An immunological cryo-ultrastructural study of a sequential appearance of proteins in placental binucleate cells in early pregnancy in the cow

Using the most sensitive immunocytochemical method available, on ultrathin frozen sections, the results in this paper demonstrate that bovine placental lactogen (bPL) is present in the earliest fetal binucleate cells found at 21 days post coitum in the trophectoderm. A second protein, the SBU-3 antigen, which is absent in the early stages of pregnancy appears abruptly in the binucleate cell granules at 30 days post coitum coincident with the start of villus development. Subsequently, the granules contain both bPL and the SBU-3 antigen. This sequential production of unlike proteins indicates that the binucleate cell has different functions depending on the stage of pregnancy and has important roles to play both at implantation and in villus development.     

Biological activity of bovine placental lactogen in 3T3-F442A preadipocytes is mediated through a somatogenic receptor.

Bovine placental lactogen (bPL) exhibited antimitogenic differentiation-promoting biological activity in 3T3-F442A preadipocytes. Competitive binding studies and affinity labelling revealed bPL activity to be mediated through a somatogenic type of receptor that recognizes human growth hormone (hGH) and bovine GH, but not ovine prolactin or human PL. The bioactivity of bPL was sixfold lower than that of hGH despite that bPL is binding to the somatogenic receptors with fivefold higher affinity. This discrepancy may result from the relatively low ability of bPL to induce post-receptoral effects such as receptor dimerization.     

Characterization of bovine placental lactogen as a glycoprotein with N-linked and O-linked carbohydrate side chains

In a previous report we showed that purified bovine placental lactogen (bPL) exists in two isoforms in the 31,000-33,000 Mr range, each with at least five isoelectric variants differing in approximately 2 orders of magnitude in isoelectric points (pI) 4-6. The multiple isoelectric variants are unique to the bovine hormone. In an effort to determine the nature of these variants endo- and exoglycohydrolase digestions were conducted to determine if this hormone was glycosylated. Analysis of peptide/N-glycosidase F and endoglycosidase F digests of radioiodinated bPL on one-dimensional gel electrophoresis showed a Mr decrease from 31,000 to 24,000 and 33,000 to 26,000 for the two isoforms. Digestion with a mixture of neuraminidase plus mixed exoglycosidases resulted in a Mr decrease of 4,000. Digestion with neuraminidase resulted in a Mr decrease of 2,000. Further analysis of peptide/N-glycosidase F- and neuraminidase-treated bPL by two-dimensional gel electrophoresis showed the isoelectric variants shifted from pI 4.4-6.3 to 4.9-8.0. The sialic acid residues on the N-linkage are responsible for the pronounced acidic character of bPL, but do not account for the residual charge heterogeneity as the different isoelectric variants persist after sialic acid removal. The apparent Mr of the protein after removal of N-linked carbohydrate residues is similar to that of PRL and GH. These enzymatic digestion results demonstrate the presence of N-linked complex oligosaccharide residues attached to the beta-amide group of an asparagine residue. Analyses of the sugar content of the molecule were consistent with the presence of one biantennary N-linked and two O-linked carbohydrate chains.     

Altered secretion of pregnancy-associated glycoproteins during gestation in bovine somatic clones

Somatic nuclear transfer (NT) in cattle is often accompanied by severe placental anomalies, hypertrophy, and hydrallantois, which induce a high rate of pregnancy losses throughout gestation. These placental deficits are associated with an abnormal increase of the maternal plasma levels of pregnancy-associated glycoprotein (PAG), produced by the trophoblastic binucleate cells (BNC) of the placenta. The objective of this study was to analyze the origin of the abnormally elevated PAG concentrations in the peripheral circulation of NT recipients during pathological pregnancies. Concentrations of PAG were measured both in maternal blood, in chorionic and cotyledonary tissular extracts from control recipients (after artificial insemination, AI, or in vitro fertilization, IVF) and clone recipients on Day 32, Day 62, and during the third trimester of gestation. Three different radioimmunoassay (RIA) systems were used. One homologous RIA for PSP60, similar to bovine PAG-1 (PAG_67kDa), and two heterologous RIA with PAG_67kDa as standard and tracer, and antisera anti-caprine PAG (AS#706 and AS#708). Circulating and tissular concentrations of bovine placental lactogen (bPL), a glycoprotein also produced by BNC, were determined by RIA at the same stages. The number of BNC in the placental tissues was determined by cell counting after immunostaining with anti PSP60 antibody on tissue sections from control and NT pregnancies. Maternal plasma PAG concentrations were not different among groups on Day 32, but they were significantly higher in NT than in control pregnancies on Day 62 with all three RIA and during the third trimester with two RIA (RIA-PSP60 and RIA with AS#708). Circulating bPL concentrations were undetectable on Days 32 and 62 and were not different in the third trimester between NT and control pregnancies. Tissular amounts of PAG on total proteins were not different between the two groups at all stages studied. No difference was determined in the percentage of PSP60-positive BNC in placental tissues between controls and NT on Day 62 and during the third trimester of pregnancy. Western blots of tissular extracts from placenta showed no major molecular weight changes of PAG in NT pregnancies compared to controls. No differences in maternal circulation concentrations or tissular content of bPL were observed between control and NT pregnancies. In conclusion, the specific increase of PAG in maternal plasma concentrations during abnormal NT pregnancies do not result from a higher proportion of BNC, or an increased protein expression of PAG and could be due to changes in the composition of terminal glycosylation which result into a clearance decrease of PAG from the circulation.     

Preparation, purification, and determination of the biological activities of 12 N terminus-truncated recombinant analogues of bovine placental lactogen.

Removal of 13 to 15 amino acids from the N terminus of bovine placental lactogen (bPL), which according to the three-dimensional structure of pGH corresponds to a nonhelical part of bPL, did not effect its secondary structure or change the monomer content of the protein preparation. However, it remarkably decreased the binding of the prolactin (PRL) type of receptors on Nb2 cells with subsequent reduction in bioactivity. The binding to the growth hormone (somatogen) receptors either did not change or was increased, resulting in an increase of somatogen receptor-mediated bioactivity. Further truncation (17-18 amino acids) resulted in a decrease of alpha-helical content and loss of binding properties and biological activity mediated through interaction of the analogues with both somatogen (3T3-F442A cells) and lactogen (PRL) receptors (NB2-11C cells). Truncation of 19-27 amino acids caused additional loss in activity, without further change in the secondary structure. Replacement of Leu28 by a more hydrophobic Phe has only minor, if any, effect on the bioactivity of bPL. Occasional point mutations due to polymerase chain reaction errors in several analogues did not seem to have any major effect on the hormone properties. It can thus be suggested that the N-terminal part of the nonhelical portion of bPL, which corresponds to the portion of the molecule that does not exist in growth hormones, is required for efficient binding to the lactogen (PRL) but not to the somatogen or unique bPL receptors. Removal of the N-terminal part of pBL changed the specificity of bPL by decreasing its PRL receptor-mediated activities and increasing its somatogen receptor-mediated activities.     

Ontogeny of somatomedin during development in the mouse. Serum concentrations, molecular forms, binding proteins, and tissue receptors

The purpose of this study was to assess the ontogeny of serum concentrations and molecular forms of somatomedin during fetal and postnatal development and to define the changes in serum binding proteins for somatomedin-C during various stages of development. The finding that fetal, placental, and decidual mouse tissues possess receptors for somatomedin suggests a role for somatomedin in fetal growth and possibly in the maintenance of pregnancy. Serum somatomedin-C was measured using a highly specific, heterologous radioimmunoassay (RIA) and a less specific membrane binding assay (MBA) which is more sensitive to the influence of somatomedins other than somatomedin-C. The assays were validated for mouse serum by showing that serum concentrations were reduced in genetically growth hormone-deficient mice and in hypophysectomized mice and were increased by growth hormone therapy. As in the human, the RIA measures only a portion of the somatomedin-C present in mouse serum. This “covering up” of somatomedin is attributed to the presence of serum binding proteins and is corrected by treatment of serum samples with acid. By both RIA and MBA, serum somatomedin concentrations are low in fetal and newborn mice, begin to rise in the fourth postnatal week, and reach adult values by 7 weeks of age. The chromatographic pattern of adult mouse serum on Sephacryl 200 is similar to that observed with human sera: The immunoreactive material elutes at apparent molecular weights of 140,000 and 30,000–40,000. The elution profile of ¹²⁵I-labeled somatomedin-C bound to components of serum is nearly identical to the pattern of endogenous activity. As with human serum, somatomedin-C in acidified mouse serum elutes at a lower molecular weight, coincident with insulin and purified somatomedin-C. Maternal serum somatomedin declines in the last half of gestation at the time when placental lactogen levels rise. Along with the absolute decline in somatomedin content is the appearance of unsaturated sites on somatomedin binding proteins. These findings are unexpected and unexplained since somatomedin rises late in pregnancy in humans and several lines of evidence suggest that placental lactogen has the capacity to stimulate somatomedin production. We previously have presented evidence that explants of multiple fetal mouse tissues synthesize somatomedin-C. The present study shows that the immunoreactive somatomedin-C in fetal mouse serum shares identical characteristics with those reported previously for media obtained from mouse liver explants. It seems possible that somatomedin's actions are exerted primarily at or near its site of production and that circulatory levels do not reflect the importance of somatomedin-C on fetal growth. While elucidation of the dramatic developmental changes in serum content and molecular forms of somatomedin-C and in somatomedin binding proteins may be essential to clarifying the role of somatomedin on fetal growth, proof that somatomedin stimulates fetal growth will depend in large part on studies of its biological actions on fetal tissues.     

Gestational profiles of rat placental lactogen-II (rPL-II) and growth hormone (GH) in maternal and fetal serum, amniotic fluid, and placental tissue.

Rat placental lactogen-II (rPL-II) and growth hormone (rGH) in maternal and fetal serum, amniotic fluid, and placental tissue were measured by a homologous radioimmunoassay during the last half of pregnancy. rPL-II appeared first in maternal circulation and the placental tissue on day 11 of pregnancy. The maternal serum rPL-II concentration increased progressively and reached the peak value (684 +/- 76 ng/ml) on day 19, and declined thereafter up to term. rPL-II content in the tissue had a similar pattern to the maternal serum profile of rPL-II, while its concentration in the tissue increased dramatically on day 12 and remained high until day 19. Fetal serum rPL-II was detected on days 17 and 18, though its concentration was much lower (ranged between 3-10 ng/ml) than that of maternal serum. rPL-II in amniotic fluid was also detectable only on days 12-14 of pregnancy, and the peak value on day 13 was 22% of the maternal serum rPL-II concentration. The rGH concentration increased gradually as pregnancy advanced with a decline on the day before parturition. Although rGH in fetal serum increased on day 20 with a decline on the following day, it was slightly detectable in amniotic fluid on the last two days of pregnancy. The molecular profile of rPL-II in amniotic fluid and maternal serum of day 13 pregnant rats were examined by Western blotting. Anti-rPL-II serum detected two proteins with molecular weights (mol wt) of 19.5K and 20.5K in amniotic fluid and one protein with a mol wt of 20.5K in maternal serum under nonreducing conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and binding properties of human fetal lung fatty acid-binding proteins

When delipidated Mr>10,000 cut-off human fetal lung cytosol was separated on gel filtration and ion-exchange chromatography on Auto-FPLC system, two fatty acid-binding proteins (FABPs) of pI 6.9 and pI 5.4 were purified to homogeneity. On Western blotting analysis with the anti-human fetal lung pI 6.9 FABP, these two proteins showed immunochemical cross reactivity with each other and with purified hepatic FABPs but not with cardiac or gut FABP. These two FABPs have identical molecular mass of 15.2 kDa, which is slightly higher than that of the hepatic proteins (14.2 kDa). Carbohydrate covalently linked to FABPs, that may substantially add to the molecular mass, was not detected in the purified protein preparations. Amino acid analysis revealed that both the proteins have same amino acid composition each containing one Trp residue that is lacking in hepatic FABP. Different isoforms of lung FABP exhibited different binding ability for their natural ligands. These proteins bind palmitoyl CoA with higher affinity than oleic acid. pI 6.9 FABP can more rapidly and efficiently transfer fatty acid than can pI 5.4 FABP from unilammelar liposomes. Thus these FABPs may play a critical role in fatty acid transport during human fetal lung development.Abbreviations AO anthroyloxy - 12-AS 12-(9-anthroyloxy)stearic acid - FABP fatty acid-binding protein - NBD-PE [N-(4-nitrobenzo-2-oxa-1,3-diazole)phosphatidylethanolamine - Pal-CoA palmitoyl coenzyme A - PITC phenylisothiocyanate - PBS phosphate-buffered saline - PtdCho phosphatidylcholine - SUV small unilamellar vesicle - Tris tris(hydroxymethyl) amino methane     

The regulation of ovine placental lactogen the role of the fetal hypothalamic-pituitary axis

The possible r?le of the fetal hypothalamic-pituitary axis in regulating the secretion of ovine placental lactogen (oPL) was investigated in chronically-catheterised ewes and fetuses in late pregnancy. Intravascular administration of agents to fetuses that significantly increased fetal prolactin concentrations (chlorpromazine 6.25 mg;thyrotrophin releasing hormone, 10 micrograms), significantly reduced fetal prolactin concentrations (bromocriptine, 0.033 mg/h), or significantly reduced fetal growth hormone (GH) concentrations (somatostatin, 2.5 micrograms/min), had no effect on maternal or fetal oPL concentrations. Mean fetal levels of prolactin or GH in late gestation could not be correlated with oPL concentrations, although fetal hypophysectomy prevented the normal prepartum fall in oPL concentrations.     

Effect of exogenous circulating anti-bPL antibodies on bovine placental lactogen measurements in foetal samples

Background  

The involvement of placental lactogen (PL) in the regulation of foetal growth has been investigated in different species by in vivo immunomodulation techniques. However, when circulating antibodies are present together with the hormone, the procedure for hormonal measurement becomes considerably complex. The aim of this study was the immunoneutralization of bovine placental lactogen (bPL) concentrations in bovine foetal circulation by direct infusion of rabbit anti-bPL purified immunoglobulins (IgG) via a foetal catheter (in vivo study). The ability of a RIA based on guinea pig anti-bPL antiserum, for the measurement of bPL concentrations in samples containing exogenous rabbit anti-bPL immunoglobulins, was also analyzed in in vitro and in vivo conditions.     

Study of the fetal and maternal renin-angiotensin system and chorio-placental steroids in the guinea pig]

1.) Total renin, active renin, prorenin, angiotensin II, estradiol and progesterone were measured in maternal, placental and fetal blood and in trophoblastic and uterine tissues of the guinea pig. Furthermore, membrane angiotensin II receptors were measured in trophoblastic tissues. 2.) Blood and tissue concentrations of total renin, active renin, angiotensin II and steroids are shown to increase with gestational age. At the full term of pregnancy (70th post-coital day), tissue concentrations of total renin in chorion (23,900 +/- 2,752 ng/g of tissue/h), maternal placenta (14,210 +/- 1,131), fetal placenta (12,475 +/- 927) and uterus (7,677 +/- 798) are 100 time higher than those observed in placental, fetal and maternal blood. Distribution of blood and tissue prorenin (inactive renin) is similar to that found for total renin. Active renin/Total renin ratio reaches 1% in uterine, placental and chorion tissues and 9.3 +/- 1.0% in maternal, placental and fetal blood. 3.) Angiotensin II levels in systemic maternal blood (690 +/- 99 pg/ml) and in uterine blood (467 +/- 84) are higher than those found in placental blood (266 +/- 39) and in different trophoblastic tissues (between 200 and 400 pg/g). Angiotensin II receptor concentrations are highest in chorion. 4.) Regarding the steroid hormones, it is noted that placental and maternal blood contain more progesterone than trophoblastic tissues. The highest concentrations of estradiol are found in chorion tissue and uterine blood. 5.) A positive correlation is observed between angiotensin II and estradiol in uterine blood (r = 0.69, P less than 0.01) and in chorion (r = 0.71, P less than 0.01). These findings indicate that angiotensin II and estradiol could, by their interactions, play an important role in the physiology of pregnancy.     

Human splenic galaptin: physicochemical characterization

Mammalian spleens were previously reported to contain beta-galactoside-binding lectins [Allen, H. J., Cywinski, M., Palmberg, R., & DiCioccio, R. (1987) Arch. Biochem. Biophys. 256, 523-533]. The aim of the present investigation was to determine the relationship of human splenic galaptin to other beta-galactoside-binding lectins identified in other human and animal tissues. Galaptin of subunit molecular mass 14.5 kDa was the only lectin of this type found in human spleen as assessed by SDS-PAGE, RP-HPLC, and Western blot analyses. Three polypeptides of pI 4.60, 4.80, and 4.85 were detected by isoelectric focusing of purified galaptin, with the major band having pI 4.85. UV spectral analysis indicated the absence of prosthetic groups and gave A1%(1cm), 280 = 5.5. Circular dichroic analysis suggested the presence of 40% beta structure, considerable random coil, and 10% alpha helix structure. The amino acid composition was very similar to that for human placental galaptin. Amino acid sequence analyses were carried out on V8 protease, CNBr, and iodosobenzoic acid digestion fragments. A total of 94 residues were identified. All sequences determined could be aligned with placental galaptin sequences. We conclude that human splenic galaptin is identical with human placental galaptin. A related polypeptide of molecular mass approximately 14.5 kDa was found to be present in several different mammalian spleens as assessed by Western blot analysis using a monospecific polyclonal anti-human splenic galaptin antiserum.     

Late but not early gestational maternal growth hormone treatment increases fetal adiposity in overnourished adolescent sheep

In the overnourished adolescent sheep, maternal tissue synthesis is promoted at the expense of placental growth and leads to a major decrease in lamb birth weight at term. Maternal growth hormone (GH) concentrations are attenuated in these pregnancies, and it was recently demonstrated that exogenous GH administration throughout the period of placental proliferation stimulates uteroplacental and fetal development by Day 81 of gestation. The present study aimed to determine whether these effects persist to term and to establish whether GH affects fetal growth and body composition by increasing placental size or by altering maternal metabolism. Adolescent recipient ewes were implanted with singleton embryos on Day 4 postestrus. Three groups of ewes offered a high dietary intake were injected twice daily with recombinant bovine GH from Days 35 to 65 of gestation (high intake plus early GH) or from Days 95 to 125 of gestation (high intake plus late GH) or remained untreated (high intake only). A fourth moderate-intake group acted as optimally nourished controls. Pregnancies were terminated at Day 130 of gestation (6 per group) or were allowed to progress to term (8-10 per group). GH administration elevated maternal plasma concentrations of GH, insulin, glucose, and nonesterified fatty acids during the defined treatment windows, while urea concentrations were decreased. At Day 130, GH treatment had reduced the maternal adiposity score, percentage of fat in the carcass, and internal fat depots and leptin concentrations, predominantly in the high-intake plus late GH group. Placental weight was lower in high-intake vs. control dams but independent of GH treatment. In contrast, fetal weight was elevated by late GH treatment, and these fetuses had higher relative carcass fat content, perirenal fat mass, and liver glycogen concentrations than all other groups. Expression of leptin mRNA in fetal perirenal fat and fetal plasma leptin concentrations were not significantly altered by maternal nutritional intake or GH. In pregnancies proceeding to term, the duration of gestation, fetal placental mass, and lamb birth weight were reduced in high-intake compared with control dams but were not significantly affected by GH treatment. In conclusion, exogenous GH has profound effects on maternal endocrinology, metabolism, and body composition when administered during early and late pregnancy. Treatment during late pregnancy has a modest effect on fetal growth independent of placental size and a profound effect on fetal adiposity, which may have implications beyond the fetal period.     

Ontogeny of neurotensin in the fetal sheep.

Neurotensin (NT) is a regulatory peptide involved in the control of gastrointestinal function. We have used the chronically cannulated ovine fetus to examine the ontogeny of circulating NT-like immunoreactivity (NTLI) in the fetus and neonatal lamb. In addition the placental transfer and clearance of NT has been determined. NTLI in the ovine fetus circulates at adult concentrations during the third trimester of pregnancy and is of fetal origin. NTLI is present in the fetal ileum, the richest source of NT, at adult concentrations and in the same molecular profile as in the adult. There is a transient increase in circulating NTLI at birth, and a small NT response to feeding in the lamb. While fetal concentrations of plasma NTLI are generally the same as in the adult and originate from the fetus, the fetus clears infused NT(1-13) twice as rapidly as the nonpregnant adult indicating a higher fetal production of NT. Thus it appears that the mechanisms involved in the production and processing of NT are mature some weeks before birth.     

Bovine placentomes contain factors which decrease progesterone secretion

We examined the effects of 20% ammonium sulfate precipitates from cytosolic extracts of whole placental tissue collected between 100-150 days of gestation on progesterone secretion by bovine granulosa cells and dispersed bovine luteal cells. These extracts produced a dose-dependent inhibition (23-92%) of progesterone synthesis by bovine granulosa cells. However, no inhibitory activity could be demonstrated in similarly prepared extracts from term placentae. Inhibitory activity could be extracted from both maternal caruncles and fetal cotyledons. In the presence of 2 mg/ml of maternal caruncle extract, basal progesterone secretion was dramatically reduced (90%), as was steroidogenesis in the presence of bovine lutenizing hormone (bLH) and 8 bromocyclic (Br)-cAMP. Moreover, coincubation of dispersed luteal cells and dispersed fetal or maternal placental cells from 100- to 150-day placentae produced a significant (50%) reduction in progesterone content of the medium. The addition of 2 mg/ml of caruncle or fetal cotyledon extract from 100- to 150-day placentae also produced 100% and 50% inhibitions, respectively, of progesterone secretion by dispersed placental cells. Thus, the inhibitory factor appears to be produced by cells of both the maternal and fetal placenta. It is heat-stable and not extractable by ether. The inhibitory substance eluted was two distinct peaks from Sephadex G-100 columns, one with a molecular weight of about 60,000 daltons and the other about 30,000 daltons. Using isoelectric focusing, several peaks of inhibitory activity were obtained, one with a pI of 3-5, the others having pIs between 6 and 9.(ABSTRACT TRUNCATED AT 250 WORDS)