Showing papers by "Fuller W. Bazer published in 1982"

Establishment of pregnancy in the pig: I. Interrelationships between preimplantation development of the pig blastocyst and uterine endometrial secretions.

Abstract: Pregnancy recognition in pigs occurs between Days 11 and 12 when blastocysts undergo transformation from the spherical to filainentous form. This study evaluated the relationship between blastocyst development and total calcium (Ca), estrone (E, ), estradiol (E2), estriol (E3), E1 sulfate (E1S), E2S, E3S, prostaglandin F (PGF), PGE2, protein (Pr) and acid phosphatase (AP) activity in uterine flushings obtained from pregnant gilts between Days 10 and 14. Data from pregnant gilts were compared to those obtained from uterine flushings collected between Days 10 and 14 of the estrous cycle. Surface and ultrastructural changes in the endometrium associated with blastocyst development were evaluated by scanning (SEM) and transmission (TEM) electron microscopy. Pregnant uterine flushings were analyzed relative to average size of blastocysts recovered: 5 mm spherical, 5-8 mm spherical, 9-50 mm tubular, and >50 mm filamentous Day 12 and Day 14. Nonpregnant gilt uterine flushings were analyzed by Day of the estrous cycle (10.5, 11, 11.5, 12 and 14). Total E2 content increased almost 4-fold in pregnant uterine flushings containing tubular blastocysts (2.3 ng) as compared with flushings with spherical blastocysts (0.6 ng) and continued to increase as the blastocysts became filamentous (4.4 ng) but had declined by Day 14 (0.4 ng). The pattern of change for total recoverable E, and E3 was similar to that for E2 and the highest values were obtained in uterine flushings with filamentous blastocysts. A concomitant increase in E, S and E2 S content also was detected in flushings with tubular and Day 12 filamentous blastocysts. Total Ca, Pr, AP, PGF, and PGE2 content increased in association with increased E2 in flushings. The increase in Pr, AP, PGF and PGE2 content in pregnant flushings continued to Day 14. However, Ca content had declined by Day 14 (0.1 mg) after a transient increase to 1.5 mg in flushings that contained tubular blastocysts (Days 11 and 12). Comparable changes in estrogens, proteins, Ca, PGF and PGE2 content in nonpregnant uterine flushings collected between Days 10.5 and 14 were not detected. Electrophoresis of protein in pregnant uterine flushings indicated that the appearance of three basic uterine proteins (Mr 32K to 60K) were associated with the increase in estrogen. These proteins were detected in flushings with tubular and filamentous blastocysts (Day 12), but were not found until Day 14 in nonpregnant gilts. A synchronized release of secretory vesicles from the glandular epithelium was observed by TEM which indicated a close association between formation of tubular blastocysts, onset of blastocyst estrogen production and increased protein in uterine flushings. Although secretion was detected in nonpregnant glandular epithelium, no synchronized release was observed. Results suggest that estrogen production by tubular and early filamentous blastocysts (Day 11.5-12.0) stimulates secretion of protein from the endometrium which may be mediated through an effect of free Ca on the uterine glandular epithelium. Increases in PGF and PGE2 were associated closely with estrogen production and blastocyst elongation.

Purification and Properties of a Major, Low Molecular Weight Protein Released by the Trophoblast of Sheep Blastocysts at Day 13-21

Abstract: Sheep blastocysts (Day 13-21) incubated in a modified Minimum Essential Medium released proteins into the medium at an approximately linear rate over a 24-h period. Single Day-16 blastocysts converted 2-8% of the radioactivity supplied (100 muCi L-[3H]leucine) into non-dialysable macromolecules which were released into the medium. Two-dimensional polyacrylamide gel electrophoresis revealed that at Day 13 there was only one major product (Protein X), consisting of three closely similar isoelectric species of (pI of denatured polypeptides about 5.5), each with molecular weights of 17 000. Between Days 14 and 21 additional proteins were detected. One of these was of high molecular weight (greater than 660 000) and did not appear on the two-dimensional gels, but Protein X continued to predominate until Day 23 when it could not be detected. Explants of chorion from Day 30 of pregnancy failed to secrete Protein X. Protein X was released in significant quantities (50-100 micrograms per 24 h) by the trophoblast but not the yolk sac of Day-14 and Day-16 conceptuses, but was present in very low amounts in the tissues. Protein X from the incubation medium of Day-14 and Day-16 conceptuses was purified by successive DEAE ion exchange and Sephacryl S-200 gel chromatography. Because Protein X and some of the other proteins are produced transiently between Days 13 and 21, it is possible that they may play a role in maternal recognition of pregnancy in sheep.

Establishment of pregnancy in the pig: II. Cellular remodeling of the porcine blastocyst during elongation on day 12 of pregnancy.

Abstract: The morphology of pig blastocysts changes dramatically just preceding initial attachment of the trophoblast to the uterine epitheliuin. Blastocysts undergo a rapid transition from spherical to tubular and elongated filamentous forms between Days 10 to 12 of pregnancy. The present study investigated rates of blastocyst development and possible mechanism(s) involved with changes in blastocyst morphology. Estimates of blastocyst growth were determined by removing the uterine horns from each animal at different intervals. Estimates of blastocyst growth were 0.25 mm/h for development of spherical blastocysts from 4 to 9 mm in diameter; however, blastocysts elongated at a rate of 30 to 45 mm/h after reaching a diameter of 10 mm. Although elongation from 10 to 150 mm occurred within a few hours, an increase in cellular hyperpiasia (mitosis) was not detected.

Metabolism of arachidonic acid in vitro by bovine blastocysts and endometrium.

Abstract: Metabolism of arachidonic acid and prostaglandin F2 alpha by bovine blastocysts and endometrial slices recovered on Days 16 and 19 postmating was studied in vitro. In Experiment 1, arachidonic acid (10 microCi tritiated and 200 micrograms radioinert) was added to blastocysts and endometrial slices prior to incubation for 24 h. [3H]arachidonic acid ([3H]AA) and metabolites in extracts of culture medium and tissue homogenates were separated on columns of Sephadex LH-20. Elution profiles of [3H]AA and metabolites in extracts of culture medium revealed that 13, 14-dihydro-15-keto-PGF2 alpha (PGFM), Pge2, PGF2 alpha, and at least four unidentified compounds were produced by Day 16 and Day 19 blastocysts. Endometrial slices from both days of pregnancy produced 3H-prostaglandins. Experiment 2 was conducted to quantify PGE2, PGF2 alpha and PGFM in aliquots of culture medium from Day 16 and Day 19 blastocyst and endometrial incubates. These tissues were incubated with 200 micrograms of radioinert arachidonic acid. Day 16 blastocysts produced less (microgram/blastocyst; P less than 0.01) of each prostaglandin than Day 19 blastocysts (PGE2, 0.7 +/- 0.4 vs. 4.2 +/- 1.0; PGF2 alpha, 2.1 +/- 0.7 vs. 22.8 +/- 4.1; PGFM, 0.03 +/- 0.01 vs. 0.5 +/- 0.2). Endometrial slices produced PGE2, PGF2 alpha and PGFM, but quantities were not affected by day postmating or uterine horncorpus luteum relationships. The third experiment was conducted to determine directly if Day 19 blastocysts and endometrial slices metabolized [3H]PGF2 alpha to [3H]PGFM. Blastocysts and endometrial slices produced [3H]PGFM. Endometrial slices metabolized 34.3 +/- 1.5% of the [3H]PGF2 alpha to [3H]PGFM, while blastocysts metabolized 7.5 +/- 1.6% of the [3H]PGF2 alpha to [3H]PGFM. Results of this study indicate that bovine blastocysts and endometrial slices can metabolize [3H]AA in vitro. It is postulated that prostaglandins of blastocyst and endometrial orgin have a role in maintenance of early pregnancy in cattle.

Nucleic acid, metabolic and histological changes in gilt mammary tissue during pregnancy and lactogenesis.

Abstract: Changes in mammary gland histology, dry weights, nucleic acids and in vitro rates of substrate oxidation in incorporation into lipid were measured in mammary biopsies of three gilts each on d 30, 45, 60, 75, 90, 105 and 112 of pregnancy, and d 1 and 4 of lactation. Histological changes noted were progressive duct growth early in pregnancy followed by rapid lobulo-alveolar development between d 75 and 90 to complete mammogenesis. Colostrum and lipid were evident by d 105 with marked distension of alveolar lumina on d 112. Complete differentiation of the secretory process was apparent on the day of parturition. Concentrtion of dry, fat-free tissue (DFFT) and DNA changed little before d 60 but increased fourfold between d 75 and 90. No further increases in DFFT or DNA were noted. RNA concentrations paralleled DNA through d 90, after which they steadily increased. Rates of acetate and glucose oxidation increased transiently during midpregnancy then declined and remained low until initiation of lactogenesis. Substrate incorporation into lipid increased slightly at midpregnancy and again at d 105, after which it increased markedly. Collectively, results indicate that mammogenesis is complete by d 90, after which lactogenesis is initiated in a two-stage process. Metabolic rates expressed on a DNA basis indicated considerable rates of oxidation, but not of lipogenesis by proliferating mammary tissue. Preferential metabolism of acetate vs glucose near parturition suggests coordination of metabolism between the mammary gland and other maternal tissues.

Placental transport and distribution of uteroferrin in the fetal pig.

Abstract: Placental transport of uteroferrin (Uf), the progesterone-induced iron transport glycoprotein, and its distribution within the fetus were investigated by the peroxidase-antiperoxidase bridge (PAP) technique. In Experiment 1, Uf was localized in endometrial and placental tissues taken from gilts on Days 60, 75, 90 and 105 of pregnancy. Uteroferrin was observed within cells of the endometrial glands and surface epithelium adjacent to placental areolae but not in endometrial surface epithelium between areolae. Heavy staining for Uf was observed in cells of the areolae and was associated with both supra- and infranuclear cytoplasmic vesicles. Vesicles located within the infranuclear cytoplasm were occasionally observed to be releasing their contents into capillaries surrounding the areolae. In Experiment 2, Uf was measured by radioimmunoassay in blood samples taken from the umbilical vein and artery of fetuses on Day 75 of pregnancy. Uteroferrin concentrations were greater (P less than 0.07) in umbilical vein blood (79.8 +/- 13.1 ng/ml) than in umbilical artery blood (43.9 +/- 13.1 ng/ml). Uteroferrin binding by Day 75 fetal liver membranes was examined in Experiment 3. Binding of 125I-Uf increased linearly with increasing quantities of membrane protein and binding of 125I-Uf was competitively inhibited by adding unlabeled Uf to the assay. In Experiment 4, urine samples were taken from Day 75 fetuses and assayed for beta-mercaptoethanol activated acid phosphatase activity which is indicative of Uf. In addition, urine proteins were analyzed for Uf by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Ouchterlony double immunodiffusion (ID). Acid phosphatase specific activity was 3.37 +/- 1.83 mumol substrate hydrolyzed/10 min per mg protein. Uteroferrin was detected in two of three urine samples by 2D-PAGE and in three of eight samples by ID. In Experiment 5, tissue distribution of Uf was determined by the PAP technique in liver and kidney tissue taken from fetuses on Day 75 of pregnancy. Staining for Uf was observed in collecting ducts and proximal tubules of kidney tissue, but staining was not observed in liver tissue. These results indicate that Uf is transported by the areolae into the chorioallantoic capillaries and to the fetus by the umbilical vein. Within the fetus Uf is either bound by the liver, probably to supply iron for hematopoiesis, or cleared by the kidney and transported within the urine to the allantoic sac to serve as a temporary iron storage reservoir.

Identification of stage-specific and hormonally induced polypeptides in the uterine protein secretions of the mare during the oestrous cycle and pregnancy

Abstract: Uterine secretions were obtained on Days 4, 8, 12, 14, 16, 18 and 20 of the oestrous cycle and early pregnancy. Acid phosphatase activity was significantly affected by day of the cycle, reaching a maximum at days 12-14 during the luteal phase and then declining to almost undetectable levels, by Day 20. In pregnant animals, activity continued to increase beyond Day 14. Two-dimensional polyacrylamide gel electrophoresis showed that albumin was a major component. However, a number of unique proteins of non-serum origin appeared in mid-cycle but had disappeared by Day 20. One of these was a basic protein indistinguishable in electrophoretic properties from the uterine acid phosphatase of the pig, uteroferrin, which is believed to be involved in iron transport from the uterine endometrial epithelium to the conceptus. These same polypeptides, including the putative uteroferrin, were also present in uterine flushings from pregnant animals until Day 20, and in flushings from ovariectomized mares treated with progesterone but not in those given only oestradiol-17 beta. Flushings from all ovariectomized animals contained a non-serum, acidic polypeptide (pI 5.3) of molecular weight 70 000. One basic polypeptide (molecular weight approximately 17 000) appeared by Day 4 of the oestrous cycle and disappeared by Day 16 but was maintained during pregnancy until Day 20. It was absent, however, in flushings from a Day 45 pseudopregnant mare. Like the sow, therefore, the mare possesses a number of proteins associated with cyclic changes in steroid hormones during the oestrous cycle and early pregnancy.

Role of uteroferrin in iron transport and macromolecular uptake by allantoic epithelium of the porcine conceptus.

Abstract: Uteroferrin (UF) is an iron-containing, progesterone-induced glycoprotein present in allantoic fluid and uterine secretions of swine between Days 30 and 105 of gestation. The role of UF in maternal-to-fetal iron transport and uptake of macromolecules by allantoicepithelium were studied in 2 experiments. In Experiment 1, 8 pregnant guts were assigned to treatment on either Day 30, 60, 90 or 105 of gestation. Three additional gifts were rendered unilaterally pregnant and assigned to treatment on Day 60. All gifts received 100 MCi "Fe injected intravenously. Twenty-four h after the "Fe treatment, guts were hysterectomized and fetal fluids and tissues collected. Uterine flushings were also collected from the nongravid uterine horn of unilaterally pregnant gifts. Fetal bone, spleen, liver, kidney and placenta accumulatedFe, but fetal spleen was the only tissue in which total " Fe accumulation was affected (P<0.05) by day of gestation. Radiolabeled UF was alsoisolated from uterine flushings (14 cpm/mg protein) and from concentrated allantotic fluid by carboxy- methyl cellulose (CMC) ion exchange chromatography. In Experiment 2, macromolecular uptake by the allantoic epithelium, both in vitro and in vivo, was examined. Sections of ailantois, chorion, amnion and fetal gut (FG) were collected from Day 60 pregnant gilts. Samples of each of the tissues were incubated in minimal essential medium (MEM) containing 1 of the following proteins labeled with fluorescein isothiocyanate (FITC): 1) FITC-'y-globuiin; 2) FITC-uteroferrin; 3) FITC-transferrin or 4) FITC-�y-globulin with iOM Na-arsenite. Uptake of these proteins by chorion and allantois, but not amnion was observed. The Na-arsenite inhibited protein uptake. When FITC-'y-globulin was introduced into the allantoic fluid on Day 60 of pregnancy, uptake by the allantois was observed. Results of this study support the concept that uteroferrin (UF) plays a major role in iron transport to the conceptus. In addition, data indicate that the allantoic epithelium is capable of transporting proteins normally found in allantoic fluid, i.e., uteroferrin and transferrin, as well as a protein not normally found in either fetal serum or allantoic fluid, i.e., 7-globulin. Failure to detect uptake of these proteins when Na-arsenite was added to the incubation medium suggests that transport of these proteins is by an active process.

High molecular weight glycoproteins released by expanding, pre-attachment sheep, pig and cow blastocysts in culture

Abstract: Summary. Blastocysts isolated from sheep (Day 14\p=n-\16),pigs (Day 16) and cows (Day 19) during the pre-attachment elongation phase were cultured for up to 30 h in a modified MEM medium in the presence of radioactive amino acids (L-[14C]leucine or L-[35S]methionine) to label protein and D-[3H]glucosamine to label complex saccharides. All the blastocysts released considerable quantities of non-dialysable radioactive material into the medium at an approximately linear rate over the course of the incubation. Ion-exchange chromatography on DEAE cellulose at pH 8\m=.\2revealed that the major glucosamine-labelled product in the medium was a non-sulphated glycoprotein which eluted early in the salt gradient. None of the blastocysts produced any detectable glycosaminoglycan-like materials such as hyaluronic acid. The glycoprotein was purified by ion-exchange and gel filtration chromatography and had a molecular weight of > 660 000. Up to 100 \g=m\gof this material could be isolated from incubations of 2 sheep conceptuses. It was relatively resistant to protease hydrolysis and consisted of approximately 50% carbohydrate and 50% protein. The main monosaccharide constituents, as revealed by gas\p=n-\liquidchromatography, were galactose and N-acetylglucosamine plus some mannose and fucose. No sialic acid was present. The linkages between the carbohydrate chains and the peptide appeared to be resistant to alkaline borohydride cleavage and were probably, therefore, N-glycosidic.

Comparison of glucose, fructose, ascorbic acid and glucosephosphate isomerase enzymatic activity in uterine flushings from nonpregnant and pregnant gilts and pony mares.

Abstract: In Experiment 1, 40 gilts and 30 pony mares were used to characterize changes in glucose, fructose, ascorbic acid and glucosephosphate isomerase (GPI) enzymatic activity in uterine flushings collected either during the estrous cycle or early pregnancy. Total recoverable glucose was greater (P less than 0.01) in uterine flushings from pregnant gilts, but pregnancy status had no effect on total recoverable glucose in pony mare uterine flushings. Fructose was undetectable in uterine flushings from nonpregnant gilts and pony mares and pregnant gilts and pony mares prior to Day 14, but occurred in increasing amounts between Days 14 and 18 or 20 of pregnancy. In Experiment 2, it was demonstrated that the porcine conceptus is the primary source, if not the sole source of fructose. Total recoverable ascorbic acid in uterine flushings was not affected by pregnancy in gilts, but was greater (P less than 0.01) in pregnant versus nonpregnant pony mares. In both species, total recoverable ascorbic acid was affected (P less than 0.01) by day of the estrous cycle and pregnancy. The GPI enzyme allows for the interconversion of glucose-6-PO4 and fructose-6-PO4. GPI total and specific activities were greater (P less than 0.01) for pregnant than nonpregnant gilts and pony mares. The periods of greatest GPI activity were temporally associated with elevated estrogens of either ovarian or blastocyst origin. Results from Experiment 3 indicated a marked increase (P less than 0.01) in GPI activity in uterine flushings from gilts treated with estradiol valerate. Results of this study indicate that glucose (gilt only), fructose, ascorbic acid and GPI activity are increased in uterine flushings of gilts and pony mares during early pregnancy. The increase in these constituents may reflect increased carbohydrate metabolism in ways which are uniquely beneficial to conceptus development in ungulates.