Production and Characterization of Monoclonal Antibodies against Bovine Luteinizing Hormone

Five mouse hybridoma cell lines producing monoclonal antibody against bovine luteinizing hormone (LH) have been established and the respective antibodies characterized by radioimmunoassay, immunofluorescence and immunoelectrophoresis. All antibodies belong to the IgG class and bind to staphylococcus protein A. Intraspecies cross-reactivity studies revealed no reaction with bovine follicle stimulating hormone (FSH). However, all antibodies showed partial cross-reaction with bovine thyroid stimulating hormone (TSH) suggesting a close conformational similarity between bovine LH and TSH. Studies on interspecies cross-reactivity (rat and human) showed that three of these five antibodies strongly react with rat LH but not at all with either rat FSH or rat TSH thus representing monospecific reagents for investigations concerning LH in this species. One of these three antibodies also strongly binds to human LH and to the same extent to human chorionic gonadotropin (CG) but not to human FSH or TSH. It was concluded that at least three different epitopes on the bovine LH molecule are recognized and that they are located on the β-chain of the hormone.     

Epitopes of Human Chorionic Gonadotropin and Their Relationship to Immunogenicity and Cross-Reactivity of β-Chain Mutants

PROBLEM: Human chorionic gonadotropin (hCG) is a placental glycoprotein hormone, a heterodimeric molecule, consisting of α and β chains. It induces the synthesis of progesterone, which is essential for the maintenance of the fertilized egg. Antibodies directed against hCG can, therefore, prevent pregnancy and serve as a vaccine. hCG belongs to the glycoprotein hormone family and shares the α chain with the other members. The β chain is a hormone-specific subunit that is unique to hCG, but still possesses 85% amino acid homology with the β chain of luteinizing hormone (LH), which means that prolonged immunization with hCG produces antibodies that cross-react with LH. METHOD OF STUDY: We have taken an approach involving the mutation of βhCG to eliminate cross-reactive epitopes without affecting the natural folding of the polypeptide chain and thus the unique βhCG-specific epitopes. RESULTS: Several mutants have been constructed that have maintained the binding to hCG-specific monoclonal antibodies (mAbs) but have lost the ability to bind to a panel of LH cross-reactive mAbs. To investigate the immunogenicity of selected mutants, mice were immunized with expression plasmid DNA, containing the gene for wild-type βhCG and two mutants: mutant 3, with four amino acid substitutions (68 Arg→Glu; 74 Arg→Ser; 75 Gly→His; 79 Val→His), and mutant 7, with a single amino acid substitution (68 Arg→Glu). CONCLUSIONS: Athough both mutants were able to elicit antibody responses in at least some animals, the levels were less than those seen with the wild-type βhCG DNA, and there seems still to be a residual cross-reacitivity with LH. Attempts to improve the immunogenicity of the mutants and to further modify the sequence to remove the cross-reacitivty are currently underway.     

A role for luteinizing hormone and follicle-stimulating hormone in the etiology of simple goiters in the pediatric population

In light of the progressive, pubertal increases in serum gonadotropin (FSH, LH) levels coinciding with the peak incidence of simple goiters; the common alpha subunits associated with the gonadotropins, TSH and hCG; the ability of LH and hCG to bind to thyroid TSH receptors and affect thyroid function; and, the probable interactions between thyroid hormones and gonadotropins, a role for gonadotropins in the etiology of simple goiters merits consideration.     

Monoclonal Antibodies Against Human Chorionic Gonadotropin (hCG): II. Affinity and Ability to Neutralize the Biological Activity of hCG

ABSTRACT: Monoclonal antibodies (MCA) against hCG have been characterized with regard to their affinity and their ability to neutralize the biological activity of hCG in vivo. The production and specificities of these reagents were described in the preceeding paper of this series. Equilibrium association constants (K_a) of the MCA, determined by radioimmunological saturation assays, ranged from less than 1 × 10⁸M⁻¹ up to 3.7 × 10⁹M⁻¹ whereas values for conventional polyclonal antisera against hCG ranged from 8.9 × 10⁹M to 1.8 × 10¹⁰M⁻¹. The ability of MCA to neutralize the biological activity of hCG was tested in a rat bioassay in vivo; 9 of 13 different MCA preparations tested could neutralize hCG. Surprisingly, this property did not correlate with affinity or specificity, and was not restricted to those MCA recognizing the hormone specific β-subunit. It could be demonstrated that determinants on each individual subunit as well as epitopes formed by both subunits are involved in the expression of the biological activity of hCG.     

Detection of gonadotropin-releasing hormone receptor in normal human pituitary cells and pituitary adenomas using immunohistochemistry

Gonadotropin-releasing hormone (GnRH), which is a well-known regulator of gonadotroph function, has recently been considered to be a paracrine factor involved in the control of somatotroph, lactotroph, and corticotroph cells. GnRH action is initiated by binding to a specific cell surface receptor, the gonadotropin-releasing hormone receptor (GnRHR), which is expressed by follicle-stimulating hormone/luteinizing hormone (FSH/LH) cells. Using in situ hybridization techniques, GnRHR messenger ribonucleic acid (mRNA) has recently been detected in normal human anterior pituitary gland and in various pituitary adenomas, including FSH/LH-cell, growth hormone (GH)-cell, adrenocorticotropic hormone (ACTH)-cell, and null-cell adenomas. However, immunohistochemical studies indicating the specific cell distribution of GnRHR in normal pituitary cells have never been reported. The aim of the present investigation was to evaluate the immunohistochemical expression of GnRHR in different types of normal pituitary cells and related tumors. Using double-label immunohistochemical techniques on formalin-fixed and paraffin-embedded tissues and specific antibodies directed against pituitary hormones and GnRHR, we found GnRHR immunoreactivity not only in FSH/LH cells, but also in GH- and thyroid-stimulating hormone (TSH) cells. GnRHR was detected in FSH/LH-cell, GH-cell, mixed GH- and prolactin (PRL)-cell, and α-subunit (α-SU)/null-cell adenomas. The findings of this study suggest that the interaction between GnRH and GnRHR may play a role in paracrine/autocrine regulation of different types of normal pituitary cells and pituitary adenomas. Received: 24 January 2000 / Accepted: 12 April 2000     

Two types of secretory granules in gonadotrophs: Discrimination by the simultaneous EM immunocytochemical localization of serotonin and β-follicle stimulating hormone

The hypothesis that the secretory granules of mammalian gonadotrophs are heterogeneous was tested. Previous studies had shown that all of the granules contain β-luteinizing hormone (β-LH) immunoreactivity, and some contain β-follicle stimulating hormone (β-FSH) or 5-HT immunoreactivity. Moreover, differential release of β-LH and β-FSH has also been demonstrated. In the current study the pituitary glands of mice were investigated immunocytochemically at the ultrastructural level with antisera directed against human growth hormone (GH), β-LH, β-FSH, and 5-HT. The immunoreactivities of β-LH, β-FSH, and 5-HT were restricted to gonadotrophs. No 5-HT immunoreactivity was seen in somatotrophs, identified by the immunoreactivity of GH in the secretory granules of these cells. Antisera to β-LH labeled all gonadotroph granules; however, anti-β-FSH and anti-5-HT sera labeled only subsets of the granules. The proportion of granules labeled could not be increased by doubling the concentration of anti-β-FSH serum. The incidence of double labeling of granules by antisera to β-FSH and 5-HT was significantly less than that predicted from the incidence of granule labeling by either reagent alone. It is concluded that β-FSH and 5-HT immunoreactivities do not co-exist in the same secretory granules of gonadotrophs; therefore, these granules are heterogeneous and there must be at least two types of granules. It is possible that the two types of granules may be responsive to different second messengers, thereby explaining the differential release of LH and FSH.     

Mutationen der Gonadotropine und Gonadotropinrezeptoren

The gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are fundamental for reproduction. They exert their action through specific receptors located exclusively in gonadal somatic cells. Mutations of the gonadotropins and their receptors are very rare but help to elucidate the mechanism of gonadotropin action. To date, only one missense mutation of the LH β-subunit and two missense mutations and one deletion of the FSH β-subunit gene have been described. These mutations lead to loss of gonadotropin function and hypogonadism. Selective loss of FSH in men caused by mutations in the β-chain result in azoospermia. For gonadotropin receptors, about two dozen loss-of-function (inactivating) and gain-of-function (activating) mutations are known. Inactivating mutations of the LH receptor cause Leydig cell hypoplasia with various degrees of hypoandrogenization up to male pseudohermaphroditism, while in females they result in primary amenorrhea. Activating mutations of the LH receptor are responsible for familial male-limited pseudoprecocious puberty (testotoxicosis) but do not cause phenotypic alterations in females. Inactivating mutations of the FSH receptor result in primary or early secondary amenorrhea in females and reduced spermatogenesis in males. Only one activating mutation of the FSH receptor has been described so far, in a hypophysectomized man with normal spermatogenesis in the absence of gonadotropins and very low endogenous testosterone levels. In addition, several polymorphisms in both gonadotropins and gonadotropin receptor genes have been identified, and their impact on gonadal pathophysiology is currently being investigated.     

Action of PMSG and asialo-PMSG on rat Leydig and granulosa cells.

Highly purified pregnant mares serum gonadotropin (PMSG) was nearly as potent as ovine luteinizing hormone (LH) and human follicle stimulating hormone (hFSH) when bioassayed in vitro in systems known to respond primarily to LH or FSH. An analogue of human chorionic gonadotropin treated with neuraminidase, galactosidase, beta-N-acetylglucosaminidase, and mannosidase (hCG) inhibited the stimulatory effects of hCG, LH, and PMGS on cAMP accumulation in rat Leydig cells but did not inhibit the stimulatory effects of FSH or PMSG on cAMP accumulation in ovarian granulosa cells obtained from immature rats fed diethylstillbestrol. Thus PMSG appeared to form functional complexes with both LH and FSH receptors and may be unique among mammalian gonadotropins. Treatment of PMGS with neuraminidase increased its potency nearly tenfold in vitro apparently by increasing its affinity for both LH and FSH receptors. Although the kinetics of PMSG binding were not investigated with radiolabeled materials, indirect functional binding studies are described that suggest that hCG more rapidly forms stable hormone-receptor complexes than PMSG, asialo-PMSG, FSH, and LH when all hormones are incubated under the same conditions.     

Clinical comparison of immunoradiometric assays for intact versus beta-specific human chorionic gonadotropin.

Monoclonal immunoradiometric assays (IRMA) for human chorionic gonadotropin (hCG) are available for either intact hCG (IhCG) or beta subunit hCG (beta hCG). The authors evaluated the clinical applications of both methods. Serum samples (N = 180) were divided into the following five clinical groups: Group 1: elevated luteinizing hormone (LH), follicular stimulating hormone (FSH), and thyroid-stimulating hormone (TSH), which share alpha subunits with hCG; Group 2: pregnancy; Group 3: trophoblastic tumors; Group 4: malignancy; and Group 5: positive rheumatoid factor or anti-nuclear antibody (ANA). The values of beta hCG versus IhCG for Group 1 showed statistical significance but no clinical significance, indicating negligible cross-reactivity in the alpha subunit group. beta hCG values, although exceeding the IhCG values, correlated well (r = 0.97) in intrauterine pregnancies; however, these values were not believed to be clinically significant. There is negligible interference by alpha subunits, elevated rheumatoid factor, ANA, or malignancy in the determination of IhCG versus beta hCG. The authors conclude that either the IhCG or beta hCG assays may be used in clinical conditions in which a potential exists for interference of alpha subunits, autoantibodies, or heterophile antibodies or in malignancy.     

Free alpha subunit of the pituitary glycoprotein hormones. Measurement in serum and tissue of patients with pituitary tumors.

A solid-phase radioimmunoassay was developed that measures the free alpha subunits of pituitary glycoprotein hormones (alpha PGpHs) and has negligible cross-reactivity with the intact hormones (less than 0.014% for thyroid-stimulating hormone [TSH], less than 0.1% for human chorionic gonadotropin [hCG], 0.8% for luteinizing hormone [LH], and 2.0% for follicle-stimulating hormone [FSH]). The assay is standardized with the alpha subunit of hCG but also reacts well with the alpha subunits of the other glycoprotein hormones (84% for alpha TSH, 77% for alpha FSH, and 64% for alpha LH). Concentrations as low as 0.3 micrograms/L can be reliably measured, and the 97.5% reference range in 27 healthy adults, including postmenopausal females, is less than or equal to 1.2 micrograms/L. Elevated preoperative alpha PGpH concentrations were found in 45 (9.4%) of 479 sera from patients with pituitary adenoma and 3 (4.5%) of 66 patients with nonadenomatous sellar lesions. Postoperative alpha PGpH levels were lower in 30 of 39 adenoma patients and 2 of 3 nonadenoma patients. In five (1%) of the patients with pituitary adenomas, alpha PGpH was the only elevated serum hormone marker. Serum values of alpha PGpH correlate weakly with alpha subunit immunocytochemical staining--95% of those with negative staining have normal alpha PGpH values, but only 18% of those with positive staining have elevated alpha PGpH values.     

Pituitary Hormones and Contact Sensitivity in Rats

Hypophysectomized (Hypo-X) rats do not develop contact sensitivity to dinitrochlorobenzene (DNCB). Daily treatment with prolactin or growth hormone completely restores the DNCB-reactivity of Hypo-X animals. Treatment of such animals with ACTH, FSH, LH, TSH or HCG has no restoring potential. Treatment with ACTH in addition to prolactin or growth hormone antagonizes restoration of Hypo-X rats. These experiments indicate that the pituitary gland has the potential of regulating contact sensitivity.     

Gonadotrophin stimulation of non-luteinized granulosa cells increases steroid production and the expression of enzymes involved in estrogen and progesterone synthesis

BACKGROUND: In regular IVF treatment, mature oocytes are collected with their luteinized granulosa cells (GCs). When in vitro maturation (IVM) of the oocytes is performed, non-luteinized GCs can be collected. We have investigated how these cells respond to gonadotrophin stimulation in culture. METHODS: GCs were collected from patients undergoing IVM treatment and compared with GCs from IVF patients. The cells were stimulated with FSH and/or hCG. After 48 h, culture media were collected for hormone analysis, and RNA was isolated for gene expression analysis. RESULTS: In IVM GCs, hCG and FSH alone and in combination induced significantly increased progesterone production, and FSH alone and in combination with hCG increased estrogen production. We also studied the gene expression of P-450aromatase and P-450scc and the receptors for FSH and LH. In non-luteinized GCs, the expression levels of P-450aromatase increased with all treatments, and P-450scc expression increased with the combined FSH and hCG treatment. LHR expression increased with FSH treatment, but the FSH receptor expression did not change with different treatments. CONCLUSIONS: Non-luteinized GCs behaved differently from luteinized GCs in culture. The data help understand the final stages of maturation of human oocytes and follicles.     

大鼠下颌下腺卵泡刺激素、黄体生成素的分布及与促性腺激素释放激素受体的共定位研究

目的研究卵泡刺激素(FSH)和黄体生成素(LH)在大鼠下颌下腺中的定位、分布及与促性腺激素释放激素受体(GnRHR)共定位的关系。方法采用邻片免疫组织化学共定位方法。结果大鼠颌下腺浆液性腺泡上皮细胞、分泌管、排泄管及颗粒曲管上皮细胞均呈FSH和LH免疫反应阳性，阳性颗粒分布在细胞质内，细胞核阴性。邻片共定位结果显示颌下腺GnRHR免疫反应阳性细胞同样呈FSH免疫反应阳性，大部分的GnRHR免疫反应阳性细胞呈LH免疫反应阳性，小部分呈免疫反应阴性。两种免疫反应阳性物质均分布在细胞质内，细胞核阴性。结论大鼠下颌下腺浆液性腺泡上皮细胞、分泌管、排泄管和颗粒曲管上皮细胞可能以自分泌或旁分泌的GnRH调节自身合成与分泌FSH和LH。     

Augmentation by thyroxine of human granulosa cell gonadotrophin-induced steroidogenesis

The objective of this study was to investigate the influenceof thyroid hormone on gonadotrophin-induced oestradiol and progesteronesecretion by human granulosa cells maintained in vitro. Granulosacells were obtained by aspiration of pre-ovulatory folliclesfrom women undergoing assisted reproductive technology. Ovulationinduction was performed with gonadotrophin-releasing hormoneagonist, human menopausal gonadotrophin and human chorionicgonadotrophin. Granulosa cells were maintained in vitro in adefined medium with added insulin. Between 48 and 72 h afterthe initiation of cell culture, oestradiol and progesteronesecretion into the medium was determined for granulosa cellsgrowing in serum-free medium with follicle-stimulating hormone(FSH)/luteinizing hormone (LH) and in serum-free medium withFSH/LH and thyroxine added in a concentration range of 10^–10–10^–7M. All concentrations of thyroxine used produced a statisticallysignificant increase in oestradiol (range 1.18–1.37 timesthe amount with FSH/LH alone) and progesterone (range 1.29–1.51times the amount with FSH/LH alone) secretion.     

A prospective, randomized comparison of ovulation induction using highly purified follicle-stimulating hormone alone and with recombinant human luteinizing hormone in in-vitro fertilization.

The commercial availability of highly purified, s.c. administered urinary follicle stimulating hormone (FSH) preparations for ovarian stimulation marked the beginning of a new era in the treatment of infertility. As these new formulations contain essentially no luteinizing hormone (LH), supplemental LH may be needed for optimal folliculogenesis. It was the aim of this pilot study to compare fertilization rates, embryo morphology, implantation rates and pregnancy outcomes prospectively in two age-matched patient groups: women who received highly purified FSH (FSH-HP) (n = 17), and women who received FSH-HP plus recombinant human LH (rhLH, n = 14) throughout ovarian stimulation. All patients received mid-luteal pituitary down-regulation with s.c. gonadotrophin-releasing hormone agonist (GnRHa) (leuprolide). Mean implantation rates were 26.9 and 11.9% in the FSH-HP only and FSH-HP + rhLH groups respectively. The mean clinical pregnancy/initiated cycle rate was 64.7 and 35.7% for the FSH-HP only and FSH-HP + rhLH patients respectively. FSH-HP patients and FSH-HP + rhLH patients achieved clinical pregnancy/transfer rates of 68.8 and 45.5% respectively. One patient in the FSH-HP + rhLH group had a spontaneous abortion; no pregnancy losses occurred in the FSH-HP only group. There were more cancellations for poor ovarian response among FSH-HP + rhLH patients (n = 3) than among FSH-HP patients (n = 1). The trend toward better pregnancy outcomes among patients who received FSH-HP without supplemental rhLH did not reach statistical significance. It is postulated that appropriate endogenous LH concentrations exist despite luteal GnRHa pituitary suppression, thereby obviating the need for supplemental LH administration.     

Effects of sulpiride isomers on the control of anterior pituitary secretion in normal man

The effects of (+)- and (-)-sulpiride on anterior pituitary hormone secretion (LH, FSH, PL, hGH, TSH) have been studied in five normal men. Both (+)- and (-)-sulpiride increase PL and TSH secretion and decrease LH and FSH secretion, whereas they do not seem to affect hGH secretion. These results are discussed in view of the different action of (+)- and (-)-sulpiride on DA receptors in the forebrain with respect to DA receptors involved in the control of anterior pituitary secretion.     

A study on the effects of interaction between naloxone and 2-Br-alpha-ergocryptine or clonidine on luteinizing hormone, follicle-stimulating hormone, prolactin and thyroid-stimulating hormone levels in normal man serum.

The effects of 2-Br-alpha-ergocryptine (2.5 mg/osM), clonidine (50 microgram, intramuscularly) and naloxone (0.4 mg, intramuscularly) as well as the interaction between naloxone and 2-Br-alpha-ergocryptine or clonidine on luteinizing hormone (LH) follicle-stimulating hormone (FSH), prolactin (PL) and thyroid-stimulating hormone (TSH) serum levels in normal man have been studied. 2-Br-alpha-ergocryptine and clonidine clearly reduce and naloxone tends to reduce PL serum levels. TSH levels are lowered by naloxone as well by clonidine plus naloxone. The results obtained point also to a possible different pattern of LH and FSH secretion after naloxone, that is after opiate receptor blockade. The clonidine effects on PL secretion are discussed in the frame of a possible adrenergic control of the release of this hormone.     

Carrier-induced epitope-specific regulation and its bypass in a protein-protein conjugate

In the course of clinical trials on a birth control vaccine, it was found that some of the immunized women responded poorly to booster immunizations. This vaccine consists of a dimer of the β chain of human chorionic gonadotropin (βhCG) and the α chain of ovine luteinizing hormone (αoLH), linked to tetanus toxoid (TT) as a carrier. Changing this carrier to diphtheria toxoid resulted in reversion to high anti-hCG antibody titers, indicating the extent to which the carrier influences anti-ligand responses in this system. The suppression of anti-hCG responses after booster immunizations was reminiscent of the phenomenon of carrier-induced, epitope-specific regulation. In a mouse model designed to test the effects of preimmunization with TT on anti-hCG responses, we found that a single preimmunization with TT causes reduced anti-hCG antibody responses in two out of four mouse strains, while anti-αoLH antibody responses were not affected by the preimmunization with TT. This is particularly interesting considering that βhCG and αoLH were not presented when linked separately to TT. In an effort to devise a strategy to circumvent this carrier-induced, ligand-specific hyporesponsiveness, we investigated the effectiveness of a synthetic T helper epitope from TT as carrier. We show that preimmunization with TT causes a less profound reduction in anti-hCG titers if the preimmunized mice are subsequently injected with αoLH-βhCG conjugated to a synthetic tetanus toxin peptide recognized by TT-induced and peptide-induced T cells.     

Suppression of LH during ovarian stimulation: effects differ in cycles stimulated with purified urinary FSH and recombinant FSH

There has been much debate about the role of luteinizing hormone (LH) during follicle stimulating hormone (FSH)-treated ovarian stimulation for assisted reproduction, where the endogenous LH is suppressed using a gonadotrophin-releasing hormone analogue. The requirement for LH in oestradiol biosynthesis is established, but other effects of 'insufficiency' are less clear, and little attention has been paid to the specific origin of the FSH used. The aim of this study was to examine the roles of profoundly suppressed circulating LH concentrations in cycles of ovarian stimulation for IVF, which were affected in two large separate cohorts of patients undergoing assisted reproduction. They were stimulated by either purified urinary FSH (MHP) or recombinant human FSH (rFSH). Within each dataset, outcomes were examined with respect to the circulating concentrations of LH in the mid-follicular phase, as plasma samples were stored prospectively, and assayed retrospectively. Patients with profoundly suppressed LH showed much reduced oestradiol concentrations at mid-follicular phase and at human chorionic gonadotrophin administration in cycles treated with either MHP or rFSH. However, gross ovarian response, as became evident by FSH dose demands, duration of stimulation, and also oocyte and embryo yields and embryo cryopreservation were influenced only in cycles treated with MHP. Furthermore, no effect upon pregnancy survival was observed. Thus, it is concluded that there is a demand for additional exogenous LH treatment only in cycles treated with purified urinary FSH where the LH is profoundly suppressed.