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The aim of this study was to determine if the changes in gonadotropin subunit gene expression following ovariectomy reflect transcriptional and/or posttranscriptional regulation by GnRH or inhibin. Subunit transcription rates were determined by recently developed quantitative RT-PCR for subunit primary transcripts (as an indicator of gene transcription), which allow us to measure both mRNA and PT from RNA extracted from a single pituitary. Following ovariectomy, LHbeta PT concentrations increased 2- to 3-fold between 72 h and 7 d, paralleling changes in serum LH and LHbeta mRNA. In contrast, serum FSH, FSHbeta mRNA, and FSHbeta PT concentrations were 6- to 9-fold greater 12-24 h after ovariectomy followed by an additional 2.5-fold increase at 72 h. Although alpha RNA was elevated at 72 h after ovariectomy, alpha-primary transcript did not change. GnRH antagonist prevented the increase in LHbeta-PT at 72 h, but had no effect on the increase in FSHbetaPT at 12 h and was only partially effective at 72 h. The acute GnRH-independent increase in FSHbeta-primary transcript after ovariectomy could be duplicated by the administration of inhibin antiserum to intact rats; inhibin-alpha antiserum did not affect LHbeta-primary transcript, but increased FSHbeta-primary transcript concentrations 8- to 11-fold. The half-disappearance rates of LHbeta and FSHbeta primary transcripts were measured after GnRH blockade or administration of recombinant human inhibin A. The half-disappearance times for LHbeta and FSHbeta primary transcripts following GnRH blockade were 13 and 17 min, respectively; the mRNAs did not change. The effects of inhibin were specific for FSHbeta; 60 min after inhibin FSHbeta-primary transcript was undetectable with a half-disappearance time of 19 min, additionally FSHbeta mRNA levels also fell with a half-life of 94 min. In conclusion, these data support previous evidence that GnRH regulates gonadotropin gene expression primarily at the level of transcription. However, the acute increase in FSHbeta-primary transcript after ovariectomy or immunoneutralization of inhibin-alpha, and the rapid fall in FSHbeta-primary transcript following rh inhibin, provide novel evidence that inhibin suppresses FSHbeta gene transcription in addition to its action in regulating FSHbeta mRNA stability.

The human glycoprotein hormones, hCG, TSH, LH, and FSH, are composed of a common alpha-subunit assembled to a hormone-specific beta-subunit. The subunits combine noncovalently early in the secretory pathway and exist as heterodimers but not as multimers. LH/FSH are synthesized in the pituitary gonadotrophs, and several of the alpha-subunit sequences required for association with either the LHbeta or FSHbeta subunits are different. Thus, it is intriguing that no ternary complexes are observed for LH and FSH in vivo (e.g. two different beta-assembled to a single alpha-subunit). To examine whether the alpha-subunit can interact with more than one beta-subunit, and to study the conformational relationships between the ligand and the receptor, we constructed a vector encoding two tandemly arranged beta-subunits fused to a single alpha-subunit gene (FSHbeta-CGbeta-alpha). This approach permitted structure-function analyses of alpha/beta domain complexes without the possibility of subunit dissociation. We reported previously that the CGbeta or FSHbeta subunit gene can be genetically fused to the alpha-gene and the resulting single chains (CGbetaalpha and FSHbetaalpha, respectively) were biologically active. Here we demonstrate that a triple-domain single chain bearing the configuration FSHbeta-CGbeta-alpha is efficiently secreted from transfected Chinese hamster ovary (CHO) cells and exhibits high-affinity receptor binding to both FSH and LH/hCG receptors, comparable to the native heterodimers. These results indicate that the alpha-subunit can interact with each beta-subunit in the same complex and that an alpha-domain fused to a beta-domain can still interact with an additional beta-subunit. The data also demonstrate the remarkable flexibility of the receptor to accommodate the increased bulkiness of the triple-domain ligand. In addition, the formation of intrachain FSH- and CG-like complexes observed in a triple-domain single chain suggests that the alpha-subunit can resonate, i.e. shuttle between alpha-beta heterodimeric intermediates during the early stages of synthesis and accumulation in the endoplasmic reticulum. Such model compounds could be useful as substrates to generate a new class of analogs in which the ratio of the LH/FSH activity is varied. This could aid in the design of analogs that could be used to mimic the in vivo hormonal profiles.

Liddle's syndrome is an autosomal dominant form of salt sensitive hypertension caused by mutations in the beta or gamma subunit of the epithelial sodium channel. Systematic mutagenesis studies revealed that a conserved PPPXY sequence (PY motif) of the C-terminus of the alpha, beta, or gamma subunits might be involved in the regulation of the channel activity. However, only two missense mutations in the PY motif of the beta subunit have been reported to cause Liddle's syndrome. We sequenced the C-termini of the beta and gamma subunits of the epithelial sodium channel in a Japanese family clinically diagnosed as having Liddle's syndrome and found a new missense mutation in the PY motif of the beta subunit, P615S. Expression studies with P615S mutant in Xenopus oocytes resulted in an about 3-fold increase in the amiloride-sensitive sodium current compared to the wild type (p = 0.001). These findings provide further clinical evidence for the hypothesis that a conserved PY motif may be critically important for the regulation of the epithelial sodium channel.

Previous studies have indicated that in addition to other glycoprotein hormones, the pituitary gland produces small amounts of hCG beta, the classical pregnancy and tumor marker. At the gene transcription level, definitive proof for hCG beta messenger ribonucleic acid transcription was still lacking, largely due to the 90% homology to hLH beta at the DNA sequence level, which renders specific hCG detection in the presence of a vast excess of LH difficult. We investigated both the presence of hCG beta messenger ribonucleic acid and the protein itself in normal human female postmenopausal (n = 4) and male pituitaries (n = 2). Reverse transcription-PCR and subsequent restriction enzyme analysis revealed that the hCG beta 3, 5, 7, and 8 genes coding for genuine hCG beta were transcribed in all pituitaries. Additionally, three alternatively spliced gene products derived from hCG beta genes 1 and 2 were detected and verified by single strand sequencing of the complementary DNAs. The most abundant fragment (244 bp) showed a point mutation (T--A) in the splice donor site for the first intron, resulting in an alternate use of exon 1 and a frame shift in the open reading frame that might give rise to a hypothetical protein, 132 amino acids in length. With regard to protein synthesis, we confirmed the pituitary as the site of production for hCG beta by reverse phase high performance liquid chromatography and subsequent immunoradiometric assays, including a monoclonal antibody directed against the unique C-terminal extension of hCG beta.

It was previously established that the administration of a potent GnRH agonist such as triptorelin (D-Trp6-GnRH) induced desensitization of pituitary gonadotropic cells, resulting in decreased expression of gonadotropin beta-subunit genes and the suppression of LH and FSH synthesis and release. Binding of GnRH to the pituitary is also affected by agonist treatment. To examine the desensitizing effects of GnRH agonist on the expression of the pituitary GnRH receptor (GnRH-R) gene, male rats were given triptorelin (long-acting formulation, 300 micrograms/kg), and levels of GnRH-R messenger RNA (mRNA) were determined by Northern and dot blot hybridization to a 32P-labeled rat complementary DNA probe. Abundances of gonadotropin alpha-subunit, LH beta, and FSH beta mRNAs were examined in parallel, using appropriate probes. A rapid time-dependent decrease in the level of GnRH-R mRNA was observed in rats after triptorelin administration. A minimum residual level of mRNA, in the range of 20-25% of the initial value, was attained as early as 5 h after treatment. Levels further stabilized to 25-30% after a small transient increase to 45% on day 5. A single injection was effective for at least 30 days, after which GnRH-R mRNA levels slowly returned to normal, suggesting a progressive abolition of agonist effects. A concomitant acute depletion of mRNA levels was observed for LH beta and FSH beta (50% decrease in about 48 and 3 h, respectively), whereas the alpha-subunit message increased (rapidly reaching a level 1.8-fold that in control rats after 1-2 days). Castration induced a 3.8-fold elevation in the amounts of GnRH-R mRNA after 3 weeks, whereas alpha, LH beta, and FSH beta mRNAs increased by 6.2-, 7.9-, and 4.2-fold, respectively, compared to corresponding values in intact animals. Administration of the GnRH agonist readily prevented, for as long as 3 weeks, the stimulatory effects of castration on the GnRH-R mRNA and mRNAs for the beta-subunit of gonadotropins, but not for the alpha mRNA, which remained at a high level. When triptorelin was administered 3 weeks postoperatively, the castration-induced increase in LH beta and FSH beta was totally abolished, and no significant effect was noted on alpha-subunit mRNA. In conclusion, these data demonstrate that expression of the GnRH-R gene is subject to regulation and depends on GnRH stimulation, in a manner that indicates susceptibility to desensitizing action by the long-acting GnRH analog, triptorelin.(ABSTRACT TRUNCATED AT 400 WORDS)

The extraembryonic coelomic fluid (EECF) represents a major compartment in the fetal-placental unit during the first trimester of pregnancy. The compartment is composed of the fluid contained between the chorionic and amniotic membranes. The levels of glycoprotein hormone free alpha-subunit and free beta-subunit in the EECF far exceed those in the amniotic fluid or maternal serum. Furthermore, the level of free alpha in this compartment is twice that of intact hCG. We purified the glycoprotein hormone free alpha-subunit from a pool of EECF. This free alpha-subunit was found to be larger in size than the alpha-subunit of intact hCG. The size difference was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reduced and denatured conditions. The carbohydrate composition of the EECF free alpha-subunit indicated a higher degree of oligosaccharide branching, as evidenced by larger amounts of fucose, sialic acid, galactose, and N-acetylglucosamine than were present on combined hCG alpha. These differences in size and carbohydrate composition argue strongly against the concept that free alpha-subunits might originate from dissociation of intact hCG or "nicked" hCG. The free subunits of the EECF were evaluated for their ability to combine with the corresponding subunit obtained by dissociation of intact hCG. EECF free beta was able to combine with hCG alpha to form intact hCG. In contrast, EECF free alpha was unable to combine with hCG beta to form intact hCG. However, after removal of the asparagine-linked glycans by treatment with N-glycanase, most of the previously uncombinable free alpha-subunits were able to combine with hCG beta. These data demonstrate that the N-linked oligosaccharide(s) of EECF free alpha function to prevent the molecule from combining with the available and combinable free beta-sub-units that coexist in the same physiological compartment during early pregnancy. In view of the large amount of free alpha that is present in the EECF and the observation that, in vitro, free alpha can stimulate uterine decidual cell PRL secretion, together with the close apposition of free alpha-producing cells to decidual cells, it is likely that EECF free alpha has a function in early pregnancy. Carbohydrate modifications generated during the biosynthesis of EECF free alpha-subunit ensure that a population of free alpha molecules can exist in the presence of substantial quantities of free beta-subunits, and correspondingly, these same carbohydrate modifications function to permit the existence of free beta-subunits in the same gestational compartment with free alpha molecules.(ABSTRACT TRUNCATED AT 400 WORDS)

The hCG beta-subunit contains a carboxy-terminal extension bearing four serine-linked oligosaccharides [carboxy-terminal peptide (CTP)], which is important for maintaining its longer half-life compared with the other glycoprotein hormones. Previously, we enhanced the in vivo half-life of FSH by fusing the CTP to the carboxy end of FSH beta coding sequence. The alpha-subunit is common to the glycoprotein family. We constructed alpha-subunit CTP chimeras, since such analogs with the appropriate O-linked glycosylation and conformation would increase the in vivo stability of the entire glycoprotein hormone family. Two chimeras were constructed using overlapping polymerase chain reaction mutagenesis: a variant with CTP at the carboxy end and another analog with the CTP at the N-terminal region of the subunit, between amino acids 3 and 4. The latter design was based on models showing that the amino-terminal region of alpha is not involved in assembly with the beta-subunit, nor is it essential for receptor binding and signal transduction. These chimeras were cotransfected with the hCG beta gene into Chinese hamster ovary cells. The chimeras were secreted and combined efficiently with the CG beta-subunit, comparable to the wild type alpha-subunit. CG dimers containing the alpha-subunit chimera with CTP at the carboxy end of the subunit had a much lower binding affinity for the hLH-hCG receptor in vitro, whereas the binding of the dimer containing the CTP at the amino-terminal end of the subunit was similar to wild type hCG. Furthermore, the in vivo activity of this analog was enhanced significantly. Moreover, regardless of the two insertion points in the alpha-subunit, the CTP sequence was O-glycosylated. These data suggest that the entire signal for O-glycosylation is primarily contained within the CTP sequence and is not dependent on the flanking regions of the recipient protein. The transfer of CTP to the alpha-subunit of hCG results in an agonist with prolonged biological action in vivo. These data further support the rationale for using the CTP as a general target to increase the potency of bioactive glycoproteins.

hCG is a dimer composed of an alpha- and a beta-subunit, joined noncovalently. In addition to the hCG dimer, uncombined alpha- and beta-subunits (free beta) and nicked hCG and free beta molecules (cleaved at 44-45 or 47-48) can be detected in the circulation. Of these circulating molecules, only the intact hCG dimer fully expresses biological activity. The pathways that dissociate, nick, and degrade hCG and beta-subunit molecules in pregnancy are unknown and could have a major role in regulating hormone levels. Immunoassays for intact (nonnicked) hCG and intact (beta-subunit (with < 1% detection of nicked molecules) and a subtractive immunoassay system for measuring nicked hCG levels have been described previously. A multiantibody scavenger assay is described here for measuring nicked beta-subunit levels (< 6% detection of intact beta-subunit). In this report we use these four assays to assess conversion of intact hCG or beta-subunit to nicked forms over time (nicking enzyme activities) in control (healthy nonpregnant), pregnant, and cancer patient serum samples. Pools of pregnancy and control sera were supplemented with intact hCG and its dissociated beta-subunit and incubated at 37 C. Intact and nicked molecule measurements were made between 0-48 h. In two different pools of control sera, no loss of intact hCG or intact beta-subunit and no significant gain in nicked hCG or nicked beta-subunit were detected over 48 h. This indicated a lack of nicking enzyme activity in control serum. In two different pools of first trimester pregnancy sera, we found no obvious loss of intact hCG or gain of nicked hCG levels over 48 h. However, we found 70% and 62% losses (pools 1 and 2) of intact beta-subunit and 51% and 39% gains of nicked beta-subunit over the same time period. We inferred that an uncombined or free beta-subunit-modifying activity was present in pregnancy serum. We repeated the pregnancy serum experiment with six different concentrations of beta-subunit (0.62-29 mg/L). A linear relationship, percent nicking against time, existed for the six concentrations for up to 6 h at 37 C (r = 0.97); after that, the rate of nicking declined. A plot of rate against concentration against revealed a classical Michaelis-Menten enzyme relationship (logarithmic regression, r = 0.96). The pregnancy serum beta-subunit nicking activity was partially purified by gel filtration. A single peak of activity emerged, eluting between the 150,000-443,000 mol wt standards.(ABSTRACT TRUNCATED AT 400 WORDS)

hCG is found in pregnancy urine and in urine from some cancer patients in a variety of forms whose concentrations have clinical importance. Recently, concerns about accurate measurement of these forms have been raised because of the finding that hCG with peptide bond cleavages within the beta-subunit is not recognized by commonly used antibodies. Such nicked forms of hCG are biologically inactive or of very low activity. They are present in normal pregnancy urine and to varying extents in the urine of patients with trophoblastic disease. International reference preparations of hCG contain nicked forms of hCG. Previously, it was not possible to separate nicked hormone from the intact form of hCG. This was a serious impediment to producing improved reference standards from natural pregnancy hormone. We now report that a simple hydrophobic purification scheme separates intact hCG from nicked hCG as well as from hCG beta core fragment. This scheme is a modification of the method of Hiyama et al. The order of elution from low to high hydrophobicity is hCG beta core fragment, nicked hCG, and lastly, intact hCG. Nicking of the putative amphipathic helix loop, hCG beta 38-57, apparently renders the hormone significantly less hydrophobic despite the equal molar content of sialic acid. The hCG CR 127 nicked preparation was only 10% as potent as the reference preparation in a heterodimer-directed assay. The nicked-depleted hCG CR 127 was 30% more potent in this assay. Improved hCG reference standards should display similar increases in immunopotency (20-30%) with most antiheterodimeric antibodies and similar increases in bio-potency assays. It should now be possible to make reference preparations of these forms of hCG directly from the raw urine of normal pregnant patients and those with trophoblastic disease.

Cyclic AMP-dependent protein kinase (cAPK) modulates synaptic transmission and influences memory and learning. Among the various isoforms of regulatory and catalytic subunits that comprise mammalian cAPK, only the regulatory type I beta (RI beta) subunit is unique to nervous tissue. The requirement for RI beta in neurons is presently unknown. Previous studies demonstrate that holoenzyme containing RI beta activates at lower concentrations of cAMP compared to other forms of cAPK. Thus, neurons that induce RI beta expression may become more sensitive to subsequent hormonal signals and maintain more long-term phosphorylation events. To further elucidate the function of this novel protein, we have begun to investigate its gene. Here we report the isolation of the mouse RI beta promoter as determined by S1 nuclease analysis and transgenic mouse expression. A beta-galactosidase fusion gene containing 1.5 kilobases of 5'-nontranscribed RI beta DNA and 2 kilobases of intron 1 was expressed preferentially in the cortex and hippocampus of the brain and within the spinal cord. In addition to mimicking the location of endogenous RI beta expression, the transgene was activated at a similar time (embryonic day 11.5) during mouse fetal development. Isolation of the RI beta promoter will help identify the elements that direct transcription in a subset of neurons and illuminate the physiological conditions that may regulate RI beta expression. This promoter can also be used to target the expression of wild type and mutant cAPK subunit genes in order to investigate synaptic plasticity in animals.

Clinically nonfunctioning pituitary adenomas represent approximately 25% of all pituitary tumors. Recent studies using a number of in vitro techniques show that the majority of such tumors produce gonadotropins. Hypersecretion of uncombined gonadotropin subunits by these tumors has also been identified raising the possibility that gonadotropin biosynthetic alterations may occur in neoplastic pituitary tissue. To determine whether underlying intracellular biosynthetic alterations lead to imbalanced secretion of the gonadotropin subunits by such tumors, we investigated 1) steady state gonadotropin-subunit messenger ribonucleic acid (mRNA) levels in tumor tissue from 49 patients with clinically nonfunctioning adenomas, 2) secretion of gonadotropins in dispersed pituitary tumor cultures, and 3) serum concentrations of gonadotropins and free subunits. Northern blots of RNA extracted from surgically obtained pituitary tumor tissue were hybridized with complementary DNA probes for FSH beta, LH beta, and alpha-subunit, and quantitative analysis was done to compare alpha- and beta-subunit biosynthesis in individual tumors. Of these tumors, 47 contained sufficient RNA for Northern analysis and 77% of these tumors contained one or more of the gonadotropin-subunit mRNAs. Steady state alpha-subunit mRNA was detected in 57% of tumors, FSH beta mRNA in 49%, and LH beta in 1 (2%). We found FSH beta mRNA in excess of alpha-subunit mRNA in one-third of tumors, including 9 tumors where alpha-subunit mRNA was undetectable. In cultured cells, FSH beta was secreted in excess of alpha-subunit in 41% of tumors. For those tumors in which both mRNA and culture data were available, FSH beta mRNA and secreted subunit levels were in excess of alpha-subunit in 64% of tumors. We conclude that clinically nonfunctioning pituitary adenomas frequently synthesize excess FSH beta subunit relative to alpha-subunit. This finding is in contrast to previous data in normal pituitary or placental tissue where alpha-subunit is present in excess of beta-subunits at both the mRNA and protein levels. The free-beta-subunit hypersecretion identified in pituitary adenomas may be due to biosynthetic abnormalities intrinsic to neoplastic gonadotrophs.

Using an activin RIA that showed limited cross-reaction with inhibin, activin immunoactivity was monitored throughout the isolation of activin from bovine follicular fluid and side-fractions during the isolation of human recombinant inhibin. Two peaks of activin immunoactivity were identified in both materials and isolated to homogeneity by dye affinity chromatography, hydrophobic interaction and gel permeation chromatography, and reverse phase HPLC. The purified proteins in all four peaks had terminal amino acid sequences identical to those of the inhibin/activin beta-subunit. The molecular masses determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing (and reducing) conditions were 25 and 15 and 15 and 15 kilodaltons (kDa) for each pair of proteins from both sources. Based on these criteria, the bovine and human recombinant 25-kDa proteins correspond to the inhibin/activin beta A-subunit dimer (activin-A), while the 15-kDa proteins correspond to the inhibin/activin beta A-subunit monomer. The activity of the monomer was 17% of the activity of the dimer in the activin RIA. Based on this level of cross-reaction and the proportion of monomer to dimer immunoactivity found after reverse phase HPLC of bovine follicular fluid, it is estimated that the levels of monomer in bovine follicular fluid are 25-60% those of the dimer. The biological activities of the human recombinant activin monomer and dimer were investigated in two different cell culture systems. In a rat pituitary cell system the activity of the activin monomer was 19% of the activity of the dimer in stimulating FSH release, while in rat thymocyte cultures the activity of the monomer was 45% the activity of the dimer in suppressing lectin-stimulated [3H]thymidine uptake. It is concluded that the beta A-subunit monomer is found in bovine follicular fluid at a level 25-60% that of the beta A-subunit dimer (activin-A). The monomer displays in vitro responses similar to those of the dimer, although the monomer is less active (18-45%) than the dimer. It is unclear if dimerization of the monomer is a necessary prerequisite for biological activity.

hCG, the hormone produced by the trophoblast throughout pregnancy, has peptide bond cleavages, or nicks, in the beta-subunit. We sought to compare the nature of these nicks in standard reference preparations of hCG, to determine the enzymes that may be responsible for generating the peptide bond cleavages, and to devise means of separating nicked from intact hormone. The standard reference preparations of hCG, which are purified from a commercial product made from large pools of pregnancy urine, were found to have varying concentrations of nicked hormone. The preceding report showed that 11 of 13 hCG preparations isolated from individual pregnancy urine samples were nicked at the beta 47-48 bond, with 2 of 13 having a second nick at beta 44-45. As shown here, all of the hCG reference standards are nicked to similar extents at both the beta 47-48 bond and the beta 44-45 bond. The percentage of peptide bond nicking in the various hCG standard preparations ranged from 10-20% and appeared higher in the more recent preparations. We showed that human leukocyte elastase is capable of specifically cleaving the beta 44-45 bond, and in extended digests it can also cleave the beta 48-49 and beta 51-52 peptide bonds. Thus, human leukocyte elastase may be the origin of some of these cleavages in the individual samples and the reference standards. Furthermore, we report that a monoclonal antibody directed to hCG alpha-beta dimer binds preferentially to nonnicked hCG and much less to nicked hCG.

Nicks, or missing peptide linkages, have been found in hCG beta-subunit between residues 44 and 45 and between residues 47 and 48. We examined the occurrence and biological and immunological activities of nicked hCG. As shown by sequence analysis, CR127 standard hCG is approximately 20% nicked, half at beta 44-45 and half at beta 47-48. Treatment with human leukocyte elastase increased the extent of nicking of CR127 standard hCG. The longer the incubation of CR127 standard with human leukocyte elastase (0, 2, and 21 h), the greater the extent of nicked hCG (20%, 46%, and 89%). As the extent of nicking increased, the receptor-binding ability diminished, as did the ability to stimulate progesterone production by rat corpus luteal cells in vitro (0.9, 0.74, and 0.29 microgram/microgram hCG, respectively). In a regression analysis, a linear relationship was indicated between the extent of nicking and receptor binding values (97% correlation) and between the extent of nicking and steroidogenic activity in vitro (99% correlation). From the intercepts of the regression lines, it was estimated that nicks reduced receptor binding by 11-fold and reduced the steroidogenic activity of hCG by 5-fold. We examined eight individual hCG preparations, three purified from pregnancy urine, three from urine from patients with hydatidiform mole, and two from urine from women with choriocarcinoma. In descending order, the eight individual hCG preparations were 100%, 100%, 85%, 76%, 42%, 41%, 0%, and 0% intact. Although no correlation was observed between the percent intact and the ability of the eight individual samples to displace 50% [125I]hCG in binding CG/LH receptor (r less than 0.5), a close correlation was noted between the percent intact and the steroidogenic activity in vitro (98% correlation). This separated the effects of nicking on receptor binding and steroidogenic activities and indicated that while multiple factors influence receptor binding, only nicking suppresses the steroidogenic activity of bound hCG. We examined the recognition of nicked hCG molecules by different hCG immunoassays. The Hybritech Tandem assay measured total hCG and did not distinguish nicked and intact hCG molecules (in a regression analysis, immunoactivity vs. percent intact hCG, r less than 0.5). In contrast, the immunometric assay using B109 hCG dimer-specific monoclonal antibody and anti-beta-peroxidase only detected the intact component of hCG (in a regression analysis, immunoreactivity vs. percent intact hCG, 98% correlation). We used these assays together to estimate the percentage of intact hCG and to deduce the extent of nicking.(ABSTRACT TRUNCATED AT 400 WORDS)

It is well documented that the hypothalamic decapeptide gonadoliberin (GnRH) controls the biosynthesis and secretion of pituitary gonadotropins; however, it is still unclear whether GnRH synthesized by the placenta plays the same role with respect to hCG. In the current study we have investigated the acute response of placenta tissue to a single GnRH pulse as well as the influence of GnRH pulses on the secretion of hCG and hCG mRNA concentrations elicited several hours after application of the peptide hormone. For this purpose we have used a superfusion culture model of first trimester placenta tissue (8-12 weeks of gestation). In the first hour after explantation of the tissue, hCG secretion was decreased, and increasing amounts of free subunits were released. Afterward, the original hCG secretion rates were recovered and maintained for several days, attended by decreased levels of free subunits in the culture medium. The superfusion model was superior to static incubations, since it showed approximately a 4-fold higher amount of hCG to be secreted within 24 h (day 3 of cultures). A single GnRH pulse (1 mumol/L; 30 min) caused a significantly increased transient release of hCG (P less than 0.0001). Two GnRH pulses (concentration range, 0.01-10 mumol/L; 30 min) applied 24 h after (first pulse) and in the interval between 36-48 h after (second pulse) the start of the superfusion culture elicited a long-lasting 2-fold increase in the hCG secretion rate, which rose approximately 6 h after the second GnRH pulse. This was correlated with increased mRNA concentrations measured by means of Northern blots of total RNA. At 0.02 mumol/L GnRH, 4-fold higher beta mRNA levels were observed. The alpha mRNA levels were 2.5-fold elevated. GnRH pulses of 0.01 and 10 mumol/L, respectively, were ineffective. A further effect of GnRH pulses was augmentation of the episodic character of hCG secretion. Our results suggest that GnRH causes different specific acute and late effects on the amount and pattern of hCG secretion as well as on hCG biosynthesis at the levels of both hCG subunit mRNAs.

This study was designed to investigate the effects of progesterone on the estradiol (E2)-induced FSH surge and FSH beta messenger RNA (mRNA) using immature rat models developed previously to demonstrate inhibition or facilitation of the LH surge by progesterone. Twenty-eight day-old rats that received E2 implants at 0900 h had FSH surges about 1700 h on day 29 (32 h). In rats treated with E2 alone, serum FSH was 15.1 +/- 1.6 ng/ml at this time, while in those animals treated concurrently with E2 and progesterone, serum FSH was significantly suppressed (8.3 +/- 0.7 ng/ml, P less than 0.001). For demonstration of progesterone facilitation, rats were primed for 24 h with E2 before progesterone treatment. This led to premature and enhanced FSH secretion: at 1400 h on day 29 serum FSH was 45.5 +/- 2.7 ng/ml compared to 6.4 +/- 0.5 ng/ml in rats treated with E2 alone. To examine the effects of these dual actions of progesterone on FSH synthesis, steady state concentrations of FSH beta mRNA were measured by Northern analysis. FSH beta mRNA generally increased in parallel with FSH release. Levels of this mRNA were about 1.5-fold higher in rats undergoing E2-induced FSH surges than in rats in which the surge was blocked by progesterone. Also, at the onset of the progesterone-facilitated FSH surge, FSH beta mRNA was about 5-fold higher in animals treated with E2 and progesterone than in those treated with E2 only. On the morning after the FSH surge (48 h after E2 treatment) FSH beta mRNA was low to undetectable. In contrast, levels of FSH beta mRNA were 7- to 8-fold higher at this time in rats in which the surge was blocked by progesterone. Serum inhibin concentrations were significantly elevated (P less than 0.05) in animals treated with E2 alone for 32 h (3077 +/- 260 fmol/ml) or 48 h (2344 +/- 148 fmol/ml) compared to those treated with E2 and progesterone in the inhibition paradigm (2469 +/- 106, 1896 +/- 114 fmol/ml, respectively). After 32 h of E2 treatment in the facilitation paradigm, serum inhibin was comparable (P greater than 0.2) in rats treated for 8 h with blank implants (2592 +/- 168 fmol/ml) and those treated for 8 h with progesterone (2720 +/- 188 fmol/ml).(ABSTRACT TRUNCATED AT 400 WORDS)

In an attempt to further study various fragments of free and combined forms of hCG beta present in biological fluids, we performed one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Western immunoblotting using antipeptide antibodies directed to the hCG beta-(111-116) portion (monoclonal antibody FB12) antiserum to the hCG beta(8-16) portion or antiserum which was specific for fragments ending at residue 47. Results observed in a crude preparation of urinary hCG demonstrated that in addition to the carboxyl-terminal part of the reduced hCG beta nicked subunit (beta NS) [hCG beta-(48-145)], three other fragments of mol wt 18,000 (F1), 16,500 (F2), and 12,000 (F3) were detectable after cleavage of disulfide bonds. Both the immunoreactivity pattern and peptide sequencing revealed that the F1 fragment was constituted of the hCG beta-(1-47) sequence, whereas the F2 fragment comprised the 6-47 portion. We then studied the beta NS in urine from either pregnant women or four patients with choriocarcinomas. Results showed that both hCG and the free beta-subunit contained beta NS. Furthermore, free hCG beta present in those urine samples appeared to be extensively, if not totally, nicked. Results observed in urine were confirmed using separation of hCG from its beta-subunit by a two-step chromatography procedure, identification of hCG and hCG beta immunoreactive peaks by specific monoclonal immunoradiometric assay, and analysis of resulting preparations by one-dimensional electrophoresis under reducing conditions, followed by Western immunoblotting with FB12. This latter protocol was also used to investigate the presence of beta NS in sera of four patients with choriocarcinoma tumors. In those sera, hCG appeared to be nicked. This study demonstrates that the beta-subunit of hCG is modified by multiple fragmentations.

hCG is a member of a family of glycoprotein hormones which share a common alpha-subunit, but differ in their hormone-specific beta-subunits. The CG beta-subunit is unique in that it contains a hydrophilic carboxyl-terminal extension with four serine O-linked oligosaccharides. To examine the role of the O-linked oligosaccharides and the carboxyl-terminal extension of hCG beta on receptor binding, steroidogenesis in vitro, and ovulation induction in vivo, site-directed mutagenesis and gene transfer methods were used. Wild-type hCG alpha and hCG beta expression vectors were transfected into an O-glycosylation mutant Chinese hamster ovary cell line to produce intact dimer hCG lacking the beta-subunit O-linked oligosaccharide units. In addition, a mutant hCG beta gene (CG beta delta T) was generated which contained a premature termination signal at codon 115. This gene was cotransfected with the hCG alpha gene into Chinese hamster ovary cells to produce hCG dimer which lacked the carboxyl-terminal amino acids 115-145 of hCG beta (truncated hCG). The O-linked oligosaccharide deficient or truncated hCG derivatives were examined for their ability to bind to the mouse LH/hCG receptor and stimulate cAMP and steroidogenesis in vitro. These studies show that the O-linked oligosaccharides and carboxyl-terminal extension play a minor role in receptor binding and signal transduction. In contrast, comparison of the stimulatory effects of truncated and wild-type hCG in a rat ovulation assay in vivo via either intrabursal or iv injection revealed that the truncated derivative was approximately 3-fold less active than wild-type hCG. These findings indicate that the carboxyl-terminal extension of hCG beta and associated O-linked oligosaccharides are not important for receptor binding or in vitro signal transduction, but are critical for in vivo biological responses.

hCG is a glycoprotein hormone composed of 2 dissimilar subunits, alpha and beta, joined non-covalently. hCG and its free beta-subunit are the principal hCG beta immunoreactivities in pregnancy serum samples, and the same plus beta-core fragment (beta-subunit residues 6-40 disulfide-linked to residues 55-92) in urine samples. Ectopic or tubal pregnancy is difficult to diagnose in emergency rooms. With the objective of finding better hCG-related assays for differentiating tubal and normal pregnancies, we tested 2 hCG, 1 free beta-subunit and 2 beta-core fragment immunoassays. Twelve urine samples were collected in the emergency room from women later shown by surgery to have tubal pregnancy. All were 38 to 80 days since last period. A further 36 urine samples were collected from the same period from those with normal intrauterine pregnancies. Using the 2 hCG assays the median level in tubal pregnancy samples was 1/38th and 1/48th of normal pregnancy concentrations. With the free beta-subunit assay tubal pregnancy levels were 1/28th of normal levels. Using 2 beta-core fragment assays (Ciba-Triton UGP kit and B204-FBT11 scavenger test), however, tubal levels were most different from intrauterine pregnancy, 1/149th and 1/800th of normal levels. A cut-off level of 100 micrograms/L was considered for the B204-FBT11 beta-core fragment test, at which a predictive value of > 98% was suggested for ectopic pregnancy. In an additional patient, levels were measured 15 days prior to the diagnosis of tubal pregnancy. At this time, results from the 2 hCG tests were 1/97th and 1/126th, from the free beta-subunit test was 1/8th and the 2 beta-core assays were 1/413th and 1/240th of median normal intrauterine pregnancy levels. While hCG levels are reduced in tubal pregnancy, beta-core fragment are reduced much further. beta-core fragment measurements may offer a major improvement over hCG in diagnosing tubal pregnancy in the Emergency Room, and in screening for this life threatening disease.

Inhibin, activin, and follistatin are three families of polypeptides originally isolated and characterized from ovarian follicular fluid based on their modulation of FSH release from pituitary cell culture. In addition to their effects on FSH synthesis and secretion, inhibin and activin have other biological functions. By contrast, the physiological significance of follistatin was obscure, until it was discovered that follistatin is a binding protein to activin. Since activin binds to follistatin, it is imperative to determine the nature of the activin/follistatin binding complex. Moreover, because inhibin contains a beta-subunit derived from activin, it is important to determine whether inhibin will also bind follistatin. Using a double-ligand blotting technique, we have determined that activin-A has two binding sites for follistatin, whereas inhibin-A has only one binding site for follistatin. Therefore, these results suggest that follistatin binds to both activin and inhibin through the common beta-subunit.

ABSTRACT. Radioimmunological studies were carried out with antisera to the alpha- and betasubunits of human thyrotropin (hTSH) and to intact hTSH. In the homologous hTSH-α assay (anti-hTSH-α and 125I hTSH-α), immunoreactivity of hCG-α was the same as that of hTSH-α. The dose-response curves of intact hTSH, hLH, hFSH, and hCG, though showing less inhibition than the alpha subunit, were parallel in this assay, thus reflecting structural similarity between the alpha subunit of hTSH and the other glycoprotein hormones. Bovine TSH-α was essentially unreactive in the homologous human alpha assay. In the homologous hTSH-β assay (anti-hTSH-β and 125I hTSH-β), the immunological specificity of the beta subunit was confirmed. Intact hCG and human menopausal gonadotropin did not crossreact; however, a low degree of cross reactivity of intact hFSH and intact hLH was observed which may be explained by contamination with small amounts of hTSH. There was a low but definite cross reactivity against bTSH-β in conf...

Several libraries of monoclonal antibodies have been produced against epitopes that reside on hCG, alpha hCG, and beta hCG. Having characterized them physically, we explored their use in the construction of highly specific and sensitive immunoradiometric assays. There were several important immunochemical considerations with respect to developing assays that accurately detect low levels of free subunits in serum in the presence of high concentrations of the native hormone. These include physical properties and specificities of the monoclonal antibodies, choice of capture antibody on the solid phase support, assay design, and purity of hormone standards. Using such assays, we found early pregnancy (in vitro fertilization) to be characterized by the sequential appearance of hCG, followed by beta hCG and then alpha hCG. Molar ratios of beta hCG to alpha hCG and beta hCG to hCG were highest in early gestation. However, there was a reversal of the beta hCG to alpha hCG ratio at 12-13 weeks gestation, and an excess of free alpha hCG was observed thereafter. Except for values obtained in very early pregnancy, the beta hCG to hCG ratio remained remarkably constant at approximately 0.5% throughout gestation. In contrast, choriocarcinoma was distinguished by absolute serum beta hCG concentrations 3-100 times greater than the maximum values observed during pregnancy and, more importantly, by exceedingly high beta hCG to hCG ratios. For comparison, we studied hCG, alpha hCG, and beta hCG levels in an additional 178 patients with nontrophoblastic tumors. Ectopic production of alpha hCG and beta hCG was rare (3%), and thus far, we have been unable to demonstrate the presence of hCG in such patients. Therefore, hCG and the free subunits appear not to be useful as serological markers for nontrophoblastic tumors.

We analyzed the immunoreactive renal metabolites of the beta-subunit moieties of unlabeled highly purified hCG, hCG beta, and desialylated hCG (as-hCG) in rats by RIA and Sephadex G-100 chromatography. Infusions of hCG beta, as-hCG, or intact hCG resulted in accumulation in the kidney of a large quantity of small mol wt peptides lacking the immunological determinants of the carboxy-terminal peptide (CTP) of the beta-subunit. In the case of as-hCG, renal accumulation of these beta-core fragments was greatly enhanced when as-hCG binding to hepatic galactose receptors was inhibited by infusion of as-fetuin. The beta-core fragments in kidney had the same immunological and G-100 chromatographic characteristics as beta-core fragments in liver, suggesting similar intracellular catabolic mechanisms in these tissues. The kinetics of beta-core fragment turnover in kidney were studied after injection of hCG beta, which is cleared from the circulation within 1 h. Loss of beta CTP immunoreactivity was the initial event in hCB beta catabolism by the kidney; most of this process occurred between 7 and 30 min after injection. This was followed by a gradual reduction of the size of accumulated hCG beta metabolites over the next 60 min. The beta-core fragments that accumulated had a Kav of approximately 0.57 and a very slow degradation rate over the next 5 h (half-life greater than 6 h). Chromatographic analysis of urine obtained 6 h after beginning a continuous infusion of hCG, hCG beta, or as-hCG displayed in each case a major peak corresponding to the infused molecule, apparently intact, and a minor peak of beta CTP immunoreactivity of small mol wt. Relative to the beta CTP fragments apparent in urine, there were few beta-core fragments. These data indicate that separate fates exist for immunoreactive fragments generated by hCG beta metabolism in the rat kidney. One appears to be intracellular and similar to the liver pathway of as-hCG degradation in that it leads to the formation of long-lived beta-core fragments. The other takes place within ready access to the urinary compartment and leads to the accumulation in urine of beta-CTP fragments.

We developed a RIA for the beta-core fragment of hCG that is excreted in the urine of pregnant women and some patients with cancer. We purified beta-core from crude commercial preparations of hCG (of which beta-core is a major constituent) derived from pregnancy urine and used this purified beta-core material to immunize rabbits. One antiserum (RW25) was particularly useful in that a RIA with purified beta-core as both radioligand and reference preparation had high sensitivity for beta-core detection and low cross-reactivity with other hCG-related molecules and glycoprotein hormones. The cross-reactivities of purified hCG (CR125), hCG beta (CR125), and hCG alpha (CR125) preparations were in each instance less than 3 x 10(-3) (wt/wt). The cross-reactivities of purified pituitary glycoprotein hormones were in each instance less than 2 x 10(-4) (wt/wt). Using the RW25 RIA, virtually all beta-core immunoreactivity in pregnancy urine eluted from Sephadex G-100 in a position coincident with that of purified beta-core. Urine from men and nonpregnant women contained very low levels of beta-core immunoreactivity (less than 6.5 micrograms/L), while urine from pregnant women and patients with testicular cancer or other neoplasms had levels of beta-core immunoreactivity ranging as high as 26,000 micrograms/L. We conclude that the improved specificity of our beta-core RIA will facilitate studies of the physiology and cancer biology of beta-core molecules.

Utilizing specific radioimmunoassay and gel chromatographic separation methods, the concentration of a glycoprotein hormone subunit and the beta subunit of hCG, hLH, and hFSH was determined. The alpha glycoprotein hormone subunit is present throughout gestation in the serum and pituitary of the human fetus; little or no subunit is apparent in contrast to that of intact LH and FSH.

Human chorionic gonadotropin (hCG) beta-subunit core fragment (beta-fragment) is present in the urine of pregnant individuals as well as those with trophoblast disease and certain other cancers at concentrations 0.8 (early pregnancy) to 7 (second trimester pregnancy)-fold greater than that of hCG. The core fragment may be directly secreted by trophoblast tissue into the circulation or possibly originates from peripheral degradation of circulating hormone by the kidney. We examined the former hypothesis. We examined 24-h organ cultures of trophoblast tissue from first, second, and third trimester pregnancy. The media from this tissue contained hCG, free beta-subunit, and beta-fragment. The amount of beta-fragment present exceeded that of hCG, as was observed in second and third trimester pregnancy urine. The beta-fragment immunoreactive material produced by trophoblast tissue was compared to a standard preparation of urinary beta-fragment. The material in medium was identical to the standard beta-fragment in its elution pattern from a gel filtration column, from a reverse-phase HPLC column, from an ion-exchange gel, and from an immobilized lectin affinity column, and also by electrophoresis and immunoblotting with fragment-reactive monoclonal antibodies. We conclude that beta-fragment can also originate directly from trophoblast tissue, and could be the principal hCG beta-immunoreactive molecule secreted.

The beta subunit of TSH (TSH-beta) usually cannot be detected (less than 0.2 ng/ml) in the serum of normal individuals, whereas patients with primary hypothyroidism exhibit elevated TSH-beta levels (0.2-9.3 ng/ml), which increase further after the administration of TRH. Two patients were found to have large TSH-beta as the only form of serum TSH-beta immunoactivity. Patient A was a euthyroid woman with a goiter; TSH and alpha subunit levels were normal (1 microU/ml and 0.6 ng/ml, respectively); TSH-beta was elevated (8-24 ng/ml). Patient B was a woman with borderline hypothyroidism, an elevated serum TSH level (19 microunits/ml), a normal serum alpha level (2.4 ng/ml), and an elevated serum TSH-beta level (1.8-3.6 ng/ml). Dilutions of both patients' sera demonstrated nonparallelism of their serum TSH-beta to standard TSH-beta. The elevated serum TSH-beta levels did not increase after TRH, although TSH and alpha subunit increased appropriately. After the administration of dexamethasone or T4 to patient B, serum TSH-beta did not decrease, although TSH and alpha decreased. Gel chromatography and rechromatography of the patients' sera on a Sephadex G-100 column showed elution of all TSH-beta immunoactivity in or near the void volume (Vo; greater than 150,000 mol wt), whereas sera of hypothyroid patients demonstrated less than 7% of TSH-beta immunoactivity in the Vo. By chromatography on a Sephadex G-200 column, the TSH-beta immunoactivity had a 160,000 mol wt in patient A and 200,000 mol wt in patient B. Incubation of labeled or unlabeled TSH-beta with serum or gamma-globulin fractions from both patients resulted in no significant increase in the binding of TSH-beta to serum components, as determined by both gel chromatography and precipitation with antihuman gamma-globulin. Large TSH-beta was stable after incubation with 6 M guanidine. Ribonuclease failed to affect the large TSH-beta. Inter-chain disulfide bonding was not demonstrated in large TSH-beta after treatment with three different reducing agents (mercaptoethanol, sodium sulfite, and dithioerythritol). Treatment with trypsin did not convert the large TSH-beta immunoactivity to standard TSH-beta. These experiments demonstrated that the large TSH-beta immunoactivity was not caused by binding of TSH-beta to an immunoglobulin or other serum protein or by aggregation of TSH-beta molecules. The significance of these apparently covalently bonded large forms of TSH-beta immunoactivity is not yet known; the presence of small amounts of a large molecular weight form in the serum of hypothyroid patients and normal pituitary extracts raises the possibility that they may be components of normal TSH biosynthesis or represent posttranslational modifications.

An assay was developed to detect tritium-labeled ovine FSH beta-subunit [( 3H]oFSH beta) secreted from primary ovine pituitary cultures. This procedure used affinity-enriched antibodies raised against reduced and carbamylmethylated oFSH beta (RCM-oFSH beta) in a two-cycle immunoextraction procedure. A discrete species with an apparent mol wt of 21,000 was detected in sodium dodecyl sulfate electrophoretic patterns of immunoextracts from culture medium. This species was identified as RCM-[3H]oFSH beta by its comigration with highly purified RCM-oFSH beta, its reduction in culture media after cultures were treated with 17 beta-estradiol, which normally decreases radioimmunoassayable oFSH; and its displacement from the extracting antibodies by excess unlabeled RCM-oFSH beta. The assay was used in a pulse-chase study to determine that [3H]oFSH beta is secreted within 1-2 h of its synthesis. Prior treatment of cultures with 17 beta-estradiol did not change this timing of secretion.

Residues 121-149 of equine LH beta (eLH beta) were removed by a simple mild acid treatment procedure. The modified eLH beta, des(121-149)eLH beta, was isolated by gel permeation chromatography on Sephacryl S-200. Recombination of des(121-149)eLH beta with eLH alpha and ovine LH alpha (oLH alpha) produced LH derivatives as efficiently as recombination with native eLH beta. In rat testicular LH radioligand assay systems employed in this study the potencies of the resulting LH preparations were, in order of decreasing potency: des(121-149)eLH beta:eLH alpha hybrid greater than eLH greater than eLH alpha + beta greater than oLH greater than des(121-149)eLH beta:oLH alpha greater than oLH alpha + eLH beta (1:0.82:0.67:0.15:0.02:0.006, eLH tracer; 1:0.88:0.67:0.21:0.02:0.006, hCG tracer). In a horse testicular LH radioligand assay with eLH tracer, only the equine LH derivatives were active, and the order of potencies was the same: des(121-149)eLH beta:eLH alpha hybrid greater than eLH greater than eLH alpha + beta (1:0.58:0.46). In a rat testicular Leydig cell steroidogenesis assay, eLH was the most active preparation, but the relative potencies of the other preparations remained unchanged: eLH greater than des(121-149)eLH beta:eLH alpha greater than eLH alpha + beta greater than oLH greater than des(121-149)eLH beta:oLH alpha greater than oLH alpha + eLH beta (1:0.61:0.55:0.27:0.004:0.003). We have previously reported that the hybrid consisting of native eLH beta and oLH alpha was inactive (less than 1%) in LH receptor and steroidogenesis assays. The data reported herein confirm this observation and demonstrate that the absence of LH activity in eLH beta:oLH alpha cannot be attributed to the C-terminal extension on eLH beta, since the des(121-149)eLH beta:oLH alpha hybrid LH is also inactive. Examination of the intrinsic FSH activity of eLH in both rat and chicken testicular FSH radioligand assays produced the following results; eLH, recombined eLH subunits, and des(121-149)eLH beta:eLH alpha were all of the same potency (13% and 0.9% as active as eFSH in rats and chickens, respectively). We conclude that the C-terminal extension on eLH and eCG beta-subunits is not involved in subunit association, LH receptor binding, or FSH receptor binding. The derivative des(121-149)eLH beta:eLH alpha provides a model compound that may be useful in determining the role, if any, of the glycoprotein hormone C-terminal extension that appears to have arisen independently at least twice in mammalian evolution.

Earlier, we reported that second trimester human fetal kidney and, to a much lesser extent, human fetal liver were capable of synthesizing and secreting the beta-subunit of hCG. Recently, we also have shown that these tissues, likewise, synthesize and secrete the alpha-subunit of hCG. The hCG produced is biologically active. To determine the cellular localization of these peptides, immunocytochemical studies were performed on human fetal tissues using antibodies against beta hCG, alpha hCG, and the intact hormone. Placental syncytiotrophoblast served as an immunopositive control. In the human fetal kidney, the ascending (thick) limb of the loop of Henle, distal convoluted tubule, and occasional cells in the collecting ducts were distinctly immunopositive for both beta hCG and the alpha-subunit. Small amounts of light positive staining occurred in only a few hepatocytes. Placental syncytiotrophoblast was routinely positive for both subunits, but fetal lung and striated muscle were negative. These immunocytochemical results indicate that immunoreactive beta hCG as well as the alpha-subunit are present in placental syncytiotrophoblast, in the distal renal nephron, and in a limited population of hepatocytes. The qualitative number and intensity of immunopositive cells closely correlate with the quantitative amounts of their hCG subunit synthesis. Taken together with our previous biosynthetic data, the immunocytochemical localization reported here indicates the probable cellular sites of alpha- and beta hCG synthesis in these tissues. The presence of comparable alpha- and beta-subunit staining in identical cell populations suggests that both hCG subunits and, therefore, perhaps intact hCG are produced at these same cellular sites during fetal life.

We measured intact hCG, free alpha hCG, and free beta hCG levels in amniotic fluid and maternal serum using specific monoclonal antibody-based immunoradiometric assays. Compared to maternal serum, amniotic fluid had low levels of intact hormone along with high levels of its free subunits. The mean amniotic fluid hCG level was 1 mg/L at 13 weeks, and it progressively decreased to 0.100 mg/L by the 23rd week. Amniotic fluid alpha hCG levels were highest at 15 and 16 weeks (0.340 mg/L) and rapidly declined to concentrations less than 0.050 mg/L at 21 weeks. Amniotic fluid beta hCG concentrations were about 0.200 mg/L between 13 and 16 weeks and progressively decreased until the 23rd week. In vitro stability studies of hCG and its subunits demonstrated that dissociation of hCG into its subunits was not responsible for these results. Both hCG and free subunits detected in amniotic fluid were indistinguishable from standard hCG and free subunit preparations, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our findings are consistent with the hypothesis that the presence of hCG and free subunits in the amniotic fluid represents secretion from the trophoblastic tissue into the amniotic fluid compartment. Furthermore, high serum hCG levels in association with low levels of its free subunits are probably the result of polarized secretion of the intact hormone from the syncytiotrophoblasts into the maternal circulation.

The amino acid sequence of the β subunit of hLH has been deduced from tryptic and thermolytic peptides of the reduced and carboxymethylated subunito hLH-β consists of 115 residues. A single tryptophan in the molecule occurs at position 8, hLH-β exhibits greater homology with hCG-B (89 identical positions) than with ovine LH-β (77 identical positions). Heterogeneity exists at the carboxy terminal portion of the molecule.

An in vivo system to study the uptake of human chorionic gonadotropin (HCG) by mouse ovaries has been developed. At various times following the injection of biologically active, radioiodinated HCG, the specific activity of various tissue specimens was measured and found to be significantly greater in ovaries than in blood, liver, or kidney. In contrast, 125I-albumin was not specifically concentrated by the ovary. The ovarian uptake of 125I-HCG has also been measured after the administration of 125I-HCG in combination with gonadotropin molecules or their derivatives. Increasing amounts of biologically active, unlabeled HCG injected with iodinated HCG progressively inhibited the uptake of 125I-HCG. Human luteinizing hormone (HLH) was also a potent competitor of 125I-HCG uptake. Asialo- HCG was less effective than native HCG in blocking the uptake of 125I-HCG. The beta subunit of HCG possessed some inhibitory effect on 125IHCG binding, but the alpha subunit had little if any effect. It is probable that some ...

The exophthalmogenic activity of the beta subunit of bovine thyrotropin is only 10% to 20% that of the thyrotropin molecule or of an exophthalmogenic factor produced by partial pepsin digestion of purified thyrotropin preparations. The alpha subunit of thyrotropin, luteinizing hormone, and both subunits of luteinizing hormone have no exophthalmogenic activity.

Immunoaffinity adsorption techniques, utilizing specific antisera for hCG and its subunits bound to Sepharose 4B, have been employed to separate hCG alpha beta dimer and free subunits of hCG. As previously reported by this and a number of other laboratories, trophoblast cells (in vivo and in vitro) produce free alpha subunit in addition to hCG dimer. We have now shown that cultured JAr choriocarcinoma cells also secrete free beta subunit: 37% of the total beta subunit (combined and free) secreted by JAr cells is in the free form. Moreover, in pooled sera from choriocarcinoma patients 15% of total beta subunit is free, and in media from placental explant cultures and in pooled first trimester pregnancy sera 11% and 6.5%, respectively, of total beta subunit are in the free form. The free beta s are all of similar mol wt to the combined forms, and associate with urinary hCG alpha to form hCG. Free alpha s, which are larger than the combined forms, are unable to associate with urinary hCG beta to form hCG. We propose that the supply of combinable alpha subunit, rather than beta, limits dimer formation.