Uteroglobin Synthesis Research Articles

Genital tract proteins in the male rabbit: I. Localization of uteroglobin.

Uteroglobin synthesis in reproductive and non-reproductive tissues of male and female rabbit was screened by means of double-diffusion tests and immunohistochemical studies. Immunoprecipitates were observed when homogenates of male accessory were incubated with a monospecific antiserum against uteroglobin; in the female the antigen occurred in the uterine secretion and in homogenates of the oviduct, six days post coitum. In nonreproductive tissue, the antigen appeared both in the male and in the female, in the homogenate of trachea and lung. The immunofluorescence technique exhibited the paraprostate gland as the site of synthesis of uteroglobin in the male genital tract. Moreover, the distribution of the fluorescence within the cells was identical to the distribution observed in the female genital tract.

Zinc Finger Proteins RUSH in Where Others Fear to Tread1

The uteroglobin (UG) gene family encodes a fascinating group of secreted proteins that includes rat prostatic steroid-binding protein subunit C3, human Clara cell 10-kDa protein, human mammaglobin, and rabbit UG. In the rabbit, UG is a progesterone-dependent preimplantation uterine protein with peak availability on Day 5 of pregnancy. Progesterone stimulates UG synthesis in estrous rabbits and in short-term (3 day to 4 wk) ovariectomized rabbits. However, with increased time after ovariectomy, the uterus becomes increasingly refractory to progesterone challenge, such that after 12 weeks, normal levels of UG synthesis could not be measured. The treatment of these long-term ovariectomized rabbits with either prolactin or progesterone resulted in a dramatic increase in the receptor for the other hormone. Sequential treatment with prolactin plus progesterone increased the endometrial UG mRNA content and stimulated UG production to a concentration equal to that found on the fifth day of pregnancy. Because the increase in UG mRNA could result from an increased rate of transcription, gel shift assays, Southwestern blots, and UV cross-linking were used to show that prolactin augments the binding of four progesterone-dependent proteins to an 85-base pair (bp) 5'-flanking region (-170/-85) of the UG gene. The cDNAs for two of these UG promoter-binding proteins, RUSH-1alpha (113 kDa) and -1beta (95 kDa), were cloned by recognition site screening and identified as new members of the SWI/SNF superfamily of nuclear receptor coactivators. RUSH-1alpha and -1beta result from alternative splicing of a 57-bp exon, and each phosphoprotein has the novel RING-finger motif near its C terminus. Competitive reverse transcription-polymerase chain reaction and HPLC analysis showed that RUSH-1alpha is the progesterone-dependent splice variant. When an endometrial cell line (HRE-H9) was transfected with either the full-length UG construct, pUG3.1-LUC, or the deletion mutant, pUG3.1 deltaRUSH-LUC, progesterone increased the transcriptional activity of pUG3.1. Transcription of pUG3.1-LUC was further increased when cells were treated with prolactin plus progesterone. Prolactin alone had no effect. Progesterone also increased the transcriptional activity of pUG3.1 deltaRUSH-LUC. However, deletion of the proximal promoter region (pUG3.1 deltaRUSH-LUC) eliminated the prolactin effect and implicated RUSH proteins as prolactin signal transducers.

Norethisterone Metabolites Modulate the Uteroglobin and Progesterone Receptor Gene Expression in Prepubertal Rabbits1

Norethisterone (NET) is a synthetic progestin, used as a contraceptive agent, that is biotransformed at target tissues into 5 alpha-NET and 3 beta,5 alpha-NET, which possess different pharmacological properties. The effects of these metabolites on the expression of uteroglobin (UG) and progesterone receptor (PR) genes, both regulated by progesterone (P4), were evaluated in the uterus of prepubertal female rabbits that were simultaneously treated with P4 (1.0 mg) for 5 consecutive days. As determined by Western and Northern blot analyses, 5 alpha-NET inhibited the P4-induced UG gene expression in a dose-dependent manner. A similar inhibition was observed with the administration of RU-486. The estrogenic agent 3 beta,5 alpha-NET and estradiol at a dose of 1.0 mg also inhibited the UG gene expression induced by P4. Both 5 alpha-NET and 3 beta,5 alpha-NET blocked the PR down-regulation induced by P4 as assessed by Western and Northern blot methods. The inhibition of UG synthesis and PR down-regulation by 5 alpha-NET and 3 beta,5 alpha-NET indicates that these NET metabolites possess antiprogestational properties.

Hormonal Responsiveness by Immature Rabbit Uterine Epithelial Cells Polarizedin Vitro*

This paper reports the development of an in vitro cell culture system of polarized uterine epithelial (UE) cells from the immature rabbit. UE cells from immature rabbit were cultured on EHS (Engelbreth-Holm-Swarm) matrix-coated semipermeable filter inserts in a serum-free, phenol red-free defined medium. The cells in primary culture attached, proliferated, formed monolayers, and exhibited structural and functional polarity. Structural differentiation was validated by ultrastructural features, such as apical microvilli, intercellular junctions, desmosomes, and polar organization of apical vs. basal membrane domains. Trans-monolayer epithelial resistance, a reflection of functional tight junctions, preferential basal uptake of [35S]methionine, polarized distribution of labeled secretory proteins, and increased secretory activity marked by preferential apical secretion (greater than 90%) of proteins were indices of functional polarity. With the development and maintenance of polarized state by the UE cells, some of the newly synthesized proteins were sorted and vectorially secreted into their distinct compartments. De novo synthesis and exclusive apical secretion of uteroglobin (a progesterone-induced protein in vivo) by the polarized UE cells occurred in response to progesterone treatment in vitro. The hormone responsiveness was maintained for a prolonged period under these conditions. A culture system in which the UE cells maintain functional and morphological polarity and sustain their hormonal responsiveness facilitates the study of regulation of specialized functions by the UE cells that are regulated by steroid hormones and their interactions with the other uterine cell types.

Effect of in vitro culture on the dynamics of uteroglobin distribution in rabbit blastocysts

The purpose of this study was to investigate the localization and transport of uteroglobin in normal rabbit blastocysts (day 4-day 6 p.c.) and in those cultured for 6-48 h in vitro, using a specific radioimmunoassay and immunocytochemistry. The results of the radioimmunoassay showed that in day 4 p.c. blastocyst tissue (based on homogenate measurements) a significant decrease of the uteroglobin content started after only 6 h of culture in vitro. A significant concomitant rise of uteroglobin was observed in the culture medium after 12 h of in vitro culture. Using immunocytochemistry it was not possible to detect uteroglobin in any compartment of the non-cultured or in vitro cultured day 4 p.c. blastocysts. The efflux of uteroglobin down a concentration gradient was confirmed by the immunocytochemistry in non-cultured and in vitro cultured day 5 p.c. and day 6 p.c. blastocysts. Uteroglobin immunoreactions were mainly detected in non-cultured blastocysts (day 5 and 6 p.c.) in large vesicles of the trophoblast cells. In addition endocytotic vesicles at the inside of the apical membrane of trophoblast cells, some cell debris within the perivitelline space and the neozona were labelled. During in vitro culture of day 5 and 6 p.c. blastocysts, uteroglobin labelling in the coverings did not change. In non-cultured and cultured day 5 and 6 p.c. blastocysts neither the compartments of the embryoblast, the endoderm cells nor the blastocyst cavity showed any uteroglobin immunoreactions. After only 6 h of in vitro culture, uteroglobin immunoreactions were no longer found within the trophoblast cells. The reaction did not reappear during the course of in vitro culture up to 48 h, suggesting a complete lack of de novo synthesis of uteroglobin by blastocysts.

Variable expression of the uteroglobin gene following the administration of norethisterone and its A-ring reduced metabolites

Enzyme-mediated A-ring reduction of norethisterone (NET) results in the transformation of a molecule with potent intrinsic progestational activity into neutral derivatives with estrogen-like effects. To ascertain whether these structural modifications of NET are able to modify the uteroglobin (U) gene (G) expression, a series of experiments assessing the UG products after the administration of NET and its reduced A-ring metabolites were conducted in prepubertal female rabbits. Synthesis of endometrial uteroglobin and its specific mRNA were studied in animals following the administration of NET, 5α-dihydro NET,3β,5α-tetrahydro NET and progesterone. Animals treated with either estradiol or vehicle alone served as controls. The uteroglobin content in uterine flushings and cytosols was determined by immunodiffusion and polyacrilamide gel electrophoresis techniques and by a specific double-antibody radioimmunoassay, while the U mRNA synthesis was assessed by its molecular hybridization to [α 32P]d-ATP uteroglobin cDNA. NET induced a significant increase of the uterine content of uteroglobin similar to that observed with progesterone with a simultaneous increase on U mRNA synthesis. On the contrary, 5α-NET and 3β,5α-NET induced very little, if any uteroglobin synthesis with a concomitantly low U mRNA production as compared with NET; thus exhibiting a similar effect to that observed in estradiol-treated animals. The overall results were interpreted as demonstrating that the enzyme mediated structural changes of NET which occur at the target organs induce variable expression of the uteroglobin gene. The data indicate that the rabbit uteroglobin gene products are suitable molecular markers to evaluate the hormonal potency of contraceptive synthetic progestins and their derivatives.

Testosterone induces the expression of the uteroglobin gene in rabbit epididymis

Uteroglobin was characterized in the rabbit epididymis by radioimmunoassay and electrophoretic determinations, as well as by analysis of its mRNA by means of 'Northern blot' and nuclease-S1 mapping. Treatment of sexually immature rabbits with testosterone during 5 days increased up to 8-fold the concentrations of both uteroglobin and its mRNA in the epididymis. The amounts of beta-tubulin mRNA, measured as reference, remained unchanged after the hormonal treatment. The synthesis of uteroglobin occurred mainly in the middle region of the epididymis, progressively decreasing toward the distal part of the organ. Uteroglobin was not detected in the testis by radioimmunoassay. The results are discussed in relation to a possible role of uteroglobin in the reproductive functions.

Uteroglobin production in the pseudopregnant rabbit uterus. Immunohistochemical studies.

Uteroglobin, a secretory protein of rabbit uterine epithelium, was localized by the direct immunoperoxidase method in control and hCG-induced pseudopregnant rabbits. In control rabbits, uteroglobin was confined to the apical cytoplasm of nearly all cells of the endometrial epithelium. The induction of pseudopregnancy resulted in a pronounced continuing increase, through 4 days post-hCG administration, in the absolute number of epithelial cells engaged in uteroglobin synthesis. Furthermore, the endoplasmic reticulum was more intensely stained for uteroglobin than in the epithelial cells of control rabbit endometrium. Thus, the increased production of uteroglobin, in response to hormonal stimulation, appears to be achieved both through an increase in the amount of uteroglobin synthesized by a given cell as well as by an increase in the number of cells involved in uteroglobin synthesis. Concurrent with the increase in the number of cells synthesizing uteroglobin, an increase in the number of unstained cells first appeared at the second day of pseudopregnancy, during the period of maximal epithelial proliferation. However, within those cells containing uteroglobin on the second day following injection with hCG, most staining was limited to the perinuclear membrane. At various times following hCG administration, a number of scattered cells, intensely stained for uteroglobin, were observed in the uterine epithelium. Based upon ultrastructural studies, failure to exclude trypan blue, and the presence of intra-mitochondrial uteroglobin, they were identified as either dead or dying cells.

Distribution of uteroglobin in the rabbit endometrium after treatment with an anti-progesterone (ZK 98.734): an immunocytochemical study.

The effect of the synthetic steroid ZK 98.734, an anti-progesterone with high affinity for the progesterone receptor, on uteroglobin distribution in the rabbit endometrium has been studied by means of immunocytochemistry. Rabbits were treated with ZK 98.734 during the second, third and fourth day of pseudopregnancy. From the fifth up to the eighth day of pseudopregnancy the uteri were processed for immunocytochemistry using the peroxidase--antiperoxidase (PAP) and protein A--gold techniques. Uteroglobin synthesis and release could be inhibited by the anti-progesterone treatment. On day 5 and 6 there was no labelling of the uterine secretions and only a few diffusely labelled non-ciliated cells could be seen in the surface and glandular epithelium. The inhibition was reversible in so far as on day 7 and day 8 the rabbit endometrium exhibits a clear labelling of the uterine secretion as well as an increase in positive reaction in the epithelial cells lining the glands. In all treated animals the intracellular uteroglobin labelling was confined to the Golgi complex and secretory vesicles with a significant increase from the fifth to the eighth day of pseudopregnancy. Together with the described morphological changes these results indicate that ZK 98.734 is capable of inducing a delayed secretion in the rabbit endometrium, which is comparable to the delay in secretion caused by post-coital oestradiol treatment. However, the antigestagen effect is probably due to a different mechanism of endocrine interference with pre-implantation. The most exciting consequence, so far, is the prolongation of progesterone action after the anti-progesterone treatment had ended.

Effect of an antigestagen (ZK 98.734) on uteroglobin synthesis and release in the rabbit endometrium

Effect of an antigestagen (ZK 98.734) on uteroglobin synthesis and release in the rabbit endometrium

Glucocorticoids induce the expression of the uteroglobin gene in rabbit foetal lung explants cultured in vitro.

In 27-day-old rabbit foetal lung explants cultured in vitro, the synthesis of the protein uteroglobin decreased progressively during several days of culture. Addition of glucocorticoids to the medium progressively induced the synthesis of uteroglobin in a dose-dependent manner without affecting the synthesis of total proteins. The glucocorticoid-mediated induction of uteroglobin appears mainly due to increased amounts of uteroglobin mRNA and seems to be independent of simultaneous cell proliferation, suggesting a glucocorticoid-triggered differentiation of pre-existing cells. The results suggest a major role of glucocorticoids in the developmental regulation of the uteroglobin gene in the lung.

Glucocorticoid-dependent uteroglobin synthesis and uteroglobulin mRNA levels in rabbit lung explants cultured in vitro.

In rabbit lung explants cultured in vitro in a synthetic medium, the synthesis of the protein uteroglobin decayed progressively becoming virtually undetectable between 24-48 h of culture. Addition of glucocorticoids to the medium maintained the synthesis of uteroglobin. This glucocorticoid effect was dose-dependent with optima at about 0.1 microM and 1 microM for dexamethasone and cortisol respectively. Estradiol, progesterone, triiodothyronine, insulin or 10% calf serum added to the medium were ineffective in maintaining uteroglobin synthesis. Actinomycin D (10 micrograms/ml) added to the medium inhibited the effect of cortisol on uteroglobin synthesis. After 24 h of culture, both the relative levels of uteroglobin mRNA, measured by molecular hybridization, and uteroglobin synthesis were correlatively higher (up to 10-fold) in glucocorticoid-treated than in control explants.

Uteroglobin mRNA from the lung of the hare ( Lepus capensis): cell-free translation and properties

1. 1. Poly(A)-containing RNA was obtained from the lung of hares ( Lepus capensis) and uteroglobin mRNA was characterized by cell-free translation and molecular hybridization to a rabbit uteroglobin cDNA probe. 2. 2. In the cell-free system, hare uteroglobin mRNA was preferentially translated as compared to the whole lung mRNA and it directed the synthesis of a precursor, preuteroglobin, containing about twenty additional amino acids. 3. 3. Hare uteroglobin mRNA was about 40 nucleotides larger than the homologous rabbit one, as analyzed by electrophoresis on agarose gel. 4. 4. Thermal stability of the hybrids formed between rabbit or hare uteroglobin mRNAs and the rabbit cDNA probe indicated differences in the nucleotide sequence of both mRNAs. 5. 5. The levels of uteroglobin mRNA and uteroglobin synthesis in lung are about two-fold higher in hare than in rabbit lung.

Isolation and characterization of uteroglobin from the lung of the hare ( Lepus capensis)

Uteroglobin has been purified from hare lung by gel filtration and chromatography on carboxymethyl-cellulose. Hare uteroglobin appears homogeneous by electrophoresis under both denaturing and nondenaturing conditions. Its chemical and immunological properties as well as its ability to bind progesterone are compared to those of rabbit uteroglobin. The two proteins have the same N-terminal residue (glycine) and both lack tryptophan but differ in amino acid composition. Sodium dodecyl sulfate-gel electrophoresis shows that hare uteroglobin is composed of two subunits of identical M r (about 7000) held together by disulfide bridges. The amino acid composition indicates a subunit composed of 65–67 residues, which is compatible with the apparent M r observed. Thus, hare uteroglobin appears to be slightly smaller than the rabbit protein. Hare uteroglobin partially reacts with anti-rabbit uteroglobin in a radioimmunoassay and also binds progesterone, although this binding is relatively unaffected by dithiothreitol. The synthesis of hare uteroglobin in the uterus appears to be rather insensitive to ovarian steroid hormones.

Regulation of uteroglobin synthesis and conservation of progesterone and estrogen receptors in immature rabbit uterus during prolonged progesterone treatment

Daily progesterone administration (1.33 mg/kg body weight) to immature rabbits brought about an initial increase in the uterine content of uteroglobin which, however, subsided when progesterone treatment was continued for 10 days. During this treatment period progesterone did not lose its own uterine receptors nor did it lose its inhibitory effect on the accumulation of occupied nuclear estrogen receptors. Since immature rabbits were used, the decrease of uteroglobin concentration cannot be explained by inhibitory effects of endogenous estrogens. The results suggest that termination of uteroglobin secretion may be a selective and inherent effect of progesterone itself.

Hormonal Regulation of Rabbit Uteroglobin Gene Transcription*

Synthesis of uteroglobin in rabbit uterine epithelial cells during early pregnancy is induced by progesterone and apparently suppressed by estrogen. To assess whether the steroid hormones exert regulatory effects on the expression of the uteroglobin gene, we performed transcriptional rate assays in endometrial nuclei prepared from ovariectomized rabbits subjected to different hormonal treatments. The isolated nuclei were incubated with [3H]UTP to assess the synthesis of newly elongated RNA transcripts by the incorporation of [3H]UMP. The percentage of labeled RNA represented by uteroglobin sequences was determined by hybridization with cloned uteroglobin DNA immobilized on nitrocellulose filters. Progesterone alone (3 mg/kg X day) for 5 days increased endometrial DNA content 16-fold, total cellular transcriptional activity 4-fold, and relative uteroglobin gene transcription about 6-fold. 17 beta-Estradiol alone (50 micrograms/kg X day) for 5 days had no significant effect on any of these three variables. Not until after 2 days of treatment did progesterone increase the relative rate of uteroglobin gene transcription, whereas total transcriptional activity per mg DNA increased rapidly on the first day of progesterone administration. Treatment of progesterone-stimulated rabbits with estradiol for 5 days reduced tissue DNA content by 80%, and a single injection of estradiol caused a small decrease in uteroglobin gene transcription. Chronic treatment with progesterone alone was sufficient to reduce uteroglobin gene transcription to control levels, without a decrease in tissue DNA content. Progesterone thus regulates uteroglobin synthesis in the uterus at least at three levels: 1) by exerting a mitogenic effect on endometrial cells, 2) by enhancing total cellular transcriptional activity, and 3) by preferential stimulation of uteroglobin gene transcription. Physiologically, the decline in uteroglobin synthesis after 5 days of pregnancy appears to be the result of a decrease in uteroglobin gene transcription in the face of the continued rise in blood progesterone, and this effect may be aided by antiprogestational actions of estradiol.

Progestin-like effects of danazol on rabbit uterus.

The action of danazol on cytosol and nuclear progestin and estrogen receptor concentrations and on the induction of uteroglobin synthesis were studied in the rabbit uterus in vivo. In addition, the relative binding affinity of danazol for the uterine progestin receptor was measured in vitro. Administration of increasing doses of danazol (10, 50, or 100 mg/kg BW daily) for 5 days to adult rabbits tended to decrease the concentrations of cytosol and nuclear progestin and estrogen receptors, whereas the uterine uteroglobin content increased with increasing doses of danazol. Progesterone (1 mg/kg BW daily) caused changes which were greater than those caused by the largest dose of danazol. Danazol was found to bind to the uterine progestin receptor in vitro with an affinity approximately 3.3% of that of progesterone. These findings suggest that danazol has a profound progestin-like activity in the rabbit uterus which may be mediated through binding to cytosol progestin receptors.

Transcriptional activity of the uteroglobin gene in rabbit endometrial nuclei during early pregnancy.

In studies on the regulation of rabbit uteroglobin production by progesterone, we have shown previously that the increase in uteroglobin secretion by the uterus in early pregnancy is preceded by an increase in uteroglobin synthesis and a parallel increase in uteroglobin messenger (m) RNA activity, which is accounted for by a rise in the steady state level of this specific mRNA in uterine epithelial cells. To investigate whether similar changes in the transcriptional activity of the uteroglobin gene occur during this period, we have transcribed endometrial nuclei in a "run-off" endogenous transcription system with [32P]uridine triphosphate as the precursor, given as a short pulse over 7 min at 25 C in vitro. Specific uteroglobin RNA transcripts were quantitated by hybridization of [32P]RNA to nitrocellulose filters containing cloned, full length uteroglobin complementary DNA. The percentage of uteroglobin RNA transcripts rose from day 0 (nonpregnant rabbits) to a peak on day 4 and declined on day 6 of pregnancy. No differences in RNA degradation and no preferential degradation of newly transcribed uteroglobin RNA were found during incubation of nuclei from the different days of pregnancy. The pattern of change in rate of transcription of the uteroglobin gene corresponds to the pattern of change in level of uteroglobin mRNA, suggesting that transcriptional controls are important in regulating the production of uteroglobin in pregnant rabbit uterus.

Implantation does not affect luminal content and synthesis of uteroglobin in the rabbit.

AbstractTo evaluate the possibility that uterine decidual reaction to the implanting blastocyst may cause a decrease in the luminal content of uteroglobin during pregnancy, experiments were designed to have unilateral pregnancy in the rabbit. The unilateral pregnancy was obtained by ligating one of the oviducts with silk sutures, with the opposite horn remaining intact for pregnancy. Since the horn with sutures did not become pregnant, it was designated as a pseudopregnant horn. The uteroglobin content and 3H-amino acid incorporation were determined in the pseudopregnant and pregnant horns on Days 5, 7, and 10 of pregnancy. Our results indicate that there was no significant difference in the concentration of uteroglobin and incorporation of 3H-amino acid into the uteroglobin between the pregnant and pseudopregnant horns on different days of pregnancy. These studies suggest that the rapid decline of the luminal uteroglobin content in pregnant rabbits compared to pseudopregnant or progesterone-treated ovari...

Glucocorticoid and developmental regulation of uteroglobin synthesis in rabbit lung.

The synthesis of uteroglobin in rabbit lung was studied after the administration of glucocorticoids to intact adult animals as well as during the late stages of rabbit development. The synthesis of uteroglobin was compared with levels of translatable uteroglobin mRNA in the lung. Uteroglobin synthesis was determined both by incorporation of [25S]methionine into the protein by lung explants incubated in vitro and by radioimmunoassay measurements of uteroglobin concentration in lung. Lung poly(A)-containing mRNA, isolated by oligo(dT)--cellulose chromatography, was translated in cell-free systems and the activity of uteroglobin mRNA was determined after immunoprecipitation. Dexamethasone administration increased about 2-fold the synthesis of lung uteroglobin compared with the controls. The effect of cortisol was more moderate. Both glucocorticoids did not affect the degradation rate of lung uteroglobin, but produced increases in the translatable levels of uteroglobin mRNA parallel to those observed for uteroglobin synthesis. During the late stages of rabbit development, both the synthesis of lung uteroglobin and the translatable levels of its mRNA increase in parallel about 12-fold in a biphasic fashion. A first increase occurred between 2 days before and 2 days after birth. Starting at 5 days of age, there was a second increase in both parameters, which at 12 days of age reached values close to those observed in adult rabbits. Our results suggest that the rate of lung uteroglobin synthesis could be mainly determined by the translatable levels of its mRNA.