Changes in ovalbumin and protein synthesis in vivo in the magnum of laying hens during the egg formation cycle.

1. The present study was carried out to investigate whether or not the rate of synthesis of total protein in various oviducal segments and ovalbumin, a major egg white protein, in the magnum fluctuated during the egg formation cycle in laying hens. 2. Synthesis of total protein and ovalbumin was measured in vivo by the incorporation of [15N]methionine after a primed continuous infusion of tracer for 3 hr. 3. Protein and ovalbumin contents in the magnum and the entire oviduct decreased sharply when an ovum moved down from the magnum to the isthmus, probably due to the secretion of egg white proteins. 4. In contrast, total protein and ovalbumin synthesis in the magnum was significantly higher when an ovum was in there than when it was in any other segments. Fluctuations of ovalbumin synthesis and total protein synthesis in the magnum were roughly parallel to those of total protein synthesis in the entire oviduct. 5. It was concluded, therefore, that the changes seen in total protein synthesis in the whole oviduct during the egg formation cycle were mainly attributable to those in magnum protein synthesis, of which a significant portion was accounted for by the synthesis of ovalbumin.     

Role of hydroxyl radical scavengers dimethyl sulfoxide,alcohols and methional in the inhibition of prostaglandin biosynthesis

Prostaglandin (PG) F_2α provoked a contraction of longitudinal tissue strips from the uterine, vaginal and isthmus regions of the chicken oviduct; no response was induced in longitudinal tissue strips from the magnum and infundibulum.PGE₂ induced a contraction of circular and longitudinal uterine strips and of circular strips from the isthmus and infundibulum. With both circular and longitudinal vaginal strips a relaxation was obtained. A dose-dependent response was observed with circular magnum strips: contraction with low doses and relaxation at higher doses. No response was obtained with longitudinal tissue strips from the isthmus, magnum and infundibulum.The possible influence of both prostaglandins in the mechanism of ovum transport and oviposition in the domestic hen is discussed.     

Smooth muscle of the quail oviduct functions as a stretch receptor during ovum transport

The present experiments were conducted to test the hypothesis that ovum transport in the quail oviduct is regulated by a time-dependent, stretch-mediated feedback cycle which alters the frequency of contractions. According to this hypothesis, a ligature preventing the forward movement of ovum should reverse the direction of the feedback cycle and an artificial ovum should be transported like the normal ovum. When the ligature was placed in the borderline between magnum and isthmus, it caused the reversal of transport direction after a delay of several minutes. Once the direction had changed, it persisted until the ovum was expulsed through the fimbrial end or until a second reversal was caused by either a second ligature or a minor mechanical impediment at the proximal end of the magnum. The ovum was transported between the ligatures at the mean speed of 1.7 +/- 0.17 mm/min (n = 7) until the ovum broke. An artificial ovum placed in the proximal magnum from which the natural ovum had been removed, was transported like the natural ova. Myoelectrical activity recorded with suction electrodes was statistically similar in both types of experiments and the direction of the frequency gradient changed when the transport direction was reversed. The frequency of the electrical activity of oviductal smooth muscle was significantly higher behind the ovum than in its front whether ova were transported in the direction of shell gland or infundibulum; in the segment maximally stretched by the ovum the activity was significantly lower than in other segments. These observations confirmed the hypothesis and suggest that the quail oviduct functions like a stretch receptor.     

Identification of precursor peptide of aculeacin A acylase as a protein with proteolytic activity

Abstract A protein with the proteolytic activity was isolated from culture filtrate of the aculeacin A acylase producing strain, Actinoplanes utahensis NRRL12052. The purified protein showed a single band of molecular mass of 87 kDa in SDS-PAGE and gel filtration using HPLC, and reacted with anti-aculeacin A acylase antiserum. The 87-kDa protein was degraded to two peptides of molecular mass of 60 kDa and 19 kDa by incubation at 37°C in the presence of 0.1% SDS and the former band also responded to the antiserum. These results indicate that the 87-kDa protein possessing the proteolytic activity is a precursor of aculeacin A acylase.     

Comparative distribution of glycosaminoglycans and proteoglycans in the reproductive tissues of the female fowl

The objective of this work was to survey and compare the composition of the parts of the reproductive system of the female fowl in glycosaminoglycans and proteoglycans. Those parts analyzed were ovary, infundibulum, magnum, isthmus, shell gland and vagina. Methods of analysis included cellulose acetate electrophoresis, infrared spectroscopy, colorimetry, amino acid determination and scanning electron microscopy. Concentrations of glycosaminoglycans were higher in vagina, ovary, infundibulum and isthmus than in shell gland and magnum. Glycosaminoglycans may be important in those parts of the reproductive tract which contribute membraneous and mucosal material to the descending egg, and where fertilization of the egg occurs.     

Ovigerous-hair stripping substance (OHSS) in an estuarine crab: purification, preliminary characterization, and appearance of the activity in the developing embryos

Ovigerous-hair stripping substance (OHSS) is an active factor in crab hatch water (i.e., filtered medium into which zoea larvae have been released). This factor participates in stripping off the egg attachment structures (i.e., egg case, funiculus, and the coat investing ovigerous hairs) that remain attached to the female's ovigerous hairs after larval release. Thus this activity prepares the hairs for the next clutch of embryos. OHSS activity of an estuarine crab, Sesarma haematocheir, eluted as a single peak on molecular-sieve chromatography, but this peak still showed two protein bands at 32 kDa and 30 kDa on SDS-PAGE. The two protein bands stained with a polyclonal antiserum raised to the active fractions from molecular-sieve chromatography. Moreover, antibodies purified from this polyclonal OHSS antiserum also recognized both the 32-kDa and 30-kDa bands. OHSS immunoreactivity and biological activity were associated with the attachment structures that remained connected to the ovigerous hairs after hatching. In developing embryos, both protein bands could be stained immunochemically at least 10 days before hatching. But OHSS biological activity appeared only 3 days before hatching. The immunoreactive protein bands were not observed in the zoea, but OHSS bioreactivity was present, though greatly reduced. The 32-kDa protein, at least, is probably an active OHSS, and the 30-kDa protein band may also be OHSS-related. The OHSS appears to be produced and stored by the developing embryo. Upon hatching, most of the material may be trapped by the remnant structures, and the remainder is released into the ambient water.     

Origin of 33 kDa protein of vitelline membrane of quail egg: Immunological studies

The inner layer of vitelline membrane is an investment of avian ovum at the time of ovulation, but its formation is poorly understood. In order to elucidate the origin of the inner layer of vitelline membrane, a 33 kDa protein, one of the components of the inner layer, was purified from quail eggs and polyclonal antibody was raised against this protein. The tissue distribution of protein interacted with the antibody was studied by Western blotting technique. No immunoreactive component could be observed in extracts of liver, kidney, heart, lung, small intestine, brain, infundibulum, albumen-secreting region of oviduct, uterus, and wall of small white follicles. The intensive band was detected in the granulosa layer, which was isolated from the large preovulatory follicles as a monolayer of granulosa cells sandwiched between the inner layer of vitelline membrane and the basal lamina. The granulosa cells isolated from the granulosa layer also reacted with this antibody. Theca layer had no immunoreactive components. The position of the band of the 33 kDa protein on SDS-PAGE was sifted to higher molecular weight in follicular tissues as compared with that in the laid eggs, indicating that the structural change of the protein occurs after ovulation. These studies indicate that the material reactive to the antibody raised against a 33 kDa protein of quail vitelline membrane is synthesized by the granulosa cells.     

Biosynthesis, glycosylation, and intracellular transport of intestinal lactase-phlorizin hydrolase in rat

The biosynthesis of rat intestinal lactase-phlorizin hydrolase was studied by pulse-labeling of jejunal explants from 5-day-old suckling rats in organ culture. Explants were either continuously labeled with [35S] methionine for 15, 30, and 60 min or pulse-labeled for 30 min and chased for various periods of time up to 6 h in the presence or absence of protease inhibitors (PI), leupeptin, phenylmethylsulfonyl fluoride, and soybean trypsin inhibitor. Lactase-phlorizin hydrolase was immunoprecipitated from microvillus membrane (MVM) and ER-Golgi fractions with monoclonal antibodies. After pulse-labeling, lactase-phlorizin hydrolase from the ER-Golgi fraction appeared on SDS-PAGE as one band of approximately 220 kDa, regardless of the presence or absence of PI in the culture media. The 220-kDa protein band could also be labeled after incubation with [2-3H]mannose. In the absence of PI, the 220-kDa band appeared in the MVM by 30 min chase, simultaneously with a 180-kDa band, and by 60 min of chase an additional band of 130 kDa was seen. With increasing time of chase, the relative intensity of the 130-kDa band increased, whereas that of the 220-kDa band decreased, suggesting a precursor-product relationship. When PI were added to the medium, the formation of the 180-kDa band was not affected, but the conversion of the 180-kDa protein to the 130-kDa protein was virtually blocked. These findings suggest that lactase-phlorizin hydrolase is initially synthesized as a glycosylated precursor of 220 kDa, which is transported to the MVM. There it undergoes the following two cleavages: first, to the 180-kDa form, which is not prevented by PI used in these experiments, and second, to the 130-kDa form inhibited by PI.     

Electrophoretic Forms of Chitinolytic and Lysozyme Activities in Ruminal Protozoa

Homogenates from a mixed ruminal protozoal population and a ruminal protozoon Entodinium caudatum were analyzed for chitinolytic and lysozyme activities by sodium dodecyl sulfate polyacrylamide gel electrophoresis. For chitinase activity, up to eight bands in mixed protozoa and seven bands in E. caudatum were detected. Estimated molecular mass ranged from 70 to 110 kDa. These enzymes did not display lysozyme activity. N-Acetyl-β-glucosaminidase activity was also detected in both samples with an estimated molecular mass of 37 kDa. Lysozyme activity in mixed protozoa was present in two major and three minor bands, where one major band displayed the same motility as chicken egg white (CEW) lysozyme, and the other had an approximate molecular mass of 17.5 kDa. The latter remained active even when denatured in the presence of dithiothreitol and renatured under anaerobic conditions. Entodinium caudatum presented one major band coincident with that of CEW lysozyme and a minor band at the 17.5-kDa point. This study showed that protozoal chitinase and lysozyme activities are originated from several enzymes and that none of these enzymes exhibited both activities.     

Post-mortem degradation of brain glutamate decarboxylase

The post-mortem stability of the GABA synthesizing enzyme glutamate decarboxylase (GAD) was studied by using SDS–PAGE and quantitative immunoblotting to measure the rates of degradation of GAD in the cerebral cortex, hippocampus, and cerebellum of rats and mice as a function of time after death. The intact 65- and 67-kDa isoforms of GAD (GAD₆₅ and GAD₆₇) disappeared gradually over a 24-h period. In both rats and mice, the degraded GAD appeared as a band with an apparent molecular mass of 55–57 kDa; no significant amounts of smaller forms were observed. The 55–57 kDa band reacted with antiserum W887, which recognizes a shared epitope at the carboxyl-terminal end of both GADs, indicating that GAD was cleaved near the amino-terminal end of the molecule. GAD₆₇ was cleaved at a site between the amino-terminus and the epitope for antiserum W883 (located within residues 79–93 of GAD₆₇), as antiserum W883 stained a 56-kDa band on the blots. The appearance of degraded GAD paralleled the loss of total GAD (GAD₆₅+GAD₆₇), and after 24 h the 55–57 kDa band accounted for 97, 88, and 59% of the intact GAD lost from rat cerebellum, cerebral cortex and hippocampus. On a percentage basis, GAD₆₇ was degraded more rapidly than was GAD₆₅ in all brain regions studied. The loss of GAD activity was greater in rat than mouse brain, even though the percent loss of intact GAD protein was similar.     

Effect of prostaglandins on oviduct tone in the domestic hen in vivo

The effect of i.v. injected prostaglandins (PG) F₂α and E₂ on intraluminal pressure of the different oviductal parts (infundibulum, magnum, isthmus, uterus and vagina) was investigated in the domestic hen. PGF₂α induced only a pressure rise in all oviduct segments. Administration of PGE₂ resulted in variable changes in oviductal tone: pressure rise in the infundibulum; pressure increase often preceded by a small decrease in the magnum, isthmus and uterus; pressure decrease in the vagina and sometimes in the uterus. Simultaneous i.v. injection of both PG's induced mostly a decrease in vaginal tone. Intraluminal administration of PGF₂α or E₂ resulted only in an increase in uterine pressure.The observed effects on oviduct tone are discussed and a possible in intervention of both PG's in the mechanism of ovum transport and oviposition in the domestic hen is proposed.     

Histochemical demonstration of the presence of serotonin in the hen (Gallus domesticus) reproductive tract

The purpose of the present study was to demonstrate visually and localize the presence of serotonin (5-HT) in the ovary and oviduct of the domestic hen using a histochemical Falck-Hillarp method. Experiments were carried out on White Leghorn laying hens with no egg in the shell gland. The specific yellow fluorescence, indicating the presence of 5-HT, was found both in the ovary and all examined oviductal parts. The strongest fluorescence was present in the ovarian stroma containing small follicles with a diameter under 4 mm. In the wall of the largest preovulatory follicle a very strong fluorescence was located mainly in the theca layer. In the oviductal parts, the intensity of 5-HT fluorescence in the infundibulum and magnum was fairly strong, whereas in the isthmus and shell gland it was weak. Fluorescence seen in the infundibulum, magnum, and isthmus was primarily localized along the luminal borders of the fold surface epithelium. In the shell gland 5-HT fluorescence was found within the uterine folds, especially in the tubular glands. Moreover, the presence of an egg in the definite oviductal segment (infundibulum or isthmus) increased the intensity of yellow fluorescence in this part.     

Ultrastructural changes in the oviduct of the laying hen during the laying cycle

The ultrastructural changes occurring in the fully functional oviduct of Isa Brown laying hens were studied during various stages of the laying cycle. Hens were killed at different positions of the egg in the oviduct. The oviduct was lined by ciliated and non-ciliated cells (also referred to as granular cells). The granular cells in the infundibulum contributed to secretion during egg formation, whereas ciliated cells showed little evidence of secretion. Ultrastructural changes were recorded in the granular and glandular cells of the distal infundibulum. In the magnum, the surface ultrastructure revealed glandular openings associated with the ciliated and granular cells. Cyclic changes were recorded in the glandular cells of the magnum. With respect to the three observed types of glands, the structure of gland type A and C cells varied at different egg positions in the oviduct, whereas type B cells represented a different type of gland cell containing amorphous secretory granules. The surface epithelium of the isthmus was also lined by mitochondrial cells. Two types of glandular cell (types 1 and 2) were recorded in the isthmus during the laying cycle. Intracisternal granules were found in type 2 cells of the isthmus. A predominance of glycogen particles occurred in the tubular shell gland. The granular cells in the shell gland contain many vacuoles. During egg formation, these vacuoles regressed following the formation of extensive rough endoplasmic reticulum; the reverse also occurred. The disintegrated material found in the vacuoles may have been derived from the disintegrating granules. The Physiology Teaching Unit, University of New England, provided financial support to K. Chousalkar for this study.     

The magnum-isthmus junction of the fowl oviduct participates in the formation of the avian-type shell membrane

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.     

Biosynthesis of the prohormone convertase mPC1 in AtT-20 cells.

A new family of mammalian subtilisin-like enzymes, probably involved in the processing of proproteins in regulated and constitutive cells at paired basic residues, has recently been discovered. Little information exists as yet concerning the biosynthesis of these endogenous subtilisin-like enzymes. In the present work the biosynthesis and release of the endogenous prohormone convertase PC1 in AtT-20 cells were studied. As predicted from mRNA studies, AtT-20 cells contain high levels of PC1 protein. Through immunoblotting, 87-kilodalton (kDa) and 66-kDa bands were detected with an amino terminally directed antiserum; however, only the 87-kDa product was detected with carboxyl terminally directed antiserum, indicating carboxyl terminal truncation. Pulse-chase experiments, using [35S]methionine/cysteine, showed that after 20 min pulse the main product in the cells was the 87-kDa protein. Cells chased for varying amounts of time exhibited a progressive increase in the intensity of a 66-kDa band, along with a corresponding decrease of the 87-kDa band. The 87-66 kDa conversion was nearly complete after 4 h of chase. This posttranslational processing was inhibited by the ionophore monensin, a Golgi disruptor, with a corresponding accumulation of the 87-kDa protein within the cell. Both the 87 kDa- and 66 kDa-labeled proteins were detected as membrane-bound rather than soluble proteins. The 87-kDa protein was the main product secreted by nonstimulated AtT-20 cells, while the 66-kDa product was only released when the cells were stimulated with CRF or BaCl2 and Bromo-cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and identification of a second form of vitellogenin from ascites of medaka (Oryzias latipes) treated with estrogen

Estrogen treatment of medaka leads to accumulation of ascites, in which vitellogenin (Vg) and choriogenins (precursors to vitelline envelope) are abundant. Besides those female-specific proteins, we detected a new component in ascites that cross-reacts with antiserum against egg yolk proteins. We tentatively named it egg yolk-related protein (YRP). YRP was purified from ascites by hydroxylapatite chromatography followed by gel filtration. Purified YRP had a molecular mass of 460 kDa in intact state while 570 kDa for Vg. The molecular weight of purified YRP on SDS-PAGE under both reducing and nonreducing conditions was 130 kDa. YRP was confirmed to be a lipoglycophosphoprotein by staining with Sudan black, periodic acid-Schiff (PAS) and methyl green. Amino acid composition of YRP resembled that of Vg except for a relatively low content of serine. A specific antiserum against YRP was raised in a rabbit. Antiserum against YRP specifically immunostained its antigen but not Vg or choriogenins. YRP was detected as a female-specific protein in serum of breeding medaka. The antiserum also cross-reacted with a band at 29 kDa in egg extracts, which is not immunoreactive to antiserum against Vg. These data show that YRP is a precursor to some egg yolk proteins with differing antigenicity from Vg (Hamazaki et al. '87). We thus conclude that YRP is a second form of medaka Vg and rename YRP as Vg 2 while formerly reported Vg as Vg 1.     

Demonstration of a Ferric Vibrioferrin-Binding Protein in the Outer Membrane of Vibrio parahaemolyticus

Under iron-restricted conditions, Vibrio parahaemolyticus produces a siderophore, vibrioferrin, accompanying expression of two major outer membrane proteins of 78 and 83 kDa. Autoradiographic analysis of nondenaturing polyacrylamide gel electrophoregrams of outer membrane preparations previously incubated with [⁵⁵Fe]ferric vibrioferrin revealed a single radiolabeled band, in which the 78-kDa protein was detected predominantly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antiserum against the purified 78-kDa protein partially inhibited Fe-VF binding to isolated OMPs. The 78-kDa protein was cleaved by the treatment of whole cells with proteinase K, indicating that a portion of this protein is exposed on the surface of the outer membrane. The treated cells lost most of their iron uptake activity mediated by vibrioferrin. These results suggest that the ferric vibrioferrin-binding protein of 78 kDa may function as the receptor for ferric vibrioferrin involved in the initial step of vibrioferrin-mediated iron uptake. Immunoblot analysis using the antiserum against the 78-kDa protein demonstrated that the molecular mass and antigenic properties of the protein were highly conserved among V. parahaemolyticus strains examined. The antiserum also recognized an iron-repressible outer membrane protein of 78 kDa from iron-restricted V. alginolyticus strains, some of which appeared to produce vibrioferrin.     

Homology model and molecular dynamics simulation of carp ovum cystatin

In this study, a homology model of carp ovum cystatin was constructed based on the crystal structure of chicken egg white cystatin. The results of amino acid sequence alignment indicate that these two proteins exhibit 36.11% of sequence identity. The resultant homology model reveals that carp ovum cystatin shares similar folds as chicken egg white cystatin, particularly in the conserved regions of Q48-V49-G52 and P98-W99 and the locations of two disulfide bonds, C67-C76 and C90-C110. However, the results of 1 ns molecular dynamics simulations show that carp ovum cystatin exhibits less structural integrity than chicken egg white cystatin in explicit water at 300 K. The relatively hydrophilic Met62 of carp ovum cystatin, corresponding to the hydrophobic Leu68 of human cystatin C and Ile66 of chicken egg white cystatin, may destabilize the hydrophobic core and form a dimeric structure more easily through domain swapping. A total of 16 positively charged residues are equally distributed on the surface of carp ovum cystatin, resulting in agglutination with the negatively charged spermatozoa via electrostatic interaction. Thus, carp ovum cystatin is considered to be important in preventing carp eggs from polyspermy.     

Avian oviduct motility induced by prostaglandin E1

Oviduct segments from infundibulum, magnum, uterus, uterovaginal junction and vagina of actively laying hens at preoviposition time were tested for their contractile reaction to prostaglandin E₁ by or methods. Maximum stimulatory response was observed from the muscular strips of the proximal oviduct segment (infundibulum) and a complete relaxation was recorded from the distal part (vagina) at molar concentrations of 1.4 × 10⁻⁷, 3.4 × 10⁻⁷ and 7.0 × 10⁻⁷. The uterine strips reacted with a stimulatory response at higher concentrations (1.4 × 10⁻⁶ and 2.8 × 10⁻⁶ moles), but lacked any significant change at lower concentrations. The uterovaginal muscular strips showed a mild but prolonged inhibitory response, while the magnum responded with a significant increase in the luminal pressure when tested . It is concluded that PGE₁ exerts a stimulatory effect on the uterus to initiate transport of the egg to subsequent segments (uterovaginal junction and vagina), which relax under PGE₁ influence and allow passage of the egg by pressure differences.     

Hatching mechanism of the Chinese soft-shelled turtle Pelodiscus sinensis

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.