Steroidogenic and maturation-inducing potency of native gonadotropic hormones in female chub mackerel, Scomber japonicus.

BACKGROUND: The gonadotropins (GtHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are produced in the pituitary gland and regulates gametogenesis through production of gonadal steroids. However, respective roles of two GtHs in the teleosts are still incompletely characterized due to technical difficulties in the purification of native GtHs. METHODS: Native FSH and LH were purified from the pituitaries of adult chub mackerel, Scomber japonicus by anion-exchange chromatography and immunoblotting using specific antisera. The steroidogenic potency of the intact chub mackerel FSH (cmFSH) and LH (cmLH) were evaluated in mid- and late-vitellogenic stage follicles by measuring the level of gonadal steroids, estradiol-17beta (Ε2) and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P). In addition, we evaluated the maturation-inducing potency of the GtHs on same stage follicles. RESULTS: Both cmFSH and cmLH significantly stimulated E2 production in mid-vitellogenic stage follicles. In contrast, only LH significantly stimulated the production of 17,20beta-P in late-vitellogenic stage follicles. Similarly, cmLH induced final oocyte maturation (FOM) in late-vitellogenic stage follicles. CONCLUSIONS: Present results indicate that both FSH and LH may regulate vitellogenic processes, whereas only LH initiates FOM in chub mackerel.

Purification and characterization of luteinizing hormone from pituitary glands of rockfish, Sebastes schlegeli.

To acquire greater knowledge of the reproductive function of luteinizing hormone (LH) in the viviparous rockfish Sebastes schlegeli, LH from the pituitary glands of mature rockfish was isolated, purified, and localized and its biological activity was characterized. The molecular mass of purified LH was estimated to be approximately 33 kDa, similar to that of known LH. When rockfish LH was purified by reverse-phase high-performance liquid chromatography, its N-terminal amino acid sequences were found to coincide with those of predicted cDNA sequences of rockfish gonadotropin α (ssGTHα) and ssLHß mature peptides. Immunocytochemical analysis using antisera against ssGTHα (molecular weight [MW], ~14.5 kDa) and ssLHß (MW, ~18.5 kDa) indicated that the LH-producing cells are mainly distributed throughout the proximal pars distalis and along the periphery of the pars intermedia. Further, in vitro ovarian follicle analysis demonstrated that purified intact rockfish LH significantly enhances E(2) secretion in a dose-dependent manner. This is the first report on the purification and characterization of LH from a viviparous teleost, and these results will enable future research and increase our understanding of the mechanisms underlying the maturation of such fish.

Divergent immunomodulatory effects of recombinant and urinary-derived FSH, LH, and hCG on human CD4+ T cells.

This study investigated the in vitro immune-modulating activities of recombinant versus highly purified urinary follicle-stimulating hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotropin (hCG) at the cellular level. CD4(+) T cells were isolated from peripheral blood mononuclear cells obtained from ten healthy women (aged 19-30 years) with regular menstrual cycles during the follicular phase of their cycle. CD4(+) T cells were stimulated with anti-CD3/CD28 monoclonal antibodies as a T cell-specific mitogen. Proliferative and cytokine responses were analyzed at standard time points (72h). Recombinant FSH (r-FSH) and LH (r-LH) alone showed a modest capacity to influence proliferation and cytokine release by CD4(+) T cells. Conversely, their addition to T cells in combination with recombinant hCG (r-hCG) induced a powerful down-modulation of T cell proliferation, decreased interferon-gamma (IFN-gamma) secretion and increased interleukin-10 (IL-10) production. These immune-modulating activities were not present when CD4(+) T cells were stimulated either in the presence of urinary-purified FSH (u-FSH) or human menopausal gonadotropin (HMG), alone or in combination with recombinant hCG. We are the first to suggest that urinary-purified gonadotropins do not display profound immune-modulating activities as compared with the recombinant preparations, despite their endocrine effects. Therefore, the use of the recombinant preparations in assisted reproductive techniques might be relevant not only for their well-documented endocrine actions but also for their impact on the transient immune tolerance known to favour embryo implantation and progression of pregnancy.

Caprine (Capra hircus) luteinizing hormone: purification and chromatographic investigation of its different isoforms.

Luteinizing Hormone (LH) from goat pituitary glands has been purified and characterized with respect to its size and subunit nature. The purification at each step was monitored by protein estimation, SDS-PAGE, and direct binding ELISA. The final product was found to be over 90 fold purified as compared to the starting pituitary extract, and the yield of the final purified LH was found to be 65.3 mg/kilogram of wet pituitary glands. Fractionation of the cLH into different charge isoforms by SP-Sephadex ion exchanger has been observed. Chromatography on immobilized Con A lectin resulted in fractionation of the purified cLH into unbound (2%), loosely bound (85%), and firmly bound (13%) fractions indicating oligosaccharide heterogeneity. The purified hormone was capable of stimulating weight increase in the seminal vesicles in immature male rats, with a biopotency equivalent to the 2200 I.U. of hCG per mg of purified cLH. The FSH content of the purified cLH was found to be less than 0.0165% as indicated by in vivo Steelman-Pohley assay.

Use of SP-Sephadexto fractionate and obtain semi-pure preparation from whole pituitaries of Indian water buffaloes (Bubalus bubalis).

Different charge isoforms of native Luteinizing hormone (LH) (dimeric form) Ve/Vo = 1.49 can be fractionated on SP-Sephadex into four different charge isoforms (LHUB, LH25, LH50, and LH100) by stepwise elution using different molarities of Na2HPO4. LHUB was found to be difficult to purify, whereas LH50 and LH100 were found to be pure and highly immunoreactive against anti-bLHbeta serum as indicated by the results obtained from direct binding ELISA and Western blot analysis. SDS-PAGE of LH50 and LH100 showed two bands corresponding to the two subunits of LH. LH25 can be purified to homogeneity by rechromatography on S-300. Purification of buLH as a highly immunoreactive preparation has also been described using SP-Sephadex. This LH preparation (SP25B), which was obtained after slight modification in the pre-existing protocol, has been found to be highly immunoreactive against anti-bLHbeta serum in direct binding ELISA. Being a very simple and reproducible method, it can be used to obtain pure LH preparation, as a reference material, in a short period of time for various immunoassays and bioassays.

A single-step chromatographic method for the purification of proteins devoid of exposed histidine residues.

The principle of the immobilized metal affinity chromatography (IMAC) is based on the differences in the affinity of proteins for metal ions bound in a 1:1 complex of iminodiacetic acid (IDA) immobilized on a chromatographic support. A single step purification was carried out for luteinizing hormone (LH) on Cu2+, Zn2+, Ni2+, and Co2+ IDA Sepharose affinity columns. Highly purified LH was obtained with a Cu2+ IDA Sepharose column. SDS-PAGE and Western blot analysis were done to confirm the purity of the hormone. Biological activity has been evaluated by radio-immunoassay. This method was found economically viable and suitable for the recovery of biologically active hormone.

Isolation and characterization of FSH and LH from pituitary glands of Atlantic halibut (Hippoglossus hippoglossus L.).

Two gonadotropins (GtH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), were isolated and characterized from pituitary glands of Atlantic halibut (Hippoglossus hippoglossus L.). Glycoproteins were extracted in 40% ethanol followed by precipitation in 85% ethanol. Subsequently, glycoproteins were fractionated by ion-exchange chromatography on a Whatman DE-52 column using a stepwise gradient of ammonium bicarbonate (50-1000 mM). Intact FSH and LH were finally purified on rpHPLC using an AsahiPak C4P-50 column with an acetonitrile gradient (10-60%). SDS-PAGE showed a molecular mass of 33 and 32 kDa for intact FSH and LH, respectively. Final purification of subunits was performed by a subsequent purification step on rpHPLC using a Phenomenex Jupiter C18 column with an acetonitrile gradient (10-60%). FSHbeta, LHbeta, and the common alpha subunit showed molecular masses of 25, 24, and 19 kDa, respectively. Subunit identity was confirmed by N-terminal amino acid sequencing. Intact FSH and LH showed gonadotropic activity by stimulating release of 11-ketotestosterone from turbot (Scophthalmus maximus L.) testicular tissue in vitro. This provides the first purification of two distinct GtHs from an evolutionary advanced pleuronectiform teleost.

Hyperexpression and purification of biologically active human luteinizing hormone and human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris.

The glycoprotein hormones, luteinizing hormone (LH), human chorionic gonadotropin (hCG), thyroid stimulating hormone (TSH), and follicle stimulating hormone (FSH), play important roles in overall physiology and reproduction. These hormones are heterodimeric molecules consisting of an identical alpha subunit non-covalently associated with the hormone-specific beta subunit. The inherent structural intricacies possessed by these hormones make them very interesting model systems for structure-function relationship studies of complex dimeric glycoproteins. The structural studies, as well as, the therapeutic applications require large quantities of biologically active hormones free of any contaminants. In this study, we report hyperexpression and purification of biologically active recombinant hLH and hCG expressed using Pichia pastoris expression system. A combination of hydrophobic interaction chromatography and ion exchange chromatography has been used to purify these recombinant hormones to homogeneity. Using a number of biochemical and immunological criteria, the recombinant hormones have been shown to be similar to the natural hormones and were equally biologically active. The preliminary data also suggested that P. pastoris cells express a low molecular weight isoform of hCG that appeared to be less glycosylated. This isoform exhibited lesser affinity for the receptor as compared to hCG, but was found to be fully biologically active.

A novel Ala(-3)Thr mutation in the signal peptide of human luteinizing hormone beta-subunit: potentiation of the inositol phosphate signalling pathway and attenuation of the adenylate cyclase pathway by recombinant variant hormone.

Upon screening for polymorphisms in the human luteinizing hormone beta-subunit (LH beta) gene, we discovered a novel mutation in the LH beta signal peptide with functional consequences for signal transduction in mouse Leydig tumour cells (mLTC-1). This G(52)A point mutation in exon 2 of the LH beta gene, detected in heterozygous form in several normal DNA samples, caused an Ala(-3)Thr amino acid substitution. Recombinant forms of wild-type (WT) and Ala(-3)Thr variant (V) LH were produced in human embryonic kidney (HEK) 293 cells and purified. The immunoreactivities of the recombinant LH were determined by immunofluorometric assays and in-vitro bioactivities in mLTC-1 cells were assessed by using cAMP, progesterone and inositol trisphosphate (IP(3)), and activation of mitogen-activated protein kinase (MAPK) as end-points. Whereas both LH forms stimulated progesterone production and MAPK in similar fashion, WT-LH was more potent in stimulating cAMP, and V-LH was more potent in stimulating IP(3) generation. Both LH forms bound to LH receptors with similar affinities. No evidence was found for influence of the signal peptide mutation on efficacy of alpha- and beta-subunit dimerization. Sequencing of the recombinant V-LH beta protein also revealed that the mutation did not interfere with signal peptide cleavage. In summary, the present findings indicate that the Ala(-3)Thr mutation in the LH beta-subunit signal peptide has functional consequences, in the form of dissociation of stimulatory potency for different signal transduction pathways in vitro.

Synthesis, purification and structural and functional characterization of recombinant form of a common genetic variant of human luteinizing hormone.

A common genetic variant (V) of luteinizing hormone (LH), with two mutations (Trp(8)Arg and Ile(15)Thr) and an extra glycosylation consensus site (Asn(13)-Ala-Thr), is associated with abnormalities of reproductive function. To address the molecular basis of the functional differences between V- and wild-type (WT)-LH, recombinant (rec) forms of WT- and V-LH were synthesized in human embryonic kidney (HEK 293) cells. The rec hormones synthesized were rigorously purified employing affinity, immunoaffinity and ion exchange chromatographies (final purity approximately 12 000 IU/mg, 180-fold purification, 28% recovery). Functional properties of the hormone preparations were compared in vitro and in vivo. The molecular size of both rec LHs was 31 kDa, as determined by SDS-PAGE. Although the mutations in V-LHbeta did not significantly affect the affinity of LH receptor (LHR) binding (Kd approximately 0.4 nmol/L), V-LH had higher in vitro biopotency than WT-LH, in terms of mLTC-1 mouse Leydig tumor cell cAMP and progesterone (P) production, and steroidogenic acute regulatory protein (StAR) expression. In addition, in HEK 293 cells expressing the human LHR, V-LH demonstrated 1.8-fold higher response of inositol trisphosphate (IP(3)) production than WT-LH. Furthermore, HEK 293 cells expressing the ElK1 trans-reporting plasmids displayed 2.7-fold greater luciferase response to V-LH than WT-LH, documenting stimulation of the mitogen-activated protein kinase (MAPK) pathway. The in vivo half-life of V-LH was clearly faster (5-9 min) than that of WT-LH (12-22 min) and human chorionic gonadotropin (hCG; 50-70 min), when injected into rat circulation. It is worth noting that analysis by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) demonstrated clear differences in structures of carbohydrate side chains attached to the two forms of rec LHs, including incomplete processing of high mannose glycans (Man(5,8,9)) in V-LH, suggesting different pathways in its intracellular trafficking. Collectively, the present findings provide the molecular basis for the qualitative and quantitative differences in LH action that are observed in carriers of the V-LHbeta allele.

Isolation of human lutropin from woman urine and comparison of its properties with pituitary hormone.

Lutropin was isolated from woman preovulatory urine by immunoaffinity chromatography using a column with monoclonal antibody (mAb) anti-beta hLH(37) coupled to Sepharose CL 4B. As it was demonstrated the isolated hormone, as well as pituitary lutropin, were separated in SDS-PAGE into several well visible fractions with 30-94 kDa molecular mass and scarcely visible fractions with 14-20 kDa. All fractions reacted only with mAb anti-alpha hCG(99)-HRP but not with mAb anti-beta hLH-HRP. Pretreatment of pituitary lutropin with PN-Gase F did not affect its electrophoretic pattern. After boiling the hormone with SDS and beta ME no fractions in SDS-PAGE were observed. No substantial differences in affinity chromatography on Con A-Sepharose between pituitary and urinary lutropin were noted. Some differences between these two hormone preparations were observed in assays performed with several ELISA variants. Using two pairs of mAbs anti-beta hLH for ELISA technique no hormone was assayed in urine samples from women, collected between 12 and 16 days of menstrual cycles.

Modulation of testicular androgen production in adolescent African catfish (Clarias gariepinus).

At 6 months of age the first spermatozoa appear in the testes of the African catfish considered to be adolescent, since the development to adulthood (12 months of age) is accompanied by further morphological and functional differentiation of Leydig cells. There are increasing plasma levels of 11-ketotestosterone (11-KT) and an increasing responsiveness to luteinizing hormone (LH) of testicular androgen secretion in vitro. Whether treatment of adolescent males with key hormones of the brain-pituitary-gonad axis [gonadotropin-releasing hormone (GnRH), LH, and 11-KT] affects the testicular steroidogenic response to a challenge with LH in vitro 7 days later has been investigated. Injection of GnRH (2.5 microg chicken GnRH-II per kilogram of body weight), LH (25 microg/kg), or a high dose of 11-KT (50 microg/kg) down-regulated basal and LH-stimulated testicular androgen secretion to a minimum of 35% of control values. Treatment with LH was, moreover, associated with changes in the ultrastructure of Leydig cell mitochondria which were either swollen and had a less electron-dense matrix or showed an elongated shape. Conversely, a moderate dose of 11-KT (20 microg/kg) enhanced LH-stimulated, but not basal, androgen secretion in vitro to a maximum of 190% of control values. In view of the generally low LH plasma levels and of the steadily increasing 11-KT plasma levels during puberty, 11-KT may be involved in the up-regulation of the testicular steroidogenic capacity observed during development to full maturity.

Isolation and partial characterization of LH, FSH and TSH from canine pituitary gland.

A new preparative procedure without using ion-exchanger is described for the efficient purification of canine LH (cLH), FSH (cFSH) and TSH (cTSH) from the pituitary gland. The hormones were extracted from the pituitary homogenate with an ammonium sulfate solution, and were separated by Concanavalin (Con) A affinity-, hydrophobic interaction-, then immobilized metal ion affinity chromatography. In the immobilized metal ion affinity chromatography, we used copper (Cu2+) as chelated metal ion with ammonium ion gradient and pH gradient in phosphate buffer to attain separation of the hormones. High purity of cLH, cFSH and cTSH was indicated as single bands in SDS-PAGE, with apparent molecular masses of 34, 36 and 37 kDA, respectively. The purified hormones showed two bands corresponding to alpha (20 kDa) and beta subunits (cLH beta: 16 kDa, cFSH beta: 22 kDa, cTSH beta: 16 kDa) under reducing condition in SDS-PAGE. The purified hormones were prepared in good recovery (LH: 53%, FSH: 34%, TSH: 36%) with high biological activity or binding activity to the receptor. Cross-contamination of the purified hormone was less than 0.5%. Examination of the hormone fraction with isoelectric focusing showed that major peaks of isoelectric isoforms were maintained throughout the purification steps of cLH and cFSH, while a few peaks were lost in Con A affinity chromatography in cTSH purification. It was concluded that the present method could prepare highly purified cLH, cFSH and cTSH which retained isoforms of the hormones and biological activity or binding affinity to the receptor.

Recombinant rat luteinizing hormone; production by Chinese hamster ovary cells, purification and functional characterization.

Rat recombinant (rec) luteinizing hormone (LH) was produced in Chinese hamster ovary (CHO) cells, to enable studies on LH physiology in this species with homologous hormone. The synthesized hormone was purified, and characterized physico-chemically and biologically in comparison with highly purified preparations of rat pituitary (pit) LH (NIDDK-rLH-I-7 and I-9) and to highly purified urinary (NIH, CR-121) and rec forms of human chorionic gonadotropin (hCG). The 33 kD molecular mass of rat recLH, as determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, was comparable with the 32 kD size of pitLH. In chromatofocusing, the isoforms of rat recLH distributed in the pI range 6.5-7.8, similar to rat pitLH. In receptor binding assays using rat testicular membranes, and physiologic salt concentration, rat recLH displayed a 5-10-fold higher affinity than rat pitLH, but about 100-fold lower affinity than hCG. In contrast, in low salt concentrations the affinities of rat recLH and rechCG to rat LH receptor were rather similar. The differences in potency in the mouse Leydig cell in vitro bioassay were in agreement with the receptor binding data at physiologic salt concentration. Neither rat recLH nor pitLH stimulated cAMP production or bound specifically to HEK 293 cells expressing the rec human LH receptor. When injected subcutaneously on four consecutive days to male rats (8.4-33.7 microg/rat/day) rat recLH did not induce seminal vesicle growth in comparison with a significant effect of human menopausal gonadotropin (hMG; 12.5-50 IU/rat/day). In contrast, ovulation was induced in 5/6 and 6/6 female rats following single injections of 3.75 and 7.5 microg of rat recLH, respectively, after pretreatment with 10 microg/kg of a GnRH-antagonist (Org 30850). In conclusion, rat recLH displays clearly lower in vivo and in vitro bioactivity than hCG. Nevertheless, it binds effectively to the rat LH receptor (with affinity dependent on salt concentration) and is bioactive in the mouse Leydig cell bioassay. This newly synthesized recombinant hormone provides a useful tool for further studies on the physiology of LH action in the rat, the most common animal model in reproduction research.

Isolation, characterization and radioimmunoassay of luteinizing hormone in the brushtail possum.

Luteinizing hormone (LH) was purified from brushtail possum (Trichosurus vulpecula) pituitary glands. The purification procedure consisted of ammonium sulfate precipitation followed by triazinyl-dye chromatography, hydrophobic interaction chromatography and gel filtration. A yield of 10 microg LH g-1 pituitary with a recovery of 20% was obtained from 1400 pituitary glands (20.3 g). Contamination with possum follicle-stimulating hormone (FSH) was < or =0.05%. The amino acid analysis and the N-terminal sequencing for 10 cycles revealed close homology with LH from other mammals. Minor amounts of LH that had been truncated near the N-terminal were also detected. No contaminating proteins were found by amino acid sequencing. The potency of possum LH was 20% that of ovine LH in a receptor assay using possum testicular receptors and 4% that of ovine LH when bovine corpora lutea receptors were used. Possum LH was able to stimulate production of cyclic adenosine 3',5'-monophosphate by bovine granulosa cells. A radioimmunoassay (RIA) for possum LH using 125I-possum LH and an antiserum raised against ovine LH was developed. The RIA has a sensitivity of 0.15 ng mL-1, a 50% displacement of 1.9 ng mL-1 and a cross-reactivity of <0.02% against possum FSH. Plasma concentrations were 0.24+/-0.04 ng mL-1 (n = 8) and 0.39+/-0.12 ng mL-1 (n = 8) in female and male possums respectively. Administration of mammalian gonadotrophin-releasing hormone (GnRH) and chicken GnRH II stimulated increases in plasma LH concentrations in male and female possums. When comparing LH responses with administration of mammalian GnRH or chicken GnRH II, plasma LH concentrations remained elevated for a longer period of time in males than in females (P < 0.01); plasma LH concentrations also remained elevated for longer after mammalian GnRH than after chicken GnRH II (P < 0.01). Gonadectomy stimulated an increase in plasma concentrations of LH in both male (P < 0.01) and female (P < 0.05) possums. The rate of increase in plasma LH concentrations in males was faster than that in females. In summary, we have purified, partially characterized, and developed a RIA for possum LH.

Isolation and partial characterization of tammar wallaby luteinizing hormone and development of a radioimmunoassay.

Tammar wallaby (Macropus eugenii) luteinizing hormone (LH) was purified from pituitaries collected from wild and captive populations by salt sequential precipitation, ion exchange chromatography and gel filtration. Pituitary tissue (5 g) yielded 1.8 mg of purified wallaby luteinizing hormone (ME-14B), as verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A heterologous radioimmunoassay has been developed for measurement of LH in plasma of marsupials using a monoclonal antibody raised against bovine LH (518B7). This assay system was able to measure basal LH concentrations in male and female tammars and detected a significant rise in plasma LH in response to oestradiol benzoate in female tammars and luteinizing hormone-releasing hormone (LHRH) in males. Parallel dose-response curves were also obtained from pituitary extracts from four other species of marsupial (brushtail possum, Trichosurus vulpecula; brown antechinus, Antechinus stuartii; kowari, Dasyuroides byrnei; and Eastern pygmy possum, Cercartetus nanus) in this assay, which suggests its usefulness in the measurement of LH in other marsupial species.

Effect of desialylation of highly purified isoforms of human luteinizing hormone on their bioactivity in vitro, radioreceptor activity and immunoactivity.

To establish whether sialic acid content is responsible for an observed 7-8-fold variability in bioactivity in vitro of highly purified human pituitary luteinizing hormone (hLH) isoforms, the bioactivity in vitro, radioreceptor activity and immunoactivity of hLH isoforms were determined before and after enzymatic desialylation. Three immunofluorometric assays with different hLH specificities allowed characterization of 13-24 pituitary hLH isoform preparations of pI 7.03-8.98 in terms of sialic acid content (1-5 sialic acid residues per LH molecule), bioactivity in vitro (4030-30,000 I.U. mg[-1]), radioreceptor activity (6420-25,400 I.U. mg[-1]) and hLH immunoactivity (2900-4400 to 18,300-27,300 I.U. mg[-1]). Significant positive correlations between sialic acid content and either immunoactivity or in vitro bioactivity were observed, whereas radioreceptor activity showed a curvilinear response. Following more than 90% removal of sialic acid, both in vitro bioactivity and radioreceptor activity were increased, although specific activity still differed between isoforms; immunoactivities were unaffected. It is concluded that the presence of the sialic acid residue(s) on hLH isoforms does partially contribute to the in vitro bioactivity and radioreceptor activity of the isoforms, but that hLH immunoactivity is independent of sialic acid content.

Structural and functional characterisation of hFSH and hLH isoforms.

Human follicle-stimulating hormone (hFSH) and luteinizing hormone (hLH) are gonadotropins which are secreted as multiple forms by the pituitary. Evidence supporting the structural and functional heterogeneity of 15 purified hFSH isoforms and 20 purified hLH isoforms from pituitary extracts will be presented. Gonadotropin isoforms were purified by a combination of preparative isoelectric focusing and ion-exchange chromatography. The protein mass of each isoform was determined by amino acid analysis, which also correlated (data for hLH) (r = 0.999, P < 0.001, n = 15) with the UV area under the curve at 280 nm of the isoforms following gel-filtration HPLC. The alpha and beta subunits of FSH and LH were shown to be intact by SDS-PAGE under reducing condition, with no evidence of proteolytic nicking or presence of contaminating proteins. hFSH radioreceptor activity varied over a seven-fold range, and a positive correlation (r = 0.85, P < 0.001, n = 9) was observed between FSH receptor activity and the sialic acid (SA) content (1.5-13.7 mol SA/mol hFSH) of the isoforms, as determined by an HPLC-based microfluorometric assay. FSH in vitro activities varied over a similar range with a high correlation (r = 0.82, n = 15) with receptor activities, suggesting that the initial association of the hormone with the receptor is the key interaction with less differences attributed to subsequent effects in the signaling pathway. A similar result was seen with the hLH isoforms. To explore FSH/LH in vivo, the circulating half-life (LH/FSH) and the in vivo bioactivity (LH) using an acute in vivo assay was investigated. The clearance of hLH and hFSH showed a bi-exponential pattern for all isoform preparations with the proportion of the slower dissociating component (t 1/2 50-60 min) increasing three-fold with increasing sialic acid content of the isoform. The more rapidly cleared component (t 1/2 approx 10 min) is attributed to hepatically cleared gonadotropin, rather than gonadotropin equilibration between body compartments. The in vivo assay procedure for LH was based on the 24 h integrated plasma testosterone levels in rats following administration of graded doses of hLH isoform or standard. A 16-fold range in vivo activities between LH isoforms (n = 14) was observed. A comparison between hLH in vitro and in vivo activities showed a good correlation (r = 0.75) with the slope of the regression line (1.39) not significantly different from unity. These results suggest that in this acute in vivo assay method, the differences in circulating half-lives between hLH isoforms although large is not a key factor in their in vivo activity. However, in chronic in vivo assay systems the differences in clearance rates between isoforms may be important in their subsequent biological response. It is concluded that structural heterogeneity of FSH and LH contributes to functional differences, with a key interaction occurring at the receptor level. The contribution of sialic acid to these activities was also investigated.

In vivo bioactivities and clearance patterns of highly purified human luteinizing hormone isoforms.

Previous studies have shown that highly purified isoforms of human pituitary LH exhibited a 20-fold range of in vitro bioactivities. The aim of this study was to determine the corresponding plasma half-lives, metabolic clearance rates (MCR), and in vivo bioactivities of these human (h) LH isoforms. Cannulated adult male rats were administered hLH isoforms as a bolus i.v. injection. For the half-life studies, blood was then serially collected over a 6-h period, and serum was assayed for hLH using a specific immunofluorometric assay. All hLH (n = 19) isoforms exhibited biexponential disappearance profiles with an initial fast half-life (t 1/2) for component A of 12.8 +/- 3.7 min, followed by a slow component B with t 1/2 of 58.9 +/- 4.4 min. The prevalence of component B in relation to component A increased significantly (r = 0.81, P < 0.001) over a 3-fold range when correlated with the sialic acid content of the isoform. Similarly, the MCR showed a significant correlation (r = 0.77, P < 0.001) with sialic acid content. The basis for the two t 1/2 components was then investigated. In the first experiment, rat plasma containing primarily component B was collected 90 min after hLH isoform administration and injected into a second animal. Only component B was observed with no evidence of component A, which indicates that the two t 1/2 components are not the product of the redistribution of the hLH isoform between body compartments. In the second experiment, component B was found to be dependent on sialic acid content, as desialylated hLH isoforms showed a rapid disappearance (t 1/2 = 8.6 +/- 3.1) with the component B proportion decreasing to < 10% of that of the nondesialylated control. This data indicates that sialic acid protects component B from rapid clearance. In addition, the proportion of the two components is dependent on sialic acid content, suggesting that the molecular location of the sialic acid on the carbohydrate moieties of hLH has a critical role in the clearance process. To determine the in vivo bioactivity of the hLH isoforms, an acute in vivo bioassay was developed in male rats. The assay was based on the hLH dose-dependent increase in total testosterone release in the same rat model as used in the plasma disappearance studies. Using the second International Standard (IS) hLH (0.3 IU-2.6 IU/kg) as standard, a linear dose-response of 24-h integrated serum testosterone levels was observed, with an index of precision of 0.11. Using this in vivo assay, a 16-fold range in in vivo bioactivities (3,200 to 51,100 IU/mg) was observed for 14 hLH isoforms. These in vivo bioactivities correlated with sialic acid content (r = 0.78, P < 0.001), MCR (r = 0.56, P < 0.05) and LH in vitro bioactivity (r = 0.75, P < 0.001) as determined using mouse Leydig cells in culture. Desialylation lead to over a 100-fold decrease in in vivo bioactivity of hLH. It is concluded that hLH isoforms are cleared in vivo by a two-component clearance mechanism, the proportion of which varies between isoforms and is dependent on sialic acid content of the isoform. These findings suggest that the molecular location of sialic acid on the hLH isoform is critical in defining the plasma disappearance of component B, whereas the mechanism of elimination of component A may well involve the hepatic GalNAc-sulphate receptor. Using an in vivo bioassay, the 16-fold difference in bioactivity between isoforms is attributed primarily to differences in their in vitro activity at the cellular level with a minor influence (< 2-fold) due to differences in in vivo clearance.