Detection of a progesterone-dependent secretory protein synthesized by cat endometrium

Uterine flushings and culture media from endometrial explants incubated in the presence of radiolabeled amino acids were analyzed using one-(1-D) and two-dimensional (2-D) gel electrophoresis to identify proteins synthesized by the endometrium and subsequently released into the uterine lumen. 1-D and 2-D analyses of uterine flushings and culture media of endometrial explants obtained from 7- to 11-day pregnant cats (pre-implantation) showed a Mr 30,000 protein that appeared on 2-D gels as a family of macromolecules with isoelectric points between 6.5 and 7.0. This family of macromolecules was also present in the culture media of implantation-site tissue obtained from 12- to 16-day pregnant cats and of nonimplantation-site endometrium obtained form 12- to 28-day pregnant cats. The Mr 30,000 protein was absent in uterine flushings and culture media from estrous and 3- to 5-day-pregnant cats. In ovariectomized, steroid-treated animals, the Mr 30,000 protein was only detected in flushings and media from those animals treated with progesterone, regardless of the presence or absence of estradiol-priming and/or simultaneous estradiol treatment. In daily flushings obtained from ovariectomized, steroid-treated cats equipped with an indwelling uterine catheter, the Mr 30,000 protein was absent during the 14 days of estradiol treatment and was first detected 3-4 days after the onset of estradiol plus progesterone treatment. This protein was not detected in serum from estrous, 9-day pregnant, ovariectomized, and ovariectomized, steroid-treated animals. This study shows that 1) a progesterone-dependent protein, with an approximate molecular weight of 30,000 and an isoelectric point of 6.5-7.0, first appears within the uterine lumen soon after the arrival of the blastocyst and continues to be present during implantation; 2) the synthesis and release of the Mr 30,000 protein is dependent on progesterone regardless of the presence or absence of estradiol; and 3) the onset of secretion of the Mr 30,000 protein requires 3-4 days of continuous progesterone treatment in the estradiol-primed cat.     

Molecular cloning and characterization of a progesterone-dependent cat endometrial secretory protein complementary deoxyribonucleic acid

In the cat, a group of low molecular weight secretory proteins have previously been shown to appear in the endometrium after progesterone (P) administration to an estrogen (E2)-primed animal. Using a polyclonal antibody to these progesterone-dependent proteins (PDP) we have isolated a recombinant cDNA clone corresponding to the mRNA for PDP from a cDNA library prepared using poly(A+) RNA from the endometrium of P-treated E2-primed cats. Comparison of Western blots using the polyclonal antibody and epitope selected antibody demonstrated that the multiple molecular weight and isoelectric forms of the PDP are immunologically related and potentially products of the same gene. Northern analysis revealed that the mRNA for the PDP in the endometrium of P-treated E2-primed cats was 1.8 kilobases in length. Using slot blot analysis, we found that the PDP mRNA levels were low in the endometrium of ovariectomized animals and undetectable in E2-treated animals. With 1 day of P treatment the PDP mRNA levels were readily detectable and they peaked after 5 to 7 days of P treatment. No PDP mRNA was detectable in myometrium, oviduct, or ovary. Sequence analysis revealed that PDP had significant homology to human, rat, and mouse cathepsin L at the nucleotide (80%, 74%, and 73%, respectively) and amino acid (68%, 65%, and 63%, respectively) level. We suggest that PDP via its collagenolytic and elastolytic activities as a cathepsin L is responsible for preparing the endometrium for blastocyst implantation.     

The detection and purification of a cat uterine secretory protein that is estrogen dependent (CUPED)

An estrogen-dependent secretory protein (CUPED) was detected and purified from uterine flushings of ovariectomized cats treated with 17 beta-estradiol. The protein was not detected in uterine flushings obtained from untreated ovariectomized animals or estrogen-primed animals treated with progesterone for 4 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of uterine flushings showed the presence of 1 or 2 protein bands with relative mobility values less than reduced and denatured thyroglobulin (Mr = 330,000). The protein was purified by differential centrifugation and gel filtration chromatography. Antiserum was raised against this purified protein in rabbits. The specificity of the antiserum to uterine fluid proteins was assessed by immunoblotting of electrophoretically transferred proteins. The antiserum cross-reacted with electrophoretically separated CUPED protein bands in uterine flushings. This protein may represent the content of the estradiol-induced secretory granules present in endometrial epithelial cells.     

The immunocytochemical localization of a cat uterine protein that is estrogen dependent (CUPED)

An estrogen-dependent polypeptide (CUPED), which was purified from uterine flushings of estrogen-treated cats, was localized in endometrial epithelial cells of cats using the peroxidase-antiperoxidase immunocytochemical staining procedure. Epithelial cells from animals treated with estradiol for 4, 7, or 14 days and estrogen-primed animals treated with progesterone for 2 days showed positive immunostaining. Staining was absent in untreated ovariectomized animals and in estrogen-primed animals treated with progesterone for 4 days. Specific cytoplasmic staining was confined to apical secretory granules in nonciliated cells of deep uterine glands. Staining was also commonly observed in the lumen of deep glands. Immunostaining was absent in the cells of the surface epithelium, stroma, and myometrium. In addition, other organs such as the oviduct, kidney, liver, pancreas, and lung showed no evidence of specific immunocytochemical staining. Therefore, the estrogen-dependent polypeptide obtained from uterine flushings of estrogen-treated ovariectomized cats is a uterine-specific secretory product that is packaged in apical cytoplasmic granules of uterine epithelial gland cells before being released into the uterine lumen.     

Quantification of an estrogen-dependent cat uterine protein (CUPED) in uterine flushings of estrogen- and progesterone-treated ovariectomized cats by radioimmunoassay

We previously reported the purification of an estrogen-dependent cat uterine protein (CUPED) and the preparation of a specific anti-CUPED serum in rabbits. Here, we describe a specific radioimmunoassay for CUPED using the purified CUPED and anti-CUPED serum that was utilized to quantify CUPED in daily uterine flushings obtained from steroid-treated ovariectomized cats. The radioimmunoassay was sufficiently sensitive to measure 0.1-100 ng CUPED. CUPED levels were low in untreated ovariectomized cats, increased within one day after the onset of treatment with estradiol, and remained elevated as long as estradiol was unopposed by progesterone. The levels of CUPED decreased when progesterone was added to the treatment regimen either 7, 14, or 28 days after the initiation of estradiol treatment. The data indicate that the presence of CUPED in the uterine flushings is dependent on the presence of estradiol and the absence of progesterone, that CUPED appears in the uterine lumen within one day after the onset of treatment with estradiol, and that the levels of CUPED are sharply reduced within one day of administration of progesterone and become nondetectable after three days.     

Detection and quantification of CUPED, an estrogen-dependent uterine protein, in uterine fluid and endometrial tissue of estrous and pregnant cats

Uterine flushings, media from cultured endometrial explants, and endometrial tissue obtained from estrous and pregnant cats were analyzed for the presence of a previously characterized, high-molecular-weight, estrogen-dependent glycoprotein (CUPED) by polyacrylamide-gel electrophoresis, Western blots, radioimmunoassay, and immunocytochemistry. Elevated levels of CUPED were present within the flushings, media, and tissue of estrous and 3-day postcoital animals. High levels of CUPED were also present in the flushings of 5-day postcoital animals; but the ability of endometrial explants to synthesize CUPED during short-term culture was greatly reduced, and only some of the endometrial glands contained CUPED secretory granules. CUPED was essentially nondetectable in the flushings, media, and tissues of animals pregnant for 7 or more days. Thus CUPED is present within the uterine lumen of the cycling cat at the time of sperm migration through the uterus and also for the first day or two that the developing blastocyst is present within the uterine lumen. The disappearance of CUPED from the tissue and flushings was correlated with the luteal production of progesterone.     

Changes in endometrial and placental protein synthesis and morphology during pregnancy and pseudopregnancy in the cat

This study was undertaken to determine the effect of the implanting cat blastocyst on endometrial morphology and protein synthesis. Placental and endometrial tissues were obtained from pregnant and pseudopregnant cats and then cultured with L-[35S]methionine and analyzed for protein synthesis by 2-dimensional polyacrylamide gel electrophoresis followed by fluorography, and also processed for light microscopy. The progesterone-dependent protein (PDP), described previously by Boomsma and Verhage (Biol Reprod 1987; 37:117-126) and Verhage et al. (Biol Reprod 1989; 41:347-354), was identified by immunocytochemical and immunoblot analysis. Attachment began after 12 days, and the deep glands contained large deposits of PDP. By 20 days the placenta was well developed, and the deep endometrial glands under the placenta had regressed and lacked deposits of PDP. The placenta continued to develop and thicken as pregnancy progressed. The surface epithelium in the non-implantation site regions developed extreme convolutions, while the well-developed deep glands with large deposits of PDP began to regress by 4 weeks, becoming similar to those in the implantation site. The endometrial glands in the pseudopregnant animals maintained deposits of PDP even though apoptotic bodies were observed between 20 and 35 days. PDP synthesis was not detected in the implantation site after 16 days, but it continued in the nonimplantation site through 5 weeks. The synthesis of nine other proteins was significantly altered by the end of implantation such that the pattern in the non-site endometrium was different from the implantation site but similar to the pattern found in the pseudopregnant endometrium. As pregnancy progressed, protein synthesis was altered in the placental/junctional zone and the non-site endometrium, but in the deep endometrial portion of the implantation site it was largely unchanged and similar to the deep portion of the non-site. Thus, the implanting cat blastocyst has a significant effect on the morphology of the implantation site and non-site endometrium, and alters the protein synthetic activity of the implantation site endometrium but apparently not the non-site region. The morphology and protein synthetic patterns of the pregnant cat uterus show regional differentiation and continue to change as pregnancy progresses.     

Secretion of a progesterone-induced inhibitor of plasminogen activator by the porcine uterus

The indirect ¹²⁵I-fibrin plate assay has been used to measure the levels of plasminogen activator (PA) in uterine flushings from pigs through the estrous cycle and during early pregnancy, and to measure the production of PA by pig conceptuses cultured in vitro. Activity in the flushings was high at the beginning and end of the estrous cycle, but only low levels were detected in mid cycle (the luteal phase). In pregnant animals, uterine PA levels became low around day 12 and did not show any further increase. Cultured day 12 blastocysts, however, released large amounts of PA into the medium in a time-dependent fashion over a 48 hr period, suggesting that this activity was inhibited in vivo. The presence of a protease inhibitor in uterine flushings has been demonstrated in cycling gilts, and follows a hormone-directed trend, with flushings taken during the luteal phase showing inhibitory activity against PA secreted early or late in the cycle. By assaying flushings from ovariectomized gilts given daily injections of progesterone, estrogen, both hormones together, or corn coil, it has been verified that the inhibitor is progesterone-induced and is also active against both PA produced by day 12 conceptuses and urokinase. It also inhibits PA, as determined using a direct fluorometric assay with glutaryl-glycyl-L-arginine-4-methyl-coumarinyl-7-amide as substrate. The PA inhibitor is acid-stable, and of low molecular weight (15,000 ± 5000), as determined by Sephacryl S-200 gel filtration. Unlike most animals, the trophoblast of the pig is not invasive in the uterus, but is invasive if transplanted to some ectopic site. The progesterone-induced inhibitor may possibly play a role in preventing invasive implantation.     

Immunocytochemical localization and messenger ribonucleic acid levels of a progesterone-dependent endometrial secretory protein (cathepsin L) in the pregnant cat uterus.

The purpose of this study was to localize immunocytochemically a progesterone-dependent protein (PDP) and to determine PDP mRNA levels during the initial stage of the implantation period. Uterine tissue was collected from Day 0-18 postcoital animals. The tissue was processed for immunocytochemical localization of PDP, and the endometrial RNA was isolated and analyzed for PDP gene expression by slot-blot hybridization. PDP was detected immunocytochemically as early as Day 5 postcoitus in the epithelial cells of the deep uterine glands, and the intensity of immunostaining appeared to peak by Day 12 postcoitus. PDP was absent in the endometrium obtained from implantation sites after Day 16 postcoitus, but the synthesis of PDP was maintained in the endometrium obtained from nonimplantation sites. Immunogold electron microscopy demonstrated that PDP was present in electron-dense granules of the glandular epithelial cells. PDP mRNA was detectable in the endometrium at Day 5 postcoitus and peaked around Day 10 postcoitus. PDP mRNA was absent in the endometrium from implantation sites after Day 16 postcoitus, but was maintained in the endometrium from nonimplantation sites. In summary, the results of this study illustrate that PDP is synthesized within the epithelial cells of the deep uterine glands, packaged within membrane-bound secretory granules, and released into the uterine lumen. Also, the process of implantation alters the gene expression in a very localized way since PDP mRNA and PDP-positive granules were absent in the endometrial glands obtained from the implantation site within 1-2 days of the onset of implantation, whereas both PDP mRNA and PDP-positive granules were maintained in the endometrial glands from nonimplantation-site regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uterine specific antigens in the ewe

Uterine flushings from ewes on days 0, 3, 6, 9, 12 and 15 of the estrous cycle were analyzed for total protein content. Flushings from days 9, 12 and 15 had greater (P<.01) amounts of protein than those from 0, 3 and 6. Antisera to uterine fluids from ewes at day 10 to 12 or day 14 to 15 of pregnancy detected two uterine-specific antigens in uterine flushings at day 7, 11 and 15 but not at days 0 and 3 of the cycle. A third uterine antigen was also detected in kidney tissue extracts. All three antigens were present in endometrial extracts at each stage examined. Progesterone, or estrogen plus progesterone, administration to ovariectomized ewes induced the appearance of the two uterine-specific antigens. The third antigen was detectable even in ovariectomized ewes. No pregnancy-specific antigens were detected in flushings from days 7, 11 or 15 of gestation. The effect of pregnancy on endometrial protein synthesis was examined in vitro . No differences were seen in the incorporation of (3)H-leucine in day 11 pregnant or nonpregnant or in day 14 pregnant or nonpregnant endometrium. No differences in total uterine lumenal protein were observed. Endometrial secretions, obtained by conditioning media with endometrial explant cultures, were evaluated to assess their effect on protein synthesis in day 11 embryos cultured in vitro . No significant effects of endometrial secretions or serum were observed.     

Effect of estradiol on the amino acid-accepting activity of uterine tRNAs and their participation in protein synthesis

Estradiol (E2) induces a complementary increase in both the amount of mRNA and the rate of translation of the mRNA in the uterus of ovariectomized mature rats. The mechanism of the translational effect was evaluated by measuring the functional capacity of uterine tRNA isolated from control, E2 (1 h)- and E2 (14 h)-treated ovariectomized rats to support amino acid acceptor activity and uterine protein synthesis. The specific amino acid acceptor activity (SAA) of deacylated tRNA for 18 individual amino acids was determined using a tRNA-dependent rat liver tRNA synthetase preparation. The SAA was the same for all amino acids for uterine tRNA from control and E2 (1 h)-treated rats but was increased for uterine tRNA from E2 (14 h)-treated rats to levels that were 1.4-4.3 times the SAA of uterine tRNA from control rats. When uterine tRNA from control and E2 (14 h)-treated rats was incubated with purified tRNA nucleotidyltransferase, the SAA for all amino acids was increased an average of 1.6-fold for control tRNA and 0.3-fold for tRNA from E2 (14 h)-treated rats. The ability of uterine tRNA to support maximal rates of protein synthesis in tRNA-dependent uterine ribosome protein synthesis assay was increased by either in vivo treatment of the rats with estradiol or by in vitro repair of the 3'-CCA terminus of this tRNA by nucleotidyltransferase. These observations suggest that E2 may increase the rate of mRNA translation in the uterus, in part, by increasing the proportion of certain tRNAs with intact and functional 3'-CCA acceptor termini.     

Progesterone-regulated secretion of the serpin-like proteins of the ovine and bovine uterus.

The uterine milk (UTM) proteins are the major progesterone-regulated proteins secreted by the sheep uterus during pregnancy. Recently, proteins related to the UTM proteins have been identified in uterine secretions of the pregnant cow and sow. The present objective was to determine the time course for induction of the UTM proteins in sheep and cattle. Twelve ovariectomized ewes received subcutaneous injections of either vehicle for 10 days or 100 mg/d of progesterone for 10 days or 30 days. The presence of UTM proteins was examined by Western blotting of uterine flushings and by immunoabsorption of radiolabeled UTM proteins from conditioned medium of endometrial explant cultures performed with [35S]methionine precursor. Uterine milk proteins were present in slight amounts in uterine flushings and endometrial-conditioned culture medium of some ewes in the control group, but amounts of proteins were greatly enhanced by progesterone after 10 or 30 days of treatment. Prolonged exposure to progesterone (30 days versus 10 days) increased amounts of UTM proteins. Immunohistochemical analysis of endometrium indicated that the major site of UTM proteins was the glandular epithelium. In the second experiment, nine ovariectomized cows were treated daily with vehicle for 12 days or 750 mg progesterone for 12 or 30 days. Uterine flushings and conditioned endometrial culture medium were examined for UTM proteins by Western blotting. Uterine milk proteins were present to some degree in cows treated with vehicle, and an enhancement in amounts of UTM proteins was not observed until after 30 days of progesterone treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of collagenolytic cysteine protease synthesis by estrogen in osteoclasts

In ovariectomized (Ovx) mice, collagenolytic cysteine protease (CCP) activity in calvaria significantly increased 7 days after ovariectomy and was about 50% of that observed in sham-operated (Sham) mice 3 weeks later. In Ovx mice, subcutaneously (s.c.) administered estradiol-17beta (E2) (10 microg/kg) for 2 weeks led to a decrease in CCP activity in calvaria to the level observed in Sham mice. In Ovx mice, though the amount of cathepsin L increased more than that of cathepsin K, cathepsin K and cathepsin L content increased by 200-400% compared with the Sham mice; cathepsin K was detected in larger amounts than cathepsin L in calvaria from both Sham and Ovx mice. The amounts of cathepsin K and cathepsin L in Ovx mice were reduced to the values seen with Sham mice after administration (s.c.) of E2 (10 microg/kg) for 2 weeks. In mouse calvarial organ culture, the increase of CCP activity and release of hydroxyproline, an indicator of degradation of type-I collagen, in the presence of 1alpha,25-(OH)(2)D(3), parathyroid hormone, interleukin (IL)-1alpha, IL-6, or tumor necrosis factor-alpha was suppressed by E2 (10(-9)-10(-7) M). In all cases, secretion of both cathepsin K and cathepsin L were suppressed by E2. In osteoclasts, expression of cathepsin K and cathepsin L was suppressed by E2 at the mRNA level. Cathepsin B was detected faintly or not at all. These results suggest that synthesis of cathepsin K and cathepsin L was negatively regulated by E2 at the mRNA level. In Ovx mice, deficiency of E2 resulted in an augmentation of cathepsin K and cathepsin L synthesis, and the cathepsins might share roles in bone resorption in vivo.     

The effects of ovarian steroids in controlling rat uterine prostaglandin F2-alpha during the peri-implantation period

Rats with delayed implantation, induced by ovariectomy or hypophysectomy, as well as those with normal pregnancy were used to examine the changes in uterine prostaglandin F2 alpha (PGF2 alpha) associated with implantation. In normal pregnant rats, while maximal uterine production of PGF2 alpha was found at 09:00, maximal catabolic enzyme activity (CEA) was seen at 17:00 of day 4. Uterine content of PGF2 alpha was high at 17:00 of day 4, but decreased by 80% within the next 24 h. There was no change in PGF2 alpha production during the first 6 h after injection of estradiol to hypophysectomized animals. There was, however, a dramatic decrease in production within the next 6 h. In contrast, CEA was not different in animals treated with estrogen than in those receiving only progesterone. In ovariectomized animals, uterine PGF2 alpha production also was lowered by estrogen but in these animals CEA was significantly elevated 18 h after injection of estradiol. Estrogen caused a greater increase in PGF2 alpha content in the hypophysectomized, compared to the ovariectomized, rats. The results are consistent with the view that ovarian steroids play an important role in controlling the changes in uterine PGF2 alpha around the time of implantation in rat.     

Gallium-67 distribution in pregnant mammals.

Studies on the distribution of 67Ga in pregnant rabbits showed a marked concentration of the isotope in uterine tissue and flushings of 5-day pregnant rabbits 24 hours after isotope injection. The isotope no longer localized throughout the uterine tissues and flushings on injection of 7- to 8-day pregnant rabbits but was specifically associated with the blastocysts and sites of implantation. As the gestation time progressed 67Ga increasingly concentrated in placental and mammary tissue while the concentration in serum and thymus tissue decreased. The marked concentration in placental tissue was readily discernible on total-body scans of pregnant rabbits. Column chromatography of uterine washings and placental extracts from pregnant rabbits and thymus extracts from estrous rabbits showed most of the isotope associated with moieties greater than or equal to 200,000 MW whereas the isotope in "milk" and extracts of mammary tissue was bound to components of 25,000-35,000 MW. Extracts of thymus from pregnant rats showed a marked, selective loss of 67Ga binding in two peaks of approximately 50,000 and 120,000 MW compared to the chromatographic profile of extracts from normal rat thymus.     

Regulation of uterine 5 alpha-reductase type 1 in mice.

A null mutation in the murine gene encoding steroid 5 alpha-reductase type 1 (5 alpha R1) leads to failure of normal parturition at term. This observation, together with the finding that mRNA levels of uterine 5 alpha R1 increase significantly at term in normal pregnant animals, indicates that 5 alpha R1 plays an important role in murine parturition. The current studies were conducted to elucidate the regulation of 5 alpha R1 in uterine tissues of nonpregnant and pregnant animals. Nonpregnant, ovariectomized ICR mice were treated with vehicle (control), 17 beta-estradiol (E(2)), progesterone (P(4) ), or E(2)+P(4) for 3 days. Thereafter, uterine tissues were obtained for histology, quantification of 5 alpha R1 specific activity, and Northern blot analysis of 5 alpha R1 mRNA expression. The 5 alpha R1 enzyme activity was significantly increased in animals treated with E(2)+P(4). However, activity was much less in uterine tissues from E(2)+P(4)-treated animals than in uterine tissues from pregnant animals near term. To evaluate further the regulation of 5 alpha R1 during gestation, mice underwent unilateral tubal ligation before timed matings. The 5 alpha R1 activity increased eightfold in uterine tissues from the fetal horn from Gestational Days 12 to 18. This temporal pattern in 5 alpha R1 activity paralleled marked increases in uterine diameter. Taken together, these studies indicate that expression of 5 alpha R1 is regulated by E(2)+P(4) in uterine tissues. Whereas E(2) alone is insufficient to induce enzyme activity, E(2) may be required to increase P(4) receptors and, thereby, mediate the effects of P(4) on 5 alpha R1 gene expression. Further increases in enzyme activity during late gestation are mediated by fetal occupancy, possibly through stretch-induced increases in endometrial growth. Thus, like other genes involved in parturition, expression of 5 alpha R1 is regulated by both hormonal and fetal-derived signaling pathways.     

Induction of cytotoxic T-lymphocyte antigen-2beta, a cysteine protease inhibitor in decidua: a potential regulator of embryo implantation

During early pregnancy, the steroid hormone progesterone induces differentiation of uterine stroma to decidual cells, which regulate embryo-uterine interactions. The progesterone-induced signaling molecules that participate in the formation and function of decidua remain poorly understood. We recently utilized high-density oligonucleotide microarrays to identify several genes whose expression is markedly altered in pregnant uterus in response to RU486, a well characterized antagonist of the progesterone receptor (PR). Our study revealed that the gene encoding cytotoxic T-lymphocyte antigen-2beta (CTLA-2beta), a cysteine protease inhibitor, is expressed during PR-induced decidualization. The spatio-temporal expression of CTLA-2beta mRNA precisely overlapped with the decidual phase of pregnancy. Interestingly, administration of progesterone to estrogen-primed ovariectomized mice failed to induce CTLA-2beta expression. A concomitant artificial decidual stimulation was necessary to trigger this expression. Uteri of PR knockout mice failed to express this mRNA, even after a combined administration of steroid hormones and artificial stimulation. The uterine expression of CTLA-2beta was, therefore, dependent on PR as well as other unknown factor(s) associated with decidual response. To identify the molecular target(s) of CTLA-2beta,we analyzed its interaction with proteins present in soluble extracts prepared from day 7 pregnant uteri containing implanted embryos. A protein affinity strategy employing recombinant CTLA-2beta helped us to determine that cathepsin L, a cysteine protease, is one of its targets in the pregnant uterus. Consistent with this finding, expression of cathepsin L was detected in the giant trophoblast cells of the ectoplacental cone on day 7 of pregnancy. Collectively, our results support the hypothesis that expression of CTLA-2beta in the decidua may regulate implantation of the embryo by neutralizing the activities of one or more proteases generated by the proliferating trophoblast.     

The effect of estrogen and progesterone on structural changes in the uterine glandular epithelium of the ovariectomized sheep.

The development of epithelial cells of the uterine glands of ovariectomized sheep in response to estradiol-17 beta (E) and progesterone (P) was studied using light and electron microscopy. Animals that had been ovariectomized for six weeks were placed in one of three experimental treatment groups. Group I animals (untreated controls) received no steroid treatment. Group II animals (E alone) received one 4-cm E implant (E approximately 5-10 pg/ml) and their uteri were removed after 2, 4, or 6 days. Group III animals (E-primed, P-treated) received an E implant (E approximately 5-10 pg/ml) for 6 days and then were treated with six 13-cm P implants (P approximately 1.5-3 ng/ml), in the continued presence of E, for 2, 4 or 6 days. Six weeks after ovariectomy the epithelial cells of the uterine glands were low cuboidal and morphologically appeared to be synthetically inactive. Following 2 days of E treatment the epithelial cells had significantly increased in cell height, and protein-synthesizing organelles were well developed. Maturation of the secretory apparatus continued throughout E treatment. The Golgi complex and rough endoplasmic reticulum (RER) were abundant. Lysosomal-like granules and granules of varying electron density were present in the cytoplasm. The chronic administration of P to E-primed animals did not result in any further increase in cell height. Elongated mitochondria, a cup-shaped Golgi apparatus, extensive apical microvilli, and irregularly shaped membranous profiles in the supranuclear cytoplasm characterized these uterine epithelial cells. Lysosomal-like granules, small vesicles, and scattered patches of glycogen were seen in the cytoplasm. These data show that the uterine epithelial cells of the ovariectomized sheep undergo morphological alterations in protein-synthesizing organelles and apical specializations that depend on the presence of E and P.     

Estrogen- and progesterone-dependent secretory changes in the uterus of the sheep.

This study was undertaken to determine the effects of 17 beta-estradiol (E) and progesterone (P) on polypeptide synthesis and release from the uterus of the sheep. Uterine flushings (UF) and endometrium were obtained from ovariectomized untreated animals, ovariectomized animals treated with E (approximately 5-10 pg/ml) for 6 days (6E) and ovariectomized animals primed with E for 6 days then treated with P (approximately 1.5-3 ng/ml), in the continued presence of E, for an additional 6 days (6EP). Endometrium was cultured (24 h) in the presence of 3H-leucine (3H-leu) or 3H-glucosamine (3H-glcN), and newly synthesized and released proteins were detected in culture media by fluorography of 10% SDS gels. The quantity of proteins in UF and radiolabeled proteins in explant culture media did not change between treatment groups (p < 0.05). Qualitative changes in the synthesis and release of proteins were observed depending on the steroid treatment. An M(r) 57,000 protein was present in UF and 3H-leu-labeled culture media obtained from animals treated only with E and an M(r) > 200,000 was present in 3H-leu-labeled culture media of endometrium obtained from 6E and 6EP animals. An M(r) 44,000 protein was present only in UF from 6EP animals but could not be detected in endometrial culture media from animals undergoing this steroid treatment. These data show that the endometrium of the ovariectomized sheep undergoes alterations in secretory protein patterns which depend on the presence of E and P.