Studies with human, baboon, and vervet monkey chorionic gonadotrophins

Vervet monkey, baboon, and human chorionic gonadotrophin(CG) were partially purified and their activity was monitored using radioimmuno- and radioreceptor-assays. They showed similar behaviour during ion exchange chromatography and isoelectric focusing and had isoelectric points between pH 4.50 and 5.13. The Stokes' radii were 32A, 34A, and 33A for vervet monkey, baboon, and human CG, respectively. Corresponding immunoassay potencies were 476.9, 1,143.1, and 9,600 IU/mg, while the bioassay estimates were 491.7, 997.8, and 8,950.0 IU/mg.     

Increase in ovarian kallikrein activity during ovulation in the gonadotrophin-primed immature rat

The ovulatory process was initiated in 25-day-old rats by injecting them with hCG (10 i.u., s.c.) 2 days after the animals had been primed with PMSG (10 i.u., s.c.). At 2-h intervals after hCG, the ovaries were extracted and assayed for glandular kallikrein activity by using a chromogenic substrate (H-D-Val-Leu-Arg-p-nitroanilide) which exhibits optical density (at 405 nm) upon hydrolysis. In 0-h control ovaries the activity was 12.5 x 10(-3) kallikrein units (KU)/mg protein and it increased to a peak of 56.6 x 10(-3) KU/mg at 12 h after hCG, when the follicles first began to rupture. The kallikrein activity was distinguishable from ovarian plasminogen activator activity on the basis of pH optima and response to trypsin inhibitor (SBTI). The activity was inhibited by a s.c. dose of indomethacin of 0.3 mg/rat, or higher, and this dosage inhibited ovulation. The results suggest that kallikrein activity contributes to the degradation of Graafian follicles during ovulation in mammals.     

Synthesis of multi-subunit domain gonadotropin complexes: a model for alpha/beta heterodimer formation

The human glycoprotein hormones chorionic gonadotropin (CG), thyrotropin (TSH), lutropin (LH), and follitropin (FSH) are heterodimers, 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. Little information is available regarding the steps associated with the assembly reaction. It is unclear if the initial alpha beta engagement results either in the formation of only mature heterodimer or if the nascent complex is reversible and can undergo an exchange of subunits or combine transiently with an additional subunit. This is relevant for the case of LH and FSH, because both are synthesized in the same cell (i.e., pituitary gonadotrophs) and several of the alpha subunit sequences required for association with either the LH beta or FSH beta subunits are different. Such features could favor the generation of short-lived, multi-subunit forms prior to completion of assembly. Previously, we showed that the CG beta or FSH beta subunit genes can be genetically fused to the alpha gene to produce biologically active single chains, CG beta alpha and F beta alpha, respectively. Studies using monoclonal antibodies sensitive to the conformation of the hCG subunits suggested that in contrast to the highly compact heterodimer, the interactions between the beta and alpha domains in the single chain are in a more relaxed configuration. That the tethered domains do not interact tightly predicts that they could combine with an additional subunit to form triple domain complexes. We tested this point by cotransfecting CHO cells with the genes encoding F beta alpha and the CG beta subunit or the CG beta alpha and FSH beta monomer. The CG beta subunit combined noncovalently with F beta alpha to form a F beta alpha/CG beta complex. Ternary complex formation was not restricted to a specific set of single chain/monomeric subunit, because a CG beta alpha/FSH beta complex was also detected implying that triple domain intermediates could be transiently generated along the secretory pathway. Monoclonal antibodies specific for the CG heterodimer recognized the F beta alpha/CG beta complex, which suggests that the epitopes unique for dimeric CG were established. In addition, media containing F beta alpha/CG beta displayed high-affinity binding to both CG and FSH receptors. The presence of CG activity is presumptive for the existence of a functional F beta alpha/CG beta complex, because neither F beta alpha nor the uncombined CG beta subunit binds to CG receptor. These data show that the alpha subunit of the tether, although covalently linked to the FSH beta domain, can functionally interact with a different beta subunit implying that the contacts in the nascent alpha beta dimer are reversible. The formation of a functional single chain/subunit complex was not restricted to the FSH single chain/CG beta subunit since CG single chain interacts with the monomeric FSH beta subunit and exhibits FSH activity. The presence of the triple domain configuration does not abolish bioactivity, suggesting that although the gonadotropins are heterodimers, the cognate receptor is capable of recognizing a larger ligand composed of three subunit domains.     

Characterization of the expression products of recombinant human choriogonadotropin and subunits

Human choriogonadotropin (hCG) is a placental glycoprotein hormone composed of a 92-amino acid alpha subunit noncovalently linked to a 145-amino acid beta subunit. We report here the expression of biologically active hCG in mouse C127 cells transfected with expression vectors containing the DNA coding for both subunits. In addition, the same cell line was used to express the alpha subunit alone. The expression products were purified by affinity chromatography using specific monoclonal antibodies to hCG or its subunits. The system secreting biologically active hCG also produced a 10-fold or greater molar excess of free beta subunit. The dimeric hormone, as well as the excess beta subunit, resembles the standard urinary hCG and beta subunit by chemical and biological criteria. In contrast, when the vector encoding for the alpha subunit was expressed alone, the alpha subunit had a higher molecular weight than both standard alpha and the alpha found in the expressed dimeric hormone. The molecular weight difference between expressed alpha subunit and standard alpha was found to reside in the alpha peptide consisting of residues 52-91 which contained all of the carbohydrate of the alpha subunit. The N-asparagine-linked carbohydrate moieties in the recombinant alpha were found to be triantennary in contrast to biantennary in urinary alpha, and this hyperglycosylation was responsible for the higher molecular weight of the alpha subunit when it was expressed alone. We found no evidence of O-threonine glycosylation at position alpha 39 reported to be present in free forms of the alpha subunit; however, the companion paper (Corless, C.L., Bielinska, M., Ramabhadran, T. V., Daniels-McQueen, S. Otani, T., Reitz, B. A., Tiemeier, D. C., and Boime, I. (1987) J. Biol Chem. 262, 14197-14203) finds a small quantity of O-glycosylation. Since the excess beta subunit appears to be of normal size and contains the expected complement of sugars, only free alpha subunit seems to be a potential substrate for addition of extra sugar moieties. No large beta subunit forms have been found by others, while large alpha subunits have been described both clinically and in tissue culture systems. These observations imply that the conformation of the free alpha subunit, in the regions of the glycosylation recognition sites, allows easier access for glycosyltransferases than those same sites in the beta subunit. When alpha is combined with beta, the local structures around the alpha glycosylation sites are apparently altered so as to make the synthesis of triantennary chains less favorable.     

Purification and characterization of the glycoprotein hormone alpha-subunit-like material secreted by HeLa cells

The protein secreted by HeLa cells that cross-reacts with antiserum developed against the alpha-subunit of human chorionic gonadotropin (hCG) has been purified approximately 30,000-fold from concentrated culture medium by organic solvent fractionation followed by ion exchange, gel filtration, and lectin affinity chromatography. The final preparation had a specific activity (by RIA) of 6.8 x 10(5) ng of alpha/mg of protein and appeared homogeneous by electrophoresis on reducing/denaturing polyacrylamide gels (SDS-PAGE). Amino acid analysis indicated that HeLa-alpha had a composition very similar to that of the urinary hCG alpha-subunit. Peptide fingerprints of the HeLa protein and hCG-alpha revealed that several of the Tyr-, Met-, and Cys-containing tryptic peptides were held in common, thus identifying the tumor protein as a glycoprotein hormone alpha-subunit with a primary structure similar to that of hCG-alpha. However, comparison of hCG-alpha and HeLa-alpha demonstrated that the tumor-associated subunit was not identical with its normal counterpart. Only two of the three Tyr-containing tryptic peptides present in hCG-alpha could be detected in HeLa-alpha after iodination with 125I. HeLa-alpha eluted prior to hCG-alpha during Sephadex G-75 chromatography, but the subunits coeluted when the tumor protein was first subjected to mild acid hydrolysis. The purified tumor protein had an apparent molecular weight greater than that of the urinary alpha-subunit when analyzed by SDS-PAGE (Coomassie blue staining), and this difference was even greater when a partially purified preparation was examined by an immunoblot technique (Western). Isoelectric focusing of the HeLa and hCG subunits demonstrated that the tumor protein had a lower pI (4.7-5.5 compared to 6.5-7.8), and removal of sialic acid by mild acid hydrolysis did not entirely eliminate this difference. Immunoprecipitation and electrophoresis of alpha-subunit from HeLa cultures labeled with [3H]fucose indicated that the tumor subunit was fucosylated, whereas analysis of hCG-alpha hydrolysates by HPLC confirmed previous reports that the placental subunit does not contain fucose. HeLa alpha-subunit was unable to combine with hCG beta-subunit to form holo-hCG under conditions where the hCG alpha-subunit was able to do so. The results indicate that, regardless of whether or not a single alpha-subunit gene is being expressed in both normal and neoplastic tissues, posttranslational modifications lead to a highly altered subunit in the tumor. The differences observed may be useful in diagnosing neoplastic vs hyperplastic conditions and may lend insight into the mechanism of ectopic hormone production by tumors     

ELISA for the measurement of serum and urinary chorionic gonadotropin concentrations in the laboratory macaque

A rapid, sensitive, enzyme-linked immunosorbant assay (ELISA) for the measurement of chorionic gonadotropin (CG) in serum and urine samples of laboratory macaques is reported. The ligand (CG) is captured by a readily available, widely used, and well-characterized monoclonal anti-body (Mab, 518B7) generated against the β subunit of bovine luteinizing hormone (LH). This Mab, while specific for LH, shows very little species specificity, and has been shown to detect LH and CG by radioimmunoassay (RIA) in both human and non-human primates. A polyclonal antiserum raised in rabbits against human chorionic gonadotropin (hCG) is conjugated to horseradish peroxidase, and is used as the second anti-body signal. This anti-hCG antiserum cross reacts with CG secreted by both the human (hCG) and macaque (mCG). The ELISA utilizes hCG as the standard, and results are based on the relative concentrations of mCG in serum and urine. Total assay time is less than 5 hours. Range of the standard curve is 0.002 to 0.5 ng hCG/well, and the least detectable concentration of hCG is 0.0023 ± 0.0007 ng/well. Pregnancy was detected in early pregnant macaques (M. fascicularis) on 9 (N = 1/16), 10 (N = 1/16), 11 (N = 1/16), 12 (N = 6/16), 13 (N = 1/16), 14 (N = 4/16), and 15 (N = 2/16) days following the pre-ovulatory urinary estrone conjugate peak. The detection of pregnancy by urinary mCG occurred approximately 24 to 72 hours after its detection in serum. Am. J. Primatol. 41:307–322, 1997. © 1997 Wiley-Liss, Inc.     

Over-expression and characterization of recombinant beta subunit of the human chorionic gonadotropin hormone synthesized in insect cells infected with a genetically engineered baculovirus.

A recombinant baculovirus, vAc beta hCG, having a replacement of the viral polyhedrin gene with the cDNA encoding the beta subunit of hCG was used to express beta hCG, an extensively glycosylated hormone, in insect cells. Virus-infected cells, 72 hr pi, secreted approximately 8.02 micrograms beta hCG/2 x 10(6) cells/ml. The recombinant beta hCG purified from insect cells exhibited increased mobility on SDS-PAGE as compared to authentic urinary beta hCG, a reflection on differences in glycosylation between insect and mammalian systems. The insect derived beta hCG, however, was identical to the native hormonal peptide in terms of immunoreactivity and bioactivity on association with alpha-subunit, as evident by its binding to rat testicular receptors and induction of steroidogenesis in a mouse Leydig cell bioassay system. The implications of using the baculovirus system to study the importance of carbohydrates for biological activity are also discussed.     

Partial purification and characterization of chorionic gonadotrophin in plasma and in culture medium of trophoblast cells from the marmoset monkey (Callithrix jacchus)

A biologically active gonadotrophin has been purified from the media of long-term cultures of trophoblast cells of the common marmoset monkey by a combination of precipitation and chromatography. Marmoset chorionic gonadotrophin (CG) is a glycoprotein which binds Concanavalin A and wheat germ agglutinin. The protein purified from culture media exists as several isoelectric species with pI in the range pH 3.5-4.5. On gel filtration it eluted with an apparent molecular weight of 68-72,000 but on PAGE migrated as if it was 58-65,000. A glycoprotein with similar characteristics has been recovered from plasma samples of pregnant marmosets. Biological activity of partly purified CG from media, as determined by a mouse testicular cell bioassay, was 1-3 i.u./mg protein.     

Contributions of arginines-43 and -94 of human choriogonadotropin beta to receptor binding and activation as determined by oligonucleotide-based mutagenesis

Members of the glycoprotein hormone family contain a common alpha subunit and a hormone-specific beta subunit. Human choriogonadotropin (hCG) beta is a 145 amino acid residue protein glycosylated at 6 positions (2 N-linked and 4 O-linked oligosaccharides). In an effort to elucidate receptor determinants on hCG beta, we have used site-directed mutagenesis to prepare and express several mutant cDNAs with replacements at arginines-43 and -94. Arg-43 is invariant in all known mammalian CG/lutropin beta amino acid sequences, and Arg-94 is conserved in 10 of the 12 sequences. Moreover, various studies involving synthetic peptides and enzymatic digestions of intact beta chains suggest that these residues may be important in hCG receptor binding. Point mutants were made in which these two arginines were replaced with the corresponding residues in human follitropin beta, Leu-43 and Asp-94. The wild-type and mutant beta chains were expressed in CHO cells containing a stably integrated gene for bovine alpha, and heterodimer formation occurred. These heterologous gonadotropins were active in assays using transformed Leydig cells, competitive binding with standard 125I-hCG, and cAMP and progesterone production, but the potency was considerably less than that associated with the hCG beta wild-type-containing gonadotropin. The double-mutant protein Arg-43 to Leu/Arg-94 to Asp also associated with bovine alpha, but the resultant heterodimer exhibited only low activity. Replacement of each arginine with lysine yielded heterodimers that were at least as potent as bovine alpha-hCG beta wild type, but the Lys-43-containing beta chain appeared to exhibit a low degree of subunit association or reduced stability relative to the expressed hCG beta wild type. These results demonstrate that arginines-43 and -94 contribute to receptor binding through a positive charge.     

The porcine LH/hCG receptor. Characterization and purification

Porcine luteal LH/hCG receptor (LH/hCG R) was solubilized with 70-80% recovery from the crude plasma membrane fraction by Triton X-100 in the presence of 25% glycerol and protease inhibitors. The solubilized receptor maintained 90% of original activity at -60 degrees C for 90 days. Equilibrium association constant (Ka) values of 1.92, 2.22, and 2.03 X 10(10) M-1 were observed for the whole homogenate, plasma membrane fraction, and solubilized LH/hCG R preparations, respectively. The specific binding capacity for the same fractions were 49, 70, 55 fmol/mg protein, respectively. Complexes of LH/hCG R and Triton X-100 were resolved into two components with approximate Mr = 2.7 X 10(5) and 5.4 X 10(5) by gel filtration on Sepharose 6B and two glycoprotein components by chromatography on concanavalin A-Sepharose. Solubilized porcine LH/hCG R was purified by two cycles of affinity chromatography on highly purified hCG-Sepharose with an overall recovery of 30-35% of the initial activity in the Triton extract. Purified porcine LH/hCG R had a specific binding capacity of 2300 pmol/mg protein and a Ka = 1.5 X 10(10) M-1. Silver staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels demonstrated that the major protein in porcine LH/hCG R preparations has Mr = 68,000. A weakly staining band at Mr = 45,000 was also observed in the purified receptor preparation. Analysis of iodinated purified LH/hCG R by autoradiography has confirmed these results. Porcine LH/hCG R was purified 40,000-fold by this method.     

Thyroidal responses to human chorionic gonadotropin in the chick and rat

The thyrotropic activity of human chorionic gonadotropin (hCG) has been examined in the chick and the rat. Uptake of 32PO4 by chick thyroid increased significantly with injection of bovine thyrotropin (bTSH) with a maximum response at 2.4 mU per chick. On the other hand, no significant stimulation of 32PO4 uptake was detected with injection of graded doses of highly purified hCG up to 0.25 mg per chick. 1 mg of partially purified hCG, equivalent in biological potency to the maximum dose of highly purified hCG used in the chick, did induce a significant increase in 32PO4 uptake. In rats, highly purified hCG stimulated a very significant release (p less than 0.001) of 125I from the thyroid and partially purified hCG had a thyrotropic activity equivalent to 0.42 microU bTSH/U hCG, identical to the value we reported in mice, 0.42 microU bTSH/U hCG. The duration of hCG action on thyroidal release of 125I in the rat was longer than that for bTSH, as it is in the mouse. hCG also induced a significant rise in the serum level of triiodothyronine in rats. We conclude that pure hCG is a weak thyrotropic substance in the rat but not in the chick. These results and other evidence suggest an inhibitory role for the densely glycosylated 30 amino acid residue C-terminal extension on the beta-subunit of hCG which limits, by steric hindrance, the interaction of the TSH-like hCG 'core' with thyrotropin receptors.     

Establishment of gonadotropin-responsive murine leydig tumor cell line

Several clonal Leydig tumor cell lines have been established by adapting the transplantable Leydig tumor, M548OP, to culture. One of these cell line, MLTC-1, has been characterized with regard to the gonadotropin-responsive adenylate cyclase system. The binding of 125I-labeled human chorionic gonadotropin (hCG) was blocked by excess unlabeled hCG and lutropin (LH) but not by follitropin, thyrotropin, or insulin, indicating the presence of specific receptors for hCG and LH. Based on the specific binding of hCG to isolated MLTC-1 membranes, the calculated dissociation constant was 1.0 +/- 0.2 X 10(-10) M. The receptors appeared identical to those from normal murine Leydig cells when analyzed by SDS PAGE and sucrose density gradient centrifugation. The molecular weight and sedimentation coefficient were 95,000 daltons and 8.5 S, respectively. MLTC-1 cells responded to hCG by accumulating cyclic AMP and producing progesterone. Cyclic AMP accumulation was time- and dose-dependent with a maximal accumulation occurring at approximately 0.2 nM hCG. At saturating levels of hCG, cAMP levels reached a maximum by 30 min and then declined very slowly. Adenylate cyclase activity in membranes prepared from MLTC-1 cells was stimulated by hCG, LH, NaF, cholera toxin, and guanyl-5'-ylimidodiphosphate, Additionally, choleragen was found to ADP-ribosylate a membrane protein of 54,000 daltons. This protein resembles the proposed guanine nucleotide regulatory component in both size and choleragen-dependent reactivity. These data suggest that MLTC-1 cells possess a gonadotropin-responsive adenylate cyclase system consisting of a specific hormone receptor, a regulatory component, and a catalytic subunit.     

Amino-terminal leucine-rich repeats in gonadotropin receptors determine hormone selectivity.

Recombinant expression of truncated receptors for luteinizing hormone/chorionic gonadotropin (LH/CG) revealed that the amino-terminal leucine-rich repeats 1-8 of the extracellular receptor domain bind human chorionic gonadotropin (hCG) with an affinity (Kd = 0.72 +/- 0.2 nM) similar to that of the native LH/CG receptor (Kd = 0.48 +/- 0.05 nM). LH/CG receptor leucine-rich repeats 1-8 were used to replace homologous sequences in the closely related receptor for follicle stimulating hormone (FSH). Cells expressing such chimeric LH/CG-FSH receptors bind hCG and show elevated cylic AMP levels when stimulated by hCG but not by recombinant human FSH (rhFSH). Similarly, a chimeric LH/CG receptor in which leucine-rich repeats 1-11 originated from the FSH receptor is activated by rhFSH but not by hCG. For this chimera, no residual [125I] hCG binding was observed in a range of 2 pM to 10 nM. Our results demonstrate that specificity of gonadotropin receptors is determined by a high affinity hormone binding site formed by the amino-terminal leucine-rich receptor repeats.     

Adenosine diphosphate sulphurylase activity in leaf tissue

1. A new method is described for the assay of ADP sulphurylase. The method involves sulphate-dependent [(32)P]P(i)-ADP exchange; the method is simpler, more sensitive and more direct than the method involving adenosine 5'-sulphatophosphate-dependent uptake of P(i). 2. ADP sulphurylase activity was demonstrated in crude extracts of leaf tissue from a range of plants. Crude spinach extract catalysed the sulphate-dependent synthesis of [(32)P]ADP from [(32)P]P(i); spinach extracts did not catalyse sulphate-dependent AMP-P(i), ADP-PP(i) or ATP-P(i) exchange under standard assay conditions. ADP sulphurylase activity in spinach leaf tissue was associated with chloroplasts and was liberated by sonication. 3. Some elementary kinetics of crude spinach leaf and purified yeast ADP sulphurylases in the standard assay are described; addition of Ba(2+) was necessary to minimize endogenous P(i)-ADP exchange of the yeast enzyme and crude extracts of winter-grown spinach. 4. Spinach leaf ADP sulphurylase was activated by Ba(2+) and Ca(2+); Mg(2+) was ineffective. The yeast enzyme was also activated by Ba(2+). The activity of both enzymes decreased with increasing ionic strength. 5. Purified yeast and spinach leaf ADP sulphurylases were sensitive to thiol-group reagents and fluoride. The pH optimum was 8. ATP inhibited sulphate-dependent P(i)-ADP exchange. Neither selenate nor molybdate inhibited sulphate-dependent P(i)-ADP exchange and crude spinach extracts did not catalyse selenate-dependent P(i)-ADP exchange. 6. The presence of ADP sulphurylase activity jeopardizes the enzymic synthesis of adenosine 5'-sulphatophosphate from ATP and sulphate with purified ATP sulphurylase and pyrophosphatase.     

Successful ovulation induction and laparoscopic intrauterine artificial insemination in the clouded leopard (Neofelis nebulosa)

Exogenous gonadotropins and a laparoscopic intrauterine artificial insemination (AI) technique were assessed for effectiveness in the clouded leopard (Neofelis nebulosa), a species difficult to breed in captivity due to severe mate incompatibility. Fourteen hormone trials using 10 female clouded leopards were performed to evaluate the ability of 50, 100, or 200 i.u. pregnant mares' serum gonadotropin (PMSG) and 75 or 100 i.u. human chorionic gonadotropin (hCG) to induce folliculogenesis and ovulation, respectively. Laparoscopic evaluation of ovarian activity was conducted at 29–48 hr after hCG administration. Time of ovulation in PMSG/hCG-treated clouded leopards was approximately 38–39 hr after hCG. Excessive follicular development was observed using the high hormone dosages (200 i.u. PMSG/100 i.u. hCG), whereas the lower dosages avoided ovarian hyperstimulation. Previous ovulation sites and mature corpora lutea were detected upon laparoscopic examinations in two of the 10 females housed alone, indicating that this species occasionally spontaneously ovulates. Five females were inseminated by depositing electroejaculated, washed sperm transabdominally into the proximal aspect of each uterine horn. One postovulatory female, previously treated with 100 i.u. PMSG and 75 i.u. hCG and inseminated in utero with 88 × 10⁶ motile sperm at 45 hr post-hCG, produced a pregnancy and two live cubs after an 89 day gestation. These results demonstrate: (1) an exquisite ovarian sensitivity to exogenous gonadotropins in clouded leopards; and (2) that artificial insemination has the potential of resulting in offspring in this species. (This article is a US Government work and, as such, is in the public domain in the United States of America.) © 1996 Wiley-Liss, Inc.     

Controlled ovarian stimulation and ultrasound guided follicular aspiration in the baboon (Papio cynocephalus anubis)

The objective of this study was to investigate whether baboon females respond to an ovarian stimulation protocol incorporating pituitary suppression with a GnRH agonist (GnRHa) and highly purified human FSH (hphFSH) with follicular development and oocyte maturation. An ovulation induction protocol was applied to 5 adult female baboons with a history of regular menstrual cycles (33-34 days). A long-acting GnRHa implant containing goserelin acetate was placed s.c. on days 22-24 of their menstrual cycle. Daily hphFSH (75 IU im) treatments were started approximately 10 days following menses. When the majority of the follicles were > or = 5 mm in diameter and the E2 levels had reached a maximum, hCG (2000 IU i.m.) was administered to induce final maturation of the oocytes and ovulation. 30 to 34 h after hCG administration, transabdominal follicular aspiration was performed using a variable frequency transvaginal transducer with ultrasound. A total of 71 oocytes were collected (average: 17). 91% of the oocytes were morphologically normal indicating that they were appropriate for in vitro insemination.     

Purification and characterization of guinea-pig chorionic gonadotrophin

A human chorionic gonadotrophin-like protein (GF-1, 1.0 g) from the placentae of 50 guinea-pigs killed at Day 26 of gestation was purified by pH and ammonium salt fractionation followed by column chromatography on DEAE-Sephadex and filtration on Sephadex G-100. Relative to the Second International hCG standard (MRC 61/6) GF-1 had an immunological potency of 21 000 i.u./mg as measured in a specific hCG-beta radioimmunoassay and, using the ovarian ascorbic acid depletion assay, an apparent biological potency of 24 064 i.u./mg. Isoelectric focusing yielded 6 bands between pH 4.4 and 5.7 and the material comprised two non-covalently linked subunits. The Stokes' radii were 3.40 nm for the native preparation, and 2.38 nm and 3.15 nm for GF-1-alpha and GF-1-beta subunits respectively. The guinea-pig placenta therefore produces a chorionic gonadotrophin which on purification has physicochemical, biological and immunological properties similar to those of hCG.     

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.     

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.