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Sensitive monitoring of luteinizing hormone (LH), a glycoprotein that regulates the synthesis of regulatory steroid hormones, can facilitate the diagnosis of various reproductive diseases. In this work, a new and highly catalytic Sulfur-doped and bimetal-coordinated CoFe(CN) 5 NO (denoted as S-CoFe(CN) 5 NO) nanoparticles are synthesized. Such material is further used to construct high performance sensing interface and coupled with primer exchange reaction (PER) and hybridization chain reaction (HCR) amplification cascades for sensitive electrochemical aptamer-based LH assay. Target LH molecules bind aptamer sequences in DNA duplex probes to liberate ssDNA strands, which initiate subsequent PER/HCR amplification cascades for the capture of many ferrocene (Fc)-tagged DNAs on sensing interface. S-CoFe(CN) 5 NO subsequently leads to catalytic oxidation of these Fc tags for yielding substantially magnified currents for realizing ultrasensitive assay of LH with the detection limit of 0.69 pM in range from 5 pM to 10 nM. Owing to the high specificity of aptamer, such sensor has high selectivity and can achieve low levels of LH assay in diluted serum samples. With the successful demonstration for detecting trace LH, such sensor can be easily extended as a universal aptamer-based electrochemical sensing method for monitoring various target analytes in the biomedical and biological fields., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The quantitative detection of luteinising hormone (LH) is critical for the study of the physiological mechanism of reproductive function and the assessment of infertility and the clinical treatment of reproductive disorders. However, conventional approaches for LH detection are mostly based on an antibody recognition module with the limitations of sensitivity, simplicity and cost. The development of robust LH sensing methods is therefore highly demanded for facilitating the diagnosis of LH-related diseases. We establish a convenient, amplified and sensitive fluorescent aptamer LH assay based on new target-triggered and cascaded autocatalytic hairpin assembly (C-aCHA) circuit amplification means via initiator sequence replication. Target LH molecules bind the aptamers in the aptamer/initiator duplexes to release the initiator sequences, which trigger CHA formation of DNA three-way junctions (TWJs) and the unfolding of fluorescently quenched signal hairpins to show amplified fluorescence. The TWJs further activate another CHA cycle for the yield of more initiator sequences to form the C-aCHA circuit amplification cycles, which lead to the unfolding of many signal hairpins to exhibit substantially magnified fluorescence recovery for detecting LH down to 8.56 pM in the range from 10 pM to 50 nM. In addition, the monitoring of trace LH in diluted serums by this sensing approach has been also verified. Our LH assay clearly outperforms current existing antibody-based methods and the C-aCHA signal amplification strategy can be easily extended as a robust means for sensitively monitoring various biomolecular markers with simple replacement of the corresponding aptamers for diverse applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In mammals, the gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are macromolecules secreted during specific reproductive phases and display strict specificity towards their cognate receptors. However, fish gonadotropins (GTH) and their receptors (GTHR) display diverse species-specific expression patterns, secretion patterns, and intra- and interspecies cross-activation. To uncover the molecular basis of this diversity, we generated and analyzed 29 in-silico models of intra- and inter-species combinations of sturgeon, carp, tilapia, and human gonadotropins with piscine receptors and analyzed the resulting receptor activation and signal transduction of these GTHR-GTH complexes in-vitro. Our results suggest that unlike humans, the surface charge on piscine FSH/LH β-seatbelt and N107 huLHCGR /K104 hFSHR homologs does not necessarily determine binding specificity. Instead, sequence and structural variations allow piscine GTHs significant conformational flexibility when binding to the receptor extracellular domain, thereby enabling cross-activation. The resulting diversity may support various reproductive strategies in different environmental niches., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Berta Levavi-Sivan reports financial support was provided by Israel Science Foundation. Berta Levavi-Sivan reports financial support was provided by European Union., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: To study the value of basal luteinizing hormone (LH) level combined with uterine volume measurement in the early diagnosis of central precocious puberty (CPP) in girls with different Tanner stages., Methods: A retrospective analysis was performed on the girls who presented with breast development before the age of 8 years and attended the Third Affiliated Hospital of Zhengzhou University from January 2017 to September 2022. According to the results of gonadotropin-releasing hormone (GnRH) agonist test, the girls with peak LH ≥5.0 IU/L and peak LH/follicle stimulating hormone ≥0.6 were enrolled as the positive group, and the other girls were enrolled as the negative group. The two groups were compared in terms of the basal LH level and uterine volume. The receiver operating characteristic (ROC) curve was used to analyze their value in the early diagnosis of CPP., Results: For the girls with Tanner B2 and B3 stages, the positive group had significantly higher basal LH level and uterine volume than the negative group ( P <0.05). The basal LH level had an optimal cut-off value of 0.325 IU/L and 0.505 IU/L respectively in the diagnosis of Tanner stage B2/B3 CPP, while uterine volume had an optimal cut-off value of 1.639 mL and 2.158 mL respectively. Basal LH level combined with uterine volume measurement had a significantly larger area under the ROC curve than uterine volume measurement alone ( P <0.001), but with no significant difference compared with that of basal LH level measurement alone ( P >0.05)., Conclusions: Basal LH level combined with uterine volume measurement is valuable in the early diagnosis of CPP in girls with different Tanner stages, which provides a basis and guiding significance for clinical diagnosis of CPP.

Background: There is still no consensus on the optimal monitoring method to evaluate the hypothalamic-pituitary-gonadal axis (HPGA) inhibition., Methods: There were 124 girls treated with triptorelin depot due to puberty disorders, including 77 central precocious puberty and 47 early puberty. After treatment, triptorelin stimulation tests were performed, and blood samples were collected at 0, 20, 40 and 60 min. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured by immunochemiluminometric assay (ICMA)., Results: Peak LH (PLH), peak FSH and estradiol in 124 girls were significantly decreased after treatment, while 2 cases had inadequate treatment efficacy. Areas under the receiver operating characteristic curves (AUC) of PLH and peak FSH after stimulation for the diagnosis of HPGA suppression were 0.984 and 0.121. When the cut-off value of PLH was ≤ 2.25 IU/L, the sensitivity was 96.7% and specificity was 100.0%. There was no difference in AUC between PLH and a single LH at 20, 40, or 60 min ( p  > 0.05). When LH were ≤ 2.34 IU/L, ≤ 2.21 IU/L and ≤ 2.00 IU/L at 20, 40 and 60 min, respectively, the sensitivity were 99.1%, 96.7% and 98.4%, and the specificity were all 100.0%. The correlation coefficients between PLH and LH at 20, 40 or 60 min were 0.947, 0.975 and 0.961., Conclusion: A single blood sample for stimulated LH at 20 min, 40 min, or 60 min assayed by ICMA during triptorelin stimulation test is useful for monitoring the treatment efficacy of triptorelin depot in girls with puberty disorders.

We encountered a 30-year-old woman with remarkably elevated luteinizing hormone (LH) levels, as measured by electrochemiluminescent immunoassay (ECLIA), and no specific symptoms. We performed the following investigations: dilution linearity test, polyethylene glycol (PEG) precipitation test, immunoprecipitation test, protein G addition test, and high-performance liquid chromatography (HPLC) analysis. The linearity of patient's serum was similar to that of a standard LH preparation, and non-specific reactions were not observed. The recovery rate of LH shown by the PEG precipitation test, immunoprecipitation test, and protein G addition test was low. Moreover, an abnormal peak in HPLC was located at a slightly larger molecular weight position than that of IgG. These results showed the presence of macro-LH, LH, and anti-LH-IgG autoantibody complex and suggested that the clearance of LH from the blood was delayed due to IgG binding, and therefore, the LH value was falsely high. We should keep the possibility of macro-LH in mind in cases of unexpectedly high LH values., (Copyright © 2021 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

We present a novel peptide sequence identified through in silico epitope design and the later generation of peptide-directed antibodies recognizing the buffalo luteinizing hormone. Peptides and antibodies, specific to reproductive hormones, are valuable tools for developing point-of-care immunodiagnostic tools. The study predicted an epitope peptide in silico from buffalo luteinizing hormone and the generation of polyclonal antibodies against this peptide sequence. In this quest, we identified a novel epitope peptide sequence (luteinizing hormone peptide, LHP) through bioinformatics tools. The peptide was further synthesized and characterized. The polyclonal antibodies (anti-LHP) were raised against the peptide in the rabbit. Thereafter, we explored a strategy for detecting buffalo luteinizing hormone (LH) using the anti-peptide antibodies developed. The affinity of the peptide, bovine lutropin beta, and crude LH (prepared from buffalo pituitary) towards the raised antibodies was established by dot blot and ELISA. Specific recognition of the luteinizing hormone by the raised polyclonal antibodies highlights the ability of the identified peptide (LHP) and developed polyclonal antibodies (anti-LHP) as suitable diagnostic reagents for sensing the buffalo luteinizing hormone. Through this work, we analyzed and translated the "-omics" information in the LH gene sequence for the development of a novel peptide and antibodies as valuable immuno-reagents., Competing Interests: Declaration of competing interest There are no conflicts to declare. The authors declare that they have no conflict of interest and no competing financial interest. A patent application with application number 1854/DEL/2015 and the title “Peptide sequence and polyclonal antibodies for the detection of cow and buffalo luteinizing hormone” has been filed at the Indian Patent Office., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Reproduction in vertebrates is controlled by the brain-pituitary-gonad axis, where the two gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) play vital parts by activating their cognate receptors in the gonads. The main purpose of this work was to study intra- and interspecies ligand promiscuity of teleost gonadotropin receptors, since teleost receptor specificity is unclear, in contrast to mammalian receptors. Receptor activation was investigated by transfecting COS-7 cells with either Fsh receptor (mdFshr, tiFshr) or Lh receptor (mdLhr, tiLhr), and tested for activation by recombinant homologous and heterologous ligands (mdFshβα, mdLhβα, tiFshβα, tiLhβα) from two representative fish orders, Japanese medaka (Oryzias latipes, Beloniformes) and Nile tilapia (Oreochromis niloticus, Cichliformes). Results showed that each gonadotropin preferentially activates its own cognate receptor. Cross-reactivity was detected to some extent as mdFshβα was able to activate the mdLhr, and mdLhβα the mdFshr. Medaka pituitary extract (MPE) stimulated CRE-LUC activity in COS-7 cells expressing mdlhr, but could not stimulate cells expressing mdfshr. Recombinant tiLhβα, tiFshβα and tilapia pituitary extract (TPE) could activate the mdLhr, suggesting cross-species reactivity for mdLhr. Cross-species reactivity was also detected for mdFshr due to activation by tiFshβα, tiLhβα, and TPE, as well as for tiFshr and tiLhr due to stimulation by mdFshβα, mdLhβα, and MPE. Tissue distribution analysis of gene expression revealed that medaka receptors, fshr and lhr, are highly expressed in both ovary and testis. High expression levels were found for lhr also in brain, while fshr was expressed at low levels. Both fshr and lhr mRNA levels increased significantly during testis development. Amino acid sequence alignment and three-dimensional modelling of ligands and receptors highlighted conserved beta sheet domains of both Fsh and Lh between Japanese medaka and Nile tilapia. It also showed a higher structural homology and similarity of transmembrane regions of Lhr between both species, in contrast to Fshr, possibly related to the substitution of the conserved cysteine residue in the transmembrane domain 6 in medaka Fshr with glycine. Taken together, this is the first characterization of medaka Fshr and Lhr using homologous ligands, enabling to better understand teleost hormone-receptor interactions and specificities. The data suggest partial ligand promiscuity and cross-species reactivity between gonadotropins and their receptors in medaka and tilapia., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Oxidative stress is an imbalance between free radicals and antioxidants which leads to reactive oxygen species (ROS) production in cells. Reactive oxygen species contains oxygen radicals that easily react with other molecules in the biological system. For decades, lead acetate (Pb(C 2 H 3 O2) 2 ) is used as an additive for many widely used chemical products such as insecticides, hair dyes, and cosmetics; however, contact with lead acetate may irritate skin, eyes, and mucous membranes.In the present study, the antioxidant and anti-inflammatory effect of using ferulic acid to inhibit lead acetate-induced toxicity in rats is investigated. Lead acetate was orally given at a dose of 20 mg/kg body weight for 10 days, either alone or with ferulic acid at dose 25 mg/kg. Serum luteinizing hormone (LH), total testosterone, and follicle-stimulating hormone (FSH) levels were measured. Also, reactive oxygen species (ROS), lipid peroxidation (LPO), total antioxidant capacity (TAC), and catalase (CAT) activities were determined. In addition, histopathological changes of testes and kidney were examined. Results showed that administration of lead acetate induced oxidative stress through attenuation of luteinizing hormone, total testosterone, and follicle-stimulating hormone levels in serum. Moreover, the kidney and testes of lead acetate-treated animals exhibited elevation of ROS level, lipid peroxide levels, as well as lysosomal enzyme activity such acid phosphatase and N-acetyl-β-glucosminidase. DNA fragmentation and histological changes were also observed in lead acetate-treated group. In contrast, ferulic acid treatment reduced the deleterious effects induced by lead acetate in both testes and kidney tissues. These results illustrated that ferulic acid has a protective action against toxicity caused by lead acetate in rats. In conclusions, ferulic acid may have future therapeutic relevance in the prevention of lead acetate-induced testicular and renal toxicity in rats.

Glycoengineering is a recently used approach to extend serum half-life of valuable protein therapeutics. One aspect of glycoengineering is to introduce new N-glycosylation site (Asn-X-Thr/Ser, where X ≠ Pro) into desirable positions in the peptide backbone, resulting in the generation of hyper-glycosylated protein. In this study, human luteinizing hormone (LH) was considered for identification of the suitable positions for the addition of new N-linked glycosylation sites. A rational in silico approach was applied for prediction of structural and functional alterations caused by changes in amino acid sequence. As the first step, we explored the amino acid sequence of LH to find out desirable positions for introducing Asn or/and Thr to create new N-glycosylation sites. This exploration led to the identification of 38 potential N-glycan sites, and then the four acceptable ones were selected for further analysis. Three-dimensional (3D) structures of the selected analogs were generated and examined by the model evaluation methods. Finally, two analogs with one additional glycosylation site were suggested as the qualified analogs for hyper-glycosylation of the LH, which can be considered for further experimental investigations. Our computational strategy can reduce laborious and time-consuming experimental analyses of the analogs., (Copyright © 2019. Published by Elsevier Ltd.)

Since the availability on the European market of the vaccine Improvac® dedicated to male pig immunological castration, the risk of misuse of this product in horses is now considered as a threat for the horseracing industry. Immunological castration is not allowed by the racing codes (immune system, Article 6). Indeed, this vaccination against the hypothalamic hormone luteinizing hormone-releasing hormone or gonadotropin-releasing hormone (GnRH) will prevent the release from the anterior pituitary of luteinizing hormone and follicle stimulating hormone, which are required for the development and activity of gonads in males (testes) and female (ovaries) and therefore all their subsequent physiological functions. This treatment will induce a strong hormonal variation resulting in a behaviour modification of the animals. In this work, four male standardbreds treated with Improvac® vaccine (two intramuscular injections within 4 weeks) were studied. Monitoring of the total scrotal width showed a decrease of the scrotum size (37%) and production of anti-GnRH antibodies was detected up to 200 days after the first injection. Anti-GnRH antibodies were detected in plasma after caprylic acid precipitation followed by an enzyme-linked immunosorbent assay (ELISA) as a rapid and efficient screening method applicable to routine analysis. These results were correlated to a switch of the sexual status from male group to gelding/female group obtained by a steroidomic approach with urine based on ten endogenous compounds. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Based on a large population of 1113 men aged 30-60 at baseline (mean: 44.1 years, standard deviation: 10.5), we investigated whether intra-individual changes in testosterone (T) and related reproductive hormones during a ten year period were dependent of marital status at baseline and follow-up. The studied men were part of a health survey in Denmark, conducted between 1982 and 1984 with a follow-up examination approximately ten years later. Data on reproductive hormones, measured in serum, and lifestyle and marital status were obtained at both time points. As expected, an age-related decline in testosterone was observed. However, independent of age and lifestyle, we observed that men who went from unmarried to married (n=81) during the study period experienced an accelerated age-related decline in testosterone (-6.6nmol/L) whereas men who went from married to unmarried (n=67) experienced an attenuated age-related decline (-2.3nmol/L). Men who were either married or unmarried at both time points (n=167, n=798, respectively) had a testosterone decline in between (-3.7nmol/L and -4.6nmol/L, respectively). Changes in T/LH ratio did not differ according to marital status indicating that the lowered T level is not compensated by increasing LH levels. This could suggest a modification of the gonadostat due to an adaptation to changing life circumstances., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In vertebrates, the regulation of gametogenesis is under the control of gonadotropins (Gth), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh). In fish, the physiological role of Gths is not fully understood, especially in species with asynchronous ovarian development. To elucidate the role of Gths in species with asynchronous ovary, we studied European hake (Merluccius merluccius) during the reproductive season. For this aim, we first cloned and sequenced both hormones. Then, we characterized their amino acid sequence and performed phylogenetic analyses to verify the relationship to their orthologues in other species. In addition, the quantification of gene expression during their natural reproductive season was analyzed in wild-caught female hake. Our results revealed that fshb peaked during the vitellogenic phase, remaining high until spawning. This is in contrast to the situation in species with synchronous ovary. lhb, on the other hand, peaked during maturation as it is also common in species with synchronous ovarian development. Finally, combining double-labeling fluorescent in situ hybridization (FISH) for Gth mRNAs with immunofluorescence for Lh protein, we evidenced the specific expression of fshb and lhb in different cells within the proximal pars distalis (PPD) of the pituitary. In addition to gonadotrope cells specific to expression of either fshb or lhb, some cells showed co-expression of both genes. This suggests either that gonadotropes with co-expression are not yet specified or they could have a plasticity that permits changes from one cell phenotype to another during certain life stages and in turn during different physiological states., (© 2017 Society for Reproduction and Fertility.)

In the reproduction process of male and female fish, pituitary derived gonadotropins (GTHs) play a key role. To be able to specifically investigate certain functions of Luteinizing (LH) and Follicle stimulating hormone (FSH) in Russian sturgeon (Acipenser gueldenstaedtii; st), we produced recombinant variants of the hormones using the yeast Pichia pastoris as a protein production system. We accomplished to create in vitro biologically active heterodimeric glycoproteins consisting of two associated α- and β-subunits in sufficient quantities. Three dimensional modelling of both GTHs was conducted in order to study the differences between the two GTHs. Antibodies were produced against the unique β-subunit of each of the GTHs, in order to be used for immunohistochemical analysis and to develop an ELISA for blood and pituitary hormone quantification. This detection technique revealed the specific localization of the LH and FSH cells in the sturgeon pituitary and pointed out that both cell types are present in substantially higher numbers in mature males and females, compared to immature fish. With the newly attained option to prevent cross-contamination when investigating on the effects of GTH administration, we compared the steroidogeneic response (estradiol and 11-Keto testosterone (11-KT) in female and males, respectively) of recombinant stLH, stFSH, and carp pituitary extract in male and female sturgeon gonads at different developmental stages. Finally, we injected commercially available gonadotropin releasing hormones analog (GnRH) to mature females, and found a moderate effect on the development of ovarian follicles. Application of only testosterone (T) resulted in a significant increase in circulating levels of 11-KT whereas the combination of GnRH + T did not affect steroid levels at all. The response pattern for estradiol demonstrated a similar situation. FSH levels showed significant increases when GnRH + T was administered, while no changes were present in LH levels., Competing Interests: The authors have declared that no competing interests exist.

Although an increase in VEGF expression and synthesis in association with LH has been established; it is unknown if all LH isoforms act similarly. This study evaluated the production of cAMP and VEGF among LH isoforms in two in vitro bioassays. The LH was obtained from hypophyses and the group of isoforms was isolated by chromatofocusing. cAMP production was assessed using the in vitro bioassay of HEK-293 cells and VEGF production was evaluated in granulosa cells. Immunological activity was measured with a homologous RIA. Immunoactivity and bioactivity for each isoform were compared against a standard, by estimating the IC50 and the EC50. The basic isoforms were more immunoactive than the standard. The neutral and the moderately acidic had an immunological activity similar to the standard. The acidic isoform was the least immunoreactive. cAMP production at the EC50 dose was similar among the basic isoforms, the moderately acidic and the standard; for the neutral and the acidic, the EC50 dose was higher. It was observed that compared with the control, VEGF production at the lowest LH dose was no different in the standard and each isoform. In the intermediate dose, a positive response was caused in the standard and the neutral and basic isoforms. Although the acidic isoform showed a dose-dependent response, it was not significant relative to the control. In conclusion, the basic isoform generated the greatest cAMP and VEGF production, similar to the reference standard, and the acidic the smallest., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objective: To evaluate the toxic effects of mixture of volatile organic compounds (VOCs) on Mice Testis related enzymes and hormones., Methods: After determining the median lethal dose (LD₅₀) of VOCs using the acute toxicity test, 40 male clean inbred Kunming mice were assigned to 1/8 LD₅₀ VOCs exposure group, 1/4 LD₅₀ VOCs exposure group, and 1/2 LD₅₀ VOCs exposure group, as well as positive control group with cyclophosphamide (60 mg/kg) and negative control group with tea oil, with 8 mice in each group. The mice were intraperitoneally injected with respective agents for 5 days. The levels of testis testosterone, estradiol, follicle stimulating hormone, and luteinizing hormone were determined by ELISA. Meanwhile, the activity of testicular marked enzymes such as lactate dehydrogenase, gamma-glutamyl transpeptidase, acid phosphatase, and glucose-6-phosphate dehydrogenase were examined., Results: Compared with the negative control group, the 1/8 LD₅₀ exposure group had a significantly increased testis coefficient (P<0.05). Both the activity of testicular marked enzymes and the levels of testicular sex hormones in all exposure groups showed significant downward trends with increasing VOC doses compared with those in the negative control group (P<0.05)., Conclusion: VOCs have obvious toxicity to mouse testis by changing the levels of testicular sex hormones and the activity of testicular marked enzymes.

Purpose: To verify if polymorphisms of LH (Trp8Arg/Ile15Thr), LH receptor (insLQ), and FSH receptor (Asn680Ser) are associated with endometriosis and infertility., Methods: This is a prospective case-control study. Sixty-seven patients with endometriosis and infertility (study group) and 65 healthy fertile patients (control group) were enrolled in the study between July 2010 and July 2013. All patients had their endometriosis diagnosis made or excluded by laparoscopic surgery; study group was submitted to the surgery for infertility investigation and control group for tubal ligation. Day-3 serum hormones were collected from all patients. Analysis of nucleotide mutations for LH polymorphisms (Trp8Arg and Ile15Thr), LHR polymorphism (insLQ), and FSHR polymorphism (Asn680Ser) were performed by PCR., Results: Day-3 FSH, estradiol and LH serum levels were not different between the groups, while CA-125 was higher in patients with endometriosis and infertility. All polymorphisms studied were in Hardy-Weinberg equilibrium. The prevalence of insLQ was significantly higher in patients with endometriosis and infertility (P = 0.005). Allele occurrence in control group was 0.10 versus 0.25 in infertile endometriosis group (P = 0.001). There was no difference regarding Trp8Arg/Ile15Thr (P > 0.05) and Asn680Ser (P > 0.05) prevalence between groups., Conclusion: This is the first time that prevalence of insLQ was shown to be higher in patients with endometriosis and infertility than in healthy fertile patients. There was no difference in LH and FSHR polymorphisms' prevalence between groups.

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 (t1/2 > 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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Quaternary structure of human, bovine and ovine Follicle-Stimulating Hormones (hFSH, bFSH and oFSH) and Luteinizing Hormone was assessed in sandwich ELISAs using monoclonal anti-oFSHβ or anti-oLHβ antibodies, respectively, for capture and a biotinylated anti-hFSHα (α4 epitope) for detection. Neither free subunit gave any signal in this assay so that it was possible to measure the residual heterodimeric fraction after thermal treatment of the gonadotropins under study. The hormones were subjected to 5-min heating between 37 and 90 °C before rapid cooling in melting ice before ELISA. The data show half-dissociation of natural and recombinant human and ovine FSH preparations between 68 and 74 °C whereas bovine FSH preparations exhibited lower stability in these conditions with half-dissociation between 61 and 64 °C. Moreover, whereas all human and bovine as well as most ovine FSH preparations were fully dissociated at temperatures above 80 °C, one natural oFSH and one recombinant hLH preparations contained an important fraction that resisted dissociation even at 93 °C and retained in vitro bioactivity. This suggests the existence of gonadotropin αβ heterodimer with covalently linked subunits. Similarly, about 20% of the recombinant hLH preparation was also found withstand heat denaturation and also probably to have cross-linked subunits. The origin and chemical nature of these inter-subunit bonds remain to be determined., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Gonadotropins extracted from the urine of post-menopausal women have traditionally been used to stimulate folliculogenesis in the treatment of infertility and in assisted reproductive technology (ART). Products, such as human menopausal gonadotropin (hMG), consist not only of a mixture of the hormones, follicle-stimulating hormone (FSH), luteinising hormone (LH) and human chorionic gonadotropin (hCG), but also other biologically active contaminants, such as growth factors, binding proteins and prion proteins. The actual amount of molecular LH in hMG preparations varies considerably due to the purification process, thus hCG, mimicking LH action, is added to standardise the product. However, unlike LH, hCG plays a different role during the natural human menstrual cycle. It is secreted by the embryo and placenta, and its main role is to support implantation and pregnancy. More recently, recombinant gonadotropins (r-hFSH and r-hLH) have become available for ART therapies. Recombinant LH contains only LH molecules. In the field of reproduction there has been controversy in recent years over whether r-hLH or hCG should be used for ART. This review examines the existing evidence for molecular and functional differences between LH and hCG and assesses the clinical implications of hCG-supplemented urinary therapy compared with recombinant therapies used for ART.

In fish, both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play important roles in reproduction. Here we explored the structure and differential specificity of tilapia (t) gonadotropins (GTHs) to delineate their physiological relevance and the nature of their regulation. We generated structural models of tGTHs and GTH receptors (R) that enabled us to better understand the hormone-receptor interacting region. In tilapia, FSH release is under the control of the hypothalamic decapeptide GnRH, an effect that was abolished by specific bioneutralizing antisera [anti-recombinant (r) tFSHβ]. These antisera also reduced the basal secretion and delayed GnRH-stimulated production of 11-ketotestosterone (11KT), and dramatically reduced LH levels. Immunoneutralization of tLH using anti-rtLHβ significantly reduced its GnRH-stimulated levels. Basal 11KT and FSH levels were also reduced. Taken together, these results suggest a feedback mechanism between FSH and LH release in tilapia., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The glycoprotein hormones are all structurally related heterodimers consisting of an α-subunit and a ligand-specific β-subunit that confers their unique biological activity. Crystal structures showed how the β-subunit surrounds a part of the α-subunit, and we showed the existence of the two mechanisms responsible for that assembly. In human choriogonadotropin, the β-subunit is folded before the subunits dock, and the α-subunit becomes incorporated into the dimer by a mechanism we termed "threading," passing between parts of the preassembled β-subunit. Here, we show that the human lutropin β-subunit is not folded completely prior to its interaction with the α-subunit and show that docking of the subunits enables the α-subunit to serve as a chaperone to the β-subunit. Based on data described here, we propose that the α-subunit facilitates formation of the human lutropin β-subunit by two mechanisms. First, the cystine knot of the α-subunit potentiates formation of the β-subunit cystine knot, and second, contacts between α-subunit loop 2 and a hydrophobic tail in the β-subunit facilitate formation of the seatbelt latch disulfide, which stabilizes the heterodimer. The primary influence of the α-subunit was seen when the hydrophobic tail was present or absent, but the secondary mechanism was required only when the hydrophobic tail of the β-subunit was present. During the evolution of human choriogonadotropin, neither of these α-subunit roles was necessary for folding of the β-subunit. The complex mechanism for lutropin assembly may be required to provide an additional control on its positive feedback function in vertebrate reproduction., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are widely recognized for their roles in ovulation and the support of early pregnancy. Aside from the timing of expression, however, the differences between LH and hCG have largely been overlooked in the clinical realm because of their similar molecular structures and shared receptor. With technologic advancements, including the development of highly purified and recombinant gonadotropins, researchers now appreciate that these hormones are not as interchangeable as once believed. Although they bind to a common receptor, emerging evidence suggests that LH and hCG have disparate effects on downstream signaling cascades. Increased understanding of the inherent differences between LH and hCG will foster more effective diagnostic and prognostic assays for use in a variety of clinical contexts and support the individualization of treatment strategies for conditions such as infertility., (Copyright © 2013 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

CGβ subunits comprise a unique carboxyl-terminal peptide (CTP) that has multiple O-linked glycans and extends serum half-life of the protein. It has evolved by incorporating a previously untranslated region of the LHβ gene into the reading frame. Although CTP-like sequences are encrypted in the LHβ genes of several mammals, the CGβ subunit developed only in primates and equids. To study this restriction in evolution, we examined whether the cryptic CTP decoded from the bovine LHβ gene (boCTP) possesses key characteristics of the human (h) CGβ-CTP. The boCTP does not impede several crucial aspects of hormone biosynthesis, but compared to the hCGβ-CTP, the stretch lacks O-glycans and determinants for circulatory survival. O-glycan deficiency and the associated incapacity to extend serum half-life is a major drawback of the boCTP. This may explain why LH did not evolve into CG in ruminants and consequently alternative mechanisms evolved to delay luteolysis early in gestation., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Hypo-glycosylated hFSH(21/18) (possesses FSHβ(21) and FSHβ(18)bands) was isolated from hLH preparations by immunoaffinity chromatography followed by gel filtration. Fully-glycosylated hFSH(24) was prepared by combining the fully-glycosylated FSHβ(24) variant with hCGα and isolating the heterodimer. The hFSH(21/18) glycoform preparation was significantly smaller than the hFSH(24) preparation and possessed 60% oligomannose glycans, which is unusual for hFSH. Hypo-glycosylated hFSH(21/18) was 9- to 26-fold more active than fully-glycosylated hFSH(24) in FSH radioligand assays. Significantly greater binding of (125)I-hFSH(21/18) tracer than hFSH(24) tracer was observed in all competitive binding assays. In addition, higher binding of hFSH(21/18) was noted in association and saturation binding assays, in which twice as much hFSH(21/18) was bound as hFSH(24). This suggests that more ligand binding sites are available to hFSH(21/18) in FSHR than to hFSH(24). Hypo-glycosylated hFSH(21/18) also bound rat FSHRs more rapidly, exhibiting almost no lag in binding, whereas hFSH(24) specific binding proceeded very slowly for almost the first hour of incubation., (Copyright © 2013 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Background: Glycosylation and glycan composition are of fundamental importance for the biological properties of FSH and LH. The aim of this study was to determine the glycosylation, sialylation, and sulfonation of serum FSH and LH throughout the normal menstrual cycle., Methods: Serum samples were collected from 79 healthy women with regular menstrual cycles. The mean numbers of anionic monosaccharide (AMS), sialic acid (SA), and sulfonated N-acetylgalactosamine (SU) residues per FSH and LH molecule were estimated for all sera with methods based on electrophoreses, neuraminidase treatments, and fluoroimmunoassays of the gonadotrophins., Results: Di-glycosylated glycoforms (FSHdi, LHdi) were detected in serum in addition to tetra-glycosylated FSH (FSHtetra) and tri-glycosylated LH (LHtri). FSHdi exhibited two peaks: one on day 5 to 7 and one, more pronounced, at midcycle. FSHtetra plateaued at a high concentration from day 5 to 15, without a midcycle peak. There were lower concentrations of LHdi than LHtri, except at midcycle when the opposite occurred. The mean numbers of SA and SU residues per molecule of FSH and LH in serum showed four different patterns during the cycle, all with highly significant (P < 0.0001) differences between levels at different phases of the cycle. The pattern of SA residues on FSH was 'M'-shaped, and that of SU on LH 'V'-shaped., Conclusion: Serum FSH and LH governing the natural ovarian stimulation process exhibited dynamic changes of glycosylation and glycan composition. This new information on the FSH and LH molecular structures may lead to more successful mono-ovulatory treatment regimens for ovulation induction in anovulatory women.

Gonadotropins bind to specific receptors in spite of sharing a high level of sequence and structural similarity. This specific binding is crucial for maintaining the reproductive health of an organism. In this study, residues that dictate the receptor binding specificity of the gonadotropins (FSH and LH) have been identified using combination of in silico methods. Docking studies (ZDOCK), based on the systematic replacement of these residues, confirmed its importance in receptor binding. An interesting observation is that the relative positioning of the residues conferring binding specificity varied for the gonadotropin-receptor complexes. This spatial difference of the key residues could be exploited for design of specific modulators. Based on the identified residues, we have rationally designed a peptidomimetic (FSHP) that displays good binding affinity and specificity for hFSHR. FSHP was developed by screening 3.9 million compounds using pharmacophore-shape similarity followed by fragment-based approach. It was observed that FSHP and hFSHâ can share the same receptor binding site thereby mimicking the native hFSHR-FSH interactions. FSHP also displayed higher binding affinity to hFSHR as compared to two reported hFSHR antagonists. MD simulation studies on hFSHR-FSHP complex revealed that FSHP is conformationally rigid and the intermolecular interactions are maintained during the course of simulation.

Luteal insufficiency affects fertility and hence study of mechanisms that regulate corpus luteum (CL) function is of prime importance to overcome infertility problems. Exploration of human genome sequence has helped to study the frequency of single nucleotide polymorphisms (SNPs). Clinical benefits of screening SNPs in infertility are being recognized well in recent times. Examining SNPs in genes associated with maintenance and regression of CL may help to understand unexplained luteal insufficiency and related infertility. Publicly available microarray gene expression databases reveal the global gene expression patterns in primate CL during the different functional state. We intend to explore computationally the deleterious SNPs of human genes reported to be common targets of luteolysin and luteotropin in primate CL. Different computational algorithms were used to dissect out the functional significance of SNPs in the luteinizing hormone sensitive genes. The results raise the possibility that screening for SNPs might be integrated to evaluate luteal insufficiency associated with human female infertility for future studies., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and their receptors play critical roles in vertebrate reproduction. In order to study intra- and interspecies ligand promiscuity of gonadotropins, COS-7 cells were transiently transfected with one of the gonadotropin receptor genes, FSHR or LHR, and tested for activation by gonadotropins from representative fish orders: Aquilliformes (eel; e), Salmoniformes (trout; tr), and Perciformes (tilapia; ta), and of mammalian origin: porcine (p), bovine (b) and human (h). The study reveals complex relations between the gonadotropin hormones and their receptors. Each gonadotropin activated its own cognate receptor. However, taLHR was also activated by hCG and eLHR was activated by hFSH, hCG, and trFSH. For FSHR, the only cross-reactivity detected was for hFSHR, which was activated by pFSH and bFSH. These findings are of great interest and applicability in the context of activation of various GTHRs by their ligands and by ligands from other vertebrates. Analysis of the three-dimensional models of the structures highlights the importance of residues outside of the currently established hormone-receptor interface region. In addition, the interface residues in taFSHR and the effect of exon duplication, which causes an insert in the LRR domain, are suggested to affect the interaction and binding of taFSH., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

In this paper, we describe a novel strategy for the fabrication of a nanosensor for detecting luteinizing hormone (LH) of sheep using a gold nanoparticle-peptide conjugate. A new peptide sequence "CDHPPLPDILFL" (leutinizing hormone peptide, LHP) has been identified, using BLAST and Clustal W analysis, to detect antibody of LH (sheep). LHP has been synthesized and characterized, and their affinity toward anti-LH was established using enzyme linked immunosorbant assay (ELISA) technique. The thiol group in LHP directly binds with gold nanoparticles (AuNPs) to yield AuNP-LHP construct. Detailed physicochemical analysis of AuNP-LHP construct was determined using various analytical techniques. Nanosensor using gold nanoparticle peptide conjugate was developed on the basis of competitive binding of AuNP-LHP and LH toward anti-LH. Nitrocellulose membrane, precoated with anti-LH, was soaked in the mixture of AuNP-LHP and sample of analysis (LH). In the absence of LH (sheep), anti-LH coated on the membrane binds with AuNP-LHP, leading to a distinctive red color, while in the presence of LH, no color appeared in the membrane due to the interaction of anti-LH with LH thereby preventing the binding of AuNP-LHP with membrane bound anti-LH. The sensor assay developed in this study can detect LH (sheep) up to a minimal concentration of ∼50 ppm with a high degree of reproducibility and selectivity. The gold-nanoparticle-peptide based nanosensor would be a simple, portable, effective, and low cost technique for infield applications.

Two closely related β1,4-N-acetylgalactosaminyltransferases, β4GalNAc-T3 and β4GalNAc-T4, are thought to account for the protein-specific addition of β1,4-linked GalNAc to Asn-linked oligosaccharides on a number of glycoproteins including the glycoprotein hormone luteinizing hormone and carbonic anhydrase-6 (CA6). We have utilized soluble, secreted forms of β4GalNAc-T3 and β4GalNAc-T4 to define the basis for protein-specific GalNAc transfer in vitro to chimeric substrates consisting of Gaussia luciferase followed by a glycoprotein substrate. Transfer of GalNAc by β4GalNAc-T3 and β4GalNAc-T4 to terminal GlcNAc is divalent cation-dependent. Transfer of GalNAc to glycoprotein acceptors that contain a peptide recognition determinant is maximal between 0.5 and 1.0 mM MnCl(2); however, transfer is increasingly inhibited by concentrations of MnCl(2) above 1 mM and by anion concentrations above 15 mM. In contrast, transfer of GalNAc to the simple sugar acceptor N-acetylglucosamine-β-p-nitrophenol (GlcNAcβ-pNP) is not inhibited by concentrations of MnCl(2) or anions that would inhibit transfer to glycoprotein acceptors by >90%. This finding indicates that interaction with the peptide recognition determinant in the substrate is sensitive to the anion concentration. β4GalNAc-T3 and β4GalNAc-T4 have similar but distinct specificities, resulting in a 42-fold difference in the IC(50) for transfer of GalNAc to chimeric glycoprotein substrates by agalacto human chorionic gonadotropin, comprising 29 nM for β4GalNAc-T3 and 1.2 μM for β4GalNAc-T4. Our in vitro analysis indicates that enzymatic recognition of the peptide determinant and the oligosaccharide acceptor are independent events.

LH and FSH are essential for control of gonadal function. They are synthesized in the same gonadotrope but differ in their mode of secretion. LH release is regulated, while FSH is secreted constitutively. One unique feature of LHβ is a carboxyl terminal hydrophobic heptapeptide. We demonstrated that deleting the heptapeptide diverted the truncated LH dimer to the constitutive pathway in vitro. To examine if the residues of this heptapeptide play a role in LH sorting, leucines 118-119 were substituted with alanine (L118A and L119A, respectively). The intracellular pool of the L118A mutant protein decreased with a corresponding increase in constitutive secretion. Moreover, immunofluorescence microscopy revealed that the L118A mutant exhibited fewer puncta as compared to wild-type LH. L119A behaved similar to wild-type LH, indicating that a single leucine residue at position 118, rather than a dileucine motif, contributes to the process that sorts LH into the regulated pathway., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The alpha (α) and beta (β) subunits of buffalo pituitary luteinizing hormone (LH) were chromatographed on Cibacron Blue 3GA agarose and their immunoreactivity was quantitated using anti-α and anti-β anti sera. Subsequent analyses showed α subunits were relatively more hydrophilic than β subunits. Further, the naturally occurring free α and β subunits were more hydrophobic than their native counterparts which were dissociated and isolated from heterodimeric LH. The lesser sugar content in freely occurring α and beta subunits may be attributed for increased hydrophobicity and consequent upon the existence of their uncombined free forms. In order to ascertain putative sugar-dye interaction, crude LH carrying free subunits, pure LH, and non-glycosylated recombinant β subunit of LH were loaded separately on Cibacron Blue. Methyl mannoside was able to elute 33% of the bound protein in case of crude and pure LH, whereas there was little (3%) elution in case of recombinant LH β subunit. This study suggests a compositional heterogeneity in free and native subunits of LH from the buffalo pituitary. In addition, our findings reveal the pseudolectin-like behavior of Cibacron Blue., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Leydig cells are responsible for the synthesis and secretion of testosterone, processes controlled by luteinizing hormone (LH). Binding of LH to a G protein-coupled receptor in the plasma membrane results in an increase in cAMP and in intracellular Ca(2+) concentration ([Ca(2+)](i)). Here we show, using immunofluorescence, that Leydig cells express ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP(3)Rs). Measurements of intracellular calcium changes using the fluorescent calcium-sensitive dye fluo-3 and confocal microscopy show that both types of receptors are involved in a calcium-induced calcium release (CICR) mechanism, which amplifies the initial Ca(2+) influx through plasma membrane T-type calcium channels (Ca(V)3). The RyRs and IP(3)Rs are functional, as judged from both their activation by caffeine and IP(3) and block by ryanodine and 2-aminoethoxydiphenyl borate (2-APB), respectively. RyRs are the principal players involved in the release of Ca(2+) from the endoplasmic reticulum, as evidenced by the fact that global Ca(2+) changes evoked by LH are readily blocked by 100 muM ryanodine but not by 2-APB or xestospongin C. Finally, steroid production by Leydig cells is inhibited by ryanodine but not by 2-APB. These results not only broaden our understanding of the role played by calcium in Leydig cells but also show, for the first time, that RyRs have an important role in determining testosterone secretion by the testis.

Background: Graves' disease (GD) is caused by thyrotropin (TSH) receptor antibodies (TSHRAbs) that bind to TSHR and activate thyrocytes. The measurement of TSHRAbs therefore has been used to assist in the diagnosis and management of GD., Methods: In this study, we evaluated the clinical significance of a newly developed bioreporter assay for the detection of TSHRAbs (Thyretain). The Thyretain bioreporter assay utilizes a chimeric receptor (Mc4), in which residues 262-335 of TSHR are replaced with a rat lutropin-choriogonadtropin receptor segment. This bioreporter is designed to specifically detect stimulating TSHRAbs (Mc4-TSHRAbs)., Results: The Mc4-TSHRAb level of sera obtained from 110 normal healthy controls, 103, 99, and 50 patients with untreated GD, painless Hashimoto's thyroiditis (PT), and subacute thyroiditis (SAT) were 27.3% +/- 11.3%, 327.8% +/- 105.9%, 48.9% +/- 48.5%, and 24.9% +/- 13.4%, respectively. Compared with the Mc4-TSHRAb levels of patients with PT and SAT, and normal healthy controls, the Mc4-TSHRAb levels of untreated GD patients were significantly higher (p < 0.01). The sensitivity and specificity of the Thyretain bioreporter assay for GD and PT were 95.1% and 96.0%, respectively, at the optimal cut-off value of 128%. Measurement of TSHRAbs with a bioassay that uses porcine thyroid cells (TSH-stimulating antibody [TSAb]) showed a positive correlation (r = 0.472, p < 0.001) with the Thyretain assay for untreated GD, and strong positive correlation (r = 0.821, p < 0.001) for the entire untreated GD, PT, and SAT population. The positive rate of Mc4-TSHRAbs for GD was significantly higher than that of TSAb (95.1% vs. 89.3%, p < 0.05) and the negative rate of PT by Mc4-TSHRAbs was also significantly higher than that of TSAb (96.0% vs. 86.9%, p < 0.01). As a result, Mc4-TSHRAbs showed statistically better (p < 0.01) diagnostic accuracy in differentiating GD from PT than TSAb., Conclusions: These data suggest that the Thyretain bioreporter assay with a chimeric TSHR (Mc4) is more useful in the differential diagnosis of GD from PT than the bioassay with wild-type TSHR on porcine thyroid cells.

Importance of the Field: A new fixed combination of recombinant human follicle stimulating hormone (r-hFSH) and recombinant human luteinizing hormone (r-hLH) at a 2:1 ratio has been recently developed to induce ovulation in patients with hypogonadotrophic hypogonadism. Whether or not this compound is useful for controlled ovarian stimulation for in vitro fertilization is still a matter of debate., Areas Covered in This Review: Description of pharmacological and clinical aspects of this new product, through the analysis of the Phase I, II and II trials and post-marketing clinical randomized trials, performed since the initial assays in 1998 to nowadays., What the Reader Will Gain: After reading this review the reader will understand the pharmacological aspects of this new compound, in terms of efficacy and safety, and will have an update of the potential role of r-LH administration in controlled ovarian stimulation for in vitro fertilization., Take Home Message: The 2:1 combination of r-hFSH and r-hLH has been seen as an optimum preparation in terms of safety and clinical efficacy in hypogonatrophic hypogonadism patients. Its use in ovarian stimulation for IVF remains controversial, as the target population that may receive a benefit of it is not well defined.

Previous studies have demonstrated that the pituitary is a main target for inorganic mercury (I-Hg) deposition and accumulation within the brain. My recent study of the US population (1999-2006) has uncovered a significant, inverse relationship between chronic mercury exposure and levels of luteinizing hormone (LH). This association with LH signifies more than its presumed role as bioindicator for pituitary neurosecretion and function. LH is the only hormone with a rare and well characterized, high affinity binding site for mercury. On its catalytic beta subunit, LH has the structure to preferentially bind inorganic mercury almost irreversibly, and, by that manner, accumulate the neurotoxic element. Thus, it is likely that LH is an early and significant target of chronic mercury exposure. Moreover, due to the role of LH in immune-modulation and neurogenesis, I present LH as a central candidate to elucidate a causal mechanism for chronic mercury exposure and associated disease., ((c) 2009 Elsevier Ltd. All rights reserved.)

Deglycosylated FSH is known to trigger poor Galphas coupling while efficiently binding its receptor. In the present study, we tested the possibility that a deglycosylated equine LH (eLHdg) might be able to selectively activate beta-arrestin-dependent signaling. We compared native eLH to an eLH derivative [i.e. truncated eLHbeta (Delta121-149) combined with asparagine56-deglycosylated eLHalpha (eLHdg)] previously reported as an antagonist of cAMP accumulation at the FSH receptor (FSH-R). We confirmed that, when used in conjunction with FSH, eLHdg acted as an antagonist for cAMP accumulation in HEK-293 cells stably expressing the FSH-R. Furthermore, when used alone at concentrations up to 1 nM, eLHdg had no detectable agonistic activity on cAMP accumulation, protein kinase A activity or cAMP-responsive element-dependent transcriptional activity. At higher concentrations, however, a weak agonistic action was observed with eLHdg, whereas eLH led to robust responses whatever the concentration. Both eLH and eLHdg triggered receptor internalization and led to beta-arrestin recruitment. Both eLH and eLHdg triggered ERK and ribosomal protein (rp) S6 phosphorylation at 1 nM. The depletion of endogenous beta-arrestins had only a partial effect on eLH-induced ERK and rpS6 phosphorylation. In contrast, ERK and rpS6 phosphorylation was completely abolished at all time points in beta-arrestin-depleted cells. Together, these results show that eLHdg has the ability to preferentially activate beta-arrestin-dependent signaling at the FSH-R. This finding provides a new conceptual and experimental framework to revisit the physiological meaning of gonadotropin structural heterogeneity. Importantly, it also opens a field of possibilities for the development of selective modulators of gonadotropin receptors.

Context: The common genetic variant of human LH has two mutations and an extra N-linked oligosaccharide chain, a modification expected to affect the half-life in the circulation., Objectives: Our objectives were to determine the half-lives of variant and wild-type forms of LH during GnRH receptor blockade in heterozygous women and to determine the time-related changes in isoform composition., Design and Participants: Serum samples were obtained from three healthy women heterozygous for variant LH before and up to 20 h after administration of the NAL-GLU GnRH antagonist., Main Outcome Measures: The half-lives were estimated by monoexponential decay. The number of sialic acid and sulfonated N-acetylgalactosamine residues per wild-type and variant LH molecule and the distribution of molecules with zero, one, two, or three sulfonated residues were measured., Results: The variant LH had a half-life that was approximately 40% longer than the corresponding forms of wild-type LH (148 vs. 108 min; P < 0.001). Variant LH had more sialic acid residues per molecule than wild type (3.6 vs. 2.4; P < 0.05), whereas the number of sulfonated residues was similar (1.0 vs. 0.98). The decline in the variant LH during GnRH receptor blockade was associated with a decrease in sulfonated and an increase in sialic acid residues similar to that for in wild-type LH. Isoforms of either variant or wild-type LH with two to three sulfonate groups per molecule had the shortest half-life., Conclusion: Variant LH remains longer in circulation than wild type during GnRH receptor blockade in heterozygous women, in accord with its higher content of sialic acid.

Objectives: A rapid and sensitive magnetic particles (MPs)-based chemiluminescence enzyme immunoassay (MPs-CLEIA) for determination of luteinizing hormone (LH) in human serum was developed and validated., Design and Methods: The method was developed based on a sandwiched immunoreaction format, in which MPs served as both the solid phase and separator. The horseradish peroxidase(HRP)-luminol-H(2)O(2) chemiluminescent system was chosen as the detection system., Results: This method had a detection limit of 0.2 mIU/mL and a linear range of 0.5-200 mIU/mL. The assay showed little cross-reactivity, and the intra- and inter-assay coefficients of variation were less than 10%. The recoveries were from 101.6% to 104.1%. The method has been successfully applied to detection of LH in human serum and showed good correlation compared with radioimmunoassay (RIA)., Conclusion: The MPs-CLEIA provided apparent advantages over RIA, and facilitated the development of high-throughput screening and automated operation systems in clinical diagnosis.

Single- and multi-analyte detection of two gonadotropic hormones (follicle stimulating hormone (hFSH) and luteinizing hormone (hLH)) was achieved by a Surface Plasmon Resonance (SPR) immunoassay on untreated human urine samples. Multi-analyte detection was accomplished using two alternative formats which are based in the individual or simultaneous immobilization of the hormones on the sensor surface. The lowest detection limit for both hormones in urine was found to be 1 ng mL(-1), which in international units (IU) in terms of the World Health Organization (WHO) standards represents 8 mIU mL(-1) of hLH and 14 mIU mL(-1) of hFSH, respectively. The reliability of the assay was demonstrated by intra- and inter-assay variabilities < 6%, chip-to-chip variabilities < 5%, recoveries in the range of 80-120% and stability of the sensor response through more than 100 measurements. The sensitivity of this biosensing methodology renders it in a useful technique for the diagnosis of reproductive disorders, as well as for fertility monitoring.

Sulfated glycoproteins are of growing importance for biomarker discovery, as well as for investigating molecular recognition processes. Mass spectrometry (MS) has become a powerful technique for the characterization of glycans and glycoproteins. However, characterization and detection of sulfated glycopeptides by MS is difficult because of the low abundance and low ionization efficiency of these molecules. To overcome this problem, we developed a novel enrichment procedure for sulfated glycopeptides. The procedure consists of anion exchange chromatography and a sulfate emerging (SE) method which controls the net charge of peptides by utilizing limited proteolyzes and modification with acetohydrazide. Using this procedure, we are able to enrich and characterize the sulfated glycopeptides of bovine luteinizing hormone (bLH). Furthermore, we demonstrate the enrichment and detection of sulfated glycopeptides from a complex mixture comprising human serum spiked with bLH at a concentration of 0.1%.

Two gonadotropins (Gths), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), control gonadal steroidogenesis and gametogenesis in vertebrates, including teleost fish. Here, we report on the production of biologically active recombinant Fsh (rec-Fsh) and Lh (rec-Lh) in Japanese eel using Drosophila S2 cells. The three subunits composing Gths, i.e., glycoprotein hormone, alpha polypeptide (Cga), follicle-stimulating hormone, beta polypeptide (Fshb), and luteinizing hormone, beta polypeptide (Lhb), were at first independently produced and were proven to be glycosylated and secreted as the mature peptides. Each beta subunit, along with its Cga, was simultaneously coexpressed to produce heterodimeric rec-Fsh and rec-Lh that were subsequently highly purified. The biological activity of rec-Gths was demonstrated in various in vitro assays. The rec-Gths differentially activated their receptors, which resulted in an increase in 11-ketotestosterone (11KT) secretion, a differential alteration of gene expression of steroidogenic enzymes in immature testis, and the induction of the complete process of spermatogenesis in vitro. The data strongly suggest that Fsh and Lh differentially play important roles in the reproductive physiology of the Japanese eel. By contrast, these rec-Gths exhibited little activity in the gonad when administered in vivo. This difference between in vitro and in vivo bioactivity is probably due to the qualitative nature of glycosylation in S2 cells, which resulted in degradation of the recombinant protein in vivo. These differences in the carbohydrate moieties need to be elucidated and ameliorated.

Luteinizing hormone (LH), produced in the anterior lobe of the pituitary, is a member of the hypothalamic-pituitary-gonad axis that is required for production of the sex hormones estradiol, progesterone, and testosterone. Perturbations in levels of hormones associated with this axis can result in defects in sexual development and maturity. LH bears unique N-linked carbohydrate units that terminate with a sulfated N-acetylgalactosamine structure (GalNAc-4-SO(4)) that mediates its clearance from the blood. To determine the significance of this terminal structure, we ablated the gene encoding the sulfotransferase responsible for sulfate addition to GalNAc on LH, GalNAc-4-sulfotransferase-1 (GalNAc-4-ST1) in mice. Mice lacking GalNAc-4-ST1 exhibited increased levels of circulating LH. In male mice, this resulted in elevated levels of testosterone and precocious maturation of testis and seminal vesicles. Female mice lacking GalNAc-4-ST1 demonstrated elevated estrogen levels and exhibited precocious sexual maturation and increased fecundity. Female mice remained in estrus for prolonged periods and produced almost 50% more litters per mouse than wild-type mice over the same period of time. Thus, sulfate modification of the terminal glycosylation of LH plays a central role in regulating the hypothalamic-pituitary-gonad axis in vivo.

The objectives of this study were to determine the efficacy of recombinant equine luteinizing hormone (reLH) in shortening the time to ovulation in cycling mares and to determine the effects of treatment on endogenous hormones and inter-ovulatory intervals. In study 1, mares of light horse breeds (3-20 years) were treated with either a vehicle, various doses of reLH, or human chorionic gonadotropin (hCG). Cycling mares were examined by palpation and ultrasound per rectum daily or every 12h from the time of treatment to ovulation. In studies 2 and 3, jugular blood samples were collected daily or every 12h from the time of treatment to ovulation for analysis of LH, follicle stimulating hormone (FSH), estradiol-17beta (E(2)), and progesterone (P(4)) by radioimmunoassays (RIA). Increasing doses of reLH (0.3, 0.6, 0.75, and 0.9 mg) showed increasing effectiveness at inducing ovulation within 48 h of treatment. Treatments with the 0.75 and 0.9 mg doses of reLH resulted in 90% and 80% ovulation rates, which were similar to hCG treatment (85.7%). Except for the early rise in LH after treatment with 0.5, 0.65, and 1.0mg of reLH, hormone profiles appeared to be similar between control and treated cycles. Inter-ovulatory intervals were similar between control and treatment cycles. In conclusion, reLH is a reliable and effective ovulatory agent that does not significantly alter endogenous hormone profiles or affect inter-ovulatory intervals.

The luteinizing hormone (LH) is secreted in multiple molecular forms into the blood stream; however, few studies have characterized the pattern of the circulating LH isoforms in domestic animals during different physiological stages. Most of the publications are related to the pattern of isoforms present in the anterior pituitary gland. This review includes recent evidence concerning the distribution of LH isoforms in the pituitary gland and serum in ruminants. The structural heterogeneity of this hormone is emphasized, including the glycosylation biosynthetic pathway, as well as the different proportions of oligosaccharides that confer particular functional characteristics to the heterodimer. Evidence for a regulating role of GnRH, estradiol and progesterone is discussed.

The relative proportion of the circulating luteinizing hormone isoforms in goats during follicular phase (pre-ovulatory peak; F) and anestrus (A) was investigated. Estrus was synchronized in six goats with a prostaglandin analogue. After estrus was detected, blood samples were taken at 1 h intervals for 24 h. Four anestrous goats received 100 microg i.v. of GnRH and blood samples were collected every 15 min for 5 h. Samples with the greatest LH concentration in follicular phase and after GnRH administration (anestrus) were analyzed by chromatofocusing and eluted with a pH gradient from 10.5 to 3.5. For quantification purposes eluted LH was grouped into basic (pH> or =7.5), neutral (pH 7.4-6.5) and acidic isoforms (pH< or =6.4) as well as by pH unit. In both physiological conditions (PC), basic and acidic isoforms were greater than the neutral. With this grouping criteria, there was an interaction between PC and pH group, with the proportion of neutral isoforms being greater (p<0.05) in A (12.0+/-0.8%) as compared with F (5+/-2%). Analysis by pH unit showed a very basic group of eluted isoforms (pH> or =10), which amounted to a percentage of 6.0+/-0.4% of the total observed during A, and 3+/-1% during F (p<0.05). Predominant isoforms in A eluted in the pH range 9.99-9.0 (42+/-3%) as compared to 7+/-3% (p<0.01) in that pH range in F. In contrast, the predominant isoforms in F eluted in the pH range 8.99-8.0, representing 55+/-8%, while in A the proportion was 11+/-2% (p<0.01). Isoforms eluted at the pH range 7.9-7 represented a significantly greater proportion during A (5.0+/-0.6%) as compared with F (3+/-1%). This is the first report on goat LH circulating isoforms. During A the LH isoforms secreted by the pituitary are more basic than during F.

To study structure-activity relationships and the role of equine gonadotropins in the normal and pathophysiology of equine reproduction, the availability of purified hormones is essential. Previous expression studies in transfected CHO cells showed inefficient assembly of the human and bovine alpha and beta subunits, resulting in low levels of recombinant LH. The ability to express a single chain bearing genetically linked alpha and beta subunits bypasses this rate-limiting assembly step. A chimera was constructed by overlap PCR in which the carboxy terminal end of the eLHbeta subunit was genetically fused to the amino end of the alpha subunit. This gene was transfected into CHO cells and the recombinant product was purified through multiple steps, including a Fractogel resin separation. Serial dilutions of pituitary derived native eLH and the single chain reLH were compared in an eLH radioimmunoassay (RIA); the concentration curves between the single chain recombinant eLH and the native eLH standard were parallel. The biological activity of the analog was determined in vitro and in vivo using homologous equine models. Testicular tissue from five colts was processed for Leydig cell cultures. Increasing doses of reLH were incubated with equine Leydig cells for 24h in vitro and testosterone production was determined by RIA. Recombinant eLH stimulated a greater than 15-fold increase in testosterone production in a dose-dependent manner. Quarter Horse breeding stallions were treated with either reLH (n=5) or saline (n=3) and plasma testosterone concentrations were measured by RIA. Recombinant eLH stimulated a four-fold increase in circulating testosterone concentrations compared to the saline control. Therefore, the single chain recombinant will be effective for a variety of structure-function analyses and for breeding management in the horse.

Chorionic gonadotropins (CG) are unique because they have a carboxyl terminal peptide (CTP) extension on their beta subunits that prolongs circulatory survival. CGbeta genes from the human being and horse have evolved from ancestral luteinizing hormone (LH) beta genes by different pathways that involve deletions that change the reading frames and yield a CTP. Here we further review our previous analysis, aimed at determining whether LHbeta genes in non-primate, non-equid species inherently possess DNA sequences that encode CTP-like domains. In multiple mammalian species, simple frame-shift mutations using either the human or equine CGbeta gene as a model can be used to construct LHbeta analogs with putative CTP domains. Furthermore, DNA sequences from mammalian LHbeta genes can be aligned to maximize similarity with CGbeta genes in order to devise more refined strategies for construction of CTP-bearing LHbeta analogs as exemplified in the bovine case. Thus, mammalian LHbeta genes have DNA sequences that can be potentially expressed in order to construct CTP-bearing glycoprotein hormone analogs.