Proteomic analysis of egg white proteins during the early phase of embryonic development

Avian egg albumen participates in embryonic development by providing essential nutrients as well as antimicrobial protection. Although various biological functions of egg white proteins were suggested during embryogenesis, global changes of these proteins under incubation conditions remained uninvestigated. This study presents a proteomic analysis on the change of egg white proteins during the first week of embryonic development. By using 2-DE, together with MALDI-TOF MS/MS, thirty protein spots representing eight proteins were identified showing significant changes in abundance during incubation. An accelerating degradation of ovalbumin was observed in a wide range of molecular weight. In addition, four protein complexes were predicted according to the detected molecular weight increase. Among these speculated protein complexes, an ovalbumin spot coupled with RNA-binding protein was detected. The absence of these protein complexes before incubation, followed by the constant increase in abundance during incubation indicates conceivable pivotal roles in embryonic development. To better understand the function of the proteins identified in this study, discrepancies of egg white protein changes between fertilized and unfertilized chicken eggs were additionally demonstrated. These findings will provide insight into the embryogenesis process to improve our knowledge of egg white proteins in regulating and supporting early embryonic development.     

Analysis of progesterone receptor in the quail oviduct. Correlation between plasmatic estradiol and cytoplasmic progesterone receptor concentrations

Specific binding sites for [3H]-progesterone are found in the cytosol fraction of the oviduct of castrated, immature and developing quails. The optimal conditions to accurately measure the total cytoplasmic concentration of this progesterone receptor are described. The dissociation constant (KD) at 0 degrees C is 3.6 +/- 0.6 x 10(-9) M (mean +/- SE) for [3H]-P and the concentration of binding sites is 13.4 +/- 2 pmol/mg DNA in immature animals. This binding capacity is not altered even 2 weeks after ovariectomy. During sexual development, although the dissociation constant remains unchanged, the number of binding sites increases to 74.5 +/- 1.6 pmol/mg DNA just before the beginning of the laying cycle. The concentration of cytoplasmic P receptor is under the inductive influence of estradiol. In castrated quails, estradiol 17 beta (E2) perfusion through the portal vein at a rate below or equal to 2 ng/min for 24 hr does not increase plasmatic E2 concentration and consequently does not change [3H]-P binding sites concentration in the oviduct. While E2 perfusion rate exceeds the metabolizing capacity of the liver (6.8 ng/min), both plasmatic E2 level and oviductal P receptor concentration are increased. When E2 is perfused through the jugular vein, plasmatic E2 level increases with the dose of E2 but P receptor concentration only increases when E2 perfusion rate reaches to 2.0 ng/min for 24 h.     

Proteomic analysis of egg white proteins during storage

Egg storage causes egg white to lose its viscous nature to form a thin liquid, commonly referred to as egg white thinning. To understand the mechanisms underlying egg white thinning, white-shell eggs were used in the present study to determine the proteome-level changes of egg white proteins occurred during storage. Egg white thinning was observed visually after 20 days of storage at ambient temperature (22 ± 2 °C) when the maximum number of proteome-level changes occurred. The proteins that showed significant changes in abundance during storage included ovalbumin, clusterin, ovoinhibitor, ovotransferrin, and prostaglandin D2 synthase. Among these, only the abundance of clusterin was observed to change continuously during the storage period. Hence, it is expected that the increase in the concentrations of clusterin and ovoinhibitor along with the change of ovalbumin content during storage might contribute to egg white thinning. Degradation of ovalbumin/clusterin during egg storage may be due to the combined effect of proteolysis and increase in pH; this may also be partly responsible for egg white thinning phenomenon.     

Augmentation of progesterone receptor concentration by progesterone and estrogen treatments in the chick oviduct

Cytosol receptors for progesterone in the chick oviduct were measured by charcoal-adsorbtion assay by using ORG 2058 as a ligand after long-term administration of progesterone and diethylstilbestrol (DES). Steroid administration was carried out by using daily injections or silastic capsules. DES treatment increased the progesterone receptor concentration (from 11500 to 21500 sites per cell, day 14). Progesterone also augmented the concentration of its own receptors (from 11500 to 29000 sites per cell, day 14). In the experiments with capsule administration the same trend was seen. This indicates that both diethylstilbestrol and progesterone are able to increase the concentration of progesterone specific cytosol receptors in the non-differentiated chick oviduct.     

Heterogeneity of progesterone receptor expression in epithelial cells of immature and differentiating quail oviduct

The localization of progesterone receptor (PR) in the quail oviduct was investigated before and after the onset of sexual maturation using an immunohistochemical technique. PR was revealed exclusively in nuclei of target cells whatever the hormonal state of the tissue (immature or not, pretreated or not with progesterone). In the immature or ovariectomized quail oviduct, PR was principally localized in the undifferentiated epithelial cells; some mesothelial cells and a very few stromal cells expressed the PR. Only 40-45% of the epithelial cells were immunoreactive. These positive cells were mainly localized in the furrows of the villi where further evagination of the epithelium will occur to form the tubular glands. The onset of sexual maturation was accompanied by an increase of the proportion of positive epithelial cells and stromal cells. In estradiol-treated animals, more than 90% of the tubular gland cells were strongly stained while only 40% of the luminal epithelial cells were immunoreactive. Our results show that there are two subpopulations of epithelial cells: those expressing the PR before the onset of sexual maturation even in ovariectomized quails (constitutive expression) and those expressing the PR during sexual maturation or after estrogen injection (inductive expression). These results, associated with previously published studies dealing with the cytodifferentiation of epithelial cells during natural development or after various hormonal treatments in ovariectomized animals, suggest that the first are the progenitors of tubular gland cells, and the second the progenitors of ciliated and goblet cells. In stromal cells, PR expression is also inducuible.     

Regulation of nuclear binding of the avian oviduct progesterone receptor. Changes during estrogen-induced oviduct development, withdrawal, and secondary stimulation

The progesterone receptors from various stages of estrogen induced oviduct development, estrogen withdrawal, and secondary stimulation with estrogen were examined. The progesterone receptors were characterized for their biological function (i.e. capacity for nuclear translocation, nuclear binding, and effects on RNA polymerase II activity) as well as certain physical properties. The progesterone receptors from the undeveloped or partially developed oviducts (0 to 8 days of estrogen treatment) displayed little or no nuclear translocation and binding in vivo or in vitro. Similarly, progesterone showed little or no effect in vivo on RNA polymerase II activity at the early stages of development. As development progressed from 8 to 12 days of estrogen treatment, the above parameters rapidly increased to maximal levels and plateaued through day 23 of estrogen treatment. A marked decrease in these parameters occurred within 1 day of estrogen withdrawal. The reverse series of events occurred during secondary estrogen stimulation of 10-day-old withdrawn chicks. While the receptor concentrations increased rapidly to maximum values by 2 days of restimulation, receptor function did not return until day 4. Similarly, the effects of progesterone on RNA polymerase II activity reached maximal values by day 4. The progesterone receptor isolated from oviducts during development, estrogen withdrawal, and restimulation, displayed similar patterns of cell-free binding to chromatin and nucleoacidic protein as that observed in vivo supporting the nativeness of the in vitro binding assay. In contrast, the cell-free binding of these same progesterone receptor to pure DNA were not similar to the in vivo binding, i.e. no patterns (differences) in progesterone receptor binding were observed. These data support that protein DNA complexes and not pure DNA represent the native acceptor sites for oviduct progesterone receptor. Comparison of the progesterone receptor between the functional and nonfunctional states revealed no differences in the steroid affinity for the receptor, in the apparent pI of the species, or in the sedimentation of the receptor under high salt conditions. However, the nonfunctional receptors consistently displayed a deficiency in one of the two monomer molecular species (the B species) as determined by isoelectric focusing. These results suggest that both monomer species of progesterone receptor are required for biological activity. Interestingly, the 7S "aggregate" species of the progesterone receptor was constantly detected even when only one of the monomer species was present.(ABSTRACT TRUNCATED AT 400 WORDS)     

Estrogen and progesterone receptors in the oviduct during egg transport in cyclic and pregnant rats

We investigated the temporal relationships between ovum transport and changes in the concentration of nuclear steroid receptors in the oviduct of cyclic and pregnant rats. A lack of parallelism between estrogen and progesterone fluctuations in plasma and their respective nuclear receptor concentrations in the oviduct predominated during egg transport. In pregnant animals, oviductal egg transport took 24 h longer than in nonpregnant animals. In both conditions, transport was initiated while the action of estrogen and progesterone on the oviduct--measured as nuclear receptor accumulation--was decreasing. Three or four days later, depending on whether the animal was pregnant, the eggs entered the uterus shortly after an increase in the nuclear receptor accumulation of both hormones. Treatment with RU486, a progesterone receptor-blocking agent known to cause premature arrival of eggs in the uterus, advanced estrogen receptor accumulation in the oviduct of pregnant rats. These data suggest that the arrival of eggs in the uterus is timed by a transitory increase in nuclear estrogen receptor in the oviduct that does not necessarily reflect a similar change of circulating estradiol. Moreover, in pregnant rats, the onset of this estrogenic action is delayed by a progesterone receptor-mediated effect that hinders nuclear estrogen receptor accumulation.     

Modulation of quail oviduct adenylate cyclase activity by estradiol and progesterone

Oviduct adenylate cyclase activity of the quail was measured by radiochemical analysis following different hormonal treatments. A single injection of estradiol benzoate (EB) to immature female quails resulted in a prereplicative surge of adenylate cyclase activity. A second surge of enzyme activity was observed during the proliferative phase induced by EB. Estradiol-17 alpha, estrone, estriol and testosterone were ineffective. Tamoxifen completely inhibits the growth-promoting effect of EB and the second surge of adenylate cyclase activity but does not inhibit the prereplicative increase of enzyme activity. This prereplicative increase of adenylate cyclase activity was also observed, even in the absence of increased plasma estradiol, when estradiol-17 beta (E2) was perfused through the hepatic portal vein. Moreover, E2 had no effect on enzyme activity when added directly to the oviduct homogenate preparation, at concentrations ranging from 10(-9) to 10(-7) M. In response to progesterone injection, oviduct adenylate cyclase activity followed a different pattern, beginning its increase after 3 h and remaining elevated up to 24 h. The activation by estradiol was independent of the presence of guanylylimidodiphosphate. Moreover, the enzyme was more sensitive to forskolin at submaximal concentration in estradiol treated birds than in control. These results demonstrate that transient activation of adenylate cyclase at the early stages of the action of estradiol does not occur through the classic nuclear receptor-gene activation pathway or a membrane receptor mediated process, but involves an indirect pathway, yet to be defined.     

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.     

Poly(adenosine diphosphate-ribose) polymerase in quail oviduct. Changes during estrogen and progesterone induction

The activities of the following enzymes have been determined in nuclei of quail oviducts in response to exogenous stimulation of the birds with diethylstilbestrol, used as an estrogen analogue and progesterone: DNA dependent DNA polymerase, DNA dependent RNA polymerase I and II and poly(adenosine diphosphate-ribose) [=poly(ADP-Rib)] polymerase.     

Estrogen-mediated cell proliferation during chick oviduct development and its modulation by progesterone

The interactions of estrogen and progesterone on mitosis were examined in the surface epithelium of the developing chick oviduct. Both of these steroid hormones can stimulate cells to divide in the unstimulated oviduct. However, progesterone treatment results in a delayed suppression of cell division in both the presence and absence of estrogen. This progesterone induced depression of estrogen-mediated cell division is observed throughout oviduct development. During oviduct development estrogen is necessary for both cell division and the differentiation of specific cell types while progesterone appears to modify the action of estrogen by blocking the progression of cells through the cell cycle.     

Modification of cell evagination and cell differentiation in quail oviduct hyperstimulated by progesterone

Quail oviduct development is controlled by sex steroid hormones. Estrogen (E) induce cell proliferation, formation of tubular glands by epithelial cell evagination and cell differentiation. Progesterone (P) strongly increases the secretory process in E-treated quails, but inhibits cell proliferation, cell evagination and differentiation of ciliated cells. The balance between E and P is critical for harmonious development of the oviduct. After 6 daily injections of two doses of estradiol benzoate (10 or 20 micrograms/d) and high doses of P (4 mg/d), tubular gland formation by epithelial cell evagination was inhibited, while epithelial cell proliferation occurred, as shown by the height of the villi and the increase in DNA. Secretory processes were strongly stimulated. Ovalbumin, a tubular gland cell marker and avidin, a mucous cell marker, were localized by immunofluorescence and immunogold labeling. Ovalbumin was localized only in the rudimentary tubular glands, whereas avidin was dispersed throughout the secretory cells. High doses of progesterone inhibited tubular gland cell proliferation, disturbed the distribution of avidin and inhibited differentiation of ciliated cells. Ovalbumin synthesis occurred only in epithelial cells which were evaginated despite the hyperstimulation. Ovalbumin gene expression appeared highly dependent upon the cell position.     

Effects of calcium on the chick oviduct progesterone receptor

The chick oviduct cytosol progesterone receptor can be transformed to a small form (Rs = 21A, S20,w:2.9) denoted "mero-receptor" by incubation in the presence of Ca2+ [8]. In the molybdate-free cytosol all the progestin binding components could be completely transformed to mero-form by 1 h treatment with 100 mM Ca2+ at 0 degrees C. If EDTA was secondarily added, the ligand was rapidly released. If molybdate (20 mM) containing cytosol was incubated with Ca2+, no radioactivity was found in the meroposition on the Agarose A 0.5 m column, but the bound steroid sedimented at 2.9 S in sucrose gradients containing Ca2+ (and no molybdate). When 20 nM molybdate was added to cytosol containing receptor activated by 0.3 M KCl, complete mero-transformation by Ca2+ was obtained also by the gel filtration criterion, indicating that molybdate does not inhibit the mero-transforming factor. Ligand-free progesterone receptor could also be completely converted to mero-form by endogenous cytosolic transforming factor and calcium. The transforming factor was completely inactivated, when cytosol was run through Agarose A 0.5 m gel. Mero-transformation was found to be irreversible. The purified progesterone receptor subunit 110 K (B) was partially converted to smaller forms by calcium alone (100 mM, 0 degrees C, 1 h) whereas addition of a small amount of cytosol allowed complete conversion to mero-form.     

Expression of nuclear progesterone receptor and progesterone receptor membrane components 1 and 2 in the oviduct of cyclic and pregnant cows during the post-ovulation period

ABSTRACT: BACKGROUND: Progesterone (P4) may modulate oviductal functions to promote early embryo development in cattle. In addition to its nuclear receptor (PR), P4 may mediate its actions through P4 receptor membrane component 1 (PGRMC1) and its relative, PGRMC2. Two successive experiments were undertaken to characterise the expression of PR, PGRMC1 and PGRMC2 in the bovine oviduct during the post-ovulation period, and to relate their expression to the presence of an embryo, the proximity of the CL and to the region of the oviduct. METHODS: In the first experiment (Exp. I), whole oviduct sections were collected from Holstein cows at Day 1.5, Day 4 and Day 5 post-ovulation (n = 2 cows per stage). The expression of PR, PGRMC1 and PGRMC2 was studied in the ampulla and isthmus by RT-PCR, western-blot and immunohistochemistry. In Exp. II, oviduct epithelial cells were collected from cyclic and pregnant Charolais cows (n = 4 cows per status) at Day 3.5 post-ovulation and mRNA expression of PR, PGRMC1 and PGRMC2 was examined in the ampulla and isthmus by real-time quantitative PCR. RESULTS: In Exp. I, PR, PGRMC1 and PGRMC2 were expressed in all oviduct samples. PGRMC1 was mainly localised in the luminal epithelium whereas PR and PGRMC2 were localised in the epithelium as well as in the muscle and stroma layers of the oviduct. The expression was primarily nuclear for PR, primarily cytoplasmic for PGRMC1 and both nuclear and cytoplasmic for PGRMC2. In Exp. II, mRNA levels for PR, PGRMC1 and PGRMC2 were not affected by either the pregnancy status or the side relative to the CL. However, the expression of PR and PGRMC2 varied significantly with the region of the oviduct: PR was more highly expressed in the isthmus whereas PGRMC2 was more highly expressed in the ampulla. CONCLUSIONS: This is the first evidence of PGRMC2 expression in the bovine oviduct. Our findings suggest that P4 regulates the functions of the bovine oviduct in a region-specific manner and through both classical and non classical pathways during the post-ovulation period.     

Nuclear origin of progesterone receptor of the chick oviduct cytosol

Summary Progesterone receptor (PR) was studied immunoelectron microscopically from fixed vibratome sections of the chick oviduct and biochemically from the fractionated oviduct homogenate. Immunoelectron microscopically both unoccupied and occupied PR were localized inside the nuclei. Only a few cells showed PR immunoreactivity in the endoplasmic reticulum which probably represents newly synthetized PR. Biochemically unoccupied PR was in the cytosol fraction. The cytosol and nuclear PR as well as the non-transformed 8S-form and the transformed 4S-forms of cytosol PR were recognized by the anti-PR antibody (IgG-RB). The lack of PR immunostaining in the cytoplasm is therefore not due to lack of recognition by IgG-RB. We propose that in the chick oviduct progesterone receptor is a nuclear protein but synthetized in the endoplasmic reticulum.     

A natural and readily available crowding agent: NMR studies of proteins in hen egg white

In vitro studies of biological macromolecules are usually performed in dilute, buffered solutions containing one or just a few different biological macromolecules. Under these conditions, the interactions among molecules are diffusion limited. On the contrary, in living systems, macromolecules of a given type are surrounded by many others, at very high total concentrations. In the last few years, there has been an increasing effort to study biological macromolecules directly in natural crowded environments, as in intact bacterial cells or by mimicking natural crowding by adding proteins, polysaccharides, or even synthetic polymers. Here, we propose the use of hen egg white (HEW) as a simple natural medium, with all features of the media of crowded cells, that could be used by any researcher without difficulty and inexpensively. We present a study of the stability and dynamics behavior of model proteins in HEW, chosen as a prototypical, readily accessible natural medium that can mimic cytosol. We show that two typical globular proteins, dissolved in HEW, give NMR spectra very similar to those obtained in dilute buffers, although dynamic parameters are clearly affected by the crowded medium. The thermal stability of one of these proteins, measured in a range comprising both heat and cold denaturation, is also similar to that in buffer. Our data open new possibilities to the study of proteins in natural crowded media.     

The effects of estrogen, insulin and dexamethasone on the synthesis and secretion of egg white proteins in primary cultured oviduct cells of laying Japanese quail (Coturnix coturnix japonica)

1. The effects of estrogen, insulin and dexamethasone on the synthesis of egg white proteins were investigated by employing primary cultured oviduct cells of laying Japanese quails. 2. It was demonstrated that oviduct cells require insulin and dexamethasone, besides estrogen, to synthesize and secrete egg white proteins maximally.