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Background Testicular torsion, which causes ischemia-reperfusion (IR) injury, is a serious urological emergency that can lead to testicular dysfunction, including infertility, primarily among newborn and pubertal males; thus, effective drugs should be administered during or after ischemia. Objectives Using a rat model of testicular IR injury, the present study investigated the protective effects of relaxin (RLN) against oxidative stress, testicular dysfunction, inflammation, histological damage, arrested spermatogenesis, and germ cell apoptosis as well as explored the usefulness of RLN as a potential protective drug for IR injury combined with surgical treatment. Materials and methods Male Sprague-Dawley rats were subjected to left testicular ischemia for 2 h, followed by 24 h of reperfusion. They were subsequently divided into three groups: sham, IR, and IR + RLN groups. Porcine RLN (500 ng/h) or saline was infused using an implanted osmotic mini-pump 90 min after inducing ischemia. The RLN dose used herein was that which resulted in serum RLN levels comparable to those in mid-pregnant rats based on previous studies. Results Testicular IR increased germ cell apoptosis and histological damage as well as promoted disorganized and arrested spermatogenesis, accompanied by a significant increase in oxidative stress and inflammation. However, RLN administration ameliorated the adverse consequences associated with IR injury by attenuating oxidative stress and mitigating apoptosis and inflammation. Discussion and conclusion The study findings clearly demonstrated that RLN exerts a protective effect against IR-induced testicular injury by attenuating oxidative stress, apoptosis, and inflammation, suggesting that RLN together with surgical treatment is a potentially efficacious approach toward ameliorating testicular dysfunction following testicular torsion. This article is protected by copyright. All rights reserved.

Order Anostraca (fairy shrimp of large branchiopods) is a primitive crustacean group, retaining ancient forms and ecology. The Holarctic family Chirocephalidae originated over 100 million years ago; it is a very long-lived freshwater taxon that has survived from the Mesozoic to the present. Thus, using this taxon as an indicator, we verified how the geographical distribution of freshwaters shifted during the ancient era. We used newly collected samples of Drepanosurus uchidai from Aomori (northern Japan) and Galaziella baikalensis and Branchinecta orientalis from Olkhon Island (the largest lake-bound island of Lake Baikal, Russia) to sequence 658 bp of COI gene. These sequences, plus those of 16S rRNA gene (~ 550-bp mt16S rDNA fragment), were compared with those retrieved from GenBank. To re-evaluate and clarify phylogenetic relationships among the Chirocephalidae that remains confused till now, six genera of the family, including Polyartemiella, Drepanosurus, Eubranchipus, Chirocephalus, Artemiopsis, and Galaziella species were used for molecular analyses. Small water bodies usually have comparatively short lives and fade away sooner or later due to growth of aquatic plants and accumulation of bottom sediments. In such environments, refugia formed on the shores of large-scale lakes were necessary for large branchiopods to survive several Ice Ages. The lake shorelines have moved with the growth or decline of the lakes, but the habitats of large branchiopods sporadically left behind can now be confirmed as a history of the shifting geographical distribution of global freshwaters. For example, two types of large branchiopod populations from island-bound water bodies on Olkhon Island are recognized: (1) populations on the northwestern coast that are closely related to the group in the Mongolian Gobi steppe region, and (2) populations in other areas distant from the coast that are highly endemic to the island. Based on fossil records and genetic distances, an absolute differentiation time of world chirocephalids can be estimated as 140 million years ago in the Mesozoic era. On the other hand, the age of Lake Baikal is only 25‒30 million years at the most. Therefore, extant large branchiopods of Olkhon Island must have first appeared near the present lake catchment after separation from their ancestral populations that had originated in Europe, and before the formation of Lake Baikal.

Thyroid hormones (THs) are essential for the correct development of nearly every structure in the body from the very early stages of development, yet the embryonic thyroid gland is not functional at these stages. To clarify the roles of the egg yolk as a source of THs, the TH content in the yolk and the expression of TH regulator genes in the yolk sac membrane were evaluated throughout the 21-day incubation period of chicken embryos. The yolk TH content (22.3 ng triiodothyronine and 654.7 ng thyroxine per total yolk on day 4 of incubation) decreased almost linearly along with development. Real-time PCR revealed gene expression of transthyretin, a principal TH distributor in the chicken, and of a TH-inactivating iodothyronine deiodinase (DIO3), until the second week of incubation when the embryonic pituitary-thyroid axis is generally thought to start functioning. The TH-activating deiodinase (DIO2) and transmembrane transporter of thyroxine (SLCO1C1) genes were expressed in the last week of incubation, which coincided with a marked increase of circulating thyroxine and a reduction in the yolk sac weight. DIO1, which can remove iodine from inactive THs, was expressed throughout the incubation period. It is assumed that the chicken yolk sac inactivates THs contained abundantly in the yolk and supplies the hormones to the developing embryo in appropriate concentrations until the second week of incubation, while THs may be activated in the yolk sac membrane in the last week of incubation. Additionally, the yolk sac could serve as a source of iodine for the embryo. ispartof: The Journal of Reproduction and Development vol:63 issue:5 pages:463-472 ispartof: location:Japan status: published

Early experience with low-quality roughage has the potential to improve its utilization in ruminants. This study determined the effects of early experience with low-quality roughage on dry matter intake (DMI), body weight (BW) and the hepatic metabolism of lambs. Ten lambs (3 month old) were randomly allocated to two treatments: the low-quality roughage group (LR) or the control group (C). The study lasted 7 months. In the first 4 months, LR was fed low-quality roughage, whereas C was fed normal roughage. In the last 3 months, both groups were fed low-quality roughage. Dry matter intake (DMI) was determined at the 1st, 3rd, 5th and 7th months (P1, P2, P3 and P4). Analysis of liver tissue metabolomics were carried out in P2 and P3. DMI was greater in LR than C in P1 (p

Early experience with low-quality roughage may induce adaptations in ruminants' metabolism. This study was conducted to explore the variation in the hepatic metabolomes of lambs fed low-quality roughage beginning early in life. Five lambs were fed Sudan grass hay (crude protein (CP): 4.6% of the dry matter (DM), neutral detergent fiber, 68.5% of DM) for 6 months during time periods P1, P2 and P3, which consisted of 2 months each. The metabolizable energy (ME) and CP intake satisfied lambs' maintenance requirements in P1 and P2, but the ME intake was 78.5% of the maintenance ME requirement in P3. Liver metabolomics was carried out in P2 and P3 by the capillary electrophoresis and time-of-flight mass spectrometry system. Eight amino acids and six amino acid metabolism-related metabolites were altered between P2 and P3. Several intermediates of glycolysis/gluconeogenesis decreased, while nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate increased in P3. Taurocholic acid and taurine increased, while glycocholic acid decreased in P3. The results suggested that amino acid utilization and the efficiency of glycolysis/gluconeogenesis might be adjusted to accommodate the low-quality roughage fed to the lambs during early stages of life. The composition of bile acids might also be optimized to promote the efficiency of lipid absorption.

The structure and mineral composition of eggshell cuticles were studied in 5 species of birds. The approximate thickness of the cuticle layer at the top of shell columns was about 1μm in the Red Junglefowl (Gallus gallus), about 130μm in the White Pelican (Pelecanus onocrotalus), about 10μm in the Japanese quail (Coturnix japonica), about 110μm in the Greater Flamingo (Phoenicopterus ruber roseus) and about 45μm in the Humboldt Penguin (Spheniscus humboldti). The matrix of the cuticle layer decalcified with EDTA was composed of vesicles in a variety of sizes in all birds. Major elements in cuticle materials detected by X-ray microanalysis were O, C, Ca and P, and their percentage numbers of atoms decreased in this order. The concentration of P was significantly higher in the cuticles of the quail, flamingo, and penguin than in those of the junglefowl and pelican. Ca mapping on electron-microscopic images showed strong signals in the shell layer and weaker ones in the cuticle layer, whereas P mapping showed that signals were mostly confined in the cuticle layer. X-ray diffraction analyses on the inside of the shell layer showed a profile of calcite crystals of calcium carbonates in all birds. In the cuticle materials, the profile was of calcite in the junglefowl, and a mixture of calcite and vaterite in the pelican. The profiles in cuticle materials of the quail, flamingo and penguin showed no specific signals, indicating that mineral compounds are amorphous in these forms. It was suggested that the diversity of mineral structures in the cuticle layer is caused by the presence of phosphorous, in addition to the structure of the cuticle matrix.

Melanopsin (OPN4) is a photoreceptive molecule regulating circadian systems in mammals. Previous studies from our laboratory have shown that co-localized dopamine-melatonin (DA-MEL) neurons in the hypothalamic premammillary nucleus (PMM) are putatively photosensitive and exhibit circadian rhythms in DAergic and MELergic activities. This study investigates turkey OPN4x (tOPN4x) mRNA distribution in the hypothalamus and brainstem, and characterizes its expression in PMM DA-MEL neurons, using in situ hybridization (ISH), immunocytochemistry (ICC), double-label ISH/ICC, and real time-PCR. The mRNA encoding tOPN4x was found in anatomically discrete areas in or near the hypothalamus and the brainstem, including nucleus preopticus medialis (POM), nucleus septalis lateralis (SL), PMM and the pineal gland. Double ICC, using tyrosine hydroxylase (TH, the rate limiting enzyme in DA synthesis)-and OPN4x antibodies, confirmed the existence of OPN4x protein in DA-MEL neurons. Also, tOPN4x mRNA expression was verified with double ISH/ICC using tOPN4x mRNA and TH immunoreactivity. PMM and pineal gland tOPN4x mRNA expression levels were diurnally high during the night and low during the day. A light pulse provided to short day photosensitive hens during the photosensitive phase at night significantly down-regulated tOPN4x expression. The expression level of tOPN4x mRNA in PMM DA-MEL neurons of photorefractory hens was significantly lower as compared with that of short or long day photosensitive hens. The results implicate tOPN4x in hypothalamic PMM DA-MEL neurons as an important component of the photoreceptive system regulating reproductive activity in temperate zone birds.

A substance interfering with the enzyme-linked immunosorbent assay (ELISA) for feline insulin concentration was investigated in healthy cats. An insulin-binding substance isolated from feline serum showed 2 bands at 25 and 50 kDa in SDS-PAGE, suggesting the presence of immunoglobulin G (IgG). Insulin-binding IgG from healthy cats indeed reduced insulin immunoreactivity in the ELISA for determining insulin concentration. The insulin-binding IgG was polyclonal/polyreactive and showed certain specificity, high affinity, and high binding capacity, which was evaluated by liquid-phase radioimmunoassay with Scatchard plot analysis. Epitope analysis revealed that the insulin-binding IgG showed significant binding at residues A1-5 and B20-30 of the insulin molecule. Removal of the antibodies from serum enabled the determination of serum insulin concentrations by ELISA. Our data indicated that serum from healthy cats contained substantial amounts of natural autoantibodies combined with insulin, and that the antibodies interfered with the heterologous immunoassay for serum insulin concentration.

The present study was conducted to determine both the site at which the eggshell matrix is produced and the critical period for its production in the oviductal uterus of the Japanese quail, Coturnix japonica. An antiserum was produced against the proteins of the 116-kDa band in electrophoretic profiles of the matrix proteins obtained from eggshells decalcified with EDTA. N-terminal amino acid sequences were very similar to those of chicken ovocleidin-116. Immunofluorescent and immunoelectron microscopic observations revealed that the 116-kDa proteins were synthesized in granular cells of the uterus mucosal epithelia, secreted between 7 and 20 h after the previous oviposition, then integrated into the structure of the eggshell matrix. Scanning electron microscopic observations revealed that 10-μm-wide posts appear on the surface of the mucosal epithelia until 21h, and disappear thereafter. The posts were less prominent in the mucosal epithelium facing the sharp end of an egg than in that facing the equator or blunt end. On the other hand, the number of pores in eggshells at the sharp end was smaller than that at the equator or blunt end. Our results indicate that air canals and/or pores are formed in the eggshell by the retreat of the posts after the establishment of eggshell.

Two calcified structures, the eggshell and sperm-associated body (SB), are present in the eggs of the Japanese quail, Coturnix japonica. X-ray diffractometry showed that calcium carbonates take the form of calcite in the eggshell and aragonite in the SB. The aim of the present study was to identify the factors that determine the morphology of calcium carbonate crystals. The matrix of EDTA-treated eggshell was a meshwork of vesicles, 200 to 500 nm in diameter, connected by fine fibers or fibrous sheets. The matrix of SB cortex was a radiation of rod-shaped projections approximately 130 nm in width. In vitro crystal formation was achieved by adding dissociated matrix substances to test solutions. When eggshell matrix material was added, formation of calcite crystals, which had many vesicular holes on their surface, was observed. When SB matrix material dissociated by sonication was added, rhombohedral calcite crystals formed at protein concentrations of 100 μg/mL or lower, and elongated and bundled crystals formed at concentrations of 150 μg/mL or higher. When SB matrix material dissociated by pipetting was added, aragonite crystals formed. These observations indicate that the matrix structure is the principal factor in determining the crystal polymorphism of calcium carbonate.

In the eggs of the quail Coturnix japonica, the limiting membrane demarcates the shell membrane at the interface with the albumen and decreases in width during the hatching process. This study was done to identify agents that affect the width of this limiting membrane. Zymography tests on extracts from extraembryonic tissues, yolk sacs, or chorioallantoic membranes, or all three, showed proteolytic activities during d 4 to 10 of incubation. Localization experiments on these activities, performed on d 5 eggs, indicated that they were located in an avascular chorion. Electron microscopic analysis showed there were secretory cells specifically located in the avascular chorion. After partial purification of d 5 avascular chorion extracts using QA52 and Sephadex G-200 column chromatography, the proteolytic activity of 20 kDa was isolated. The protease showed a high level of activity toward succinyl-Gly-Pro-Leu-Gly-Pro-4-methylcoumaryl-7-amide. It had an optimal pH of 9 and digested the limiting membrane. These enzymatic activities were inhibited moderately by EDTA and strongly by leupeptin and aprotinin. It was concluded that it is the 20-kDa protease, showing collagenase-like activity produced by the avascular chorion, that affects the limiting membrane.

Time-resolved fluoroimmunoassay (TR-FIA), which has recently been developed as a non-isotopic immunoassay, was intended for assay of gonadotropins (LH and FSH). Ovine LH or FSH was labeled with N1-(p-isothiocyanatobenzyl)-diethylenetriamine-N1, N2, N3, N3-tetraacetic acid Eu-chelate (DTTA), and competitive binding assay was performed using a 96-weel titer plate previously coated with a second antibody, followed by measurement using time-resolved fluorometry. TR-FIA for standard ovine LH (NIDDK, LH-I-3) or FSH (NIDDK, FSH-I-1) had a sensitivity of about 25 pg/50 microliters sample. The assay system was applied to heterologous assay of porcine gonadotropins. Linearity was obtained by the dilution test using medium from primary culture of porcine anterior pituitary cells. Intra- and inter-assay coefficients of variation of LH and FSH determination in 31 different porcine samples were satisfactorily low, between 3.5 and 8.1% (intra-assay) and between 1.7 and 13.1% (inter-assay). Correlation coefficients between radioimmunoassay and TR-FIA were calculated to be 0.945 for LH and 0.978 for FSH. Stimulation of LH and FSH release with GnRH was observed by TR-FIA. This non-isotopic TR-FIA thus provides as good sensitivity, reproducibility and accuracy as conventional RIAs.

The present study was conducted to determine both the site at which cuticle materials are produced and the critical period for their production in the oviductal uterus of the Japanese quail, Coturnix japonica. An antiserum was produced against the 32-kDa band in electrophoretic profiles of cuticle materials obtained from eggshells decalcified with EDTA. Immunofluorescence and immunoelectron microscopic observations revealed that the 32-kDa protein was synthesized in luminal ciliated epithelial cells of the uterus until 21 h after the previous oviposition (the first phase) and then secreted during the 4 h before the next oviposition (the second phase). Scanning electron microscopic observations revealed that 10-microm-wide posts appear on the surface of the luminal epithella during the first phase, and that they disappear during the second phase. During the second phase, air canals are formed in the eggshell by the retreat of the posts, and a cuticle layer forms on the eggshell. Our results indicate that the cuticle may function as a lubricant that facilitates egg rotation in the uterus.

Electron microscopic observations have revealed an electron density gradient in the chalaziferous layer of avian egg. The density is highest in the innermost sub-layer, which is bound with the vitelline membrane, and is thus traditionally referred to as the vitelline membrane outer layer (VMO). After high-concentration salt treatment, two proteins, VMO-I and VMO-II, are liberated from the egg envelopes of quail (Coturnix japonica), which results in the separation of the chalaziferous layer from the vitelline membrane. VMO-I and VMO-II were purified through gel-filtration and subjected to antisera produced in rabbits. The molecular sizes of these two proteins were estimated to be 9 to 15kDa for VMO-II and 18kDa for VMO-I. Their amino acid sequences were very similar to those of chickens. Immunofluorescent and immunoelectron micrographs showed that VMO-II was produced by the luminal epithelium and VMO-I by both the luminal and glandular epithelia of the infundibulum. Immunogold particles for the labeling of both proteins were distributed throughout the chalaziferous layer, as well as in the chalazae, with a density gradient similar to that mentioned above. In salt-treated envelopes, immunogold labeling was completely absent when stained with anti-VMO-II antiserum and weak with anti-VMO-I antiserum. Immunohistochemical studies revealed that purified VMO-II bound with vitelline membranes of salt-treated envelopes, and ligand blotting tests revealed that ZP1 and ZP3 were the molecules bound by VMO-II; the binding was inhibited by various kinds of sugars. VMO-II might play a role in the binding of the chalaziferous layer with the vitelline membrane, a process that leads to the anchoring of the chalaza cord.

The objective of this study was to compare the feed intake, digestibility and metabolism in lambs fed low-quality roughage with those of lambs fed normal roughage from an early stage of their life. The study consisted of two treatments [low-quality roughage group (LR) and control group (C)] over three time periods (P1, P2 and P3; 2 months each). Four lambs (4 months old) were allocated to each treatment. In P1 and P2, LR was fed sudangrass hay (CP: 5.1% DM; NDF: 70.4% DM), whereas C was fed timothy hay (CP: 8.4% DM; NDF: 60.3% DM). In P3, all lambs were fed sudangrass hay. Although the feed intake was significantly greater (p

Avian eggs possess a shell membrane in the shape of an asymmetrical ellipsoid and with a limiting membrane that is a smooth layer of homogeneous, dense materials. We describe the role of the magnum-isthmus junction (MIJ) of the oviduct in the formation of the avian-type shell membrane in the domestic fowl Gallus domesticus. The narrow width of the lumen at the MIJ indirectly participates in the determination of the asymmetrical ellipsoid shape of eggs that are encased by the egg-white layer and subsequently by the peri-albumen layer (PL) and the shell membrane. The PL reacts with Alcian blue and exists between the egg white and the limiting membrane. It is added to the ovulating egg at the MIJ and covers the outermost surface of the egg-white layer. The function of the PL is to provide a smooth surface by covering the irregular surface of the egg-white layer. The materials of the PL consist of an Alcian blue-positive polysaccharide (or glycoprotein) of 240 kDa and five proteins of 135, 116, 72, 49, and 46 kDa. The isolated materials have an affinity to bind with the egg-white mass. An antiserum against quail PL materials stains the domestic fowl PL and secretory cells of the luminal epithelium at the MIJ, and cross-reacts with the molecules of 240, 135, and 116 kDa.

Although a number of immunohistochemical studies have been carried out on the differentiation of chicken gonadotropes during embryogenesis, the temporal and spatial properties of appearance of gonadotropes are not clear. In this study, we studied the appearance and morphological characteristics of gonadotropes in the embryonic and adult chicken anterior pituitary glands using RT-PCR, in situ hybridization and immunohistochemistry. For this purpose, we raised specific antisera against chicken follicle-stimulating hormone beta-subunit (cFSHbeta) and chicken luteinizing hormone beta-subunit (cLHbeta) based on each putative amino acid sequence. RT-PCR analysis revealed that cFSHbeta mRNA was expressed from embryonic day 7 (E7). Chicken FSHbeta mRNA-expressing (-ex) and -immunopositive (-ip) cells started to appear in the ventral part of the caudal lobe in the anterior pituitary gland at E8. Chicken LHbeta-ip cells were also first observed there at E8, but cLH mRNA expression was confirmed from E4 by RT-PCR analysis. The distribution of these chicken gonadotropin-ex and -ip cells spread from the ventral part to dorsal part in the caudal lobe around E10 and subsequently expanded to the cephalic lobe from E12 to E20. These cells were morphologically classified into two types (round- and club-shaped cells). It was found that the density of gonadotropin-ip cells in the caudal lobe was always higher than that in the cephalic lobe throughout the period of development. To the best of our knowledge, this is the first report focusing on the differentiation of chicken gonadotropes by assessment of both protein and mRNA of chicken gonadotropin.

Thyrotropin or thyroid-stimulating hormone (TSH) secretion in the chicken is controlled by several hypothalamic hormones. It is stimulated by thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH), whereas somatostatin (SRIH) exerts an inhibitory effect. In order to determine the mechanism by which these hypothalamic hormones modulate chicken TSH release, we examined the cellular localization of TRH receptors (TRH-R), CRH receptors type 1 (CRH-R1) and somatostatin subtype 2 receptors (SSTR2) in the chicken pars distalis by in situ hybridization (ISH), combined with immunological staining of thyrotropes. We show that thyrotropes express TRH-Rs and SSTR2s, allowing a direct action of TRH and SRIH at the level of the thyrotropes. CRH-R1 expression is virtually confined to corticotropes, suggesting that CRH-induced adrenocorticotropin release is the result of a direct stimulation of corticotropes, whereas CRH-stimulated TSH release is not directly mediated by the known chicken CRH-R1. Possibly CRH-induced TSH secretion is mediated by a yet unknown type of CRH-R in the chicken. Alternatively, a pro-opiomelanocortin (POMC)-derived peptide, secreted by the corticotropes following CRH stimulation, could act as an activator of TSH secretion in a paracrine way.

The unavailability of homologous antibodies to avian thyrotropin (TSH) has been a major hindrance in the study of avian reproduction and metabolism. Here we report the production and characterization of an anti-peptide antibody specific to chicken TSH β subunit. According to the cDNA sequence of chicken TSH β subunit, a synthetic peptide corresponding to the amino acid residues 35-50 (Tb3550; Asp-Ser-Asn-Gly-Lys-Lys-Leu-Leu-Leu-Lys-Ser-Ala-Leu-Ser-Gln-Asn) was prepared to produce polyclonal antibody in rabbits. This peptide sequence is highly specific to avian TSH β and was designed for the antibody to show no cross-reaction to pituitary and other chicken proteins with known amino acid sequences. Anti-Tb3550 recognized a 18.2-kDa band on immunoblot of the cephalic lobe homogenate, but not of the caudal lobe homogenate, of chicken anterior pituitary gland. Positive immunohistochemical staining with anti-Tb3550 was restricted to the cephalic lobe, whereas the cells positive to anti-chicken luteinizing hormone were scattered throughout the anterior pituitary. Anti-Tb3550 did not bind either to purified chicken luteinizing hormone or to follicle stimulating hormone preparation. These results indicate that this antibody specifically recognizes chicken TSH β subunit. This antibody should be useful for immunochemical study of avian TSH and for tracing TSH activity during the purification of the hormone.

Levels of pituitary thyrotropin messenger RNA and circulating hormones during molting induced by feed and water deprivation were determined by reverse-transcription polymerase chain reaction and radioimmunoassays. Plasma levels of luteinizing hormone and estradiol decreased from the next day (Day 1), and progesterone from Day 3, of deprivation treatment, and were associated with the cessation of egg laying on Day 4. The plasma thyroxine level of experimental hens increased on Day 5 and remained high until Day 17, whereas the plasma concentration of triiodothyronine was lower than the control level throughout the experimental period. The messenger RNA level of the pituitary thyrotropin β subunit of experimental hens decreased markedly at first, but then increased showing a peak on Day 3 followed by a gradual decrease until Day 17. The serum prolactin level decreased on Day 3 and remained low until Day 17. These results suggest that (1) a decline in hormones associated with egg laying is followed by activation of the pituitary-thyroid axis, (2) thyrotropin triggers the increase in thyroxine but is not necessarily responsible for the maintenance of the high thyroxine level, and (3) prolactin might be involved in molting induced by feed and water deprivation.

An aromatase inhibitor, Fadrozole (CGS16949A), was administered into chick embryo on the 5th day of incubation to clarify the roles of endogenous estrogen in the regulation of circulating lipoprotein levels during the development. The plasma of 10-, 16- and 20-day embryos and neonatal chicks were separated by polyacrylamide gel electrophoresis. Amounts of plasma lipoproteins, high density lipoprotein (HDL), low density lipoprotein (LDL) and very low density lipoprotein (VLDL) were then measured using a densitometer and expressed as a percentage in total lipoproteins. Changes in HDL percentages of the groups treated with the aromatase inhibitor (AI group) and propylene glycol (Vehicle group) showed similar patterns during the experimental period. This was also the case in the changes in LDL percentages. The 16-day embryos of the AI group, in which endogenous estrogen synthesis was inhibited, showed a distinctly high VLDL percentage, whereas plasma VLDL in the Vehicle group showed only a little developmental changes. The plasma concentration of triglyceride (a major component of VLDL), phospholipid and total cholesterol in the AI group showed a profile similar to that of VLDL, with the maximum level in the 16-day embryo. These results suggest that endogenous estrogen of the embryo plays an important role in the utilization and resulting clearance of plasma lipids from the circulation, in addition to its well-documented role in sex differentiation.

産卵との関連におけるLHとPRLの合成の動態を明らかにしようとして,産卵鶏と休産鶏において,一日のいろいろな時間に下垂体前葉中のLH-βsubunitとPRLのmRNA濃度をSolution hybridization-RNase protection assayを用いて測定するとともに,下垂体前葉と血中におけるLHとPRL濃度をラジオイムノアッセイにより測定した。排卵周期中において,下垂体前葉中のLH-βsubunit mRNA濃度は排卵18-19時間前から順次増加し,排卵9-10時間前に最も高い値を示した。下垂体前葉中のLH濃度は排卵9-10時間前に有意なピ-クが見出された。血中LH濃度は排卵6-7時間前に有意に高い値が認められた。これに対して,休産鶏においてはこれらいずれに関しても一日の内で有意な変動は認められなかった。一方,下垂体前葉中のPRL mRNAとPRL濃度は,産卵鶏と休産鶏においてともに,一日の内で有意な変動は認められなかったが,血中PRL濃度は暗期後半の午前4-5時に有意なピ-クが見出された。これらの結果から,排卵前に認められるLHサ-ジに先立ってLH合成が促進されると推察され,また,PRL合成は一日の内で顕著な変動を示さないが,PRL放出は排卵とは無関係に明暗周期と関連して暗期後半に多くなるような様相で日内変動していることが明らかとなった。

This study investigates whether chicken lutropin (LH) specifically binds to rat ovarian follitropin (FSH) receptor and exerts FSH-like bioactivity. Glycoprotein fraction, prepared from the chicken anterior pituitary gland, was fractionated using isoelectric focusing within a pH range of 3.5-11. Analysis of the focused fractions, by a radioreceptor assay (RRA) specific for FSH in rats using rat ovarian homogenate as receptor source, and 125I-labeled rat FSH as radioligand, detected a large component having an isoelectric point of 10.25. This focusing profile obtained by RRA was quite similar to that obtained by a specific radioimmunoassay (RIA) for chicken LH, but clearly different from that obtained by a specific RIA for chicken FSH, indicating this RRA specifically recognizes chicken LH. Chicken LH fraction prepared from the electrofocused material was used for further studies. The chicken LH preparation was three times more potent than rat FSH in the RRA in displacing the radioligand bound to rat ovarian receptor, while chicken LH facilitated an 8-fold less production of estradiol in dispersed rat granulosa cells than rat FSH. These results suggest that chicken LH acts like rat FSH in rat ovarian FSH receptor, but receptor-binding activity is much higher than biological activity.

Crude extracts were prepared from the anterior pituitary glands of male and female birds of 8- and 10-week-old Japanese quails and fractionated by isoelectric focusing. Focused fractions were subjected to a radioreceptor assay using homogenized rat ovary as receptor preparation and 125 I-labeled rat follicle stimulating hormone as radioligand. This radioreceptor assay was previously characterized for measuring mammalian follicle stimulating hormone. Components having basic Isoelectric points (pI) were detected in both male and female birds of 8- and 10-week old. The component was most basic (pI 12.2) and largest (1.6μg/fraction/pituitary gland) in 10-week old female birds. The component in males was larger in 10-week-old birds than in 8-week-old birds. The present findings suggest that changes in pI and amounts of Isoelectric components may be related to sexual maturation in Japanese quails.

Thyroid-stimulating hormone (TSH) beta subunit cDNA was obtained from Japanese quail (Coturnix japonica) by PCR and its nucleotide sequence was determined. The cDNA encodes a putative signal peptide and a mature protein consisting of 20 and 114 amino acids, respectively. The amino acid sequence of quail TSHbeta subunit shows homologies of 67-69% in mammalian species, 58% in amphibian and 43-49% in teleost fish. Comparison of the amino acid sequence with TSHbeta subunits of other species reveals some differences in several regions responsible for its biological functions and characteristic features of the avian TSHbeta subunit, suggesting that the functional domains have diverged cooperatively between the hormone and its receptor during evolution.

Anti-insulin immunoglobulin G (IgG) has been found in the sera of healthy cats. To determine the concentrations of these antibodies, an enzyme-linked immunosorbent assay (ELISA) for anti-insulin IgG was developed. ELISA maintained the linearity of a standard concentration line between 67.5 and 2160 ng/ml. The coefficients of variances (CVs) of intra-assays in two different plasma samples were 4.0% and 3.7%, respectively. The inter-assay CVs in two different plasma samples were 5.1% and 6.9%, respectively. The dilution curves of two samples were rectilinear. Anti-insulin IgG was detected in all 84 of the healthy cats that were tested. Plasma anti-insulin IgG concentrations ranged from 80 to 1578 μg/ml, with a median concentration of 221 μg/ml, and this value correlated positively with total plasma IgG concentrations (r=0.383, p

12時間照明12時間暗黒(12L:12D,6時点灯18時消灯)から2種類の昼夜逆転光周期,連続照明,連続暗黒,8L:8D,18L:18Dおよび24L:24Dの光周期条件に各々移した時の幼若鶏や成鶏における血漿メラトニン濃度の変化をラジオイムノアッセイ法によって測定した.12L:12Dの後,14,82および450日齢のニワトリを12時間照明下で,また14,72および400日齢のニワトリを12時間暗黒下で飼育し,再び12L:12D条件下に戻した場合の血漿メラトニンリズムは,14日齢での処理1日目の場合を除いて2種類の昼夜逆転光周期に同調した.連続照明の場合,14日齢のニワトリの血漿メラトニンリズムは,光によって処理1日目のみ抑制されたが,その後はリズムが観察された.しかし,70および150日齢の場合では,処理3日間を通して暗期に有意に高い濃度を示すフリーランニングリズムを認めた.連続暗黒の場合,14,70および150日齢における血漿濃度は,24時間よりも長いフリーランニングリズムを示した.なお,連続照明または連続暗黒の場合のリズムの振幅は12L:12Dの光周期のものと同じであった.8L:8D条件下での成鶏の血漿濃度は,暗期予定時間帯にのみ顕著に増加した.18L:18D条件下では,処理後1サイクルにおいてのみ濃度が上昇したが,その後は光周期に同調しなかった.24L:24D条件下では,リズムはなく12L:12Dの昼夜の間の濃度を維持した.これらのことから,成鶏を24時間よりも長い光周期で飼育した時の血漿メラトニンリズムは不明確で,砂時計型であることが明らかとなった.また,24時間よりも短い光周期では,明暗刺激がメラトニンの概日リズムを速やかに変化させることや,明期開始ではなく暗期開始が,ニワトリのサーカディアンリズムを同調させる最も重要な生理学的信号になるものと考えられた.

Localization of melatonin in the pineal glands of 1, 21, 56 and 560dayold chickens kept under 12hr light and 12hr dark photoperiodic conditions was examined. Pineal parenchymal cells of the chicken were immunostained using a primary antimelatonin antiserum at dilutions up to 1:5, 000. The antiserum recognized melatonin specifically in situ, although crossreactivity with related compounds particularly with 5-methoxytryptamine hydrochloride was observed in vitro. Immunoperoxidase method for the demonstration of melatonin was applied to paraffin sections of icecold 10% neutral buffered formalin or BOUIN's fixation in the chicken pineal gland. A more intense immunostaining was found at 12:00 (midday) in the follicular zone as compared with the parafollicular zone in the pineal gland of al dayold chick, but no difference between these two divided zones was observed in immunoreactivity after 21 days of age. In both follicular and parafollicular cells at 24:00 (midnight), intense immunostaining in almost all the parenchymal cells was observed at all ages examined in our study. The number of immunoreactive melatonin granules in these two regions of pineal tissue thus showed daily changes. The amount of immunoreactive melatonin in the pineal parenchyma increased both at midday and midnight with advancing age. These results suggested that the regression of the photosensory function paralleled by crushing follicular structure resulted in the progression of the competence of melatonin biosynthesis with advancing age.

The mechanism by which the embryo hatches out of the egg envelope, the vitelline membrane and egg white, was studied in the Chinese soft-shelled turtle Pelodiscus sinensis. The cDNA of the turtle hatching enzyme (HE) was 1555 bp-long and a mature enzyme of 321 amino acids. The mature HE was composed of an astacin protease domain of 200 amino acids and a CUB domain of 121 amino acids, and the estimated molecular size was 35,311. The protease domain contained two active site consensus sequences, HExxHxxGFxHExxRxDR and MHY. An immunoblotting test of an extract of allanto-chorions revealed a 40-kDa band by cross-reaction with the anti-Xenopus HE antiserum. The first change in the envelopes was the appearance of a hole, 1 mm in diameter, at the location around the animal pole of day 8 incubation eggs. A cluster of tall cells, forming a circle in the avascular chorion of day 8 embryos and facing the edge of the hole, had various sizes of inclusion bodies and secretory granules that were labeled by immuno-electron microscopic staining with the antiserum. The egg envelopes were degraded gradually from the animal pole side towards the vegetal pole side in accordance with translocation of the avascular site of the chorion in the same direction. Labeled cells degenerated, presumably when the chorion was underlain by allantois in succeeding developmental stages. The vitelline membrane and egg white were totally digested, presumably by secreted HE, during the hatching period and were consumed for embryonic growth.

To develop a homologous radiolnlniunoassay (RIA) for a hormone of a small or rare animal often meets difficulty in collecting a large anmount of purified antigen required for antibody production. On the other halld, to employ a heterologous RIA to estimatc the horm me oftell gives poor scnsilivity, To overcome this difficulty, a “hetero-antibody” RIA was studied. In a hetero-antibody RIA system, a purified preparation of a hormone is used for radioiodination and standardization and a heterologous antibody to the hormone is used for the first antibody. Canine motilin and rat LH were selected as examples, and anti-porcine motilin and anti-hCC, and-hCGβ or and-ovine LHβ was used as the heterologous antibody. The sensitivities of the hetero-antibody RIAs were much higher than those of heterologous RIAs in any case, showing that these hetero-antibody RIA systems were suitable for practical use. To clarify the principle of hetero-antibody RIA, antiserum to porcine motilin was fractionated on an affinity column where canine motilin was immobilized. The fraction bound had greater constants of affinity with both porcine and canine motilins than the rest of the antibody fractions. This fraction also reacted with a synthetic peptide corresponding to the C-terminal sequence common to porcine and canine motilins in a competitive binding test with labeled canine motilin. These results suggest that an antibody population having high affinity and cross-reactivity is present in polyclonal antiserum and indicate that the population can be used in hetero-antibody RIA at an appropriate concentration.

In accordance with chicken mtDNA sequences, three pairs of primers were designed for the cytochrome b (Cyt b) gene. Three mtDNA fragments were amplified via PCR from silky fowl total genomic DNA in blood. The PCR products were used for detecting the DNA sequences by the direct-sequencing method. The silky fowl cytochrome b gene consisted of 1143bp nucleotides, encoded for a 380 amino acid protein, and showed 99.4% and 86.8% homology with White Leghorn and Japanese quail, respectively. The homology of the amino acid sequence of the Cyt b gene of silky fowl and White Leghorn birds was 100%, with a corresponding 93.1% between silky fowl and quail. The sequences reported in this paper have been deposited in the GenBank database (accession no. AF 102874).

Molecular mechanisms regulating animal seasonal breeding in response to changing photoperiod are not well understood. Rapid induction of gene expression of thyroid-hormone-activating enzyme (type 2 deiodinase, DIO2) in the mediobasal hypothalamus (MBH) of the Japanese quail (Coturnix japonica) is the earliest event yet recorded in the photoperiodic signal transduction pathway. Here we show cascades of gene expression in the quail MBH associated with the initiation of photoinduced secretion of luteinizing hormone. We identified two waves of gene expression. The first was initiated about 14 h after dawn of the first long day and included increased thyrotrophin (TSH) beta-subunit expression in the pars tuberalis; the second occurred approximately 4 h later and included increased expression of DIO2. Intracerebroventricular (ICV) administration of TSH to short-day quail stimulated gonadal growth and expression of DIO2 which was shown to be mediated through a TSH receptor-cyclic AMP (cAMP) signalling pathway. Increased TSH in the pars tuberalis therefore seems to trigger long-day photoinduced seasonal breeding.

The present study was conducted to determine the mechanism of chalaza formation in eggs of the Japanese quail Coturnix japonica and to determine the production site of chalaza materials in the oviduct. Electrophoretic profiles of the chalaza materials showed six bands of 480, 320, 210, 180, 96, and 58 kDa following Coomassie Blue staining and one band of 600 kDa after immunoblotting. An antiserum was produced against the 180-kDa band. This antiserum and an antiserum generated against the 600-kDa protein were used as probes to detect chalaza materials. Immunofluorescent and immunoelectron-microscopic observations revealed that chalazae and chalaziferous layers overlaid to approximately 40 microm upon the vitelline membrane of the ovum were composed of the same materials as those produced by both types of secretory cells in the luminal and glandular epithelia at the infundibulum. We propose that the mechanism of chalaza formation is as follows: (1) chalazae first appear as fine filaments at the presumptive sharp and blunt ends of the ovum at the infundibulum; (2) these filaments are twisted into a lead fiber while the ovum is rotating and descending in the magnum; (3) at the posterior end of the magnum, the lead fiber is anchored to the thick egg white and lifted outward with the chalaziferous sublayers when the inner egg white is liquefied by absorbing water; (4) the lead fiber and chalaziferous sublayers are twisted further into the chalaza in the uterus.

The present study describes the biological meaning of the asymmetrical shape in avian reproduction using quail. During the incubation of eggs, water was gradually lost and the air chamber which appeared in between the inner and outer shell membranes at the blunt end expanded, so that the angle made by the long egg-axis and the horizontal line increased, presumably because the centre of gravity of the egg contents moved toward the sharp end. The increase in angle occurred in both fertile and infertile eggs, suggesting that this phenomenon occurs irrespective of fertility and is due to the asymmetrical shape. The increase in the volume of the air chamber resulted in an increase in the area of the inner shell membrane at the chamber to satisfy the amount of gas exchange needed by the developing embryo for better hatching. We isolated a 300-kDa protein from the inner shell membrane. It was produced by cells in the luminal epithelium of the oviductal isthmus and was found in the cortex of the fibres of shell membranes and a lining surrounding the air chamber. The lining comprised a medial layer between the inner and outer shell membranes in uterine eggs. The asymmetrical ellipsoid produces the air chamber at the blunt end of the avian egg during its sojourn in the oviductal isthmus, to maintain the blunt end up after oviposition and to raise that end during incubation in a dry environment, leading to high hatchability.

We investigated the mechanism for non-proteolytic activation of human prorenin using five kinds of antibodies. Each of the antigens, L1PPTDTTTFKRI11P, T7PFKRIFLKRMP17P, I11PFLKRMPSIRESLKER26P, M16PPSIRESLKER26P, and G27PVDMARLGPEWSQPM41P, was designed from the tertiary structure of predicted prorenin. These antibodies were labeled anti-01/06, anti-07/10, anti-11/26, anti-16/26, and anti-27/41, respectively, for their binding specificities. Inactive recombinant human prorenin (0.1 nM) bound to various concentrations of anti-01/06, anti-11/26, and anti-27/41 antibodies at 4 degrees C with equilibrium dissociation constants of 138, 41, and 22 nM, respectively. However, intact prorenin (0.1 nM) did not show significant binding to 200 nM anti-07/10 and anti-16/26 antibodies for 20 h. Ninety percent of prorenin (0.1 nM) was found to be non-proteolytically activated by incubation with anti-11/26 antibodies (200 nM) at 4 degrees C for 20 h. Prorenin was not active even under complex with either anti-01/06 or anti-27/41 antibodies. Prorenin was also reversibly activated at pH 3.3 and 4 degrees C for 25 h. The acid-activated prorenin bound to anti-07/10 and anti-16/26 antibodies as well as to anti-01/06, anti-11/15, and anti-27/41 antibodies at neutral pH and 4 degrees C in 2 h. Their dissociation constants were 13, 40, 8.6, 3.6, and 14 nM, respectively. The acid-activated prorenin was re-inactivated by incubation at pH 7.4 and 4 degrees C in 50 h. Anti-07/10 and anti-11/26 antibodies inhibited such re-inactivation at 25 degrees C by more than 90% and 50%, respectively, whereas other kinds of antibodies did not prevent the re-inactivation at 25 degrees C. These results indicate that prorenin has "gate" (T7PFKR10P) and "handle" (I11PFLKR15P) regions critical for its non-proteolytic activation.