The expression of gonadotropin-releasing hormone and its receptor in endometrial cancer, and its relevance as an autocrine growth factor.

The presence of a direct extra-pituitary action of gonadotropin-releasing hormone (GnRH) via specific receptors in endometrial cancer (EC) has been suggested as an explanation for the therapeutic effect of GnRH analogue (GnRHa) in recurrent disease. We have sought the expression of the GnRH peptide and functional GnRH receptor (GnRH-R) in human tissues and cell lines to investigate the possibility of an autocrine growth regulation mechanism. Using reverse transcription-PCR, differing GnRH mRNA transcripts were detected in two EC cell lines (Ishikawa and HEC-1A), a choriocarcinoma (JEG3) cell line, and tissues from endometrium and placenta. However, secretion of immunoreactive GnRH could be detected by RIA in only 1 of 10 EC tissues in primary culture, and in none of the cell lines. Low levels of GnRH-R mRNA expression were found in the same cells, which were only detectable by reverse transcription-PCR and Southern blotting of the PCR product. In radioligand binding assays using GnRHa goserelin, no pituitary-like, high-affinity GnRH binding sites could be found in either EC cell lines or tissues. Low affinity binding (Kd = 1.0 - 3.1 x 10(-7)M) was detected in three of eight (37%) EC tissues. Furthermore, receptor signal transduction measurements carried out in these cells showed no increases in either total inositol phosphate, cyclic AMP production, or cytosolic Ca2+ in response to either GnRH or GnRHa. Finally, no effect of either GnRH or GnRHa on the growth of EC cell lines was detected in vitro, under estrogen-free conditions, assessed by DNA content. Our data suggest that although there is a potential for autocrine activity for GnRH in EC as judged by the presence of mRNA for peptide and receptor, no functional receptor activity could be detected in vitro. Alternative mechanisms should be studied to explain the in vitro action of GnRHa.

De novo synthesis of placental protein-14 (PP14) and not PP12 by monolayer cultures of glandular epithelium of gestational endometrium.

Nine monolayer cell cultures of glandular epithelium from gestational endometrium were established from six apparently healthy women undergoing elective termination of pregnancy (7-11 weeks gestation). Radiolabel incorporation studies showed increasing incorporation of [35S]methionine into proteins present in supernatant and cytosol fractions over 48 h. The secreted proteins represented approximately 20% of the total incorporation of methionine into cytosolic proteins. De novo synthesis and secretion of placental protein-14 (PP14) and not PP12 was identified by a novel combination of line immunoelectrophoresis and autoradiography. All monolayer cultures demonstrated the presence of radiolabeled PP14, but not PP12, in the culture supernatants. These observations suggest that the glandular epithelial cells are the major site of synthesis and secretion of PP14 in human gestational endometrium.

Synthesis of placental protein 12 by decidua from early pregnancy.

The synthesis and secretion of placental protein 12 (PP12) by early pregnancy decidua and trophoblast were studied in vitro from tissues obtained by curettage during elective termination of pregnancy (weeks 8-14). The tissue explants were incubated in Ham's F-10 medium for a 27-h period, and the PP12 levels in media and tissue homogenates were measured by RIA. De novo synthesis of PP12 was assessed by measuring the incorporation of radioactivity into PP12 after 20 h of incubation of tissues with 20 microCi/ml [35S]methionine. PP12 from the culture medium was immunoprecipitated with anti-PP12(A) antiserum, and the immunoprecipitate was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The initial tissue content of PP12 was 10- to 72-fold higher in decidua than in trophoblast. The total amount of radioimmunoassayable PP12 released into medium by decidual explants during the 27-h incubation period together with that present in the tissues at the end of incubation exceeded the initial tissue content by 242.7 +/- 63.7% (mean +/- SE). Only small amounts of PP12 were detected in media from trophoblast cultures. During the first 7 h of incubation, inclusion of cycloheximide had no effect on PP12 release by decidual explants in three of four experiments. Between 7 and 27 h, the amount of PP12 released by cycloheximide-treated tissues was 20.0 +/- 7% of that released by control tissues (P less than 0.01). Cycloheximide had no effect on PP12 release by trophoblasts. Decidual explants incorporated [35S]methionine into PP12, but trophoblasts did not. In sodium dodecyl sulfate-gel electrophoresis, the newly synthesized PP12 comigrated with the major band of purified PP12 corresponding to mol wt 29,000. These data clearly confirm that PP12 is a protein of decidual rather than trophoblastic origin, and indicate that decidua from early pregnancy has the ability to synthesize it.

The selection of antibodies with defined desorption properties from precipitated immune complexes for use in immunoadsorption procedures.

A general method for preparing immunosorbents with preselected antibody avidity is described. The method, which is a modification of a method described previously, also includes immunospecific purification of the ligand prior to coupling on the gel matrix. Polyclonal anti-alpha-1-fetoprotein antibodies in precipitated immune complexes were separated according to their avidity (low, intermediate and high) by dissociation with agents of increasing efficiency. After solid-phase coupling the antigen binding activity of the separated antibody preparations was examined according to recovery, capacity and binding strength. Antibodies of intermediate avidity derived from the immune complexes demonstrated optimal properties for preparative affinity chromatography.

Demonstration of antigenic determinants specific for the split products of the third complement factor, C3.

Crossed anti-C3c immunoelectrophoresis of a plasma sample with in vivo complement activation revealed a pronounced 'spur' formation towards the cathodic region of immunochemical interaction between native C3 and C3c. In situ absorption of the antibody preparation against C determinants of the 3rd complement factor using fresh normal EDTA plasma as antigen demonstrated the presence of C3 split product specific determinants.

Molecular heterogeneity of pregnancy specific beta 1 glycoprotein: the effect on measurement by radioimmunoassay and electroimmunoassay.

A comparison of methods for the quantification of circulating pregnancy specific beta 1 glycoprotein in late pregnancy was performed to assess the influence of the presence of a high molecular weight glycoprotein with alpha 2 electrophoretic mobility (SP1 alpha) which is immunochemically identifiable with pregnancy-specific PBETA 1 glycoprotein (SP1 beta). Serum samples from 47 volunteers in the 3rd trimester of pregnancy were subjected to measurements of pregnancy specific beta 1 glycoprotein in rocket immunoelectrophoresis, quantitative crossed-immunoelectrophoresis and radioimmunoassay. Rocket immunoelectrophoresis gives a result which reflects the total SP1 content, i.e. SP1 alpha and SP1 beta while quantitative crossed-immunoelectrophoresis permits differentiation between the two molecules. Radioimmunoassay predominantly measures authenic SP1, i.e. SP1 beta in the presence of physiological amounts of SP1 alpha.

Influence of time, temperature and coagulation on the measurement of C3, C3 split products and C4.

Quantitative and qualitative immunoelectrophoretic analyses of circulating C3, C3 split products and C4 were performed in matched EDTA plasma and serum obtained from 5 normal subjects and stored for up to 48 h at room temperature (18 degrees C-22 degrees C) and 4 degrees C. Fluctuations in apparent levels of C3 were greater in serum than plasma stored at room temperature, a fall in levels seen by 24 h being followed by a significant increase. By contrast, levels of C3 did not alter if stored at 4 degrees C. C4 levels in both EDTA plasma and serum remained unchanged for 24 h, a slight decrease being seen at 48 h. Levels of C4 remained constant if samples were stored at 4 degrees C. Crossed immunoelectrophoresis revealed a significant progressive decrease in C3 levels and a simultaneous increase in C3c occurring after 4 h in serum and 8 h in EDTA plasma, stored at room temperature. In studies conducted at 4 degrees C, similar but delayed fluctuations were seen. A progressive and significant increase in C3d levels was seen in both plasma and serum samples stored at room temperature, levels rising to 276% (plasma) and 308% (serum) of levels seen at zero time. At 4 degrees C marginal increases in C3d levels only were observed. These results suggest that in vitro degradation of C3 and C4 are readily facilitated by temperature, time and coagulation, and that conditions of collection and storage of samples must be optimized for the accurate definition of activation of the complement cascade.

Development of a radioimmunoassay for adrenal specific protein: its measurement in biological fluids and tissues.

A radioimmunoassay for a new human adrenal specific protein (ASP) is described. The assay has a lower limit of detection of approximately 50 micrograms/l. The ASP extracted and purified from adrenal glands was used for standardization, radio-iodination and immunization of rabbits. Adrenal specific protein can be detected in normal human serum and plasma (240 +/- 80 and 190 +/- 60 micrograms/ml (means +/- S.D.) respectively), tissue levels were found in extracts of adrenal medulla, with appreciable amounts in the adrenal cortex, pituitary gland, seminal vesicle and testis, and low levels in all other tissues examined. The highest concentrations in biological fluids were found in seminal plasma and to a lesser extent in milk and menstrual fluid.

Changes in the concentration of 5-methoxytryptophol in the circulation at different phases of the human menstrual cycle.

The pineal indole 5-methoxytryptophol (ML) has been shown to have an antigonadal activity when administered to experimental animals, but data on its normal pattern of secretion have been lacking. Using a new gas chromatography-mass spectrometry assay, the concentration of ML at various phases of the human menstrual cycle has been studied. Daily samples were obtained throughout the month from five women with a normal cycle and two women taking an oral contraceptive. In women with a normal cycle levels of ML were found to be significantly lower in the last third of their cycle; this change was not seen in women taking an oral contraceptive who had low levels throughout the month. The changes in concentration of ML did not correlate with the changes in concentration of gonadotrophins.

The ratio between molecular variants of pregnancy-specific beta 1 glycoprotein (SP1) in the peripheral and retroplacental circulation during later pregnancy.

The ratio between two molecular variants of pregnancy-specific beta 1 glycoprotein (SP1) in the peripheral and retroplacental circulation was analysed in nine women during late pregnancy. The ratio between the two molecular variants, i.e. SP1 beta and SP1 alpha, was found to be identical in the peripheral and retroplacental circulation in all women examined. Comparisons were also made between SP1 beta as measured by radioimmunoassay and a normal serum protein, alpha 2 macroglobulin. A positive concentration gradient from peripheral to retroplacental circulation was found for SP1 beta whereas the gradient was less pronounced for alpha 2 macroglobulin. These findings add weight to the concept that SP1 alpha is a unique molecular species and not a genetic variant or a product of SP1 beta.

Specific and reversible interaction between pregnancy-associated plasma protein A and heparin.

An interaction between heparin and circulating pregnancy-associated plasma protein A (PAPP-A) has been observed. This interaction could be reversed by the addition of the specific heparin antagonist protamine sulphate. In the presence of heparin, crossed immunoelectrophoretic analysis revealed an alteration in the electrophoretic mobility of PAPP-A, while a significant increase (22 to 64 per cent, P less than 0.02) in measured levels of PAPP-A was seen in electroimmunoassays. These findings are discussed in relation to the optimal conditions for the precise measurement of PAPP-A.

Examination of placental proteins in charge-shift immunoelectrophoresis.

Three pregnancy-associated proteins, pregnancy-specific beta1-glycoprotein (SP-I), pregnancy-associated plasma protein A(PAPP-A), and placental lactogen (hPL) were examined by charge-shift immunoelectrophoresis. PAPP-A and hPL exhibited hydrophilic properties whereas SP-I was amphiphilic. These observations suggest that SP-I is a cell membrane protein.

Immunohistochemical localization of two endometrial proteins in the early days of human pregnancy.

Insulin-like growth factor binding protein-1 (IGFBP-1) [also known as placental protein 12(PP12)] and placental protein 14 (PP14) have been identified by specific immunostaining in early pregnancy specimens obtained 13-35 days of gestation. PP12 was evident in a discrete number of stromal decidual cells at the deciduotrophoblastic interface and under the endometrial surface epithelium. These cells did not have the rounded appearance of classic decidual cells but most often displayed cytoplasmic expansions. Staining for PP14 was strictly localized to the glandular epithelium of the endometrium. Implantation of the conceptus may be an important mechanism in the early expression of PP12 but not PP14.

Simultaneous autoradiography and line immunoelectrophoresis (ARLIE): a novel combination to identify de novo protein synthesis by pregnancy tissues.

A novel combination of two conventional techniques (autoradiography, AR and line immunoelectrophoresis, LIE; ARLIE) for identification of specific proteins synthesized de novo by explants is described. The incorporation rate of [35S]-methionine was linear in proteins derived from cytosol fractions and supernatants of first trimester human trophoblast and gestational endometrium for up to 18 h. SDS-PAGE analysis of these fractions provided further evidence of the protein synthesis and secretion by the tissue explants. The ARLIE system was evaluated by investigating the synthesis and secretion of five test proteins (PP12, PP14, hPL, FA-1 and FA-2) by trophoblast and gestational endometrium. The synthesis P12 and PP14 could be demonstrated by gestational endometrium only. Similarly the synthesis of hPL could be demonstrated by the trophoblast alone. The synthesis of the fetal proteins (FA-1 and FA-2) could not be demonstrated by either tissue. The control procedure, Protein A assisted immunoprecipitation, yielded similar results for PP14 but not hPL. This novel combination (ARLIE) provides a simple technique with which to study the de novo synthesis of several proteins simultaneously which is independent of the subclass and species of origin of antibodies.

Investigations into the molecular heterogeneity of pregnancy-specific beta 1-glycoprotein (SP1).

The molecular heterogeneity of pregnancy-specific beta 1-glycoprotein (SP1) was examined by analytical immunoelectrophoresis and a radioimmunostaining technique of immunoelectrophoretic plates. Analytical crossed immunoelectrophoretic analysis and radioimmunostaining of these plates demonstrated the presence of normal human serum components in the alpha-mobile precipitate previously considered to be exclusively the high molecular pregnancy-specific protein, SP1 alpha. This observation suggested that two molecular populations were contributing to the alpha-mobile precipitate. Following fractionation of late-pregnancy serum by size chromatography, the radioimmunostaining technique further demonstrated the presence of normal serum components in the intermediate fraction but not in authentic SP1 (i.e., SP1 beta) or the high molecular weight form (SP1 alpha). We suggest that SP1 antigenic determinants are distributed in three different fractions of pregnancy serum, one of which (intermediate fraction) is a complex of authentic SP1 (SP1 beta) and a normal serum protein, whereas non-pregnancy serum components were not demonstrable in the remaining two (i.e., SP1 beta or SP1 alpha).

Purification of pregnancy-associated plasma protein-A by a two step affinity chromatographic procedure.

A high molecular weight pregnancy specific protein (PAPP-A) was purified by immunospecific affinity chromatography. The purification procedure was based on a new method for affinity chromatography using direct coupling of precipitable immune complexes to the gel matrix. The procedure was performed in two steps: a positive immunospecific affinity chromatography followed by a negative affinity chromatography in which balanced amounts of anti-contaminant antibodies were used as ligant. The purified material at a concentration of 300 microgram/ml was tested by immunoelectrophoretic methods and no contaminants were detectable. WHO beta-1 SP1 reference material 78610 contained 45 microgram PAPP-A/ml.

Pregnancy-associated plasma protein A levels in maternal serum, extraembryonic coelomic and amniotic fluids in the first trimester.

First trimester maternal serum levels of pregnancy-associated plasma protein A(PAPP-P) are reduced in women with a Down's syndrome pregnancy. We have examined the concentration of this molecule in the amniotic (AF) and extra-embryonic coelomic (EECF) fluids surrounding the developing fetus. Maternal serum levels of PAPP-A were elevated in all samples and steadily rose from a median of 480 mIU/1 at 8 weeks to a median of 6375 mIU/1 at 14 weeks gestation. Levels of PAPP-A were low in EECF and undetectable in the AF until 14 weeks gestation. This pattern of distribution is in contrast to that of most other trophoblast-associated antigens. This may reflect PAPP-A physiology and its specific production by the syncytiotrophoblast.

Pregnancy-associated plasma protein-A and placental protein 5 in human ovarian follicular fluid.

By sensitive and specific radioimmunoassays PAPP-A and PP5 were detected in follicular aspirates obtained from women undergoing ovarian hyperstimulation for oocyte harvest prior to in vitro fertilization and embryo transfer. Follicular and pregnancy-derived PAPP-A were immunologically and physicochemically indistinguishable. Similarly, pregnancy- and nonpregnancy-derived PP5 were immunologically indistinguishable. However, in addition to the 18- and 36-K species, a larger species having a molecular size greater than 140K was found in the follicular fluid. Mean follicular PAPP-A and PP5 concentrations were 727 mIU/L and 1376 mAU/L, respectively, with no significant correlation between follicular PAPP-A, PP5, and steroid concentrations. There was, however, a significant but negative relationship with follicular volume. Preliminary in vitro studies indicated that both proteins were synthesized by granulosa cells in preparation for follicular rupture. Follicular PP5, like antithrombin III, interacted reversibly with heparin and thrombin affinity matrices, suggesting a potential biological role as a follicular anticoagulant, whereas PAPP-A, a specific and potent inhibitor of leukocyte elastase, contributes to the maintenance of proteolytic homeostasis and the protection of spermatozoa and embryo against proteolytic attack originating from the maternal leukocytes.

Pregnancy-associated plasma protein-A and placental protein 5 in human seminal plasma.

Human seminal plasma contains two glycoproteins which are physiochemically and immunologically indistinguishable from pregnancy-associated plasma protein-A and placental protein 5. Seminal concentrations of both glycoproteins did not correlate with clinical assessment of semen quality. Furthermore, analysis of split ejaculates indicated a nontesticular origin for both proteins, which are possibly secreted into the distal portions of the tract by the accessory glands (prostate gland and seminal vesicles). The physiological significance of these findings has yet to be determined. However, it is suggested that pregnancy-associated plasma protein-A, a known potent inhibitor of leukocyte elastase, protects the deposited sperm against proteolytic attack originating from the localized leukocyte reaction within the female reproductive tract, thus contributing towards sperm survival within this immunologically hostile environment and enabling fertilization to occur.

Effect of smoking on maternal blood SP1 levels in late pregnancy.

Maternal blood levels of pregnancy-specific beta 1-glycoprotein (SP1) were measured in 1669 samples from 1078 patients at 31 to 40 weeks' gestation. Mothers who smoked throughout pregnancy showed a general reduction in infant birth weight and blood SP1 levels. Further analysis of these findings showed that SP1 levels were directly related to fetal weight, and suggested that smoking affects fetus and placenta as a single unit.