Donryu rats were investigated. Dams were administered BPA (0, 0.006 and 6 mg/kg/day) daily by gavage from gestation day 2 up to the day before weaning (postnatal day 21 at offspring). The serum levels of BPA were significantly elevated in the dams receiving 6 mg/kg/day, however, BPA levels in the milk of dams, and those in the serum and liver of offspring were similar between control and treated groups. The treatment did not exert any influences on uterine development including weight, gland genesis and estrogen receptor alpha expression, vaginal opening and gonadotropin secretion in the female offspring up to puberty. After maturation, no effects were evident with regard to estrous cyclicity in female offspring treated with BPA. In addition, the treatment had no effects on age-related morphological changes of the reproductive and endocrine organs and uterine carcinogenesis until 15 months of age. The results demonstrate that maternal exposure to BPA at levels comparable to human exposure did not have any effects on the female reproductive system of offspring in rats. In addition, BPA was also found in the serum, milk and liver of control dams and pups, and low levels of BPA were detected in drinking water and pellet diet. The present study showed that the experimental animals were also exposed to environmental BPA in the animal room.     

Effect of bread baking on the bioavailability of hydrogen-reduced iron powder added to unenriched refined wheat flour

Elemental iron powders are widely used to fortify flour and other cereal products. Our objective was to test the hypothesis that baking enhances the bioavailability of elemental iron powders by oxidizing Fe(0) to Fe(2+) or Fe(3+). An in vitro digestion/Caco-2 cell culture model and a piglet model were used to measure bioavailability. Bread flour, either unfortified or fortified with hydrogen-reduced (HR) iron powder or FeSO(4) (300 mg Fe/kg flour), was baked into bread. For the in vitro studies, bread samples were treated with pepsin at pH 2, 3, 4, 5, 6, or 7 and subsequently incubated with pancreatic enzymes at pH 7 in a chamber positioned above monolayers of cultured Caco-2 cells. Ferritin formation in the cells was used as an index of iron bioavailability. Ferritin formation in cells fed HR Fe bread was similar to cells fed FeSO(4) bread when the peptic digestion was conducted at a pH 2 but lower when the peptic phase was conducted at pH 3, 4, 5, 6, or 7 (P < 0.05). Pig diets containing 35% dried bread were prepared and fed to cross-bred (Hampshire x Landrace x Yorkshire) anemic pigs in two studies. The rate of increase in hemoglobin Fe over the feeding period was used to calculate relative biological value (RBV), an index of iron bioavailability. In the first pig study, RBV of HR Fe added to flour prior to baking was 47.9% when compared to FeSO(4) fortified flour (P < 0.05). In the second pig study, a third treatment consisting of unfortified bread with HR iron added during diet mixing (after bread baking) was included. RBVs of the HR Fe diet (Fe added after baking) and HR Fe diet (Fe added before baking) were 40.1% and 53.5%, respectively, compared to the FeSO(4) diet. Differences in RBV between the HR Fe (before and after baking) and FeSO(4) (before baking) treatment groups were significant, but the difference between the before and after HR treatment groups was not significant. We conclude that bread baking does not enhance the bioavailability of elemental iron powders.     

Expression of inhibins, activins, insulin-like growth factor-I and steroidogenic enzymes in the equine placenta

In this study, the expression patterns of inhibins, activins, insulin-like growth factor-I (IGF-I) and steroidogenic enzymes in equine placentae recovered during the latter two-thirds of gestation were examined. Concentrations of inhibin A and inhibin pro-alphaC in endometrial and fetal placental tissue homogenates were very low during the period examined, whereas these tissues contained high concentrations of activin A. In both maternal endometrial and fetal placental tissues, activin A levels decreased as pregnancy progressed. Expression of inhibin alpha-subunit was not observed in the placenta using either immunohistochemistry or in situ hybridization. Inhibin/activin betaA-subunit and its mRNA were confined to maternal endometrial glands, whereas immunopositive betaB-subunit was not detected in either endometrial glands or microcotyledons. Cytochrome P450 side chain cleavage enzyme was detected by immunohistochemistry in both endometrial glands and microcotyledons, whereas cytochrome P450 17alpha-hydroxylase/lyase was absent in these tissues. Immunopositive signals for 3beta-hydroxysteroid dehydrogenase and cytochrome P450 aromatase were localized in microcotyledons but not in endometrial glands. Immunohistochemistry revealed that IGF-I was highly expressed in microcotyledons around Day 130, and decreased as pregnancy progressed. Changes in the expression of IGF-I were correlated with the number of PCNA positive cells in the placenta. The present study demonstrated the presence and localized the site of expression of activin, IGF-I and steroidogenic enzymes in equine placental tissues during the latter two-thirds of gestation; the results suggest that activin and IGF-I may be involved in the regulation of placental development.     

Effect of Pueraria mirifica on the sexual skin coloration of aged menopausal cynomolgus monkeys

To investigate the estrogenic effect of Pueraria mirifica (PM), a Thai herbal plant that contains many phytoestrogens, sexual skin coloration was studied in cynomolgus monkeys. Aged menopausal monkeys were divided into three groups. Each group (n=3) was fed 10, 100, or 1,000 mg of PM daily. The treatment schedule was divided into three periods, a 30-day pre-treatment period, 90-day treatment period, and 60-day post-treatment period. The results show that the sexual skin exhibited reddish coloration within 24 h after PM-treatment and remained this way for the first half of the PM-feeding period. The changes in sexual skin coloration were not dose-dependent. The present results indicate that PM had estrogenic action by increasing reddish sexual skin coloration in aged menopausal monkeys.