Recombinant Hormone Research Articles

CONSTRUCTION AND EXPRESSION OF AN OVINE SINGLE CHAIN TRIPLE-DOMAIN CHIMERIC GONADOTROPIN: TANDEM LINKAGE OF THE GENES ENCODING THE α-AND FSHβ AND LHβ SUBUNITS

Members of the glycoprotein hormone family (LH, FSH, TSH and CG) are heterodimers composed of common α and hormone-specific β subunits that are non-covalently associated. Both subunits are glycosylated, containing asparagine (N)-linked oligosaccharides and, in the case of the CGβ subunit, O-linked carbohydrates are also present in a cluster of amino acids at the carboxy-terminus. The ovine α subunit consists of 120 amino acids and the mature protein sequence is identical with the corresponding bovine sequence. The ovine FSHβ and LHβ genes encode proteins of 129 and 141 amino acids, respectively and they are most closely related to their bovine counterparts (96% and 98%, respectively). To examine the structure-activity relationships of the ovine gonadotropins, and their role in follicle development and ovulation, the availability of purified hormones is essential. The purity and potency of recombinant gonadotropins is greater than tissue-derived gonadotropins. Thus, the recombinant gonadotropins may modulate the gonadal function of sheep in a more precise and controlled manner. In addition, recombinant hormones are likely free of contaminants and disease causing pathogens and, therefore, may improve overall herd health and productivity. Previous studies in transfected Chinese hamster ovary (CHO) cells demonstrated that the assembly of α and the β subunits is inefficient, resulting in low rates of formation of recombinant human, bovine and equine LH. A number of reports have now shown that covalently linking the β and the α subunits in a single chain results in biologically active analogs. The ability to express a single chain bearing genetically linked α and β subunits by-passes the rate-limiting assembly step, often resulting in a greater yield of secreted analog. Recently, we also reported that a covalently fused triple-domain gonadotropin analog containing human FSHβ, LHβ and α subunit was secreted efficiently and its bioactivity was similar to the heterodimer, suggesting productive interactions between the domains in the tether. Here, we generate ovine triple-domain single chain gonadotropin with the following structure: FSHβ–LHβ–α. This chimera was constructed using bovine cDNAs encoding FSHβ, LHβ or α subunits as templates and PCR mutagenesis to generate the corresponding ovine subunits. Stable cell lines expressing the single chain were metabolically labeled with [35S]cysteine. Lysates (intracellular) and media (secreted) were immunoprecipitated with polyclonal human CGβ antiserum (cross reacts with ovine LHβ subunit) and analyzed by SDS– PAGE. The secreted form of the analog was also analyzed on Western blot under non-reducing conditions. Collectively, these data show that the ovine triple-domain analog is synthesized and secreted from CHO cells. The apparent molecular mass of the secreted form is 80 kDa. The ability to construct dually active single-chain analogs could permit more effective control of the half-life and duration of gonadotropin actions. This should aid in the design of analogs that could be used to promote superovulation and/or out-of-season breeding in sheep. (poster)

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

The effectiveness of a CpG motif-based adjuvant (CpG ODN 2006) for LHRH immunization

A recombinant ovalbumin-luteinizing hormone-releasing hormone (ova-LHRH) antigen has been developed for immunocontraception. In this study, a novel immunostimulant for ova-LHRH immunization, CpG oligodeoxynucleotide (ODN) 2006, was compared against Mycobacterium butyricum. Also, the immunogenicity of ova-LHRH after lyophilization and exposure to organic solvents was assessed. Rats received either ova-LHRH solubilized in urea; lyophilized ova-LHRH; lyophilized ova-LHRH exposed to methylene chloride; or lyophilized ova-LHRH exposed to ethyl acetate. Immunogenicity of lyophilized ova-LHRH was reduced compared with solubilized ova-LHRH. Exposure to ethyl acetate further decreased immunogenicity of ova-LHRH. CpG ODN 2006 was a more effective immunostimulant than M. butyricum for LHRH immunization.

Could bone tissue be a target for luteinizing hormone/chorionic gonadotropin?

Ovariectomy (OVX) and Zoladex administration to adult rats gave conflicting results with respect to the excretion of total urinary hydroxyproline (OH-Pro), a valuable indicator of bone collagen catabolism. Whereas OVX culminated in early (1 week) increases in OH-Pro, the use of Zoladex actually lowered OH-Pro and showed no sign of increasing over controls for a 2-month period. Since both OVX and Zoladex produce a state of estrogen deficiency we reasoned that the differential effects of the two procedures on OH-Pro were attributed to LH status. Receptors for luteinizing hormone (LH)/human chorionic gonadotropin (hCG) have been identified in many, non-gonadal, estrogen sensitive sites and although bone is receptive to estrogen what effects LH/hCG might have upon bone metabolism have received scant attention. Treatment of osteoblasts in culture with a urinary derived formulation of hCG resulted in increased alkaline phosphatase (ALP) activity, raised matrix mettaloproteinase-2 (MMP-2) levels and increased expression of type I collagen. Further studies, using murine calvaria, supported a bone-resorbing effect of hCG. Taken together our initial findings suggested that raised hCG and/or LH might lead to an overall increase in bone matrix turnover as reported for puberty, pregnancy and the menopause. However, when the urinary derived preparation of hCG was replaced with recombinant hormone no changes in osteoblast activity were found implying the presence of contaminating agents in the urine derived hCG. Herein we describe that epidermal growth factor (EGF) could account for the changes observed for urinary derived hCG in osteoblast cultures and that the effects of LH/hCG on bone tissue are probably indirect.

Biological activities of recombinant Manchurian trout FSH and LH: their receptor specificity, steroidogenic and vitellogenic potencies

Gonadotropins (GTHs), FSH and LH, play central roles in vertebrate reproduction. Here, we report the production of biologically-active recombinant FSH (r-mtFSH) and LH (r-mtLH) of an endangered salmon species, Manchurian trout (Brachymystax lenok), by baculovirus in silkworm (Bombyx mori) larvae. The biological activities of the recombinant hormones were analyzed using COS-7 cell line transiently expressing either amago salmon FSH or LH receptor. The steroidogenic potency of the r-mtFSH and r-mtLH was examined by a culture system using rainbow trout follicles in vitro. In vivo, bioactivity was assessed by measuring ovarian weight, oocyte diameter, and plasma steroid hormone levels in female rainbow trout. Moreover, inducing potency of milt production were examined in vivo using goldfish. Our results demonstrated that the r-mtFSH and r-mtLH were successfully produced in the baculovirus-silkworm system and recognized by their cognate receptors specifically in vitro. The production of estradiol-17beta (E2) and testosterone (T) was stimulated by the r-mtFSH and r-mtLH respectively, from the full-grown follicles of rainbow trout, whereas both E2 and T were increased by relatively higher doses of the recombinant hormones from the follicles of the maturing stage. In in vivo assay, injection of the r-mtFSH but not r-mtLH increased ovarian weight, oocyte diameter, and plasma E2 levels in immature rainbow trout. Injection of both r-mtFSH and r-mtLH induced milt production in male goldfish. In conclusion, the present study strongly suggests that the r-mtFSH and r-mtLH have distinct biological properties, such as a specific responsiveness for the cognate receptor, steroidogenic, and vitellogenic activities for ovarian follicles in salmonids. These recombinant FSH and LH may be applied for future studies on the gonadal development and maturation in fishes as well as the endangered salmon species.

Biological Properties of a Novel Follicle-Stimulating Hormone/Human Chorionic Gonadotropin Chimeric Gonadotropin

A chimeric recombinant human gonadotropin, termed C3, demonstrates both follitropic and lutropic bioactivities. The alpha-subunit construct for C3 is comprised of the recombinant wild-type human glycoprotein hormone alpha-subunit. The beta-subunit DNA construct for C3 encodes residues 1-145 from human chorionic gonadotropin (hCG)-beta with the exceptions that FSH beta amino acid 88 (D) is substituted for hCG beta amino acid 94 (R) and FSH beta amino acids 95-108 (TVRGLGPSYCSFGE) are substituted for hCG beta amino acids 101-114 (GGPKDHPLTCDDPR). C3 is a potent FSH and LH agonist able to bind and to signal through FSH and LH receptors in vitro. In in vivo bioassays optimized to quantify each type of activity, C3 was found to have lutropin and follitropin potencies at levels similar to those of recombinant human LH and recombinant human FSH, respectively. In immature rats, C3 was sufficient to support the maturation of normal ovarian follicles. Moreover, a significant portion of follicles matured by C3 ruptured in response to an ovulatory hCG stimulus and gave rise to morphologically normal oocytes. Furthermore, a low dose of C3 promoted weight gain in the rodent uterus, suggesting it also supported preparation for implantation without histological evidence of excessive luteinization of the ovary. In summary, the biological properties of C3 indicate that its chimeric nature has resulted in a fully functional, dual-acting human gonadotropin.

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Is there a Need for Recombinant Human Luteinizing Hormone (Lutropin Alfa) Supplementation in Ovarian Stimulation for Assisted Reproduction?

Luteinizing hormone is now available as the recombinant product, lutropin alfa for the treatment of female infertility. It is necessary in the natural process of follicular growth and maturation. It is not yet clear which patients really benefit from the addition of this medication to conventional gonadotropin stimulation procedures in infertility treatment. Certainly, it has a proven benefit in patients suffering from hypogonadotropic hypogonadism (WHO I). Others may be older patients, patients with a profound gonadotropin suppression stimulated in long gonadotropin-releasing hormone agonist protocols, or patients with poor ovarian response to conventional stimulation strategies. The available data are reviewed herein.

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Plasmid-Based Expression Technology Using Growth Hormone Releasing Hormone: A Novel Method for Physiologically Stimulating Long-Term Growth Hormone Secretion

Novel DNA-based technologies were recently introduced for various purposes, such as screening of targets identified from genomic projects, shuffled molecules for vaccination, or to direct the in vivo production of hormones and other peptides for therapeutic or preventative applications. We have used a plasmid-based technology to deliver growth hormone releasing hormone (GHRH) to various animal species for screening, toxicology and therapy. A single intramuscular injection of a low dose of plasmid followed by electroporation can ensure that the target species will produce physiological levels of GHRH for extended periods of time, which would replace costly, frequent injections of the recombinant hormone and improve the quality of life and compliance of patients. This therapeutic modality is of particular importance in circumstances requiring long-term administration of small molecules with naturally short half-life (e.g. treatment of anemia and cachexia associated with renal failure, cancer or other chronic disability). A similar technique was used to create, test and validate protease-resistant analogs of GHRH with significantly longer half-life. Analysis of the characteristics of each of the plasmid components and tissue-specific transcription factors and the choice of target tissue is imperative when designing plasmids for therapeutic applications. Using the species-specific sequences of GHRH or other molecule along with the appropriate choice of plasmid backbone and expression cassette components can result in long and steady expression of the transgene product.

In vivo biological activity of recombinant goldfish gonadotropins produced by baculovirus in silkworm larvae

Recombinant gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) of goldfish Carassius auratus were produced by baculovirus in silkworm Bombyx mori larvae. The induction of gonadal maturation is an important aspect of fish aquaculture and the possible biotechnological production of recombinant hormones was examined. cDNAs encoding goldfish GTH subunits (FSHβ, LHβ and common α) were introduced into the baculovirus, which was infected into silkworm larvae after propagation of the recombinant virus in B. mori culture cells. Hemolymph of silkworm larvae containing recombinant FSH (rFSH) or LH (rLH) was collected, and biological activities of recombinant hormones were analyzed in vivo using goldfish and the bitterling Rhodeus ocellatus ocellatus. Injection of hemolymph containing rFSH or rLH induced milt production in male goldfish. Plasma testosterone levels were increased by rLH in male goldfish and plasma estradiol levels were increased by rFSH in female goldfish. rLH induced ovulation in female bitterling, but rFSH showed weak potency. In conclusion, biologically active goldfish recombinant GTHs were able to be produced in a baculovirus-silkworm larvae system and it is suggested that recombinant fish GTHs produced by this system could be applied for the induction of gonadal development in aquaculture fishes.

674. Administration of rAAV2 and rAAV5 to the Parotid Gland of Non-Human Primates

Previous studies from our laboratory showed that therapeutic levels of transgene expression were obtained from mouse submandibular glands after retro-ductal administration of relatively low doses (109 particles/animal) of a recombinant serotype 2 adeno- associated viral vector (rAAV2) encoding human erythropoietin (hEPO). The recombinant hormone was preferentially secreted into the bloodstream rather than into saliva. Serum hEPO levels steadily increased for an 8–12 week period and remained relatively stable until the end of the experiment (54 weeks; Voutetakis et al, PNAS, 2004).

Production of recombinant hormones and growth factors for use in aquaculture

Progress in recombinant DNA technology and development of new expression systems has provided valuable means to produce large quantities of hormones and growth factors from various fish species that otherwise required sacrifice of enormous numbers of fish to obtain only small amounts of the native protein. The motivation to produce recombinant hormones and growth factors for application to fish culture can be attributed to (a) the low amounts of pep- tides in producing tissues available for purifica- tion and characterization by classical methods, i.e., follicle stimulating hormone (FSH = GtH-I) in the pituitary or insulin-like growth factor-I (IGF-I) and IGF-II in the liver, (b) the similarity in chemical structure between two hormones that does not permit efficient separation (FSH and LH = GtH-II), (c) the need for large quanti- ties of hormones for growth enhancement by growth hormone (GH), and (d) interest in studying structure/function relationships, i.e., single or multiple amino acid deletion, amino acid substitution, etc.

Degradation of teriparatide by gastro-intestinal proteolytic enzymes

Teriparatide, a recombinant parathyroid hormone (1–34) is the first approved agent for the treatment of osteoporosis that stimulates new bone formation. Currently, the drug is administered daily by s.c. injection. Because of the obvious advantages of oral teriparatide administration, the development of such a delivery system would be of great benefit. Besides other barriers, the enzymatic barrier caused by gastro-intestinal (GI) proteolytic enzymes is believed to be responsible for negligible teriparatide oral bioavailability. It was therefore the aim of the study to evaluate the stability of teriparatide towards a variety of GI proteases under physiological conditions. Results indicate that teriparatide is entirely degraded by trypsin, chymotrypsin and pepsin within 5 min. In contrast, even after 3 h of incubation with elastase about 85% of undegraded teriparatide could still be detected. Within an incubation period of 3 h in the presence of rat small intestinal mucosa, approximately half of the teriparatide was degraded. Experiments with isolated aminopeptidase N demonstrated that this membrane bound peptidase is primarily involved in the degradation process. Results gained from and recorded in this study provide a precise characterisation of the enzymatic barrier for oral teriparatide administration and represents a prerequisite for the development of oral teriparatide delivery systems.

AC-015 Comparison of clinical outcome by stimulation with recombinant luteinizing hormone (rLH, Luveris) or human menopausal gonadotrophin (HMG) in IVF–embryo transfer treatment

AC-015 Comparison of clinical outcome by stimulation with recombinant luteinizing hormone (rLH, Luveris) or human menopausal gonadotrophin (HMG) in IVF–embryo transfer treatment

Effects of Anticatabolic and Anabolic Therapies at the Tissue Level

The recent decade has seen the emergence of a wide variety of new effective therapies for osteoporosis. Although hormone replacement therapy and calcium supplementation were the only available therapies 20 yr ago, we now have a wide variety of anticatabolic (antiresorptive) therapies (bisphosphonates, calcitonin, selective estrogen receptor modulators [SERMs]) and anabolic therapies in the form of recombinant parathyroid hormone [PTH(1–34) and PTH(1–84)] approved and commercially available. Our initial perceptions around these therapies were quite primitive, being mainly based on bone mineral density measurements. However, recent progress in imaging technology and structural and histological evaluation of bone has yielded important new insights into the mechanism of action of the various treatments. This article summarizes current knowledge about both anticatabolic and the more recent anabolic therapies, with special emphasis on the results obtained from histological and structural analyses of bone biopsies. The evidence currently available indicates that anticatabolic therapies exert their significant antifracture efficacy through a pronounced reduction of bone turnover. This reduction in remodeling activity causes preservation of trabecular structure and a decrease in cortical porosity, both effects that will preserve bone biomechanical strength. Although anticatabolic drugs preserve bone architecture, bone-forming (anabolic) therapies, in this context exemplified by PTH, are able to reverse the deterioration of cancellous and cortical bone architecture seen during age-dependent bone loss and osteoporosis. Recent analyses using techniques enabling analysis of bone matrix constituents suggest that both anticatabolic and anabolic therapies also alter the properties of bone tissue components like mineralization and collagen crosslinking.

The use of recombinant luteinizing hormone in down-regulated patients

The use of recombinant luteinizing hormone in down-regulated patients

Standardisation des immunodosages de la TSH : production de nouveaux calibrateurs et harmonisation des tests

Discordances among TSH measurements have been long documented over the past two decades but are still poorly understood at the molecular level. This review presents an overview of TSH structural polymorphism in the pituitary stock compared to various recombinant preparations and the circulating hormone. Comparative immunochemical analysis of pituitary TSH and a recombinant hormone produced in mammalian cells demonstrate that several epitopes are differentially expressed as a result of alteration in glycosylation. Consequently, variable antibody recognition may be generated between blood samples and internal calibrators among assays. There is thus a need for designing new calibrators which best mimick circulating TSH for developing harmonization of TSH tests.

Expression and purification of a biologically active recombinant rabbitfish ( Siganus guttatus) growth hormone

Recombinant rabbitfish growth hormone (rfGH) protein was expressed in Escherichia coli, BL21(DE3) cells. The cDNA encoding the mature protein of rfGH was first cloned in pGEM-Teasy vector and then transferred to pET-3d expression vector. Expression in E. coli cells was then induced by IPTG (0.4 mM). Inclusion bodies (IB) containing the expressed protein were purified by treating bacterial cells pellet with lysozyme followed by repeated washings in cold water containing Triton X-100, sonication, and centrifugation. IB were then solubilized in 4.5 M urea, refolded at pH 11.3 in the presence of catalytic amounts of cysteine and purified by Q-Sepharose column. Gel filtration on Superdex column showed the purified protein to be a monomeric GH. Based on SDS–PAGE, the purity of the recombinant rfGH preparation is approximately 98%. The recombinant rfGH was tested for its biological activity both in vitro, by its ability to stimulate IGF-I mRNA expression in the liver, and in vivo, by its ability to accelerate growth in rabbitfish fry injected with the hormone. A significant increase in growth was observed in rabbitfish fry given the recombinant hormone. Polyclonal antibody raised against the native rfGH immunoreacted with the recombinant rfGH in Western blots and in ELISA, indicating the suitability of these reagents for future quantification of GH in rabbitfish plasma.

A case of stunning of lung and bone metastases of papillary thyroid cancer after a therapeutic dose (3.7 GBq) of131I and review of the literature: implications for sequential treatments

Thyroid stunning is usually defined as the inhibition or suppression of iodide trapping by remnant thyroid tissue or by functioning metastases following a diagnostic dose of 131I. The risk of stunning increases progressively with larger doses. Because the threshold above which this effect occurs in thyroid remnants seems to be between 37 MBq and 111 MBq of 131I, therapeutic 131I doses of 3.7 GBq may cause stunning. We describe stunning of papillary thyroid cancer lung and bone metastases after a therapeutic dose of 131I (3.7 GBq). A T1 bone metastasis and bilateral lung metastases were diagnosed by post-therapeutic dose whole-body scan. Nuclear MRI detected another lesion at T4, whose 131I fixation was not obvious. An additional 0.7 GBq were given after recombinant TSH, 37 days after the therapeutic dose; 24 h later, uptake by the lung and T1 metastases had disappeared, but trapping was again seen 6 months later on the post-therapeutic scan. This re-appearance is evidence in favour of the transitory and reversible character of stunning, and confirms its correspondence to the decreased ability of viable thyroid cells to trap iodine and not to their destruction. A better understanding of stunning would make it possible, in the event of rapidly progressing disease and in conjunction with recombinant thyroid stimulating hormone (TSH), to give several therapeutic doses of 131I in close succession without each dose hampering the effectiveness of the subsequent one.