Gonadotropin beta subunits determine the rate of assembly and the oligosaccharide processing of hormone dimer in transfected cells

The glycoprotein hormones lutropin (LH) and chorionic gonadotropin (CG) share a common structure consisting of an identical alpha subunit noncovalently linked to a hormone-specific beta subunit. While LH is produced in the anterior pituitary, CG is synthesized in placenta. To compare the assembly, processing, and secretion of human LH and CG in the same cell type, we have expressed their subunits, individually and together, in mouse C-127 mammary tumor cells. Analysis of transfected clones revealed an unexpected difference in the secretion of individually expressed subunits. Whereas alpha and CG beta subunits were rapidly and quantitatively secreted, only 10% of newly synthesized LH beta subunit reached the medium. The remaining subunit was found in an intracellular, endoglycosidase H (endo H)-sensitive pool that had a turnover rate of approximately 8 h. Coexpression with alpha subunit resulted in "rescue" of LH beta subunit by formation of LH dimer, which was efficiently secreted. However, combination of LH beta with alpha was slow, with an overall efficiency of only 50% despite the presence of excess alpha. In contrast, CG beta was rapidly assembled with the alpha subunit after synthesis. The two beta subunits also differed in their influence on the N-linked oligosaccharide processing of combined alpha. The oligosaccharides of LH dimer were endo H resistant, while those of CG dimer remained partially endo H sensitive. Thus, despite a high degree of homology between LH beta and CG beta, the two subunits differ in their secretion as free subunits, their rate of assembly with alpha subunit, and in their effect on the N-linked oligosaccharide processing of combined alpha.     

The recombinant equine LHβ subunit combines divergent intracellular traits of human LHβ and CGβ subunits

The pituitary LHβ and placental CGβ subunits are products of different genes in primates. The major structural difference between the two subunits is in the carboxy-terminal region, where the short carboxyl sequence of hLHβ is replaced by a longer O-glycosylated carboxy-terminal peptide in hCGβ. In association with this structural deviation, there are marked differences in the secretion kinetics and polarized routing of the two subunits. In equids, however, the CGβ and LHβ subunits are products of the same gene expressed in the placenta and pituitary (LHβ), and both contain a carboxy-terminal peptide. This unusual expression pattern intrigued us and led to our study of eLHβ subunit secretion by transfected Chinese hamster ovary and Madin–Darby canine kidney cells. In continuous labeling and pulse-chase experiments, the secretion of the eLHβ subunit from the transfected Chinese hamster ovary cells was inefficient (medium recovery of 16%–25%) and slow (t_1/2 > 6.5 hours). This indicated that, the secretion of the eLHβ subunit resembles that of hLHβ rather than hCGβ. In Madin–Darby canine kidney cells grown on Transwell filters, the eLHβ subunit was preferentially secreted from the apical side, similar to the hCGβ subunit secretory route (∼65% of the total protein secreted). Taken together, these data suggested that secretion of the eLHβ subunit integrates features of both hLHβ and hCGβ subunits. We propose that the evolution of this intracellular behavior may fulfill the physiological demands for biosynthesis of the LH and CG β-subunits in the pituitary and placenta, respectively.     

Human chorionic gonadotropin beta-core fragment is directly produced by cancer cells

The present study was undertaken to investigate whether hCG beta-core fragment (hCGbeta cf) was directly produced by cancer cells. Fifteen cell lines, including four choriocarcinoma and five ovarian cancer cell lines, were tested, and immunoreactivity of hCGbeta cf was present in the culture media of five of the cell lines. It was also present in the culture media of Chinese hamster ovary (CHO) cells transfected with hCGbeta gene. In addition to hCGbeta cf, gel chromatography and Western blot analysis of the culture media showed the presence of an hCGbeta cf immunoreactive material with a molecular weight of approximately 40 kDa. In an in vivo study, hCGbeta cf immunoreactivity was detected in the sera of the mice transplanted with NaUCC-3 choriocarcinoma cells, although the ratios of hCGbeta cf/hCG and hCGbeta cf/free hCGbeta were lower than those in the culture medium. Incubation experiments of purified hCGbeta cf in the serum showed no substantial decrease in its values, ruling out the possibility that formation of a macromolecule with serum components may mask hCGbeta cf immunoreactivity in the serum. Taken together, these results indicate that hCGbeta cf immunoreactive materials are directly produced by cancer cells and hCGbeta cf is not a urinary metabolite of hCG or hCGbeta alone. Also, reduced levels of hCGbeta cf in the serum compared with that of intact hCG or free hCGbeta are likely due to its short half-life.     

Expression of biologically active human chorionic gonadotropin and its subunits by recombinant vaccinia virus

Vaccinia virus (VV) expression vector was used to clone the genes for coding alpha and beta subunits of human chorionic gonadotropin (hCG). Recombinant viruses VSL3 and VSS1 containing these genes were selected as blue coloured plaques on the basis of co-expression of Escherichia coli beta-galactosidase in the infected cells. CV-1 cells when infected with VSL3 or VSS1 secreted 2.4 and 1.8 micrograms of alpha and beta hCG subunits, respectively, per 3 x 10(6) cells after 24 h of infection. The subunit proteins expressed individually had immunoreactivity with monoclonal and polyclonal antibodies specific to hCG. The subunit hormonal peptides associated with each other during co-infection to form the complete hCG dimer, which was biologically active as evident from the induction of steroidogenesis in a mouse Leydig cell system.     

Purification and properties of baboon chorionic gonadotrophin

Baboon CG from the urine and placenta (Days 33-45 of gestation) was purified by ammonium acetate/alcohol extraction followed by ionic exchange, gel filtration and affinity chromatography. The CG activity was monitored using a double-antibody radioimmunoassay utilizing anti-baboon CG and hCG both as the labelled ligand and standard. Biological activity was measured by the rat luteal cell radioreceptor assay. The purified preparation exhibited heterogeneity in terms of its behaviour during ionic exchange chromatography and isoelectric focussing. Like hCG, baboon CG was made up of two non-covalently linked subunits: the Stokes' radii were 36.5, 29.0 and 22.0 X 10(-10) m for native CG, the beta subunit and the alpha subunit. The material focussed between pH 4.2 and 5.5. Relative to the second international hCG standard the biological potency of the purified urinary baboon CG was 4058 i.u. whilst the immunological activity was 4364 i.u. It is concluded that baboon and human CG are very similar with respect to their physicochemical properties and that baboon CG can be purified by the methods that have been developed for hCG.     

Immunosuppressive activity of human chorionic gonadotrophin preparations in vivo: evidence for gonadal dependence

Human chorionic gonadotrophin preparations (hCG), when injected ip daily for 4 days, suppress the delayed-type hypersensitivity (DTH) response of mice to sheep red blood cells. Preparations of crude hCG, purified hCG subunits, and hCG that was formed by recombining the purified subunits showed immunosuppressive activity in accord with their gonadotrophic activity. The immunosuppressive effects in male and female mice were comparable. However, removal of the gonads completely abrogated the immunosuppressive activity of hCG in both males and females, suggesting that the effect of hCG is mediated by a factor released from the gonads. We conclude that the hCG molecule itself exhibits immunosuppressive activity in vivo in both male and female mice and that the gonads are required for the expression of this activity.     

Mutagenesis and gene transfer define site-specific roles of the gonadotropin oligosaccharides

Human chorionic gonadotropin (hCG), luteinizing hormone (LH), follicle-stimulating hormone and thyroid-stimulating hormone are a family of glycoprotein hormones that share a common alpha subunit but differ in their hormone-specific beta subunits. Using site-directed mutagenesis and gene-transfer, we analyzed the role of the N-linked oligosaccharides of alpha and chorionic gonadotropin (CG)beta in the secretion, assembly, and biologic activity of hCG. Absence of carbohydrate at alpha asparagine (Asn) 52 decreased combination with CG beta but did not alter monomer secretion. Absence of the alpha Asn78 oligosaccharide increased the degradation of the alpha subunit, but the presence of CG beta stabilized this alpha mutant in an efficiently formed dimer complex. Alternatively, absence of both alpha oligosaccharides slowed both secretion and dimer formation but allowed an intermediate level of alpha secreted or dimerized compared to the single-site mutants. Analysis of the CG beta glycosylation mutants revealed that absence of the Asn30 oligosaccharide, but not Asn13, slowed secretion but not assembly, whereas absence of both oligosaccharides slowed both secretion and dimer formation. Analysis of the receptor binding of the hCG glycosylation mutants showed that absence of any or all of the hCG N-linked oligosaccharides had only a minor effect on receptor affinity of the derivatives. However, the absence of alpha Asn52, but not the alpha Asn78 or the CG beta carbohydrate units, reduced the steroidogenic effect, unmasked differences in the beta oligosaccharides, and converted the deglycosylated derivatives into antagonists.     

Yoked complexes of human choriogonadotropin and the lutropin receptor: evidence that monomeric individual subunits are inactive

Human choriogonadotropin (hCG) contains an alpha-subunit, common to other members of the glycoprotein hormone family, and a unique beta-subunit that determines hormone specificity. It is generally thought that heterodimer formation is obligatory for full hormonal activity, although other studies have indicated that individual subunits and homodimeric hCGbeta were capable of low affinity binding to the LH receptor (LHR) and subsequent activation. Previously, we constructed two yoked hormone (hCG)-LHR complexes, where the two hormone subunits and the heptahelical receptor were engineered to form single polypeptide chains, i.e. N-beta-alpha-LHR-C and N-alpha-beta-LHR-C. Expression of both complexes led to constitutive stimulation of cAMP production. In the present study, we investigated whether the human alpha-subunit and hCGbeta can act as functional agonists when covalently attached to or coexpressed with the LH receptor. Our initial results showed that hCGbeta, but not alpha, was able to activate LHR with an increase in intracellular cAMP in human embryonic kidney 293 cells but not in Chinese hamster ovary or COS-7 cells. Further examination of this apparent cell-specific agonist activity of hCGbeta revealed that low levels of endogenous alpha-subunit were expressed in human embryonic kidney 293 cells, thus enabling sufficient amounts of active heterodimer to form with the transfected hCGbeta to activate LHR. The studies in Chinese hamster ovary and COS-7 cells clearly demonstrate that, even under experimental conditions where hormone-receptor interactions are maximized, individual subunits of hCG can not act as functional agonists, at least in their monomeric form.     

A single-chain tetradomain glycoprotein hormone analog elicits multiple hormone activities in vivo

We previously demonstrated that genetically linking one or more of the glycoprotein hormone-specific beta subunit genes to the common alpha subunit resulted in single-chain analogues that were bioactive in vitro. The ability of such large structures to bind their cognate receptors with high affinity supported the hypothesis that extensive flexibility exists between the ligand and receptor to establish a functional complex. To further characterize the extent of this conformational flexibility, we engineered a single-chain analogue that consists of sequentially linked thyroid-stimulating hormone (TSH) beta, follicle-stimulating hormone (FSH) beta, and chorionic gonadotropin (CG) beta subunits to the alpha subunit and expressed this chimera in transfected CHO (Chinese hamster ovary) cells. Because the four subunits are genetically linked and expressed as a single-chain, this analogue presumably lacks significant native structural features of the individual heterodimers. However, it exhibited FSH, CG, and TSH activities in vitro. Here, we test whether this nonnative structure would be stable in vivo and thus biologically active. Using a variety of bioassay protocols, we demonstrate that the analogue elicits multihormone activities when injected in vivo. First, treatment with the analogue caused increases in ovarian and uterine weights and resulted in elevated serum estradiol. Second, the analogue-stimulated ovarian follicle growth and pharmacologically rescued in vivo FSH deficiency similar to recombinant human FSH or equine CG (eCG) as confirmed by induction of aromatase in the ovaries of FSHbeta knockout mice. Third, in a superovulation protocol, when primed with eCG, the analogue elicited a dose-dependent ovulatory response comparable with that by native heterodimeric human CG. Finally, the analogue-stimulated thyroxin production in hypothyroid mice similar to the pituitary-derived human TSH standard. Based on these data, we conclude that a single-chain tetradomain glycoprotein hormone analogue, despite its presumed altered conformation, is stable and biologically active in vivo. Our results establish the permissiveness and conformational plasticity with which the glycoprotein hormones are recognized in vivo by their target cell receptors.     

Site-specific mutagenesis defines the intracellular role of the asparagine-linked oligosaccharides of chorionic gonadotropin beta subunit

The beta subunit of human chorionic gonadotropin contains two asparagine (N)-linked oligosaccharides. To examine the structural and functional roles of these oligosaccharide units in vivo, we constructed mutant genes containing alterations in either the asparagine or threonine codons of the two glycosylation consensus sequences and inserted them into a eukaryotic expression vector. Wild-type and mutant CG beta proteins were expressed in Chinese hamster ovary cells alone or in the presence of native alpha subunit. Pulse-chase analysis of the beta-expressing clones showed that absence of the second N-linked sugar but not the first slows secretion 1.6-1.8-fold; absence of both N-linked units slows secretion 2-2.4-fold. Analysis of dimer clones reveals that greater than 80% of the native and glycosylation mutant CG beta subunits are secreted as dimer. However, pulse-chase analysis of these clones also reveals that the mutants completely devoid of N-linked sugars but not the single site mutants are slow to assemble with the alpha subunit. Thus, in vivo the two N-linked oligosaccharides of CG beta are critical for efficient secretion and assembly with the alpha subunit and are likely important for proper folding of the CG beta subunit.     

Effect of human chorionic gonadotropin derivatives on leydig cell function.

BACKGROUND: Several human chorionic gonadotropin (hCG) derivatives have been detected in healthy human subjects, indicating that they may play a role in cell function. These hCG derivatives include deglycosylated hCG, proteolytic digestion products of hCG and free alpha and beta subunits of the hormone. It is well documented that testicular Leydig cells are responsive to luteinising hormone (LH) or its analogue hCG. These hormones have high affinity for LH/hCG receptors on the plasma membrane. METHODS: We designed functional and binding studies to compare the effects of native hCG and several hCG derivatives on a rat Leydig cell system. The molecular weight of the hCG derivatives was determined by SDS-PAGE and the binding affinity to LH/hCG receptors was measured by a radioligand assay. In addition, their ability to produce testosterone, cyclic AMP and arachidonic acid release was also studied. RESULTS: These hCG derivatives, with the exception of the free beta subunit, were able to bind to LH/hCG plasma membrane receptors with different affinities than that of native hCG. In addition, hCG derivatives did not increase intracellular cAMP levels or arachidonic acid release. However, they did increase testosterone production. CONCLUSION: Taken together, the results of this study lead us to suggest that these hCG derivatives may regulate the action of the native hormone in Leydig cells and are, thus, molecules of physiological relevance.     

Transgenic models to study gonadotropin function: the role of follicle-stimulating hormone in gonadal growth and tumorigenesis.

The role of FSH in gonadal tumorigenesis and, in particular, in human ovarian cancer has been debated. It is also unclear what role the elevated FSH levels in the inhibin-deficient mouse play in the gonadal tumorigenesis. To directly assess the role of FSH in gonadal growth, differentiation, and gonadal tumorigenesis, we have generated both gain-of-function and loss-of-function transgenic mutant mice. In the gain-of-function model, we have generated transgenic mice that ectopically overexpress human FSH from multiple tissues using a mouse metallothionein-1 promoter, achieving levels far exceeding those seen in postmenopausal women. Male transgenic mice are infertile despite normal testicular development and demonstrate enlarged seminal vesicles secondary to elevated serum testosterone levels. Female transgenic mice develop highly hemorrhagic and cystic ovaries, have elevated serum estradiol and progesterone levels, and are infertile, mimicking the features of human ovarian hyperstimulation and polycystic ovarian syndromes. Furthermore, the female transgenic mice develop enlarged and cystic kidneys and die between 6-13 weeks as a result of urinary bladder obstruction. In a complementary loss-of-function approach, we have generated double-homozygous mutant mice that lack both inhibin and FSH by a genetic intercross. In contrast to male mice lacking inhibin alone, 95% of which die of a cancer cachexia-like syndrome by 12 weeks of age, only 30% of the double-mutant male mice lacking both FSH and inhibin die by 1 yr of age. The remaining double-mutant male mice develop slow-growing and less hemorrhagic testicular tumors, which are noted after 12 weeks of age, and have minimal cachexia. Similarly, the double-mutant female mice develop slow-growing, less hemorrhagic ovarian tumors, and 70% of these mice live beyond 17 weeks. The double-mutant mice demonstrate minimal cachexia in contrast to female mice lacking only inhibin, which develop highly hemorrhagic ovarian tumors, leading to cachexia and death by 17 weeks of age in 95% of the cases. The milder cachexia-like symptoms of the inhibin and FSH double-mutant mice are correlated with low levels of serum estradiol and activin A and reduced levels of aromatase mRNA in the gonadal tumors. Based on these and our previous genetic analyses, we conclude that elevated FSH levels do not directly cause gonadal tumors. However, these results suggest FSH is an important trophic modifier factor for gonadal tumorigenesis in inhibin-deficient mice.     

Structure, pathology and function of the N-linked sugar chains of human chorionic gonadotropin

Human chorionic gonadotropin (hCG) contains five acidic N-linked sugar chains, which are derived from three neutral oligosaccharides by sialylation. Each of the two subunits (hCGalpha and hCGbeta) of hCG contain two glycosylated Asn residues. Glycopeptides, each containing a single glycosylated Asn, were obtained by digestion of hCGalpha with trypsin, and of hCGbeta with chymotrypsin and lysyl endopeptidase. Comparative study of the sugar chains of the four glycopeptides revealed the occurrence of site-directed glycosylation. Studies of the sugar chains of hCGs, purified from urine of patients with various trophoblastic diseases, revealed that choriocarcinoma hCGs contain sialylated or non-sialylated forms of eight neutral oligosaccharides. In contrast, hCGs from invasive mole patients contain sialyl derivatives of five neutral oligosaccharides. The structural characteristics of the five neutral oligosaccharides, detected in choriocarcinoma hCGs but not in normal placental hCGs, indicate that N-acetylglucosaminyltransferase IV (GnT-IV) is abnormally expressed in the malignant cells. This supposition was confirmed by molecular biological study of GnT-IV in placenta and choriocarcinoma cell lines. The appearance of tumor-specific sugar chains in hCG has been used to develop a diagnostic method of searching for malignant trophoblastic diseases. In addition, a summary of the current knowledge concerning the functional role of N-linked sugar chains in the expression of the hormonal activity of hCG has been presented.     

Clinical utility of hyperglycosylated hCG in serum taken before hydatidiform mole evacuation to predict persistent trophoblastic disease

OBJECTIVE: Human chorionic gonadotropin (hCG) is widely used in the management of hydatidiform mole and persistent trophoblastic disease (PTD). Studies on hyperglycosylated human chorionic gonadotropin (invasive trophoblast antigen, ITA) in PTD are limited. In serum samples taken before evacuation of molar pregnancies we measured the concentrations of free hCG beta-subunit (free hCGbeta), "total" hCG (hCG+hCGbeta) and ITA, and determined whether ITA, the two other hCG analytes, or the calculated ratios of hCGbeta/hCG+hCGbeta, hCGbeta/ITA and hCG+hCGbeta/ITA could predict the later development of PTD. DESIGN: A retrospective study based on blood specimens collected in the Dutch Central Registry for Hydatidiform Moles. The study group comprised 97 patients with hydatidiform moles who did not develop PTD after mole evacuation and 33 patients who did develop PTD. Methods: Serum samples from 130 patients with hydatidiform mole with or without PTD were assayed using specific (radio)immunoassays for free hCGbeta, total hCG, and ITA. From these analytes we also calculated the ratios hCGbeta/hCG+hCGbeta, hCGbeta/ITA, and hCG+hCGbeta/ITA. To predict the development of PTD from these analytes and parameters we performed receiver-operating characteristic (ROC) curve analysis, resulting in areas under the curve (AUCs) that represented the diagnostic accuracy which was rated in a range from excellent (AUC >0.9 or <0.1) to poor (AUC 0.4-0.6). Results: The diagnostic accuracy of ITA was moderate (0.618) and not different from that of free hCGbeta (0.610) and hCG+hCGbeta (0.622). CONCLUSIONS: ITA as well as the other analytes and parameters in serum taken prior to evacuation from patients with molar pregnancies cannot be used to predict the subsequent development of persistent trophoblastic disease.     

Site-specific processing of the N-linked oligosaccharides of the human chorionic gonadotropin alpha subunit

Two forms of the gonadotropin alpha subunit are synthesized in placenta and in human chorionic gonadotropin (hCG)-producing tumors: an uncombined (monomer) form and a combined (dimer) form. These forms show differences in their migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The slower migration of the monomeric form on sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been attributed to a different glycosylation pattern. Previous studies demonstrated different roles of each of the two alpha N-linked glycosylation sites (Asn-52 and Asn-78) in secretion of the uncombined subunit and the biologic activity of hCG dimer. To assess the influence of formation of dimer on the processing pattern at the individual sites, we characterized the N-linked oligosaccharides of monomer and dimer forms of recombinant human choriogonadotropin alpha subunit. Two approaches were employed. First, site-directed mutagenesis was used to alter the two N-linked oligosaccharide attachment sites, thus allowing the expression of alpha subunits containing only one glycosylation site. Second, tryptic glycopeptides of the wild-type subunits were examined. Concanavalin A (ConA) binding and sialic acid content indicated that the oligosaccharides at each glycosylation site of the uncombined alpha subunit are processed differently. Oligosaccharides present at Asn-52 are almost exclusively ConA-unbound and contain three sialic acid residues. The majority of Asn-78-linked oligosaccharides are ConA-bound and disialylated. Both sites are processed independently because no significant differences were observed between the oligosaccharides at the same sites in wild-type and mutant monomeric alpha subunits. By contrast, the majority of the oligosaccharides at both glycosylation sites of the dimer alpha are bound to ConA. Thus, combination primarily affects the processing pattern of the Asn-52-linked species. Because glycosylation at this site is essential for hCG assembly and signal transduction, these data imply a critical link between the site-specific processing and hormone function.     

Gonadotropin alpha subunit. Differential processing of free and combined forms in human trophoblast and transfected mouse cells

The gonadotropins luteinizing hormone, follicle-stimulating hormone, and human chorionic gonadotropin are composed of two noncovalently linked subunits, alpha and beta. The alpha subunit, identical in all three hormones, is produced in excess over the unique beta subunits by pituitary and placenta, and is secreted as uncombined, or free subunit. Free alpha subunit from both tissues has a larger molecular weight than the dimer form. In bovine pituitary an extra O-linked oligosaccharide is added to free alpha subunit, and this modification has recently been detected at an analogous position (threonine 39) on human alpha subunit secreted by choriocarcinoma cells. To assess the contribution of N-linked and O-linked oligosaccharides to the heterogeneity of human free alpha subunit, we have compared free alpha with human chorionic gonadotropin alpha secreted by explants and cultured cytotrophoblasts of human first trimester placenta. We have also examined the free and combined forms of human alpha subunit expressed in transfected C-127 mouse mammary tumor cells. Processing of the alpha subunit in placental and C-127 cells was similar. Tryptic mapping of placental-derived and transfected alpha subunits indicated that O-glycosylation at threonine 39 was not a major modification. In the presence of the oligosaccharide processing inhibitor swainsonine the difference in size between the free and combined forms of alpha was eliminated in both placental and C-127 cells, indicating that the two forms of alpha differed in their N-linked oligosaccharides. Furthermore, the oligosaccharides of free alpha subunits from placental and transfected cells were resistant to endoglycosidase H, but the combined forms of alpha were partially sensitive to the enzyme. Thus, in human first trimester placenta and mouse C-127 cells, combination of alpha with human chorionic gonadotropin beta alters the processing of N-linked oligosaccharides on alpha subunit.     

A method for assessing the reassembly of a multisubunit glycoprotein by western blotting

The Western blot procedure has been adapted to detect the reassembly of a two-subunit glycoprotein, urinary human chorionic gonadotropin (hCG), directly on the nitrocellulose. This glycoprotein is composed of two nonidentical subunits, alpha and beta. A simple procedure using immunoblotting has been developed to detect reassembly of the monomers to dimer. Three monoclonal antibodies were required for the development of this method: A109, which binds the alpha subunit or hCG; B105, which binds the beta subunit or hCG; and B107, specific for the intact hCG dimer. The alpha subunit and beta subunit of hCG were each electrophoresed and transferred to nitrocellulose, and the transfer was then incubated with the appropriate complementary subunit; reassembly of the dimer was determined by the binding of the monoclonal antibody B107. Evidence that the reassembly occurs directly on the nitrocellulose comes from the fact that B107 immunoreactivity is detected at the molecular weight position of the subunit and not at the dimer molecular weight. A genetically expressed recombinant form of the alpha subunit was also tested for its ability to recombine with the opposite subunit to produce the dimer. The recombinant alpha subunit was determined to have additional carbohydrate which interfered with the binding of the beta subunit. N-Glycanase digestion of the recombinant alpha subunit produced a form which, when incubated with the beta subunit, did recombine on the nitrocellulose and could be recognized by B107.     

Differential regulation of hCG alpha and beta subunit mRNAs in JEG-3 choriocarcinoma cells by 8-bromo-cAMP

The coordinate regulation of human chorionic gonadotropin (hCG) subunit synthesis by JEG-3 choriocarcinoma cells was studied at the pretranslational level. The responses of the hCG alpha and beta mRNAs were measured during stimulation with the potent cAMP analog 8-bromo-cAMP (8-Br-cAMP) using 32P-labeled hCG alpha and beta cDNA probes. The hCG alpha mRNA (850 bases) and beta mRNA (1050 bases) from JEG-3 cells were identical in size to that of their respective mRNAs from placenta, by Northern blot analysis. After 48 h of stimulation with 2 mM 8-Br-cAMP, production of immunoreactive alpha and beta subunits increased 25- and 52-fold, respectively; corresponding levels of the alpha and beta mRNAs increased 36- and 43-fold, respectively, in a dot blot hybridization assay. Total cellular protein, DNA content, and messenger RNA pools were not altered by treatment with 8-Br-cAMP. The temporal coordination of the expression of the hCG alpha- and beta-subunit genes was examined by comparing the time course of stimulation of the respective mRNAs and the production of immunoreactive subunits. The kinetic responses of the alpha and beta mRNAs differed: the increase in hCG alpha mRNA preceded the increase in hCG beta mRNA, while levels of free alpha subunit and intact hCG increased in parallel with the increase in beta mRNA. hCG alpha mRNA levels increased rapidly between 8 and 24 h after the addition of 8-Br-cAMP, and approached a plateau by 48 h. The levels of hCG beta mRNA increased steadily throughout the 8-48 h period. These results demonstrate that the cAMP analog 8-Br-cAMP differentially regulates hCG subunit biosynthesis in JEG-3 cells at a pretranslational level, and that the stimulation by 8-Br-cAMP in this system appears to be relatively selective for hCG subunits.     

Transgenic offspring by transcaryotic implantation of transgenic ovaries into normal mice

Female transgenic mice may be unable to reproduce successfully if the product encoded by the transgene results in pathological changes or affects the fertility of the mouse. To approach this problem, we have produced chimaeras by transferring the ovaries of transgenic mice into normal mice of the same strain. Such chimaeras will be an ideal tool for investigating the interactions between transgenic ovaries and normal mice or vice versa. Here we show that, using this method, we were able to get large numbers of transgenic offspring even from founder transgenic female mice that were themselves infertile as a result of the overexpression of growth hormone genes. Although none of the ovary recipients were given immunosuppressant treatment, 60% of the recipients had biologically active ovaries over a mean period of about 100 days.