Isolation of an insulin-like peptide from the Asian malaria mosquito, Anopheles stephensi, that acts as a steroidogenic gonadotropin across diverse mosquito taxa.

Many insulin-like peptides (ILPs) have been identified in insects, yet only a few were isolated in their native form for structural and functional studies. Antiserum produced to ILP3 in Aedes aegypti was used in a radioimmunoassay to monitor the purification of an ILP from heads of adult An. stephensi and recognized the ILP in other immunoassays. The structure of the purified peptide matched that predicted for the ILP3 in this species. The native form stimulated ecdysteroid production by ovaries isolated from non-blood fed females. Synthetic forms of An. stephensi ILP3 and ILP4 similarly activated this process in a dose responsive manner. This function was first established for ILP3 and ILP4 homologs in Aedes aegypti, thus suggesting their structural and functional conservation in mosquitoes. We tested the extent of conservation by treating ovaries of An. gambiae, Ae. aegypti, and Culex quinquefasciatus with the An. stephensi ILPs, and both the native and synthetic ILP3 were stimulatory, as was the ILP4. Taken together, these results offer the first evidence for ILP functional conservation across the Anophelinae and Culicinae subfamilies.

[Mexican National Consensus on Assisted Reproduction Treatment]. / Consenso Nacional Mexicano de Reproducción Asistida.

BACKGROUND: It is estimated that 15% of couples living in industrialized countries are infertile, ie have failed to conceive, reproductive age, after 12 months ormore of regular intercourse without contraception. During the past decade has increased the demand for fertility treatments because they believe are moreeffective now. OBJECTIVE: To unify the therapeutic approach and service to patients and set a precedent for a Mexican Official Standard respect and support for the legislation of these procedures. METHOD: Consensus by technical experts group panel with the participation of 34 national centers accredited for use in assisted reproduction. He organized seven workshops with the following themes: 1) selection of patients for assisted reproduction treatment, 2) schemes controlled ovarian stimulation for assisted reproduction techniques of high complexity, 3) preparation and egg retrieval technique, 4) transferembryo; 5) luteal phase supplementation; 6) indications and techniques of cryopreservation and 7) informed consent. Each table had a coordinator who wrote and presented the findings to the full, it made a number of observations until they reached unanimity of criteria, which are reflected in this document. RESULTS: Patient selection for assisted reproduction techniques is the first step of the process. Proper selection lead to success, in the same way that a bad pick up for failure. In the case of egg donation the most important recommendation is that only one to two embryos transferred in order to reduce multiple pregnancy rates and maintaining high pregnancy rates.

Production of recombinant Japanese eel gonadotropins by baculovirus in silkworm larvae.

Recombinant follicle-stimulating hormone (reFSH) and luteinizing hormone (reLH) of the Japanese eel Anguilla japonica were produced by baculovirus in silkworm Bombyx mori larvae. cDNAs encoding Japanese eel gonadotropin subunits (i.e., FSH beta, LH beta, and common alpha) were introduced into the baculovirus, which was infected into silkworm larvae after propagation of the recombinant virus in B. mori culture cells. A 100ml solution of pooled hemolymph from silkworm larvae containing reFSH or reLH were obtained from approximately 250 infected larvae. Ten milliliters of hemolymph were applied to Ni-affinity choromatography, and 5.6 and 3.5mg of partially purified reFSH and reLH were obtained, respectively. Using Western blot analysis concentrations of reFSH and reLH in the original hemolymph was estimated to be 2.2 and 1.1mg/ml, respectively. Biological activities of reFSH and reLH were assessed in vitro and in vivo. Purified reFSH and reLH induced eel oocyte maturation in vitro, and administration of hemolymph containing reFSH or reLH induced spermatogenesis in vivo in sexually immature Japanese eel. The present study indicates that a baculovirus-silkworm system could produce large amounts of biologically active recombinant fish gonadotropins for use in investigations in reproductive endocrinology and/or aquaculture of fish.

Production, characterization, and applications of mouse monoclonal antibodies against gonadotropin, somatolactin, and prolactin from common carp (Cyprinus carpio).

The gonadotropin alpha subunit (cGTH alpha), gonadotropin II beta subunit (cGTHII beta), somatolactin (cSL), and prolactin (cPRL) were isolated from the pituitaries of common carps, purified by traditional chromatographic analysis, identified by mass-chromatographic analysis, and used as immunogens in the B-lymphocyte hybridoma technique. Totally, 7, 11, 17, and 8 hybridoma cell lines were established, which were able to stably secrete monoclonal antibodies (mAbs) against cGTH alpha, cGTHII beta, cSL, and cPRL, and designated as FMU-cGTH alpha 1-7, FMU-cGTHII beta 1-11, FMU-cSL 1-17, and FMU-cPRL 1-8, respectively. The isotype, titer, and specificity were identified by enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemical staining, respectively, and application of these mAbs in the aforementioned tests has been proved. Furthermore, sensitive sandwich-ELISA systems for quantitative detection of the hormones mentioned above were also developed.

Gonadotropin preparations: past, present, and future perspectives.

This Educational Bulletin offers past, present and future perspectives on gonadotropin preparations.

Isolation of Atlantic halibut pituitary hormones by continuous-elution electrophoresis followed by fingerprint identification, and assessment of growth hormone content during larval development.

Continuous-elution electrophoresis (CEE) has been applied to separate putative hormones from adult Atlantic halibut pituitaries. Soluble proteins were separated by size and charge on Model 491 Prep Cell (Bio-Rad), where the homogenate runs through a cylindrical gel, and protein fractions are collected as they elute from the matrix. Protein fractions were assessed by SDS-PAGE and found to contain purified proteins of molecular size from 10 to 33 kDa. Fractions containing proteins with molecular weights of approximately 21, 24, 28 and 32 kDa, were identified as putative growth hormone (GH), prolactin, somatolactin and gonadotropins, respectively. These were analyzed further by mass spectrometry and identified with peptide mass protein fingerprinting. The CEE technique was used successfully for purification of halibut GH with a 5% yield, and appears generally well suited to purify species-specific proteins often needed for research in comparative endocrinology, including immunoassay work. Thus, the GH obtained was subsequently used as standards and iodination label in a homologous radioimmunoassay, applied to analyze GH content through larval development in normally and abnormally metamorphosing larvae. As GH is mainly found in the pituitary, GH contents were analyzed in tissue extracts from the heads only. The pituitary GH content increases proportionally to increased larval weight from first feeding to metamorphic climax. No difference in relative GH content was found between normal and abnormal larvae and it still remains to be established if GH has a direct role in metamorphosis.

Batch-to-batch consistency of human-derived gonadotrophin preparations compared with recombinant preparations.

Different gonadotrophin preparations derived from human urine or manufactured by recombinant technology are currently used in clinical practice for the treatment of infertility. It has been widely assumed that gonadotrophin products manufactured by recombinant technology have better batch-to-batch consistency compared with human-derived preparations and that this potentially will be shown to provide a more constant clinical response, but there is little evidence for either statement. This study compared the batch-to-batch consistency between urinary-derived and recombinant manufactured gonadotrophin preparations using standard analytical techniques, as well as a novel in-vitro follicle bioassay to evaluate the consistency of the biological response at the target organ. Oligosaccharide isoform profiling, immunoassay testing, size exclusion chromatography analysis and in-vitro bioassay testing of urinary derived gonadotrophin preparations (MENOPUR and BRAVELLE) confirm that these products display a high degree of batch-to-batch consistency, similar to recombinant FSH (GONAL-f) either filled by mass or bioassay. The data also suggest that the batch-to-batch variation is independent of the manufacturing procedure (filled-by-bioassay or filled-by-mass) for the recombinant preparation (Gonal-f), but that the total FSH bioactivity delivered from a single dose preparation after reconstitution differs between the two manufacturing procedures.

The use of urinary gonadotrophins should be discouraged.

In view of concerns regarding the potential presence and infectivity of prion proteins in human urinary gonadotrophin preparations, together with the availability of both recombinant FSH and recombinant LH, it is argued that the use of urinary gonadotrophins should be discouraged.

Is there a risk of prion disease after the administration of urinary-derived gonadotrophins?

Concern has been raised recently about the possibility of prion proteins appearing in the urine of animals and, possibly, humans affected by prion disease [scrapie, bovine spongiform encephalopathy (BSE) and Creutzfeldt Jakob disease (CJD)]. A debate has started in which the suggestion has been made that the purification of human urine for the provision of gonadotrophins should be discontinued. The alternative would be to use recombinantly-derived gonadotrophin preparations. The recombinant products, however, rely upon bovine serum during the cell culture process and could potentially also be exposed to abnormal prion proteins. It is reassuring that the different types of gonadotrophin preparations that are currently available are produced with either urine or bovine serum that is sourced from countries that at the present time appear to be free of BSE and new variant CJD. We can therefore be reassured that the gonadotrophins that we use therapeutically appear to be equally safe.

Risk of infection is not the main problem.

The risk of infection from prion proteins in urinary preparations of human gonadotrophins is uncertain--and is of lesser importance than the risk of multiple pregnancies and issues of cost.

Purification of mummichog (Fundulus heteroclitus) gonadotropins and their subunits, using an immunochemical assay with antisera raised against synthetic peptides.

To detect mummichog gonadotropins (GtHs) and their subunits immunochemically, fragment peptides with amino acid sequences corresponding to cDNA data were synthesized, and antisera were raised against them. In the case of GtH-IIbeta, large loops such as the second loop and the "seat belt" structure (deduced from the hCG 3D structural data) were considered to be favorable regions for antigen, although further examination is needed to determine if this is the case of GtH-Ibeta and GtH-alpha. In the purification process, glycoprotein was extracted from acetone-dried mummichog pituitary and separated by various liquid chromatography procedures. Each fraction was assayed by immunoblotting with the appropriate antisera against synthetic peptides. Subunits (GtH-alpha, GtH-Ibeta, and GtH-IIbeta) were obtained through gel filtration, anion-exchange chromatography, and reverse-phase HPLC. Intact bioactive GtH-I and GtH-II were obtained through gel filtration, anion-exchange chromatography, and hydrophobic chromatography. Both GtH-I and GtH-II dissociated into subunits under acidic conditions. Nominal MW of each subunit was estimated from SDS-PAGE as 23,000 for GtH-alpha from GtH-I, 22,000 for GtH-alpha from GtH-II, 18,000 for GtH-Ibeta, and 21,000 for GtH-IIbeta.

Ovarian stimulation: from basic science to clinical application.

Treatment for infertility, including ovarian stimulation, was first introduced almost 100 years ago. At this time, radiation therapy became an established treatment, and it was only some decades later that the problem of radiation-induced cancer emerged. Non-human gonadotrophins, such as pregnant mare serum gonadotrophin (PMSG), and human pituitary gonadotrophins (HPG), were commonly used for hormonal stimulation procedures. However, use of PMSG led to antibody formation, and it was therefore only useful for the first treatment cycle. HPG produced good results, but its use came to an end in the late 1980s when it was linked to the development of Creutzfeldt-Jakob disease. The first hormonal product from human menopausal urine to be used was human menopausal gonadotrophin (HMG), followed later by purified preparations of this product. All of these preparations contained a high percentage of unknown urinary proteins, which interfered with batch-to-batch consistency. This changed with the introduction of recombinant gonadotrophins, produced from an immortalized/standardized mammalian cell line (CHO). More recent developments include the introduction of long-acting gonadotrophin formulations. The development of gonadotrophin-releasing hormone (GnRH) analogues and more recently the use of GnRH antagonists has helped to improve ovarian stimulation protocols by optimizing their efficacy, and making them easier to administer.

[Preparative isolation of gonadotropin and urokinase on carboxylic cation exchangers]. / Preparativnoe vydelenie gonadotropina i urokinazy na karboksil'nykh ka tionitakh.

Low-pressure ion-exchange chromatography was used for sequential isolation of chorionic gonadotropin and urokinase (EC 3.4.99.26) from urine of pregnant women. Theoretical analysis of electrostatic interactions of protein macromolecules with the sorptive surface of a carboxylic cation exchanger was performed. This analysis allowed us to optimize the pH values for selective sorption and reversible desorption of the target component on a cation exchanger with a selected acidity of functional groups. The use of carboxylic cation exchangers KM-2p and Biokarb-GM allowed us to obtain the preparations with purity not inferior to commercially available preparations.

Effects of urinary gonadotrophin preparations on human in-vitro immune function.

Among commercially available urinary human menopausal gonadotrophin (HMG) material, gonadotrophins comprise <5% of the total protein content. Thus, during a typical ovarian stimulation cycle with HMG, several milligrams of non-relevant proteins are administered that may lead to unwanted side effects, including allergic or other hypersensitivity reactions. The effects of two recombinant and four urinary gonadotrophin preparations of different purity upon the function of T cells from healthy blood donors were studied. Only one of the HMG preparations significantly enhanced the spontaneous proliferation of peripheral blood mononuclear cells. Phytohaemagglutinin-induced proliferation was not modified by any preparation, while two preparations significantly increased proliferation in the mixed lymphocyte reaction. Three of the HMG preparations induced the release of interleukin (IL)-1. Highly purified FSH, either urinary or recombinant, showed no effect. None of the preparations induced detectable IL-2 production, whereas only one HMG preparation tended to decrease IL-2 secretion. No major changes in CD25 expression were induced by any of the gonadotrophins. Cytokine measurement by immunoassays detected only IL-1beta in two commercially available preparations. The various effects exhibited by the crude urinary preparations were not a result of the gonadotrophin content and differed from product to product, suggesting that the contaminants present in these preparations are not identical. This could contribute to unpredictable clinical manifestations of allergic or other immune reactions.

Naturally occurring analogs of Lymantria testis ecdysiotropin, a gonadotropin isolated from brains of Lymantria dispar pupae.

Lymantria testis ecdysiotropin (LTE) was isolated from the most prominent peptide peak corresponding to an active fraction obtained by high pressure liquid chromatographic (HPLC) separation of a homogenate of 13,000 Lymantria dispar pupal brains. In this work we examined the other active fractions from this separation as well as a second HPLC separation of an additional 2,300 pupal brains. Bioassay of the ecdysteroidogenic effects of each peak on L. dispar testes allowed detection of 20 peptide peaks with testis ecdysiotropic activity in addition to LTE. Of these, ten peptides were purified and sequenced. All of them were comparable to LTE in molecular weight. The amino acid sequences of five of the peptides were similar enough to LTE to be considered to be members of an LTE family. However, the other five peptides had no significant homology with LTE or with each other. A BLAST database search indicated LTE family homology with portions of inhibitory peptides such as those inhibiting cytolysis. In contrast, non-LTE ecdysiotropic peptides, in which undetermined residues designated X were assumed to be cysteine, were strikingly homologous to portions of vertebrate and invertebrate zinc finger peptides and to vertebrate and invertebrate virus proteins.

Chemical synthesis of a 7 kDa insect gonadotropic neurohormone.

An original insect neurohormone of 65 residues was synthesized by the solid-phase methodology using t-Boc strategy and Boc-Val-PAM-resin. The purification, conducted by several steps of liquid chromatography having mass, polarity or charge as separative criteria, yielded the product with the correct molecular weight of 6922 Da determined by mass spectrometry. The synthetic peptide had both the same affinity for the anti-native neurohormone serum and the same biological activity as the native neurohormone.

Isolation of gonadotropin subunits and evidence for two distinct gonadotropins in Atlantic croaker (Micropogonias undulatus).

Gonadotropic hormones (GTHs) were purified from Atlantic croaker (Micropogonias undulatus) pituitary glands collected in December, when the gonads were regressed. GTH was identified during the purification by an in vitro ovarian steroidogenesis bioassay and by radioimmunoassay using an Atlantic croaker maturational GTH antiserum. Two gonadotropic fractions were separated by anion-exchange chromatography of gel-filtered pituitary extracts. Reverse-phase HPLC followed by n-terminal amino acid sequencing of the GTH subunits revealed that a common alpha-subunit was present in both fractions. However, the two gonadotropic fractions contained different forms of the beta-subunit which differed both in their n-terminal amino acid sequences and in their amino acid compositions. The differences in the amino acid compositions between the two croaker GTH beta-subunits were similar to those previously reported between salmon GTH I and II beta-subunits (Kawauchi et al., 1989, Fish Physiol. Biochem. 7, 29-38). The n-terminal sequence of one of the croaker beta-subunits (beta-II) resembled that of the salmon GTH II beta-subunit. The other subunit (beta-I) had only a low degree of homology in its n-terminal sequence with the salmon GTH I beta-subunit but had a similar amino acid composition. In addition, the n-terminal end of beta-I had 40% sequence identity with the GTH I beta-subunit of bonito (Kawauchi et al., 1991, In "Proceedings of the Fourth International Symposium on Reproductive Physiology of Fish (A. P. Scott, J. P. Sumpter, D. E. Kime, and M. S. Rolfe, Eds.), pp. 19-21. Univ. of East Anglia, Norwich, UK). Moreover, the beta-I-subunits from all three species had cysteine residues at positions 4 and 8. These results indicate that a dual gonadotropin system exists in the Atlantic croaker as in the salmon and bonito and support the concept that such a system is ubiquitous in teleosts.

Maturational gonadotropin from the African catfish, Clarias gariepinus: purification, characterization, localization, and biological activity.

A gonadotropic hormone of the African catfish, Clarias gariepinus, was was purified and chemically characterized. Its biological activity was tested and its localization in the gonadotropic cells of the pituitary demonstrated. An ethanolic extract of 500 pituitaries of adult male and female African catfish was subjected to ion-exchange chromatography on DE-52. The 31- to 38-kDa fraction was further purified on Sephadex G-75. On rpHPLC over an ODS 120T column two major components appeared as single bands after SDS-PAGE. From the amino acid composition and sequence analysis of these fractions, compared with those of salmon and carp GTH II-alpha and salmon GTH II-beta it was concluded that they represent catfish GTH alpha- and II-beta-subunits. The biological activity of the complete hormone (the 31- to 38-kDa fraction from the G-75 column) was tested on the production of 11 beta-hydroxyandrostenedione and 17 alpha-hydroxy-20 beta-dihydroprogesterone by catfish testis in vitro. Polyclonal antibodies were raised against the purified beta-subunit. Immunocytochemical study using these showed them to bind specifically to hypophysial gonadotropic cells. To date only one form of GTH has been demonstrated in the African catfish.

Purification of gonadotropes and intracellular free calcium oscillation. Effects of gonadotropin-releasing hormone and interleukin 6.

The intracellular free calcium concentration ([Ca2+]i) in single gonadotropes was measured with a calcium-sensitive fluorescent dye indo-1 or fura-2 and a digital imaging fluorescence microscopic system to determine how interleukin-6 (IL-6) increases release of gonadotropins. IL-6 induced an increase in the basal [Ca2+]i or the amplitude of spontaneous oscillation of [Ca2+]i in gonadotropes in a mixed population. Gonadotropin-releasing hormone (Gn-RH) induced a biphasic increase in [Ca2+]i, a transient increase, and then a prolonged increase. These effects were inhibited by the absence of extracellular calcium or pretreatment with calcium channel blockers, cobalt or nifedipine. Next, purified gonadotropes were prepared by fluorescence-activated cell sorting and argon laser treatment of the cells. Gonadotropes labeled with anti-luteinizing hormone antibody were sorted by fluorescence-activated cell sorting and then cultured as monolayers for 24-48 h. In this way, gonadotropes were concentrated from 5-10% to 70-85% from whole pituitary cells. After relabeling with anti-luteinizing hormone antibody, 100% purified gonadotropes were obtained by killing other types of cells with an argon laser. Gonadotropin-releasing hormone induced almost the same responses of [Ca2+]i in the purified cell population as in the mixed cell population, but IL-6 did not affect [Ca2+]i in the purified gonadotropes. These results suggest that IL-6 affects calcium mobilization in gonadotropes indirectly via paracrine pathways.