Molecular cloning and temporal expression during pregnancy of the messenger ribonucleic acid encoding uteroferrin, a progesterone-induced uterine secretory protein

A lambda gt11 expression library containing cDNA inserts prepared from porcine endometrial mRNA was immunologically screened by using an antiserum developed against porcine uteroferrin (Uf), a glycoprotein that has been strongly implicated in transplacental iron transport in the pregnant pig. Antibody reactive clones (lambda 4a3, 13.1, and 2.2) were isolated after screening 1.5 x 10(5) recombinant phages. Clones 4a3 and 13.1 expressed Uf antigenic determinants in beta-galactosidase fusion proteins and specifically selected antibody which reacted with Uf in immunoblots prepared from uterine cytosolic extracts. In addition, all three cDNA clones collectively contained DNA sequences that encoded an 85-amino acid peptide which corresponded to a region within the carboxyterminal portion of the Uf protein. Northern blot hybridization of these cDNAs to RNAs extracted from whole uterine tissue of pregnant pigs revealed a single uterine poly(A)+ RNA of approximately 1.7 kilobases in length, which was not found in liver and mammary tissue RNAs. The concentration of the Uf mRNA changed in a temporal fashion during pregnancy in a manner that was distinct from that of the progesterone receptor mRNAs. Highest levels of Uf mRNA were found at mid and late pregnancy (days 45-110) and were about 50-fold greater than at day 30 of pregnancy. By contrast, RIA analysis of the uterine tissue extracts showed that maximum amounts of Uf were present at day 60 and then declined sharply. Thus the pattern of Uf mRNA present in the uterus did not parallel the amount of Uf polypeptide that could be recovered from the tissue. The tissue specific and temporal regulation of Uf gene expression emphasizes that the protein plays an important role in uterine activity and/or fetal development.     

Regulation of synthesis of uterine secretory proteins: evidence for differential induction of porcine uteroferrin and antileukoproteinase gene expression

Mechanisms regulating the expression of two pregnancy-associated proteins of the porcine uterus, namely the iron-transport protein uteroferrin (UF) and the lysosomal serine proteinase-inhibitor antileukoproteinase (ALP), were investigated by comparing the effects of estrogen (E), progesterone (P4), and conceptuses on the steady-state levels of their mRNAs. For UF, the expression of mRNA with production and secretion of the corresponding protein was also investigated. Progesterone increased the levels of endometrial UF mRNA and of secreted UF, but did not affect the levels of ALP mRNA in ovariectomized gilts that had received P4 treatment for 8 days. Estrogen inhibited the accumulation of endometrial UF mRNA, increased UF secretion in these gilts, but had no effect on levels of ALP mRNA. Administration of E to gilts on Day 11 of the cycle slightly diminished UF mRNA levels at 1 h post-E; had no effect at 6, 12, and 24 h post-E; and increased levels of secreted UF in uterine luminal fluids 24 h post-E. The presence of conceptuses increased levels of endometrial ALP mRNA and decreased UF protein in uterine luminal fluids, but did not affect levels of endometrial UF mRNA. Myometrium, endometrium, and placenta from Day 75 and Day 105 pregnant gilts were also evaluated for ALP and UF mRNA expression to determine regional expression of these steroid-regulated genes. Myometrium and endometrium expressed comparable levels of UF and ALP mRNAs within Days 75 or 105, but placenta did not express detectable levels of mRNA for either protein. Within the myometrium, UF protein is immunolocalized mostly to the inner circular and to a lesser extent to the outer longitudinal layer of smooth muscle. These results indicate that E, P4, and presence of conceptuses differentially affect endometrial expression of ALP and UF mRNAs and secretion of UF.     

Peroxidase activity in the mouse uterus, placenta and fetus during pregnancy

Changes in peroxidase activity during pregnancy were examined in CD-1 mice. Peroxidase activity was measured with guaiacol as the substrate in uterine extracts of nonpregnant mice and in uterine, placental, and fetal extracts of pregnant mice on days 9, 12, 14, 16, and 18 of gestation. Uterine peroxidase activity in nonpregnant mice was high, but declined logarithmically to only 0.2% by day 18 of pregnancy. In contrast to this decline, a concomitant 50-fold logarithmic increase in fetal peroxidase activity was observed between day 12 and 18. Activity in placental extracts did not change significantly throughout the gestational period examined. These results suggest that membrane bound peroxidase in mouse uterus and fetus undergoes major shifts during pregnancy.     

Hyaluronate and CD44 expression patterns in the human placenta throughout pregnancy

Hyaluronan (HA) and CD44 are involved in several processes such as cell migration and differentiation. In the present study, we examined the expression and distribution of both hyaluronan and its cell surface receptor (CD44) in the human placenta, which is a rapidly growing and differentiating organ that plays a fundamental role in fetal life. Hyaluronan was detected by a specific biotinylated binding probe, termed b-PG. In the first half of gestation, HA was strongly expressed in the stroma of the mesenchymal villi which have been previously identified as responsible for the growth and differentation of the villous trees. The other villous types showed an intense staining only in the fetal vessel walls and in the connective tissue closely underlying the trophoblastic cover. In addition, hyaluronan positive staining was also apparent in a restricted rim of villous stroma directly apposed to extravillous cytotrophoblastic cell islands and cell columns. In full term placentas, all villi expressed HA in their stromal tissue with a more homogenous staining than in the first half of gestation. In contrast to hyaluronan, in the first trimester CD44 was restricted to some of the Hofbauer cells which may be able to internalize hyaluronan, thus playing a significant role in its removal in early pregnancy. CD44 was primarily expressed starting from the 16th week of gestation. At the end of pregnancy it was expressed in the various villous types, especially in stem villi. Moreover, the plasma membrane of some extravillous cytotrophoblastic cells in the basal plate and the large majority of the decidual cells showed a positive immunostaining for this receptor. Taken together, these data suggest that HA is strongly involved in early villous morphogenesis, whereas CD44 seem to be play an important role in tissue remodelling later in gestation.     

Resonance Raman scattering from uteroferrin, the purple glycoprotein of the porcine uterus.

Uteroferrin, a purple-colored, iron-containing glycoprotein, purified from the uterine fluid of progesterone-stimulated pigs, owes its natural purple color to a broad absorption band centered at 545 nm. Laser excitation within the visible absorption band of uteroferrin results in an intense resonance Raman spectrum which bears a striking resemblance to that reported for Fe(III)-transferrin, the iron transport protein of serum. Excitation profiles for the four resonance-enhanced bands of uteroferrin were obtained from 4579 A to 6741 A, using lines from Ar+ and Kr+ lasers. Each of the profiles have maxima near 545 nm. The spectral similarities of uteroferrin and Fe(III)-transferrin lead to the belief that the Fe(III) binding sites of the two proteins must be, at least in some respects, quite similar. In particular, it is concluded that, as in Fe(III)-transferrin, the metal binding site of uteroferrin contains a tyrosine ligand and that the visible absorption spectrum of uteroferrin results from a phenolate to Fe(III) charge transfer.     

Synthesis of 'decidualization-associated protein' in tissues of the rat uterus and placenta during pregnancy.

The synthesis of a soluble protein (referred to as 'decidualization-associated protein', DAP), has been examined in uterine and placental tissues of rats during pregnancy by means of polyacrylamide gel electrophoretic analysis of [3H]leucine-labelled soluble proteins. No synthesis of the protein was detected in non-implantation regions of the uterus. In implantation site tissue, no synthesis was detected on Days 6 or 7 of pregnancy. Only slight synthesis was present in the endometrium on Day 8, but synthesis rose rapidly from Days 9 to 12 in both the endometrium and myometrium although differences in the rates of increase were observed. Synthesis fell from Day 12 to 14 in both tissues. Synthesis by the myometrium was entirely localized in the mesometrial region, which contains the metrial gland. After Day 12, when the endometrium is represented by the chorioallantoic placenta, synthesis was examined in the labyrinthine and the decidua basalis/basal zone placenta tissues. No synthesis of 'DAP' was detected in the labyrinthine placenta from Day 16 of pregnancy. Synthesis observed in the decidua basalis/basal zone placenta fell dramatically from Day 14 to 20. The pattern of synthesis of 'DAP' during pregnancy suggests a role in the establishment of the chorioallantoic placenta and metrial gland in the rat.     

Progesterone induction of the uterine milk proteins: major secretory proteins of sheep endometrium

Progesterone induction of the uterine milk proteins (UTMP), the major secretory products of the ovine uterus during pregnancy, was studied in ovariectomized ewes given physiological levels of progesterone for 0, 2, 6, 14, or 30 days. Western blotting of uterine flushes and of endometrial explant culture medium, endometrial RNA analyses on dot and Northern blots, and immunocytochemistry performed on uterine tissue sections demonstrated the presence of low levels of UTMP mRNA and UTMP protein after 6 days of progesterone therapy, and increasing levels of UTMP production and secretion after 14 days. Highest activity was observed at Day 30. The induction of the UTMP progressed from small amounts of antigen present in the supranuclear region of a few epithelial cells in deep and middle-depth regions of uterine glands in the Day 6 progesterone-treatment group to large amounts detected in epithelial cells spread throughout the length of the glands in later groups. UTMP production was also identified in the uteri of intact ewes at Day 16 (but not earlier) of the estrous cycle and during early pregnancy (Days 14 to 22). Production of a protein similar to the UTMP was also noted in the uterus of a pregnant cow. The UTMP provide a good model of a progesterone-responsive secretory protein in a mammal whose synthesis is increased gradually over a period of weeks.     

TRPV6 and Calbindin-D9k-expression and localization in the bovine uterus and placenta during pregnancy

ABSTRACT: BACKGROUND: Transient receptor potential channel type 6 (TRPV6) and Calbindin-D9k (CaBP-9 k) are involved in the active calcium (Ca2+) transport mechanism in many tissues including placenta and uterus, suggesting a role in the establishment and maintenance of pregnancy. Moreover, TRPV6 and CaBP-9 k seem to support the materno-fetal Ca2+ transport that is crucial for fetal Ca2+ homeostasis, bone growth and development. However, it is unknown if these proteins are also involved in the aetiology of pathologies associated with parturition in cows, such as retained fetal membranes (RFM). The aim of the current study was to create an expression profile of uterine and placentomal TRPV6 and CaBP-9 k mRNAs and proteins during pregnancy and postpartum in cows with and without fetal membrane release. METHODS: Uteri and placentomes of 27 cows in different stages of pregnancy and placentomes of cows with and without RFM were collected. Protein and mRNA expression of TRPV6 and CaBP-9 k was investigated by real-time PCR, immunohistochemistry and Western blot. RESULTS: In the uterine endometrium, highest TRPV6 and CaBP-9 k expression was found in the last trimester of pregnancy, with a particular increase of protein in the glandular epithelium. In the placentomes, a gradual increase in TRPV6 mRNA was detectable towards parturition, while protein expression did not change significantly. Placentomal CaBP-9 k expression did not change significantly throughout pregnancy but immunohistochemistry revealed an increase in staining intensity in the maternal crypt epithelium. Immunohistochemical, stronger placental CaBP-9 k signals were seen in animals with RFM compared to animals with an undisturbed fetal membrane release, while protein levels, measured by Western blot analyses did not change significantly. CONCLUSIONS: The results of the present study demonstrate a dynamic expression of TRPV6 and CaBP-9 k during pregnancy in the bovine uterine endometrium and placentomes, suggesting a functional role for these proteins in Ca2+ metabolism during pregnancy. The temporal and spatial expression patterns indicate that TRPV6 and CaBP-9 k may be involved in materno-fetal Ca2+ transport, mainly through an interplacentomal transport, and that both proteins may participate in physiological processes that are crucial for fetal and placental development. However, neither TRPV6 nor CaBP-9 k seem to be causative in the retention of fetal membranes.     

Role of secretory protease inhibitor SPINK3 in mouse uterus during early pregnancy

Successful embryo implantation depends on intricate epithelial-stromal cross-talk. However, molecular modulators involved in this cellular communication remain poorly elucidated. Using multiple approaches, we have investigated the spatiotemporal expression and regulation of serine protease inhibitor Kazal type 3 (SPINK3) in mouse uterus during the estrous cycle and early pregnancy. In cycling mice, both SPINK3 mRNA and protein are only expressed during proestrus. In the pregnant mouse, the expression levels of both SPINK3 mRNA and protein increase on days 5-8 and then decline. Spink3 mRNA is expressed exclusively in the uterine glandular epithelium, whereas SPINK3 protein is localized on the surface of both luminal and glandular epithelium and in the decidua. Moreover, SPINK3 in the decidua has been observed in the primary decidual zone on day 6 and the secondary decidual zone on days 7-8; this is tightly associated with the progression of decidualization. SPINK3 has also been found in decidual cells of the artificially decidualized uterine horn but not control horn, whereas Spink3 mRNA localizes in the glands of both horns. The expression of endometrial Spink3 is not regulated by the blastocyst according to its expression pattern during pseudopregnancy and delayed implantation but is induced by progesterone and further augmented by a combination of progesterone and estrogen in ovariectomized mice. Thus, uterine-gland-derived SPINK3, as a new paracrine modulator, might play an important role in embryo implantation through its influence on stromal decidualization in mice.     

Biochemical characterization and biosynthesis of the uterine milk proteins of the pregnant sheep uterus

The uterine milk proteins (UTM-proteins), a pair of basic glycoproteins with similar isoelectric points and molecular weights (57,000 and 55,000), are secreted by the endometrium of the pregnant ewe. Peptide mapping of the two species of UTM-proteins demonstrated them to be structurally related. Furthermore, pulse-chase and continuous-labeling experiments indicated that both are produced from a common precursor of lower molecular weight. Purified UTM-proteins were found to be rich in basic amino acids, low in tyrosine, and apparently lacking in tryptophan. The proteins were about 5.6-5.7% carbohydrate by weight and bound the lectin, concanavalin A. UTM-proteins synthesized in vitro incorporated D-[3H]glucosamine. Analysis of [3H]glucosamine-labeled glycopeptides of Pronase-digested UTM-proteins by gel filtration indicated that most radioactivity is associated with one size class of oligosaccharide. UTM-proteins secreted by the endometrium in the presence of tunicamycin, an N-glycosylation inhibitor, were of lower molecular weight than those from control endometria, indicating that sugar chains are attached to the protein core via N-linkages to asparagine. UTM-proteins synthesized in culture incorporated [32P]orthophosphate, and tunicamycin inhibited this incorporation. Analysis of hydrolyzed UTM-proteins by paper chromatography indicated that much of the 32P was associated with mannose 6-phosphate. Because this moiety is the so-called lysosomal recognition marker and is present on uteroferrin, the acid phosphatase of porcine uterine secretions, we tested UTM-proteins for several enzymatic activities characteristic of lysosomes, but none was found. In conclusion, the UTM-proteins are related glycoproteins that, like porcine uteroferrin, contain mannose 6-phosphate, a result which suggests that secretion of glycoproteins with phosphorylated oligosaccharide chains may be a common feature of the progestational uterus.     

Vitamin D, the placenta and pregnancy

Impaired vitamin D status is common to many populations around the world. However, data suggest that this is a particular problem for specific groups such as pregnant women. This has raised important questions concerning the physiological and clinical impact of low vitamin D levels during pregnancy, with implications for classical skeletal functions of vitamin D, as well as its diverse non-classical actions. The current review will discuss this with specific emphasis on the classical calciotropic effects of vitamin D as well as the less well established immunological functions of vitamin D that may influence pregnancy outcome. The review also describes the pathways that are required for metabolism and function of vitamin D, and the various clinical complications that have been linked to impaired vitamin D status during pregnancy.     

Immunolocalisation of arabinogalactan proteins and pectins in Actinidia deliciosa pollen

Summary. The cell wall composition of germinating pollen grains of Actinidia deliciosa was studied by immunolocalization with monoclonal antibodies against arabinogalactan proteins (AGPs) and pectins. In ungerminated pollen, the JIM8 epitope (against a subset of AGPs) was located in the intine and in the cytoplasm, while the MAC207 epitope (against AGPs) was only located in the exine. After germination, the JIM8 and MAC 207 epitopes were located in the cytoplasm and in the pollen tube wall. The Yariv reagent that binds to AGPs was added to the germination medium inducing a reduction or inhibition in pollen germination. This indicates that AGPs are present in the growing pollen tube and play an important role in pollen germination. To identify the nature of the pectins found in pollen grains and tubes, four monoclonal antibodies were used. The JIM5 epitope (against unesterified pectins) was located in the intine, more intensely in the pore region, and along the pollen tube wall, and the JIM7 epitope (against methyl-esterified pectins) was also observed in the cytoplasm. After germination, the JIM5 epitope was located in the pollen tube wall; although, the tube tip was not labelled. The JIM7 epitope was located in the entire pollen tube wall. LM5 (against galactans) showed a labelling pattern similar to that of JIM5 and the pattern of LM6 (against arabinans) was similar to that of JIM7. Pectins show different distribution patterns when the degree of esterification is considered. Pollen tube wall pectins are less esterified than those of the pollen tube tip. The association of AGPs with pectins in the cell wall of the pollen grain and the pollen tube may play an important role in the maintenance of cell shape during pollen growth and development.Correspondence and reprints: Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal.     

Lineage, maturity, and phenotype of uterine murine dendritic cells throughout gestation indicate a protective role in maintaining pregnancy

Dendritic cells (DCs) are known to play a major role in the induction, maintenance, and regulation of immune responses. Recently, DCs have been described to be present at the feto-maternal interface in human decidua. However, only limited information is available about DC presence, phenotype, and--more importantly--function throughout gestation. Thus, we analyzed local (uterine) and systemic (blood) DCs in a murine model. DBA/2J mated CBA/J females with vaginal plugs were separated and killed on Gestation Days (GDs) 1.5, 3.5, 5.5, 6.5, 7.5, 8.5, 10.5, 13.5, 15.5, or 17.5. Frequency of uterine and blood CD11c+ DC, phenotype (coexpression of CD8alpha and major histocompatibility complex class II [MHC II] antigens), and presence of intracellular cytokines (interleukins 12 and 10) were determined by flow cytometry. The morphology of DC in the pregnant uterus was evaluated by immunohistochemistry. In uterus, the relative number of CD11c+ cells increased from GD 5.5, reaching a plateau on GD 9.5 until GD 17.5, while a transient peak of systemic CD11c+ cells was found on GD 8.5 and 10.5. The vast majority of uterine DCs were CD8alpha- and thus, belonged to the myeloid lineage. Interestingly, a significant peak of lymphoid DC was present on GD 1.5 and 5.5. Further, significantly more intracellular interleukin 10 than interleukin 12 was present in CD11c+ cells. Interestingly, mature DCs (MHC II+) were diminished from GD 5.5 to 8.5. Characterization of CD11c+ cell kinetics in uterus and blood reveals variation of phenotype during pregnancy, pointing toward an eminent immunoregulatory role of DCs throughout gestation at the feto-maternal interface.     

Spatiotemporal expression of the serine protease inhibitor,SERPINE2, in the mouse placenta and uterus during the estrous cycle,pregnancy, and lactation

Background  

SERPINE2, also known as glia-derived nexin or protease nexin-1, belongs to the serine protease inhibitor (SERPIN) superfamily. It is one of the potent serpins that modulates the activity of the plasminogen activator (PA) and was implicated in tissue remodeling. In this study, we investigated the expression patterns of SERPINE2 in the mouse placenta and uterus during the estrous cycle, pregnancy, and lactation.     

Binding of progesterone to the proteins of the uterine luminal fluid. Identification of uteroglobin as the binding protein.

1. The uterine luminal fluid of rabbits treated with estradiol and progesterone contains a protein factor with high affinity for [3-H] progesterone which is not present in the uterine secretion of control rabbits treated with estradiol. 2. This progesterone dependent factor is shown by gel filtration and polyacrylamide gel electrophoresis to be identical with the uterus specific protein uteroglobin, which seems to be required during the preimplantation phase. Uteroglobin specific antiserum, prepared in guinea pigs, completely inhibits the progesterone binding activity of the proteins of the uterine fluid. 3. Progesterone binding to uteroglobin is dependent upon millimolar concentrations of dithioerythritol. At saturation, one molecule of progesterone binds per uteroglobin molecule and the apparent association constant is 2 x 10-6 M-1 at 0 degrees C. 4. The progesterone binding species of uteroglobin exhibits a molecular weight of around 12 000 on polyacrylamide gels containing dodecylsulfate, and of 15 000 upon gel filtration, indicating a non-globular shape. This molecule is compased of two subunits of similar molecular size which are held together by a disulfide bridge among other forces.     

Transrectal Doppler sonography for evaluation of uterine blood flow throughout pregnancy in 13 cows

Once weekly from 30 to 270 days of gestation in 13 cows, Doppler ultrasound scanning (triplex Doppler system) was done to assess blood-flow parameters of both median uterine arteries. Resistance, velocity and volume indices were measured. Resistance index values were negatively correlated to all other blood-flow parameters (P<0.05), but there were positive correlations between velocity and volume indices (P<0.05). Resistance indices were lower, and velocity and volume indices were significantly higher in the median uterine artery ipsilateral versus contralateral to the fetus. Resistance indices decreased continuously during the first 36 weeks of pregnancy. Velocity values rose three-fold, whereas the area increased 20-fold and the volume increased 17-fold by the end of gestation (P<0.05). Birth weight of calves was positively correlated with blood-flow volume (r=0.34) but negatively correlated with the resistance index (r=-0.45). There were no significant differences between male versus female calves (at any stage of gestation) in the resistance, time-average maximum velocity, and volume indices (P>0.05). In conclusion, arterial blood flow was monitored with transrectal Doppler sonography in both median uterine arteries weekly throughout pregnancy in cattle; this could be very valuable for monitoring pregnancies at high risk for abnormalities of the placenta, fetus or both, e.g. cloned calves.