Determination of doping peptides via solid-phase microelution and accurate-mass quadrupole time-of-flight LC-MS.

A complete analytical protocol for the determination of 25 doping-related peptidic drugs and 3 metabolites in urine was developed by means of accurate-mass quadrupole time-of-flight (Q-TOF) LC-MS analysis following solid-phase extraction (SPE) on microplates and conventional SPE pre-treatment for initial testing and confirmation, respectively. These substances included growth hormone releasing factors, gonadotropin releasing factors and anti-diuretic hormones, with molecular weights ranging from 540 to 1320Da. Optimal experimental conditions were stablished after investigation of different parameters concerning sample preparation and instrumental analysis. Weak cation exchange SPE followed by C18 HPLC chromatography and accurate mass detection provided the required sensitivity and selectivity for all the target peptides under study. 2mg SPE on 96-well microplates can be used in combination with full scan MS detection for the initial testing, thus providing a fast, cost-effective and high-throughput protocol for the processing of a large batch of samples simultaneously. On the other hand, extraction on 30mg SPE cartridges and subsequent target MS/MS determination was the protocol of choice for confirmatory purposes. The methodology was validated in terms of selectivity, recovery, matrix effect, precision, sensitivity (limit of detection, LOD), cross contamination, carryover, robustness and stability. Recoveries ranged from 6 to 70% (microplates) and 17-95% (cartridges), with LODs from 0.1 to 1ng/mL. The suitability of the method was assessed by analyzing different spiked or excreted urines containing some of the target substances.

Aptamer based peptide enrichment for quantitative analysis of gonadotropin-releasing hormone by LC-MS/MS.

Over recent years threats to racing have expanded to include naturally occurring biological molecules, such as peptides and proteins, and their synthetic analogues. Traditionally, antibodies have been used to enable detection of these compounds as they allow purification and concentration of the analyte of interest. The rapid expansion of peptide-based therapeutics necessitates a similarly rapid development of suitable antibodies or other means of enrichment. Potential alternative enrichment strategies include the use of aptamers, which offer the significant advantage of chemical synthesis once the nucleic acid sequence is known. A method was developed for the enrichment, detection and quantitation of gonadotropin-releasing hormone (GnRH) in equine urine using aptamer-based enrichment and LC-MS/MS. The method achieved comparable limits of detection (1 pg/mL) and quantification (2.5 pg/mL) to previously published antibody-based enrichment methods. The intra- and inter-assay precision achieved was less than 10% at both 5 and 20 pg/mL, and displayed a working dynamic range of 2.5-100 pg/mL. Significant matrix enhancement (170 ± 8%) and low analytical recovery (29 ± 15%) was observed, although the use of an isotopically heavy labelled GnRH peptide, GnRH (Pro(13)C5,(15)N), as the internal standard provides compensation for these parameters. Within the current limits of detection GnRH was detectable up to 1h post administration in urine and identification of a urinary catabolite extended this detection window to 4h. Based on the results of this preliminary investigation we propose the use of aptamers as a viable alternative to antibodies in the enrichment of peptide targets from equine urine.

Combined Labelled and Label-free SERS Probes for Triplex Three-dimensional Cellular Imaging.

Cells are complex chemical systems, where the molecular composition at different cellular locations and specific intracellular chemical interactions determine the biological function. An in-situ nondestructive characterization of the complicated chemical processes (like e.g. apoptosis) is the goal of our study. Here, we present the results of simultaneous and three-dimensional imaging of double organelles (nucleus and membrane) in single HeLa cells by means of either labelled or label-free surface-enhanced Raman spectroscopy (SERS). This combination of imaging with and without labels is not possible when using fluorescence microscopy. The SERS technique is used for a stereoscopic description of the intrinsic chemical nature of nuclei and the precise localization of folate (FA) and luteinizing hormone-releasing hormone (LHRH) on the membrane under highly confocal conditions. We also report on the time-dependent changes of cell nuclei as well as membrane receptor proteins during apoptosis analyzed by statistical multivariate methods. The multiplex three-dimensional SERS imaging technique allows for both temporal (real time) and spatial (multiple organelles and molecules in three-dimensional space) live-cell imaging and therefore provides a new and attractive 2D/3D tracing method in biomedicine on subcellular level.

Solid-phase extraction of small biologically active peptides on cartridges and microelution 96-well plates from human urine.

Currently liquid chromatography - mass spectrometry (LC-MS) analysis after solid-phase extraction (SPE) on weak cation-exchange cartridges is a method of choice for anti-doping analysis of small bioactive peptides such as growth hormone releasing peptides (GHRPs), desmoporessin, LHRH, and TB-500 short fragment. Dilution of urine samples with phosphate buffer for pH adjustment and SPE on weak cation exchange microelution plates was tested as a means to increase throughput of this analysis. Dilution using 200 mM phosphate buffer provides good buffering capacity without affecting the peptides recoveries. SPE on microelution plates was performed on Waters Positive Pressure-96 Processor with subsequent evaporation of eluates in nitrogen flow. Though the use of smaller sample volume decreases the pre-concentration factor and increases the limits of detection of 5 out of 17 detected peptides, the recovery, linearity, and reproducibility of the microelution extraction were comparable with cartridge SPE. The effectiveness of protocols was confirmed by analysis of urine samples containing ipamorelin, and GHRP-6 and its metabolites. SPE after urine sample dilution with buffer can be used for faster sample preparation. The use of microelution plates decreases consumption of solvents and allows processing of up to 96 samples simultaneously. Cartridge SPE with manual рН adjustment remains the best option for confirmation. Copyright © 2015 John Wiley & Sons, Ltd.

The endocrinology of the menstrual cycle.

The ovulatory menstrual cycle is the result of the integrated action of the hypothalamus, pituitary, ovary, and endometrium. Like a metronome, the hypothalamus sets the beat for the menstrual cycle by the pulsatile release of gonadotropin-releasing hormone (GnRH). GnRH pulses occur every 1-1.5 h in the follicular phase of the cycle and every 2-4 h in the luteal phase of the cycle. Pulsatile GnRH secretion stimulates the pituitary gland to secrete luteinizing hormone (LH) and follicle stimulating hormone (FSH). The pituitary gland translates the tempo set by the hypothalamus into a signal, LH and FSH secretion, that can be understood by the ovarian follicle. The ovarian follicle is composed of three key cells: theca cells, granulosa cells, and the oocyte. In the ovarian follicle, LH stimulates theca cells to produce androstenedione. In granulosa cells from small antral follicles, FSH stimulates the synthesis of aromatase (Cyp19) which catalyzes the conversion of theca-derived androstenedione to estradiol. A critical concentration of estradiol, produced from a large dominant antral follicle, causes positive feedback in the hypothalamus, likely through the kisspeptin system, resulting in an increase in GnRH secretion and an LH surge. The LH surge causes the initiation of the process of ovulation. After ovulation, the follicle is transformed into the corpus luteum, which is stimulated by LH or chorionic gonadotropin (hCG) should pregnancy occur to secrete progesterone. Progesterone prepares the endometrium for implantation of the conceptus. Estradiol stimulates the endometrium to proliferate. Estradiol and progesterone cause the endometrium to become differentiated to a secretory epithelium. During the mid-luteal phase of the cycle, when progesterone production is at its peak, the secretory endometrium is optimally prepared for the implantation of an embryo. A diagrammatic representation of the intricate interactions involved in coordinating the menstrual cycle is provided in Fig. 1.

Capillary electrophoresis in classical and carrier ampholytes-based background electrolytes applied to separation and characterization of gonadotropin-releasing hormones.

Capillary zone electrophoresis (CZE) in classical buffer-based background electrolytes (BGEs) and carrier ampholytes-based capillary electrophoresis (CABCE) using narrow pH cuts of carrier ampholytes (CA) as constituents of quasi-isoelectric BGEs have been applied to separation and characterization of synthetic human and salmon gonadotropin-releasing hormones (GnRH) and their derivatives and fragments. The selectivity, separation efficiency, resolution and speed of CZE and CABCE analyses have been compared within a wide pH range of the BGEs (3.50-9.75) using two mixtures of structurally related GnRH peptides as model analytes. A baseline separation of mixture 1 (human GnRH and its three fragments) was achieved in CA-based BGEs at pH 3.5 and in both classical and CA-based BGEs within the pH range 7.00-9.75. Full separation of mixture 2 (salmon GnRH, its two fragments and human GnRH fragment) was obtained in both types of BGEs at acidic pH values 3.5 and 4.00 and at neutral pH 7.00. In addition to the separation of related GnRHs, their effective electrophoretic mobilities were determined and from the dependences of mobilities on pH, the isoelectric points (pI) of analyzed peptides were estimated. The pI values obtained by CABCE were in a good agreement with those determined by CZE in classical BGEs but in some cases rather different from those predicted by theoretical calculations.

Characterization and putative role of a type I gonadotropin-releasing hormone in the cephalochordate amphioxus.

GnRH, originally isolated from mammalian hypothalamus, is a key player in the control of vertebrate reproduction. Employing reverse-phase chromatography, we purified a peptide of relative molecular mass of 1182.60 Da from the cephalochordate amphioxus Branchiostoma lanceolatum. We found that its amino acid sequence (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH(2)) was identical to that of mammalian GnRH. The highest concentrations (4.04 +/- 0.3 microg/g tissue), localized in the anterior part of the body, occurred in November, a time when amphioxus gonads prepare for the seasonal spawning. Furthermore, the biological activity of amphioxus GnRH was investigated by examining its capability to elicit LH release from the rodent pituitary gland. The origins of GnRH can be traced back to the origins of chordates. The seasonal variations of amphioxus GnRH also suggest an ancient role of this peptide in the control of reproduction in chordates, even before the evolution of a proper pituitary gland.

Origins of gonadotropin-releasing hormone (GnRH) in vertebrates: identification of a novel GnRH in a basal vertebrate, the sea lamprey.

We cloned a cDNA encoding a novel (GnRH), named lamprey GnRH-II, from the sea lamprey, a basal vertebrate. The deduced amino acid sequence of the newly identified lamprey GnRH-II is QHWSHGWFPG. The architecture of the precursor is similar to that reported for other GnRH precursors consisting of a signal peptide, decapeptide, a downstream processing site, and a GnRH-associated peptide; however, the gene for lamprey GnRH-II does not have introns in comparison with the gene organization for all other vertebrate GnRHs. Lamprey GnRH-II precursor transcript was widely expressed in a variety of tissues. In situ hybridization of the brain showed expression and localization of the transcript in the hypothalamus, medulla, and olfactory regions, whereas immunohistochemistry using a specific antiserum showed only GnRH-II cell bodies and processes in the preoptic nucleus/hypothalamus areas. Lamprey GnRH-II was shown to stimulate the hypothalamic-pituitary axis using in vivo and in vitro studies. Lamprey GnRH-II was also shown to activate the inositol phosphate signaling system in COS-7 cells transiently transfected with the lamprey GnRH receptor. These studies provide evidence for a novel lamprey GnRH that has a role as a third hypothalamic GnRH. In summary, the newly discovered lamprey GnRH-II offers a new paradigm of the origin of the vertebrate GnRH family. We hypothesize that due to a genome/gene duplication event, an ancestral gene gave rise to two lineages of GnRHs: the gnathostome GnRH and lamprey GnRH-II.

The presence and distribution of gonadotropin-releasing hormone-liked factor in the central nervous system of the black tiger shrimp, Penaeus monodon.

The distribution and presence of gonadotropin-releasing hormone (GnRH) in the central nervous system (CNS) of Penaeus monodon were examined by immunocytochemistry, high performance liquid chromatography (HPLC), and radioimmunoassay (RIA). We demonstrated the existence of octopus (oct)GnRH-liked immunoreactivity (ir-octGnRH) and lamprey (l)GnRH-III-liked immunoreactivity (ir-lGnRH-III) in cell bodies of medium-sized neurons of the anterior part (protocerebrum) of the supraesophageal ganglion (brain). In addition, only the ir-octGnRH was detected in the nerve fibers located in the brain and segmental ganglia (subesophageal, thoracic, and abdominal ganglia). Moreover, some branches of these fibers also innervated the neurons in the middle (deutrocerebrum), posterior (tritocerebrum) brain and segmental ganglia. There was no ir-lGnRH-I and ir-salmon (s)GnRH detected in the shrimp CNS. The results from HPLC and RIA showed ir-GnRH in the CNS using anti-lGnRH-III, but not with anti-mammalian (m)GnRH. The data from immunocytochemistry, HPLC and RIA suggest that ir-GnRH in shrimp may be more similar to octGnRH and lGnRH-III than the other forms. These findings support the hypothesis that GnRH-liked factor(s) may be an ancient peptide that also exists in this decapod crustacean.

Separation and investigation of structure-mobility relationship of gonadotropin-releasing hormones by capillary zone electrophoresis in conventional and isoelectric acidic background electrolytes.

Capillary zone electrophoresis (CZE) has been applied to qualitative and quantitative analysis, separation and physicochemical characterization of synthetic gonadotropin-releasing hormones (GnRHs) and their analogs and fragments. Structurally related peptides were separated in conventional and isoelectric acidic background electrolytes (BGEs), pH 2.18-2.50. Best separation was achieved in isoelectric BGE composed of 200 mM iminodiacetic acid, pH 2.32. The effective electrophoretic mobilities, m(ep), of GnRHs in five BGEs were determined and four semiempirical models correlating effective mobility with charge, q, and relative molecular mass, M(r), (m(ep) versus q/M(r)(k), where k is related to the molecular shape) were tested to describe the migration behavior of GnRHs in CZE. None of the models was found to be quite definitively applicable for the whole set of 10 GnRHs differing in size (tetrapeptide-decapeptide) and positive charge (0.91-3.00 elementary charges). Nevertheless, for the dependence of m(ep) on q/M(r)(k), the highest coefficient of correlation, R=0.995-0.999, was obtained for k close to the value 0.5 in all five acidic BGEs. This indicates that the most probable structure of GnRHs in these BGEs can be predicted as a random coil.

Cloning, expression, and purification of a highly immunogenic recombinant gonadotropin-releasing hormone (GnRH) chimeric peptide.

To design an anti-gonadotropin-releasing hormone (GnRH) vaccine capable of eliciting strong immunogenicity, a gene fragment encoding a chimeric peptide was constructed using polymerase chain reaction and ligated into a novel expression vector for recombinant expression in a T7 RNA polymerase-based expression system. The chimeric peptide called GnRH3-hinge-MVP contained three linear repeats of GnRH (GnRH3), a fragment of the human IgG1 hinge region, and a T-cell epitope of measles virus protein (MVP). The expression plasmid contained the GnRH3-hinge-MVP construct ligated to its fusion partner (AnsB-C) via an unique acid labile Asp-Pro linker. The recombinant fusion protein was expressed in an inclusion body in Escherichia coli under IPTG or lactose induction and the target peptide was easily purified using washing of urea and ethanol precipitation. The target chimeric peptide was isolated from the fusion partner following acid hydrolysis and purified using DEAE-Sephacel chromatography. The purified GnRH3-hinge-MVP was determined to be highly homogeneous by IEF analysis and the N-terminal sequencing. Further, immunization of female mice with the recombinant chimeric peptide resulted in generation of high-titer antibodies specific for GnRH. The results showed that GnRH3-hinge-MVP could be considered as a candidate anti-GnRH vaccine.

A new strategy utilizing electrospray ionization-quadrupole ion trap mass spectrometry for the qualitative determination of GnRH peptides.

Numerous forms of the neurotransmitter GnRH have been discovered in vertebrates and invertebrates. Methods used for identification of these peptides are laborious and often require the application of multiple, confirmatory techniques. In this study, we investigate whether HPLC-MS/MS and de novo sequencing techniques applied to whole peptide analysis can provide a simpler approach to GnRH characterization. Experiments were performed with six GnRH forms (chicken I, chicken II, lamprey III, mammalian, salmon and seabream) to determine whether MS/MS spectra would be dominated by proline-directed fragmentation to the detriment of obtaining sufficient fragmentation for sequencing. While the expected b8 fragment was prominent, sufficient ion series were obtained for the six GnRH peptides to provide sequence identification. On the basis of the patterns observed for six model peptides, similar fragmentation patterns are expected for other GnRH forms. To confirm the applicability of the method, extracts from Sprague-Dawley rat brains were examined. These experiments confirm the presence of mammalian GnRH and a posttranslationally modified form of mammalian GnRH, hydroxyproline9 GnRH, in Sprague-Dawley rat brains and demonstrate that ESI-MS/MS techniques provide a valuable addition to existing qualitative methods.

Determination of purity degree and counter-ion content in lecirelin by capillary zone electrophoresis and capillary isotachophoresis.

Capillary zone electrophoresis (CZE) and capillary isotachophoresis (CITP) were applied for the determination of peptide purity degree and counter-ion content in lecirelin, the synthetic analogue of luteinizing hormone-releasing hormone (LHRH). CZE analyses were carried out in acidic background electrolyte (100 mM H3PO4, 50 mM Tris, pH 2.25) in bare fused silica capillary using UV-absorption detection at 206 nm. CITP analyses were performed in the electrophoretic analyzer with column coupling, equipped with contactless conductivity detectors both in preseparation capillary and in analytical capillary, and with UV-absorption detector (220 and 254 nm) in analytical capillary. Determinations of peptide purity were carried out in cationic mode with leading electrolyte (LE), 10 mM KOH/AcOH, pH 4.5, and terminating electrolyte (TE), 10 mM beta-alanine (BALA)/AcOH, pH 4.4. Degree of peptide purity determined by both CZE and CITP was in the range 60.1-80.9% for crude preparations of lecirelin and in the range 96.4-99.9% for HPLC purified batches. Concentrations of contaminating counter-ions, the anions of trifluoromethanesulfonic acid (TFMSA), trifluoroacetic acid (TFA) and acetic acid (AcOH), were determined by CITP analyses in anionic mode with LE 10 mM HCl/His, pH 6.0, and TE 10 mM 2-(N-morpholino)-ethanesulfonic acid (MES), pH 4.0, by the calibration curve method. Mass percentages of the counterion contents in the analyzed lecirelin batches varied from zero to ca. 9% (TFMSA), 3% (TFA) and 11% (AcOH), respectively.

Expression of pejerrey gonadotropin-releasing hormone in three orders of fish.

Molecular variants of GnRH were characterized by reverse-phase, high-performance liquid chromatography from brain extracts of fish in three different orders: Synbranchiformes (swamp eel [Synbranchus marmoratus]), Cyprinidontiformes (platyfish [Xiphophorus maculatus] and green swordtail [X. helleri]), and Atheriniformes (Patagonia pejerrey [Odontesthes hatchery]). Also, pituitary gland extracts from the pejerrey O. bonariensis (Atheriniformes) were characterized. Eluted fractions were tested in radioimmunoassays with antisera specific to GnRH, including both antisera that detected only one form of GnRH and those that detected several forms. The results show that brain extracts obtained from all species contained the same three molecular forms of GnRH, which were immunologically and chromatographically undistinguishable from chicken GnRH-II, pejerrey GnRH (pjGnRH), and salmon GnRH. This study supports the hypothesis that expression of these three forms is common in different fish orders and that pjGnRH is the main regulator of pituitary function in these fish.

Isocratic liquid chromatographic assay for monitoring the degradation of luteinizing hormone releasing hormone by extracts from the gastrointestinal tract of possums.

A simple HPLC method to separate human luteinizing hormone releasing hormone (LHRH) from its metabolites using an isocratic elution is described. Intact LHRH and five metabolites were separated in 11.4 min. The calibration curve (peak area versus concentration) was linear over the concentration range 1.25-35 microg/ml (r(2)=0.99) with the intercept not significantly different from zero (P>0.05). Intra-day and inter-day variability of the assay was less than 5% for repeat injections of 5, 14.5 and 29 microg/ml. The method was applied to evaluate the susceptibility of LHRH to enzymes present in the lumen and mucosal extracts of the gastrointestinal tract of possums. The major degradation products of LHRH were identified by HPLC separation, amino acid analysis and mass spectrometry as LHRH (1-5), LHRH (1-4), LHRH (1-3) and LHRH (3-4).

Exonic splicing enhancer-dependent splicing of the gonadotropin-releasing hormone premessenger ribonucleic acid is mediated by tra2alpha, a 40-kilodalton serine/arginine-rich protein.

In an earlier study, we found that excision of the first intron (intron A) from the rat GnRH primary transcript is attenuated in non-GnRH-producing cells. This attenuation can be partially relieved by exonic splicing enhancers (ESEs) located in GnRH exons 3 and 4. In the present study, we confirmed that intron A of the mouse GnRH pre-mRNA was not excised in a HeLa nuclear extract (NE) in vitro or in COS-7 cells in vivo. Intron A could, however, be partially removed when exon 3 and/or 4 were linked to exon 2. In the presence of an ESE in exon 4 (ESE4), an addition of GT1 NE further increased the excision rate of intron A, whereas the addition of KK1 (a non-GnRH-producing cell) NE decreased it. To define the GnRH neuron-specific splicing activity, GT1 NE was fractionated by ultracentrifugation and ammonium sulfate precipitation. A 50-90% ammonium sulfate pellet (ASP50-90) fraction was further precipitated with 20 mM MgCl(2) to isolate a serine/arginine-rich (SR) protein fraction. Among the ASP fractions, ASP40-50 significantly increased the excision rate of intron A in the presence of HeLa NE or SR protein-rich fraction. However, the ASP40-50 fraction alone could not remove intron A. This result suggests the presence of a cofactor protein(s) in the ASP40-50 fraction that may mediate the interaction between a 3' spliceosome complex and the ESE4-SR protein complex. UV cross-linking and gel mobility shift analysis revealed that Tra2alpha but not other SR proteins tested, specifically binds to ESE4. Moreover, Tra2alpha stimulated intron A excision in a dose-dependent manner. These results imply that Tra2alpha and a cofactor protein in the ASP40-50 fraction are involved in mediating the GnRH neuron-specific excision of intron A from the GnRH primary transcript.

Isolation and characterization of a GnRH-like peptide from Octopus vulgaris.

Gonadotropin-releasing hormone (GnRH) is the key peptide in the hypothalamo-hypophysial-gonadal axis, the core of regulation of reproduction in vertebrates. In this study, an octopus peptide with structural features similar to vertebrate GnRHs was isolated from brains of Octopus vulgaris. This peptide showed luteinizing hormone-releasing activity in quail anterior pituitary cells. A cDNA encoding the precursor protein was cloned. The RT-PCR transcripts were expressed in the supraesophageal and subesophageal brains, peduncle complex, and optic gland. The presence of the peptide in the different brain region was confirmed with enzyme-linked immunosorbent assay and time-of-flight mass spectrometric analysis. Immunoreactive neuronal cell bodies and fibers were observed in the subpedunculate lobe that controls the optic-gland activity. Optic gland nerves and glandular cells in the optic gland were immunostained. The isolated peptide may be octopus GnRH that contributes to octopus reproduction not only as a neurohormone but also as an endocrine hormone.

Presence of two neuropeptides in the fusiform ganglion and reproductive ducts of Octopus vulgaris: FMRFamide and gonadotropin-releasing hormone (GnRH).

We have found evidence of FMRFamide-like and cGnRH-I-like immunoreactivity in the central nervous system (CNS) and in the reproductive ducts of both female and male cephalopod Octopus vulgaris. Cell bodies and fibers were immunolocalized in the fusiform ganglion from which the nerves that reach the female and male reproductive ducts arise. FMRFamide-like and cGnRH-I-like immunoreactive nerve endings were present in the oviduct, and in the oviducal gland of the female and in the seminal vesicle of the male. The GnRH-like peptide from the reproductive ducts has been partially characterized by HPLC. The retention time of the Octopus vulgaris GnRH-like peptide was similar to the retention time of cGnRH-I. Based on these observations we suggest that FMRFamide-like and a novel GnRH-like peptide are involved in the control of reproductive ducts of Octopus vulgaris. One possibility is that the peptides affect gamete transport. Another possibility is that they regulate secretory products such as mucus and mucilaginous substances from the oviducal gland and the seminal vesicle. Our data provide further evidence to support the hypothesis of the existence of a central and peripheral peptidergic control of reproduction of Octopus vulgaris.

Structure and biological activity of gonadotropin-releasing hormone isoforms isolated from rat and hamster brains.

Rat and hamster brain tissues were used to investigate the possible existence of a follicle stimulating hormone (FSH)-releasing factor with similar characteristics to the lamprey gonadotropin-releasing hormone III (lGnRH-III) form proposed in previous reports. The present studies involved isolation and purification of the molecule by high-performance liquid chromatography (HPLC), identification by radioimmunoassay, sequence analysis by automated Edman degradation, mass spectrometry and examination of biological activity. Hypothalamic extracts from both species contained an HPLC fraction that was immunoreactive to GnRH and coeluted with lGnRH-III and 9-hydroxyproline mGnRH ([Hyp(9)]GnRH). Determination of primary structure from purified total brain material demonstrated that the isolated molecule was [Hyp(9)]GnRH. This is the first report showing the presence of the posttranslationally modified form already known as [Hyp(9)]GnRH by primary sequence analysis. The biological activity of distinct GnRH peptides was also tested in vitro for gonadotropin release using rat pituitary primary cell cultures. The results showed that [Hyp(9)]GnRH stimulated both luteinizing hormone and FSH release, as already reported, whereas lGnRH-III had no action on the secretion of either gonadotropin.

The similarity of FSH-releasing factor to lamprey gonadotropin-releasing hormone III (l-GnRH-III).

To validate further the existence of a specific hypothalamic follicle stimulating hormone releasing factor (FSHRF), stalk-median eminence (SME) fragments from sheep and whole hypothalami from male rats were purified by gel filtration on Sephadex G-25, and the gonadotropin-releasing activity on hemipituitaries of rats incubated in vitro was determined by bioassay and compared with the radioimmunoassayable luteinizing hormone releasing hormone (LHRH) and lamprey gonadotropin releasing hormone (l-GnRH) activities in the fractions. The FSH-releasing fractions eluted in the same sequence of tubes from the Sephadex column found earlier by in vivo bioassay and were clearly separated from the immunoassayable and bioassayable LHRH. The radioimmunoassay (RIA) for l-GnRH recognized equally l-GnRH-I and -III but had negligible cross-reactivity with LHRH. Fractionation of rat hypothalamic extract by gel filtration on Sephadex G-25 revealed three peaks of l-GnRH determined by RIA, all of which eluted prior to the peak of LHRH. Only the second peak had FSH-releasing but not LH-releasing activity. To determine if this FSH-releasing activity was caused by the presence of l-GnRH in the fraction, the pituitaries were incubated with normal rabbit serum or the l-GnRH antiserum (1:1000), and the effect on the FSH- and LH-releasing activity of the FSH-releasing fraction and the LH-releasing activity of LHRH was determined. The antiserum had no effect on basal release of either FSH or LH but eliminated the FSH-releasing activity of the active fraction without altering the LH-releasing activity of LHRH. Since l-GnRH-I has little activity to release FSH or LH, and its activity is nonselective, whereas previous experiments have shown that l-GnRH-III highly selectively releases FSH with a potency equal to that of LHRH to release LH, the results support the hypothesis that the FSH-releasing activity observed in these experiments was caused by l-GnRH-III or a closely related peptide.