Immunohistochemical localization of three GnRH systems in brain and pituitary of Japanese flounder

ABSTRACT:   To clarify the possible roles of gonadotropin-releasing hormone (GnRH) in the reproduction of Japanese flounder Paralichthys olivaceus , localization of salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and sea bream GnRH (sbGnRH) immunoreactive (ir) cell bodies and fibers in the brain and pituitary were examined together with follicle stimulating hormone (FSH) and luteinizing hormone (LH)-ir cells in the pituitary by immunohistochemistry. sGnRH-ir cell bodies were localized in the ventromedial part of the rostral olfactory bulb and cGnRH-II-ir cell bodies were restricted to the midbrain tegmentum, while sbGnRH-ir cell bodies were evident in the preoptic area. sGnRH-ir fibers were distributed throughout the brain, especially abundant in the forebrain. cGnRH-II-ir fibers were also scattered in many areas of the brain with abundance in the midbrain, but sbGnRH-ir fibers were observed in the preoptic–hypothalamic area and innervated the pituitary. In the pituitary, neither sGnRH-ir fibers nor cGnRH-II-ir fibers were found, but sbGnRH-ir fibers were profuse in the neurohypophysis and invaded the proximal pars distalis, targeting FSH and LH cells. These results suggest that three GnRH systems can play different physiological roles in the brain of Japanese flounder. Among them, sbGnRH is considered to be involved in reproduction by stimulating gonadotropin secretion, while sGnRH and cGnRH-II can function as a neurotransmitter and/or neuromodulator within the brain in this species.     

Structure and Steroidogenesis of the Placenta in the Antarctic Minke Whale (Balaenoptera bonaerensis)

There are few reports describing the structure and function of the whale placenta with the advance of pregnancy. In this study, therefore, the placenta and nonpregnant uterus of the Antarctic minke whale were observed morphologically and immunohistochemically. Placentas and nonpregnant uteri were collected from the 15th, 16th and 18th Japanese Whale Research Programme with Special Permit in the Antarctic (JARPA) and 1st JARPA II organized by the Institute of Cetacean Research in Tokyo, Japan. In the macro- and microscopic observations, the placenta of the Antarctic minke whale was a diffuse and epitheliochorial placenta. The chorion was interdigitated to the endometrium by primary, secondary and tertiary villi, which contained no specialized trophoblast cells such as binucleate cells, and the interdigitation became complicated with the progress of gestation. Furthermore, fetal and maternal blood vessels indented deeply into the trophoblast cells and endometrial epithelium respectively with fetal growth. The minke whale placenta showed a fold-like shape as opposed to a finger-like shape. In both nonpregnant and pregnant uteri, many uterine glands were distributed. The uterine glands in the superficial layer of the pregnant endometrium had a wide lumen and large epithelial cells as compared with those in the deep layer. On the other hand, in the nonpregnant endometrium, the uterine glands had a narrower lumen and smaller epithelial cells than in the pregnant endometrium. In immunohistochemical detection, immunoreactivity for P450scc was detected in most trophoblast cells, but not in nonpregnant uteri, suggesting that trophoblast epithelial cells synthesized and secreted the sex steroid hormones and/or their precursors to maintain the pregnancy in the Antarctic minke whale.     

Low-dose recombinant canine interferon-γ for treatment of canine atopic dermatitis: An open randomized comparative trial of two doses

The purpose of this study was to investigate the minimum effective dose of recombinant canine interferon-γ (rCaIFN-γ) for the treatment of dogs with atopic dermatitis (AD). Thirty-four dogs with AD from 17 animal hospitals in Japan were administered half or one-fifth of the approved rCaIFN-γ dose of 10 000 units/kg, three times a week for 4 weeks, followed by once weekly for an additional 4 weeks. Pruritus, excoriation, erythema and alopecia were evaluated and scored by the investigators on weeks 2, 4, 6, 8 and 12. The efficacy rate (number of excellent cases + number of good cases/total number of cases) at week 8 in the 2000 units/kg group was 36.4% for pruritus, 36.4% for excoriation, 45.5% for erythema and 36.4% for alopecia. In contrast, in the 5000 units/kg group, the efficacy rate was 64.3% for pruritus, 57.1% for excoriation, 78.6% for erythema and 78.6% for alopecia. The efficacy rate of the 5000 units/kg group was high for all signs evaluated and comparable to that of the 10 000 units/kg group reported in a previous study. The results of this study showed that 2000 units/kg of rCaIFN-γ is less effective than 5000 units/kg to treat dogs with AD, and the efficacy of the 5000 units/kg dose is comparable to that of 10 000 units/kg at week 8.     

Characterization of the D/C mosaic neurotoxin produced by Clostridium botulinum associated with bovine botulism in Japan

Clostridium botulinum types C and D are related to avian and mammalian botulism. Bovine botulism occurred at various farms from 2004 to 2007 in Japan. Since culture supernatants of isolates from cases of bovine botulism were neutralized completely and partially with type D and C antitoxins, respectively, we attempted to confirm the nucleotide sequences of the neurotoxin gene in isolates. The neurotoxin gene comprised two-thirds of the type D neurotoxin gene and one-third of the type C neurotoxin gene, indicating that the neurotoxin of bovine isolates is a mosaic of type D and C neurotoxins, D/C mosaic neurotoxin. We prepared four sets of primers to differentiate the genes of the mosaic and authentic forms with PCR. The results showed that all bovine botulism-related isolates possess the gene for the D/C mosaic form. Pulsed-field gel electrophoresis analysis demonstrated that isolates from bovine botulism which had occurred between 2004 and 2007 were genetically homologous, except for the isolate from one area. We further examined the biological and antigenic properties of the D/C mosaic neurotoxin, which was found to exhibit the highest lethal activity in mice compared with other types of neurotoxins. In the D/C mosaic neurotoxin, three epitopes recognized by monoclonal antibodies that specifically react to and neutralize the toxin were located in the carboxyl-terminal domain of the heavy chain. These results indicate that D/C mosaic neurotoxin is a pathogenic agent causing bovine botulism and has unique characteristics different from other type C and D neurotoxins.     

Involvement of sex steroids,luteinizing hormone and thyroid hormones in upstream and downstream swimming behavior of land-locked sockeye salmon <Emphasis Type="Italic">Oncorhynchus nerka</Emphasis>

The involvement of testosterone (T), estradiol-17β (E₂), 11-ketotestosterone (11-KT), 17,20β-dihydroxy-4-pregnene-3-one (DHP), luteinizing hormone (LH), thyroxine (T₄), and triiodothyronine (T₃) in the regulation of downstream and upstream movement (swimming behavior) was investigated in land-locked sockeye salmon Oncorhynchus nerka, using an artificial raceway. During the downstream migratory period, T implant resulted in high plasma T levels and inhibited the occurrence of downstream swimming behavior (negative rheotaxis) in yearling (1+) immature smolts. In terms of upstream behavior, 2-year-old (2+) males exhibited high plasma T and 11-KT levels, while 2+ females had elevated T and DHP levels. In 1+ immature fish, a T implant induced upstream swimming behavior (positive rheotaxis). In experiments 1 and 3, the plasma T₄ and T₃ levels of non-migrants tended to be higher than those of migrants. In contrast, no marked changes in plasma and pituitary LH were found in both downstream and upstream migrants. These results suggest that sex steroids, such as T, play significant roles in the regulation of downstream and upstream swimming behaviors in land-locked sockeye salmon.     

Gossypol inhibits LH‐induced steroidogenesis in bovine theca cells

Gossypol, a polyphenolic aldehyde found in cottonseed, has been shown to perturb steroidogenesis in granulosa and luteal cells of rats, pigs and cattle. However, little is known about the direct effect of gossypol on theca cell functions in any species. The present study was conducted to investigate the effect of gossypol on the steroidogenesis and the expression of genes involved in it in cultured bovine theca cells. Theca cells were isolated from healthy preovulatory follicles and were cultured in the presence of luteinizing hormone (LH) for up to 7 days. During the culture period, main steroid products of the theca cells shifted from androstenedione (A4) at day 1 to progesterone (P4) from day 2 onward. At days 1 and 7, theca cells were treated with gossypol (0‐25 μg/mL) for 24 h. Gossypol inhibited LH‐stimulated theca cell A4 and P4 production in a dose‐dependent manner at both occasions. The viability of theca cells was not affected by gossypol at any doses used. Gossypol down‐regulated expressions of steroidogenic enzymes CYP11A1, HSD3B1 and CYP17A1, but not that of LHR. These results indicate that gossypol inhibits thecal steroidogenesis through down‐regulating gene expressions of steroidogenic enzymes but without affecting cell viability in cattle.     

Disease resistance induced by nonantagonistic endophytic Streptomyces spp. on tissue-cultured seedlings of rhododendron

Of 82 strains of endophytic actinomycetes isolated from rhododendron plants, 12 were not antagonistic against Pestalotiopsis sydowiana, which is the causal agent of Pestalotia disease. Of these 12, MBR-37 and MBR-38 (identified as Streptomyces spp.) grew on IMA-2 medium. Tissue-cultured seedlings of rhododendron treated with these nonantagonistic strains showed less wilting and/or smaller lesions to P. sydowiana, although the degree of resistance was a little lower than that conferred by antagonistic Streptomyces galbus strain R-5. These seedlings accumulated the anthocyanin(s), suggesting that resistance induced by these strains could depend on activated defense responses associated with the phenylpropanoid pathway rather than with antibiosis.     

Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum

Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36–37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum.