The effect of estrogen on phosphorylation of prolactin in the mouse pituitary gland

Several studies have indicated that prolactin (PRL) assumes oligomeric, proteolytically cleaved, phosphorylated and glycosylated forms. Phosphorylated PRL (PPRL) is considered to be the most important posttranslationally modified form in the rat. In the present study, we examined whether or not PRL is present in the mouse pituitary gland in the phosphorylated form. Mouse pituitary PRL was digested with acid phosphatase, resolved by two-dimensional gel electrophoresis, stained with Coomassie brilliant blue, and then immunoblotted against the anti-PRL, anti-phosphoserine and anti-phosphothreonine antibodies. We also examined whether PRL is phosphorylated by protein kinases and semi-quantified the ratios of PPRL to PRL in the pituitary gland. The results indicated that three types of PRL are present in the pituitary glands of both male and female mice. One was non-phosphorylated (isoform 1), and the other two were immunoreactive to anti-phosphoserine (isoform 2) and/or anti-phosphothreonine (isoform 3) antibodies. The ratio between isoforms 2 and 1 of the 30-day-old female mice was higher than that of the 20-day-old female mice. However, the ratios among the three isoforms in the male pituitary glands did not differ with age. The ratio of PPRL to isoform 1 was obviously reduced after ovariectomy (OVX), and it recovered with estrogen replacement. These results suggest that estrogen influences PRL phosphorylation in female mice.     

Combined effects of treatment with trientine, a copper-chelating agent, and x-irradiation on tumor growth in transplantation model of a murine fibrosarcoma

Combined effects of treatment with trientine, a copper-chelating agent, and X-irradiation on development of fibrosarcoma using a murine transplantation model in vivo and on cellular survival in vitro were examined. Copper contents in the tumors and serum of trientine-treated mice were significantly lower than those of untreated mice. The tumor volumes of mouse fibrosarcoma QRsp-11 cells increased more slowly in the trientine-treated and the X-irradiated mice than in the control mice from 10 to 24 days postinoculation. The extent of inhibition of tumor growth by X-irradiation at 3 Gy was similar to that obtained by treatment with trientine. A combination of trientine and X-irradiation at 3 Gy showed inhibitory effects on tumor growth similar to those obtained by X-irradiation at 6 Gy. The results showed that trientine and X-irradiation interacted additively in inhibition of tumor growth. When QRsp-11 cells and mouse and bovine endothelial cells were treated with trientine after X-irradiation, the surviving fractions of the cells with combined treatments were essentially consistent with the products of the surviving fractions of trientine-treated cells and those of X-irradiated cells. When the cells were pretreated with trientine and X-irradiated, the surviving fractions of the pretreated cells were lower than those of cells without treatment.     

Evaluation of Intestinal Absorption of Dietary Halocynthiaxanthin,a Carotenoid from the Sea Squirt Halocynthia roretzi

Halocynthiaxanthin is an acetylenic carotenoid mainly found in Halocynthia roretzi. To date, several bioactivities of halocynthiaxanthin have been reported, but its mechanism of digestion and absorption in mammals has not been studied yet. In this study, we evaluated the intestinal absorption of halocynthiaxanthin in mice. The halocynthiaxanthin-rich fraction was prepared from the tunicate Halocynthia roretzi. Mice were orally administered the fraction at a dose of 5 mg/kg body weight. The halocynthiaxanthin levels in the plasma, liver, and small intestine, were quantified using HPLC-PDA, 1, 3, 6, and 9 h after ingestion. The halocynthiaxanthin-rich fraction mainly consisted of the all-trans form and a small amount of cis forms. These three isomers were detected in the plasma of mice 3 h after ingestion. Time-course changes after the ingestion of this fraction were found, with cis isomers being more abundant than the all-trans isomer in the mouse plasma and liver. In the small intestine, however, the all-trans isomer was primarily detected. The possibility that cis isomers might be absorbed rapidly from the small intestine cannot be denied, but our results suggest that dietary all-trans-halocynthiaxanthin might be isomerized to the cis isomer after intestinal absorption.     

Structure of the intratesticular duct system for sperm emission in the starspotted smooth-hound Mustelus manazo

We conducted histological observation of male germ cells and reproductive organs of the starspotted smooth-hound Mustelus manazo in Tokyo Bay to reveal any abnormality in male reproductive traits, as part of a study to elucidate the factors causing recent fluctuation in abundance of the population. Spermatogenesis proceeded in spermatocysts from the germinal zone in the ventral part of the testis to the degenerative zone in the dorsal part, where the spermatozoa were conveyed into the ciliated lumina of the attached terminal branches of the intratesticular ducts. The intratesticular ducts were classified from their terminal ends into branch, stem, and collecting tubules. The ducts formed in the germinal zone and grew as the spermatocysts developed. An opening formed through the wall of each of the most mature spermatocysts into a branch tubule; bundles of spermatozoa were evacuated through this opening into the branch and then the stem tubule and subsequently into the collecting tubules in the rete testis and the efferent duct connected to the epididymis. Spermatocysts that were unable to emit sperm because of failure of adhesion to the branch tubules were disorganized in situ, as were their spermatozoa. The collapsed spermatocysts seem to be cleared by hemophagocytosis with lymphocytes and leukocytes, which may have been recruited from the epigonal organ. There were no specific abnormalities in the spermatogenesis or the morphological structure of testes, which suggested that an abnormality of male reproductive traits was not the major cause of the recent fluctuation in the population abundance of this species. Details of the intratesticular duct system for sperm emission to the epididymis are the first findings in elasmobranchs worldwide.     

Effects of mangrove deforestation on fish assemblage at Pak Phanang Bay,southern Thailand

Daytime sampling using a seine net was conducted at Pak Phanang Bay (Nakhon Si Thammarat Province, Thailand) in February and July 2006, to determine differences in fish assemblage structures between a mangrove site and an adjacent site completely cleared of mangroves. The overall numbers of fish species and individuals were significantly higher at the mangrove site than the cleared site in both months. Although benthic crustacean feeders showed more species and individual numbers at the mangrove site in both months, the opposite was found for zooplankton feeders. A cluster analysis, based on the abundance of each species, demonstrated that the fish assemblage structures were distinctly different between the two sites. In addition, significant differences in length frequency distributions for each of the four most abundant species were found between the sites in February and/or July. Small individuals of Scatophagus argus, Ambassis nalua, and Tetraodon nigroviridis were more common at the mangrove site, and of Chelon subviridis at the cleared site. These results suggest that mangrove deforestation exerts marked effects on fish assemblages.     

Thermal behavior of beta-1 subunits in lignin: pyrolysis of 1,2-diarylpropane-1,3-diol-type lignin model compounds

1,2-Diarylpropane-1,3-diol-type lignin model compounds, 1,2-bis(4-hydroxy-3-methoxyphenyl)propane-1,3-diol (1) and 1-(3,4-diethoxyphenyl)-2-(4-methoxyphenyl)propane-1,3-diol (2), were pyrolyzed at 500 degrees C for 4 s to clarify the thermal behavior of beta-1 subunits in lignin. Products were monitored by gas chromatography/mass spectrometry. The cleavage of the Calpha-Cbeta bond to produce benzaldehydes such as 4-hydroxy-3-methoxybenzaldehyde (9) and phenylethanals as the counterparts such as 4-hydroxy-3-methoxyphenylethanal (10) occurred in pyrolyses of both 1 and 2. In pyrolysis of 1, an oxetane pathway leading to the formation of Z/E-stilbenes without the gamma-CH2OH group such as Z/E-4,4'-dihydroxy-3,3'-dimethoxystilbene (3) was predominant. In pyrolysis of 2, the oxetane pathway was minor, while pathways producing a dimer with a =CgammaH2 group by loss of water and a dimer with an alpha-carbonyl group were predominant. Pyrolysis of Japanese cedar wood provided 3 and 10 in approximately 0.8% and 0.6% yields, respectively, based on the Klason lignin content, while pyrolysis of a guaiacyl bulk dehydrogenation polymer gave them in a very small amount.     

Intratubal insemination with fresh semen in dogs

The number of spermatozoa required to obtain conception by intratubal insemination in dogs was examined. Three groups consisting of 5, 8 and 8 dogs received 0.5 x 10 (6), 2.0 x 10(6) and 4.0 x 10(6) spermatozoa, respectively, into each uterine tube. No conception occurred in the 5 animals inseminated with 0.5 x 10(6) spermatozoa, but conception occurred in 6/8 (75.0%) and 3/8 (37.5%) dogs inseminated with 2.0 x 10(6) and 4.0 x 10 (6) spermatozoa, respectively. Among the pregnant animals, three aborted (33.3%) and the mean number of newborns was small, 2.5 +/- 0.5 (SE). One acardiacus anceps was observed with normal fetus in one animal with a Caesarean delivery.     

Lectin-binding characteristics and capacitation of canine epididymal spermatozoa

Cross sections of the testes and the caput, corpus and cauda epididymides removed from 12 dogs were stamped on glass slides, and the sperm on the slides were stained with 6 different FITC-lectins (Con A, DBA, PNA, PSA, SBA, and WGA) to examine the characteristics of the surface glycoproteins (GPs) on canine epididymal sperm. The corpus epididymal sperm were washed three times by centrifugation, and their lectin-binding characteristics were investigated. The washed sperm from the corpus and cauda epididymides were incubated for 24 hr, and the fertilizing capacity of the sperm was evaluated by calculating the percentages of actively motile sperm (%MO), hyperactivated sperm (%HA), and acrosome-reacted sperm (%AR), and the number of canine zona-pellucida (ZP)-binding sperm. The testicular sperm did not stain with SBA lectin, but the SBA lectin fluorescence was observed on the surface of the entire heads of the caput epididymal sperm. Although all of the entire heads or acrosomal regions of the corpus epididymal sperm stained with all 6 FITC-lectins, the heads and acrosomal regions of the cauda epididymal sperm did not stain with DBA or SBA lectins. Washing the sperm from the corpus epididymis resulted in loss of the fluorescence of the FITC-DBA and -SBA lectins. The mean %MO, %HA, %AR, and ZP-binding number of the cauda epididymal sperm after 24 hr of incubation were higher than the values for the corpus epididymal sperm. All of the mean values for the washed sperm from the corpus and cauda epididymides were higher than the values for the unwashed sperm from the corpus and cauda, and with the exception of %AR, the values from the washed sperm from the corpus epididymis were significantly higher (P<0.05, 0.01). The results indicate that DBA- and SBA-lectin-binding GPs on the surface of canine epididymal sperm are associated with the fertilizing capacity and may be decapacitation factors.     

Factors affecting transuterine migration of canine embryos

Dogs, cats, and pigs have a bicornuate uterus, and transuterine migration of embryos occurs in 40% or more of pregnant animals. However, the mechanism of the transuterine migration has not been elucidated in dogs. Thus, we investigated the occurrence of transuterine migration of embryos when embryos were retained in an unilateral uterine tube with more ovulated ova (Experiment 1), when one ovary was excised (Experiment 2), and when ova ovulated from the right and left ovaries were fertilized with sperm from male dogs with different blood types (Experiment 3). Transuterine migration of embryos was observed in 7/8 (87.5%), 10/10 (100%), and 11/17 (67.4%) fertilized animals in Experiments 1, 2, and 3, respectively. In Experiment 3, intrauterine embryo mixing reported in pigs did not occur. These findings suggest that transuterine migration of embryos occurs due to the number of embryos that enter the uterus but that differences in the number of ovulated ova between the right and left ovaries or the number of embryos retained in the uterine tube do not affect the migration.     

Performance of using opposing homozygotes for paternity testing in Japanese Black cattle

Genome-wide single nucleotide polymorphism (SNP) markers in Japanese Black cattle enable genomic prediction and verifying parent–offspring relationships. We assessed the performance of opposing homozygotes (OH) for paternity testing in Japanese Black cattle, using SNP genotype information of 50 sires and 3,420 fattened animals, 1,945 of which were fathered by the 50 genotyped sires. The number of OH was counted for each sire–progeny pair in 28,764 SNPs with minor allele frequencies of ≥0.05 in this population. Across all pairs of animals, the number of OH tended to increase as the pedigree-based coefficient of relationship decreased. With a threshold of 288 (1% of SNPs) for paternity testing, most sire–progeny pairs were detected as true relationships. The frequency of Mendelian inconsistencies was 2.4%, reflecting the high accuracy of pedigree information in Japanese Black cattle population. The results indicate the utility of OH for paternity testing in Japanese Black cattle.