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Endocrinology Vol. 143, No. 5 1602-1612
Copyright © 2002 by The Endocrine Society

NEUROENDOCRINOLOGY

Guinea Pig GnRH: Localization and Physiological Activity Reveal That It, Not Mammalian GnRH, Is the Major Neuroendocrine Form in Guinea Pigs

Danielle Grove-Strawser, Stacia A. Sower, Paul M. Ronsheim, Jane B. Connolly, Cari G. Bourn and Beverly S. Rubin

Department of Anatomy and Cellular Biology (D.G.-S., P.M.R., B.S.R.), Tufts Medical School, Boston, Massachusetts 02111; and Department of Biochemistry and Molecular Biology (S.A.S., J.B.C., C.G.B.), University of New Hampshire, Durham, New Hampshire 03824

Address all correspondence and requests for reprints to: Beverly S. Rubin, Ph.D., Department of Anatomy and Cellular Biology, Tufts Medical School, 136 Harrison Avenue, Boston, Massachusetts 02111. E-mail: . beverly.rubin{at}tufts.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The isolation of GnRH cDNA from guinea pig hypothalamus predicted a novel form of GnRH with two unique amino acid substitutions relative to all known forms of this essential decapeptide. The predicted substitution at amino acid 2 in guinea pig (gp) GnRH was particularly intriguing because of the proposed importance of position 2 for binding and activation of the GnRH receptor. In the present study, gpGnRH was synthesized, and a specific antibody was generated and used to assess translation of the gpGnRH transcript. The localization of intensely labeled gpGnRH-positive cell bodies and processes in tissue sections through the preoptic area and hypothalamus argue that gpGnRH is the major neuroendocrine form of GnRH in guinea pigs. Guinea pig GnRH stimulated LH release in guinea pigs and increased LH output from guinea pig pituitary fragments, thus demonstrating biological activity in this species. In contrast, gpGnRH demonstrated little ability to stimulate LH release in rats, a species known to possess the highly conserved mammalian GnRH receptor. These findings suggest that: (1) the amino acid substitutions in gpGnRH impede binding to and/or activation of the mammalian GnRH receptor, and (2) the unique amino acid substitutions in gpGnRH are accompanied by changes in the guinea pig GnRH receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH, ALSO REFERRED TO AS LH releasing hormone, is the primary hypothalamic signal that regulates pituitary gonadotropin release. GnRH release is pulsatile (1), and the parameters of this pulsatile release influence GnRH receptor gene expression and gonadotropin subunit gene expression in the pituitary as well as the ratio of circulating titers of LH and FSH (2). In males, GnRH is essential for testosterone production and spermatogenesis, and in females it is important for the LH surge required for ovulation.

Our interest in the steroid regulation of GnRH gene expression led us to the guinea pig (Cavia porcellus) as an attractive animal model for these studies. Guinea pigs, like primates and unlike other lab rodents, have a relatively long reproductive cycle (16–17 d) with a true luteal phase (3). Thus, they afford the opportunity to examine GnRH gene expression during progesterone dominant (luteal phase) and E2 dominant (follicular phase) endocrine states. Our initial studies in the guinea pig isolated GnRH cDNA from an expression library generated from guinea pig hypothalamus (4). Surprisingly, the nucleotide sequence of the guinea pig GnRH (gpGnRH) transcript predicted a GnRH decapeptide that differed from the expected mammalian form by two amino acid substitutions. Moreover, the predicted amino acid substitutions were unique among all currently known forms of GnRH. The high levels of gpGnRH transcript measured in hypothalamic extracts from individual guinea pigs suggested that gpGnRH replaces mammalian GnRH (mGnRH) as the main neuroendocrine form of the decapeptide in this species. This serendipitous finding challenged the prevailing belief that mGnRH is the primary neuroendocrine form of GnRH in all mammals.

Remarkably, the predicted amino acid sequence of gpGnRH indicates that it is the first peptide of the family (Fig. 1Go) in which histidine is not present in position 2 of the decapeptide (various forms of GnRH are reviewed in Ref. 5 , also see Refs. 6 and 7). The predicted nonconservative substitution of tyrosine for histidine occurs in what has been considered the invariant N-terminal sequence of the decapeptide. In addition, in gpGnRH valine replaces the leucine present in position 7 of mGnRH. This conservative change represents a second unique substitution relative to all forms of GnRH identified to date. However, unlike the change in amino acid 2, some variation in amino acid 7 has been documented previously among the currently known forms of GnRH decapeptide (Fig. 1Go).



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  		Figure 1. The 15 currently identified forms of GnRH. Guinea pig GnRH has unique amino acid residues in position 2 and 7 (boxed) relative to all known forms of the decapeptide. Amino acid residues that vary from mammalian GnRH are underlined and bold. As depicted, amino acid residues in positions 1, 4, 9, and 10 are invariant in all forms of GnRH.

 
The substitution of tyrosine for histidine at amino acid 2 in gpGnRH is of particular interest because all vertebrate GnRH receptors are thought to require the basic conserved NH2 and COOH terminal sequences of GnRH for receptor binding and activation (8). The relationship among biological activity, receptor affinity, and residue substitution has been investigated in detail for mGnRH (9). Data from these studies have suggested that the histidine in position 2 of the decapeptide may be important for binding and/or activation of the mGnRH receptor (8). Reduced activity of synthetic forms of GnRH that contain substitutions at amino acid 2 have been demonstrated. The limited data available regarding tyrosine2 analogs have revealed reduced LH-releasing activity in rat pituitary cultures (10) and a decrease in binding affinity at the mGnRH receptor (11).

In the present study, we describe a specific antiserum generated to gpGnRH (TF 60) that is used to assess translation of the previously identified gpGnRH transcript and to localize the peptide in the guinea pig brain. Moreover, the relative abilities of gpGnRH and mGnRH to stimulate LH release are compared in guinea pigs and rats, a species known to possess the highly conserved mGnRH receptor, using in vivo and in vitro paradigms.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Twenty-one male Hartley White guinea pigs were purchased from Elm Hill Breeding Laboratories (Chelmsford, MA) (500–550 g), and 26 male Sprague Dawley rats (Crl:CD(SD)IGS BR) were purchased from Charles River Laboratories, Inc. (Wilmington, MA) (200–225 g). Animals were individually housed in a facility managed by the Division of Laboratory Animal Medicine. Guinea pigs were exposed to a 12-h light/12-h dark cycle (lights on at 0600 h). Guinea pig chow and water were available ad libitum, and their diet was periodically supplemented with fresh vegetables. Rats were exposed to a 14-h light/10-h dark cycle (lights on at 0400 h) with rat chow and water available ad libitum. Protocols for these studies were approved by the Institutional Animal Research Committee, and all experiments were carried out according to the Guide for the Care and Use of Laboratory Animals.

Peptide synthesis and antibody generation
Guinea pig GnRH synthesis was performed using Fast Moc TM chemistry on a model 431A, version 1.12 software, peptide synthesizer (Applied Biosystems, Foster City, CA). The peptide was cleaved from the resin using a mixture of phenol, ethanedithiol, thioanisole, and trifluoroacetic acid. Samples were purified, reconstituted, and sent for Matrix-assisted laser desorption/ionization mass spectrum (HT Laboratories, San Diego, CA) for conformation of peptide synthesis. Guinea pig GnRH was purified to greater than 95% by HPLC in the Protein Core Facility in the Department of Physiology at Tufts Medical School. An extended form of gpGnRH, containing lysine coupled to the C terminus, was conjugated to BSA via glutaraldehyde following the procedures described by Coligan (12). This preparation was then shipped to Cocalico Biologicals (Reamstown, PA) for polyclonal antibody production. Two male New Zealand white rabbits housed at their facility were injected with the extended peptide, boosted, and bled (13). All bleeds were sent to us for characterization of the polyclonal antisera.

Localization of gpGnRH in brain
Characterization of antiserum to gpGnRH-RIA.
RIA was performed as previously described (14, 15, 16). Guinea pig GnRH was iodinated using the chloramine T method (14). Iodinated mGnRH was purchased from NEN Life Science Products (Boston, MA). Binding curves were set up with various dilutions of the two polyclonal antisera generated to gpGnRH (antisera TF 59 and TF 60 generated at Cocalico Biologicals) and an antiserum generated to mGnRH (Ab-R1245 obtained from Terry Nett, Colorado State University, Fort Collins, CO). Because antiserum TF 60 exhibited a very high level of specificity for gpGnRH (in RIA at a dilution of 1:400,000, cross-reactivity with mGnRH was calculated at <0.001% for TF 60 and 28% for TF 59), it was used for the studies described. Standard curves were set up with synthetic gpGnRH or mGnRH and antiserum TF 60 to gpGnRH (1:50,000 working dilution, 1:400,000 final dilution) or antiserum to mGnRH (Ab-R1245, 1:30,000 working dilution, 1:120,000 final dilution). Antiserum TF 60 was also used in RIA to assess levels of gpGnRH present in hypothalamic extracts from guinea pigs and rats.

Characterization of antiserum to gpGnRH: immunocytochemical protocols.
Six guinea pigs and three rats were deeply anesthetized with Nembutal (100 mg/kg, ip, Abbott Laboratories, North Chicago, IL). They were perfused intraventricularly with a fixative containing 2% acrolein (Polysciences, Inc., Warrington, PA) and 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). Brains were removed from the skull, postfixed for an additional 30 min and coronally sectioned at 50 µm on a Vibratome (Technical Products International, St. Louis, MO). Brain sections were placed sequentially into one of six vessels. This collection method allowed adjacent sections to be incubated with antisera for gpGnRH (TF 60) at dilutions of 1:70,000 and 1:150,000 and mGnRH (DiaSorin, Inc., Stillwater, MN) at a dilution of 1:30,000. For controls, sections were incubated with gpGnRH antiserum (1:70,000) or mGnRH antiserum (1:30,000) preabsorbed with synthetic gpGnRH or mGnRH at a concentration of 0.05 mM (0.0591 mg/ml).

The details of the immunocytochemical protocol have been described previously (17). In brief, following a pretreatment to remove residual aldehydes and decrease background staining, tissues were incubated with antisera or preabsorbed antisera for 48–72 h at 4 C. Bound antibody was detected with a Vectastain Elite ABC kit, rabbit IgG (Vector Laboratories, Inc., Burlingame, CA) followed by the chromagen diaminobenzidine (0.25 mg/ml in 0.05% hydrogen peroxide).

The number of cells labeled with antisera to gpGnRH (TF60 at a dilution of 1:70,000) or mGnRH (DiaSorin, Inc. at a dilution of 1:30,000) were counted in sets of five matched adjacent sections through the preoptic area and 12 matched adjacent sections through the anterior, medial, and posterior hypothalamus of the guinea pig brain.

Assessment of physiological activity
Catheterization.
The catheterization protocol was similar to that described previously in guinea pigs (17) and rats (18). Guinea pigs were approximately 6–9 wk old at the time of surgery. They were anesthetized with xylazine (5 mg/kg, im, Burns Veterinary Supply, Inc., Rockville Center, NY) and ketamine hydrochloride (50 mg/kg, im, Fort Dodge Animal Health, Fort Dodge, IA). Rats were approximately 7–12 wk old at the time of surgery. They too were anesthetized with xylazine (6–6.7 mg/kg, im) and ketamine (75–85 mg/kg, im) and received 0.1 mg/kg buprenorphine hydrochloride (im Reckitt \|[amp ]\| Colman Products Ltd., Hull, UK) to ease postoperative pain. A beveled SILASTIC brand catheter (0.020" inner diameter, 0.037" outer diameter, Dow Corning Corp., Midland, MI) was inserted into the right jugular vein, threaded into the right atrium of the heart, and the free end of the catheter exited from the back of the neck. Catheters were flushed daily (with heparinized saline) to maintain patency and accustom the animals to the blood sampling routine.

In vivo GnRH administration and blood sampling
A cross-over design was used for GnRH administration such that each animal was tested with equivalent doses of gpGnRH and mGnRH. On the day of infusion, fresh aliquots of gpGnRH and mGnRH were diluted in saline so that the desired dose was delivered in a volume of 0.2 ml. Animals were allowed a minimum of 48 h to recover after surgery before GnRH infusion and sample collection. A minimum of 6 d for guinea pigs and 3 d for rats was allowed between the first and second GnRH infusion.

Baseline blood samples were collected at least 1 h before infusion of GnRH. For guinea pigs, blood samples (0.5 ml) were collected at 10, 20, 40, and 60 min after GnRH infusion. For rats, blood samples (0.3 ml) were collected at 15, 30, 60, and 120 min after GnRH infusion. After each blood sample, the catheter was flushed with saline and heparinized saline. Blood samples were centrifuged for 20 min at 1000 x g at 4 C and plasma was stored at -30 C until subsequent LH RIA.

In vitro perifusion
Guinea pigs were killed for perifusion experiments a minimum of 14 d after the last GnRH infusion. Because of their large size (723 ± 38 g body weight), they were anesthetized with Nembutal before decapitation. The pituitary was rapidly removed, placed in a dish with medium that had been warmed and oxygenated, and the anterior pituitary was dissected into eight fragments (19). Tissues were perifused with Medium 199 without phenol red (no. 11043–023, Life Technologies, Inc., Rockville, MD) with the addition of 25 mM HEPES (15630-080, Life Technologies, Inc.), 0.5% BSA (A-7030, Sigma-Aldrich Corp., St. Louis, MO), 90 U/ml bacitracin (B-0125, Sigma), and 25 µg/ml gentamicin (no. G-1272, Sigma). Pituitary fragments from a single animal were placed into a 500-µl chamber in the AcuSyst-S cell culture system (Cellex Biosciences Inc., previously Endotronics, Inc., Coon Rapids, MN) with warmed (37 C) and oxygenated (95% O2/5%CO2) medium flowing at a rate of 0.2 ml/min (12 ml/h). Effluents were collected every 5 min on ice and stored at -30 C until LH RIA. On each day of perifusion, two guinea pigs were killed and two chambers were run. One received pulses of gpGnRH, and the other received equivalent pulses of mGnRH. Pulses lasted 4 min and were administered at 60 min (0.5 µg/ml; 4.2 x 10-7 M), 120 min (1 µg/ml; 8.4 x 10-7 M), and 180 min (5 µg/ml; 4.2 x 10-6 M) after the pituitary fragments were placed in the chambers. At 240 min, tissues received a 30-min exposure to 60 mM KCl to confirm tissue viability.

The procedure for rats was similar to that for guinea pigs with a few exceptions. Rats were lightly anesthetized with metofane before decapitation. Tissues were exposed to three 3.5-min GnRH pulses at hourly intervals containing 0.1 µg/ml (8.4 x 10-8 M, pulses 1 and 2) and 0.2 µg/ml (1.7 x 10-7 M, pulse number 3). Exposure to KCl was limited to 5 min.

LH RIA
The RIA for guinea pig LH was optimized previously in the lab (20). Briefly, the LH antiserum (CSU 120, generously provided by Dr. Terry Nett, Colorado State University) was used at a final dilution of 1:100,000. Guinea pig pituitary powder (21) was used as a reference prep for the standard curve, and iodinated rat LH was purchased from Covance Laboratories, Inc. (Vienna, VA). Plasma samples were assayed in singlets and perifusion samples were diluted and assayed in duplicate. The limit of detection for the LH assay was 11.2 U/tube (88.2% B/Bo) and the midrange of the assay was 59.7 U/tube. Intraassay variability ranged from 3.2–8.3% and interassay variation was calculated at 6.5%.

Rat LH was measured as previously described (18), using the rat LH assay kit provided by the NHPP and NIDDK containing LH antiserum NIDDK-anti-rLH-S-11 and reference preparation LH NIDDK-rLH-RP3. The limit of detection of the assay was 21 pg/tube (89% B/Bo), and the midrange of the assay was 121 pg/tube. Intraassay variation ranged from 2.7% to 6.8%, and the interassay variability was 4.8%.

Statistics
Comparisons of GnRH cell numbers in sections through the guinea pig brain incubated with antiserum to gpGnRH vs. antiserum to mGnRH were analyzed by a paired t test. In vivo response to infusions of gpGnRH and mGnRH were assessed by ANOVA. Comparisons of the in vivo and in vitro responses to the two forms of GnRH were made using two-way ANOVA with repeated measures and post hoc t tests and Fisher’s protected least significant difference (PLSD; StatView, Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of gpGnRH antiserum
Characterization by RIA.
Initial binding curves demonstrated marked specificity of antiserum TF 60 for gpGnRH. As mentioned previously, the cross-reactivity of antiserum TF 60 with mGnRH in RIA at a final dilution of 1:400,000 was calculated at less than 0.001%. Therefore, antiserum TF 60 was used for the studies described. Displacement curves (Fig. 2AGo) demonstrate that iodinated gpGnRH bound to antiserum TF 60 is displaced by synthetic gpGnRH but not by synthetic mGnRH. In contrast, iodinated mGnRH bound to mGnRH antiserum Ab-R1245 is displaced by both synthetic mGnRH and synthetic gpGnRH (Fig. 2BGo), indicating that Ab-R1245 binds gpGnRH. RIA results with antiserum TF 60 revealed the presence of significant levels of gpGnRH in hypothalamic extracts from guinea pigs (Fig. 2CGo). No evidence of gpGnRH was detected in hypothalamic extracts from rats (Fig. 2CGo).



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  		Figure 2. Binding curves reveal specificity of gpGnRH antiserum TF 60. A, Iodinated gpGnRH bound to gpGnRH antiserum TF 60 is displaced by increasing amounts of synthetic gpGnRH ({circ}) but not synthetic mGnRH ({square}). B, Iodinated mGnRH bound to mGnRH antiserum Ab-R1245 is displaced by increasing amounts of both synthetic mGnRH ({square}) and synthetic gpGnRH ({circ}). C, Iodinated gpGnRH bound to gpGnRH antiserum TF 60 is displaced by extracts from guinea pig hypothalami ({circ}) but not by extracts from rat hypothalami ({square}).

 
Characterization by immunocytochemistry.
Incubation of tissue sections through the guinea pig brain with antiserum TF 60 (1:70,000 and 1:150,000 dilutions) revealed intense reaction product in cell bodies and processes. Examples of these tissue sections through the preoptic area and the medial basal hypothalamus and caudal median eminence are shown in Figs. 3Go, A and B, and 4Go, A, B, and C. Adjacent tissue sections incubated with a commercially available antiserum to mGnRH (DiaSorin, Inc.: 1:30,000 dilution; Figs. 3EGo and 4Go, D, E, and F) revealed fewer cell bodies and processes and less intense reaction product relative to that observed with antiserum TF 60. There was, however, no convincing evidence to suggest that specific subgroups of the population of GnRH neurons were revealed with gpGnRH antiserum and not mGnRH antiserum. In tissue sections through the preoptic area, mGnRH antiserum revealed approximately 60% of the number of GnRH positive cell bodies detected in adjacent tissue sections incubated with gpGnRH antiserum (P = 0.009) (Fig. 5Go). In more caudal tissue sections through the hypothalamus, mGnRH antiserum revealed approximately 75% of the number of GnRH-positive cell bodies detected with gpGnRH antiserum TF 60 (P = 0.102, NS). Preabsorption of the TF 60 antiserum with synthetic gpGnRH before incubation eliminated reaction product in the guinea pig brain (Fig. 3CGo). In contrast, preabsorption of TF 60 with mGnRH did not eliminate immunoreactive cell bodies and fibers (Fig. 3DGo). Preabsorption of the mGnRH antiserum with synthetic gpGnRH (Fig. 3FGo) completely eliminated immunoreactivity in the guinea pig brain.



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  		Figure 3. Localization of gpGnRH and mGnRH in the guinea pig brain. GnRH neurons are shown in coronal sections through the rostral preoptic area at the level of the optic chiasm in guinea pigs. Incubation with the antiserum to gpGnRH, TF 60, at dilutions of 1:70,000 (A) or 1:150,000 (B) revealed intensely labeled cell bodies and processes. Preabsorption of TF 60 (1:70,000) with synthetic gpGnRH eliminated all immunoreactivity (C). Preabsorption of TF 60 (1:70,000) with mGnRH did not eliminate immunoreactivity (D). Incubation with a commercially available antiserum to mGnRH (DiaSorin, Inc., at 1:30,000) revealed immunoreactive cell bodies and processes (E), but preabsorption of the mGnRH antiserum with gpGnRH eliminated the reaction product (F). III V, Third ventricle; oc, optic chiasm. Scale bar, 100 µm.

 


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  		Figure 4. GnRH immunoreactivity in the hypothalamus. Examples of GnRH immunoreactivity are shown in adjacent coronal sections through the caudal median eminence. Incubation with the antiserum to gpGnRH TF 60 at 1:70,000 dilution (A, B, C) revealed more intense immunoreactivity than that revealed with the antiserum to mGnRH (DiaSorin, Inc.) at 1:30,000 dilution (D, E, F). Rectangles in A and D indicate the regions of higher magnification shown in B, C, E, and F. Examples of GnRH-positive neurons in the medial basal hypothalamus are shown in B and E. Immunoreactive axons and terminals in the caudal median eminence are shown in C and F. III V, Third ventricle. Scale bar, 100 µm.

 


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  		Figure 5. Mean number of GnRH-positive cell bodies. Data from cell counts of 5 matched sections through the preoptic area (rostral cells) and 12 matched sections through the hypothalamus (caudal cells) are shown. In each case, more GnRH-positive cell bodies were detected with antiserum TF 60 to gpGnRH relative to the number detected with the antiserum to mGnRH. Differences in positive cell number were statistically significant in sections through the preoptic area (rostral cells; *, P = 0.009).

 
In the rat brain, pale labeling of some cell bodies and processes was observed in tissue sections incubated with gpGnRH antiserum, TF 60, at a dilution of 1:70,000 (Fig. 6AGo). Immunoreactivity was completely lost when the antiserum was further diluted to 1:150,000 (not shown) or when it was preabsorbed with either mGnRH (Fig. 6BGo) or gpGnRH (not shown). Intensely labeled GnRH-positive cells and processes were observed in adjacent rat brain sections incubated with the mGnRH antiserum (Fig. 6CGo). The mGnRH antiserum clearly revealed more GnRH-positive cell bodies and processes than were observed following incubation with the gpGnRH antiserum TF 60.



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  		Figure 6. Localization of GnRH in the rat. GnRH neurons are shown in coronal sections through the preoptic area in the rat. Tissue sections incubated with antiserum to gpGnRH, TF 60, at a dilution of 1:70,000 revealed only faint reaction product (A, arrowheads point to cell bodies). Preabsorption of the gpGnRH antiserum, TF 60 with mGnRH eliminated reaction product in the rat brain (B). Adjacent sections incubated with antiserum to mGnRH (DiaSorin, Inc.) at 1:30,000 revealed intensely labeled cell bodies and processes (C). Scale bar, 100 µm.

 
Localization of gpGnRH in guinea pig brain
The pattern of immunoreactivity observed with gpGnRH antiserum TF 60 was examined in every sixth 50-µm section through the guinea pig brain extending from the diagonal band of Broca rostrally through the caudal hypothalamus. Antiserum TF 60 revealed intensely labeled GnRH-positive cell bodies throughout the diagonal band of Broca, rostral preoptic area, preoptic area (Fig. 3Go, A and B), and septal nuclei, and within the anterior, medial (Fig. 4Go, A and B), and posterior divisions of the hypothalamus. Within the hypothalamus, some GnRH-positive cell bodies appeared to be located within the arcuate nucleus, whereas other positive cell bodies were located in lateral, ventral, and dorsal aspects of the hypothalamus. Immunoreactive processes were noted in the regions mentioned above as well as in the amygdala and the median eminence (Fig. 4Go, A and C).

Physiological activity of gpGnRH
Activity of gpGnRH in guinea pigs.
In vivo studies demonstrated the ability of gpGnRH to release LH in guinea pigs. Guinea pigs infused with 0.3, 1, and 10 µg of synthetic gpGnRH exhibited a dose-dependent response to the treatments (Fig. 7AGo). Following infusion of the two lower doses, LH levels peaked at 10 min and returned to baseline by 40 min. After infusion of the highest dose, LH levels peaked at 10 min and returned to baseline by 60 min.



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  		Figure 7. In vivo bolus infusion of gpGnRH vs. mGnRH in guinea pigs. Plasma LH levels (U/ml) measured in sequential blood samples taken from guinea pigs 10, 20, 40, and 60 min after infusion of gpGnRH (A) or mGnRH (B). The doses of GnRH are as follows: {diamond} = 0, saline vehicle; {triangleup} = 0.3 µg; {circ} = 1 µg; {square} = 10 µg. LH measurements are displayed as the mean ± SEM. Each point represents LH measurements from five to six animals.

 
In contrast to the response to gpGnRH, all three doses of mGnRH resulted in a similar LH response at 10 min (Fig. 7BGo, P = 0.8278, NS), and LH levels failed to return to baseline at 60 min. Following the 10-µg infusion, LH levels at 40 and 60 min were significantly increased relative to the levels measured at the 10-min time point (ANOVA F = 19.844, P < 0.0001; Fisher’s PLSD, P = 0.0006, 10 min vs. 40 min; P = 0.00075, 10 min vs. 60 min). Comparisons of the response to the 1-µg infusion of mGnRH and gpGnRH (Fig. 7Go, A and B) revealed that, although both peptides release similar amounts of LH at 10 min, LH levels differed at the 20-, 40-, and 60-min time points (ANOVA, F = 4.629, P = 0.0041; Fisher’s PLSD, p20 min = 0.0117, p40 min = 0.0004, and p60 min = 0.0026). Comparison of the response to 0.3-µg infusions of the two forms of GnRH (7, A and B) suggests that the LH response to mGnRH is more robust in guinea pigs (ANOVA F = 4.447, P = 0.0057; Fisher’s PLSD, response to gpGnRH vs. mGnRH P < 0.01 at 10, 20, 40, and 60 min).

In vitro perifusion studies verified the ability of gpGnRH to release LH from guinea pig pituitary fragments (Fig. 8Go). The LH response to equivalent doses of gpGnRH and mGnRH were not significantly different. Pituitary fragments exposed to mGnRH appeared to demonstrate higher baseline LH levels relative to those exposed to gpGnRH.



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  		Figure 8. In vitro perifusion of guinea pig pituitary fragments exposed to gpGnRH vs. mGnRH. Compiled profiles of LH output from guinea pig pituitary fragments exposed to gpGnRH ({circ}) or mGnRH ({square}). The mean LH value and SEM were calculated for each 5-min fraction. Fragments were exposed to GnRH at fractions 12, 24, and 36 (arrows). Stimulation with KCl occurred at fraction 48. An asterisk indicates a significant difference (P < 0.05) between the mean LH values measured in fragments treated with gpGnRH, compared with mGnRH. The data are compiled from three perifusion experiments.

 
Activity of gpGnRH in rats
Guinea pig GnRH did not effectively stimulate LH release in rats (Fig. 9AGo). Rats infused with 0.1, 0.3, and 1 µg of gpGnRH showed only minimal increases in LH release (Fig. 9AGo) relative to the levels released in response to equivalent doses of mGnRH (Fig. 9BGo). Infusion of 0.05 µg of gpGnRH (Fig. 9Go, A and B) did not stimulate LH release (ANOVA, P = 0.7311, NS), although the same dose of mGnRH elicited a robust LH response (ANOVA, F = 14.986, P < 0.001).



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  		Figure 9. In vivo bolus infusion of gpGnRH vs. mGnRH in rats. Plasma LH levels (ng/ml) measured in sequential blood samples taken from rats 15, 30, 60, and 120 min after infusion of gpGnRH (A) or mGnRH (B). The doses of GnRH are as follows: {diamond}, broken line = 0.05 µg; {triangleup}, solid line = 0.1 µg; {circ}, broken line = 0.3 µg; {square}, solid line = 1 µg. LH measurements are displayed as the mean ± SEM. Each point represents LH measurements from five to seven animals.

 
When rat pituitary fragments were stimulated with gpGnRH, the levels of LH released were only slightly higher than baseline levels (Fig. 10Go). In contrast, administration of mGnRH induced a robust LH response. Moreover, baseline levels of LH release from pituitary fragments that received gpGnRH were lower than those that were exposed to mGnRH (Fig. 10Go). As shown in Fig. 10Go, KCl failed to stimulate LH release in rat pituitary fragments that received gpGnRH. These data are consistent with the lack of response of the rat pituitary to stimulation by gpGnRH. Previous data from Elskus et al. (22) revealed that the ability of male rat pituitary fragments to respond to KCl stimulation after several hours of perifusion was dependent on prior stimulation with mGnRH.



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  		Figure 10. In vitro perifusion of rat pituitary fragments exposed to gpGnRH vs. mGnRH: compiled profiles of LH output from rat pituitary fragments exposed to gpGnRH ({circ}) or mGnRH ({square}). The mean LH value and SEM were calculated for each 5-min fraction. Fragments were exposed to GnRH at fractions 12, 24, and 36 (arrows). Stimulation with KCl occurred at fraction 48. An asterisk indicates a significant difference (P < 0.05) between the mean LH values measured in fragments treated with gpGnRH, compared with mGnRH. The data are compiled from three perifusion experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Localization of gpGnRH in guinea pigs
The generation of a specific antiserum to gpGnRH enabled us to demonstrate that the previously identified gpGnRH transcript (4) is translated in guinea pig brain. Use of the antiserum in RIA revealed the presence of high levels of gpGnRH in hypothalamic extracts from guinea pig brains. Moreover, immunocytochemical analyses enabled the precise localization of gpGnRH positive cell bodies and processes.

As described, immunoreactive cell bodies were localized within the diagonal band of Broca; preoptic area; septal nuclei; and the anterior, medial, and posterior hypothalamus. Immunoreactive processes were apparent in these same regions as well as in the amygdala. As expected, labeled axons and terminals were most abundant in the caudal median eminence. The localization of gpGnRH-positive cells and processes are in general agreement with early descriptions of immunoreactive GnRH in the guinea pig brain (23, 24); however, the intensity of the immunoreactivity and number of GnRH-positive cell bodies and processes observed in the present study appear to be significantly greater than that described in earlier studies (23, 24) and relative to previous observations in our own laboratory (17, 20). Both more sensitive immunocytochemical protocols and the availability of a specific antiserum to gpGnRH undoubtedly enhanced the level of immunoreactive GnRH observed in the guinea pig brain in the present study.

The two antisera to mGnRH used in the present studies (Ab-R1245, DiaSorin, Inc.) demonstrated significant cross-reactivity with gpGnRH. This was not surprising because mGnRH antisera have routinely been used to detect GnRH in guinea pigs (20, 23, 24, 25, 26, 27). The level of immunoreactivity observed in guinea pig tissues incubated with gpGnRH antiserum was clearly increased relative to that observed with antiserum to mGnRH. The number of GnRH neurons counted, intensity of the fiber networks observed, and density of axons and terminals in the median eminence were all markedly increased in tissue sections incubated with gpGnRH antiserum relative to adjacent sections incubated with an antiserum generated to mGnRH.

Although the available data cannot definitively rule out the presence of mGnRH in the guinea pig hypothalamus, both the RIA data and the immunocytochemical data argue that gpGnRH is the predominant neuroendocrine form of GnRH in guinea pigs. When the gpGnRH antiserum was preabsorbed with gpGnRH, no immunoreactivity was detected in the guinea pig brain. In contrast, when the gpGnRH antiserum was preabsorbed with mGnRH, GnRH-positive cell bodies and processes were clearly evident. The intensity of labeling was somewhat reduced in tissue sections incubated with gpGnRH antiserum preabsorbed with mGnRH relative to those incubated with gpGnRH antiserum alone. A review of the immunocytochemical data presented suggests that the reduced level of immunoreactivity might be explained by cross-reactivity of some component of the polyclonal gpGnRH antiserum with mGnRH at the dilution used rather than the presence of mGnRH in guinea pig brain. These data are consistent with the results of the immunological analyses of Gao et al. (28) that demonstrated that the major form of GnRH in guinea pig hypothalamic extracts differed from mGnRH.

Localization of gpGnRH in rats
Immunocytochemical analyses revealed little convincing evidence of the presence of gpGnRH in the rat brain. Incubation of tissue sections through the rat brain with antiserum to gpGnRH at a dilution of 1:70,000 revealed only pale labeling of cell bodies and a low level of reaction product in axons and terminals of the median eminence. Further dilution of the gpGnRH antiserum (1:150,000) or preabsorption of the gpGnRH antiserum with mGnRH completely eliminated immunoreactivity in the rat brain. As discussed above, these data too would be consistent with a cross-reactivity of some component of the polyclonal gpGnRH antiserum with mGnRH at the lower dilutions used in immunocytochemistry. Consistent with this interpretation, hypothalamic extracts from rat brain failed to reveal detectable levels of gpGnRH in RIA.

Physiological activity of gpGnRH in guinea pigs
Intra-atrial infusions of gpGnRH stimulated in vivo release of LH in guinea pigs confirming the physiological activity of this unique form of GnRH in this species. The magnitude of the LH response was clearly dose dependent. At each of the three doses, a rapid elevation of LH was noted at 10 min, and LH levels subsequently returned to baseline by the end of the sampling period.

As would be expected based on data from previous studies (29, 30, 31, 32, 33), guinea pigs did respond to mGnRH; however, the LH response to infusion of mGnRH differed from that of gpGnRH. The magnitude of the LH response to all three doses of mGnRH was similar at the 10-min time point, and mGnRH administration resulted in a prolonged elevation of circulating LH titers. LH levels failed to return to baseline during the sampling period even in response to the lowest dose of mGnRH. These data suggest that the LH response to mGnRH may be more robust than the response to the species-appropriate form of GnRH in guinea pigs.

The direct measurements of circulating LH levels in the present study are in agreement with the indirect assessments of circulating LH levels reported by Gao et al. (28) using the guinea pig Leydig cell bioassay (34). As in the present study, the bioassay data suggested an increased sensitivity of guinea pigs to mGnRH relative to gpGnRH and a prolonged elevation of LH levels after infusion of mGnRH. The dramatic rise in LH levels noted 40 and 60 min after administration of the highest dose of mGnRH to guinea pigs in the present study was not observed by Gao et al. (28). Whether this variation in the data may be attributed to a distinction between LH immunoreactivity and LH bioactivity remains to be determined.

Whereas the LH profile observed in guinea pigs after stimulation with mGnRH may relate to an inability of guinea pigs to effectively degrade mGnRH, this explanation seems unlikely because the enzymes that degrade GnRH are not specific for the decapeptide. A more likely explanation may be that the prolonged LH response reflects altered binding kinetics of mGnRH at the gpGnRH receptor. Formal studies of binding kinetics await characterization of the GnRH receptor in guinea pigs.

Guinea pig GnRH and LH release in rats
Guinea pig GnRH demonstrated little ability to release LH in rats, a species known to possess the highly conserved mGnRH receptor (8). This finding was not entirely surprising, given the amino acid substitutions in gpGnRH relative to mGnRH. As mentioned previously, amino acids 1 and 2 of the N-terminal segment of the decapeptide are conserved in all 15 currently known forms of GnRH with the exception of gpGnRH. Numerous studies of the ligand-receptor interaction for mGnRH have indicated that the histidine at residue 2 is one of three important amino acids (histidine2, tryptophan3, arginine8) for the binding and activation of the mGnRH receptor (8, 35). An early study that examined the effects of replacing histidine2 of mGnRH with tyrosine reported a 95% reduction in LH-releasing activity in rat pituitary cultures (10). A more recent study examined the mGnRH receptor and the changes in binding affinity that occur with substitutions in mGnRH (11). GnRH analogs in which histidine2 was replaced by tyrosine required seven times more agonist than mGnRH to give an equivalent half-maximal response, suggesting that the amino acid substitution decreased affinity for the mGnRH receptor. Evidence of decreased binding affinity was also suggested when phenylalanine or tryptophan were substituted for histidine2 (11). These latter findings are consistent with reports of significant reductions in the activity of mGnRH analogs with these same substitutions (10, 36, 37). It is interesting to note that substitutions at amino acid 2 are a common feature of the GnRH peptide antagonists in current clinical use (38).

Of potential relevance with regard to gpGnRH is the finding that a mGnRH analog in which leucine7 was replaced with valine (the amino acid present in position 7 of gpGnRH) favored FSH release (39). In addition, the lamprey III form of GnRH, which also contains a substitution at position 7 relative to mGnRH, has been shown to preferentially stimulate FSH release at the mammalian GnRH receptor (40, 41). These data suggest that future studies should explore the FSH-releasing activity of gpGnRH at the mGnRH receptor.

The gpGnRH receptor
Comparisons of the relative abilities of gpGnRH and mGnRH to release LH in rats and guinea pigs suggest that the gpGnRH receptor underwent a change in response to or concomitant with the alteration in the GnRH decapeptide. Rats possess the highly conserved mGnRH receptor (8), and they exhibited only a minimal LH response to gpGnRH. Although the GnRH receptor in guinea pigs has not yet been characterized, following infusion of gpGnRH, guinea pigs exhibited a marked increase in LH levels. Therefore, it is likely that in addition to a unique form of GnRH, guinea pigs possess a unique GnRH receptor with significant changes from the mGnRH receptor. It is important to note that the putative changes in the gpGnRH receptor do not eliminate its ability to bind or be activated by mGnRH. Rather as previously discussed, guinea pigs exposed to mGnRH demonstrated a robust and prolonged LH response. Given that guinea pigs appear to be more sensitive to mGnRH than gpGnRH, is it possible that guinea pigs actually maintain the mGnRH receptor and express a second receptor that binds gpGnRH? These issues await further clarification in future studies that identify and characterize the GnRH receptor(s) in guinea pigs and that examine GnRH ligand receptor-binding kinetics in this species.

Is gpGnRH the sole form of GnRH in guinea pigs?
The present study concentrated on the localization of the neuroendocrine form of gpGnRH. Whether guinea pigs synthesize additional forms of GnRH remains to be determined. It is likely that additional forms of GnRH will be identified in guinea pigs because multiple forms of GnRH have been identified in most vertebrates that have been examined (42, 43, 44, 45, 46, 47, 48, 49, 50).

A close relative of the guinea pig, the capybara (Hydrochaeris hydrochaeris), reportedly synthesizes three forms of GnRH including mGnRH, salmon GnRH, and chicken II GnRH (50, 51). The identification of mGnRH in this species resulted from the analysis of HPLC extracts from the capybara preoptic-hypothalamic region. These extracts revealed a peak that eluted with mGnRH and was capable of binding antisera generated to mGnRH (50). Because it is clear from the data presented here that some antisera to mGnRH demonstrate a significant level of cross-reactivity with gpGnRH, assessment of the possibility that the capybara synthesizes gpGnRH may require additional analysis. In this regard, it should be noted that the initial HPLC studies of GnRH in guinea pig hypothalamus (27) demonstrated a dominant form of GnRH in guinea pig tissue extracts that eluted with synthetic mGnRH. This form of GnRH was presumed to be mGnRH because it was immunoreactive with antiserum B6, a sequential-type GnRH antiserum generated to mGnRH in the Sherwood laboratory (27). Antiserum B6 is directed toward the last six amino acids of mGnRH. As discussed by the authors of the study, given its specificity, antiserum B6 would be capable of binding to forms of GnRH with alterations in amino acids 2, 3, or 4. Therefore, the substitution in amino acid 2 would not be expected to interfere with the ability of gpGnRH to bind antiserum B6.

The results presented here provide evidence of another example of an endocrine anomaly in guinea pigs (52) that may relate to the isolation of this species during evolution (53, 54). Whether this unique form of GnRH is also present in close relatives of the guinea pig that belong to the same suborder (Hystricopmorpha) remains to be determined. Moreover, potential novel and distinct properties of this unique, naturally occurring form of GnRH remain to be explored. The eventual identification and characterization of the gpGnRH receptor will provide an important opportunity for further elucidation of GnRH ligand-receptor interactions.


   Acknowledgments
 
The authors would like to thank Janet Yu and Dr. Andrew P. Laudano for expert technical assistance. We would also like to thank Dr. Parlow, the NIDDK, and the National Hormone and Peptide Program for supplying reagents for LH measurement, and Dr. Terry Nett (Colorado State University) for providing us with antisera generated to LH (CSU 100) and to mGnRH (Ab-R1245).


   Footnotes
 
This work was supported by National Science Foundation Grants IBN 9818049 (to B.S.R.) and 0090852 (to S.A.S.).

Abbreviations: gpGnRH, Guinea pig GnRH; mGnRH, mammalian GnRH; PLSD, protected least significant difference.

Received October 5, 2001.

Accepted for publication January 23, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Levine JE 1998 GnRH pulse generator. In: Knobil E, Neill JD, eds. The encyclopedia of reproduction. San Diego: Academic Press; 478–483
  2. Marshall JC, Dalkin AC, Haisenleder DJ 1992 Regulation of gonadotropin gene expression by gonadotropin releasing hormone. In: Crowley WFJ, Conn PM, eds. Modes of action of GnRH and GnRH analogs. New York: Springer-Verlag; 55–68
  3. Everett JW 1961 The mammalian female reproductive cycle and its controlling mechanisms. In: Young WC, ed. Sex and internal secretions. Baltimore: Williams & Wilkins Co.; 497–555
  4. Jimenez-Linan M, Rubin BS, King JC 1997 Examination of guinea pig luteinizing hormone-releasing hormone gene reveals a unique decapeptide and existence of two transcripts in the brain. Endocrinology 138:4123–4130[Abstract/Free Full Text]
  5. Yoo MS, Kang HM, Choi HS, Kim JW, Troskie BE, Millar RP, Kwon HB 2000 Molecular cloning, distribution and pharmacological characterization of a novel gonadotropin-releasing hormone ([Trp8] GnRH) in frog brain. Mol Cell Endocrinol 164:197–204[CrossRef][Medline]
  6. Okubo K, Amano M, Yoshiura Y, Suetake H, Aida K 2000 A novel form of gonadotropin-releasing hormone in the medaka, Oryzias latipes. Biochem Biophys Res Commun 276:298–303[CrossRef][Medline]
  7. Montaner AD, Park MK, Fischer WH, Craig AG, Chang JP, Somoza GM, Rivier JE, Sherwood NM 2001 Primary structure of a novel gonadotropin-releasing hormone in the brain of a teleost, pejerrey. Endocrinology 142:1453–1460[Abstract/Free Full Text]
  8. Sealfon SC, Weinstein H, Millar RP 1997 Molecular mechanisms of ligand interaction with the gonadotropin-releasing hormone receptor. Endocr Rev 18:180–205[Abstract/Free Full Text]
  9. Karten MJ, Rivier JE 1986 Gonadotropin-releasing hormone analog design. Structure-function studies toward the development of agonists and antagonists: rationale and perspective. Endocr Rev 7:44–66[Abstract/Free Full Text]
  10. Rees RWA, Foell TJ, Chai SY, Grant N 1974 Synthesis and biological activities of analogs of the luteinizing hormone-releasing hormone (LH-RH) modified in position 2. J Med Chem 17:1016–1019[CrossRef][Medline]
  11. Flanagan CA, Rodic V, Konvicka K, Yuen T, Chi L, Rivier JE, Millar RP, Weinstein H, Sealfon SC 2000 Multiple interactions of the Asp2.61(98) side chain of the gonadotropin-releasing hormone receptor contribute differently to ligand interactions. Biochemistry 39:8133–8141[CrossRef][Medline]
  12. Coligan J 2000 Peptides. In: Coligan J, Kruisbeek A, Margulies D, Shevach EM, Stober W, eds. Current protocols in immunology. New York: John Wiley & Sons, Inc.; 2:9.0.0–9.8.15
  13. Calvin JL, Slater CH, Bolduc TG, Laudano AP, Sower SA 1993 Multiple molecular forms of gonadotropin-releasing hormone in the brain of an elasmobranch: evidence for IR-lamprey GnRH. Peptides 14:725–729[CrossRef][Medline]
  14. Stopa EG, Sower SA, Svendsen CN, King JC 1988 Polygenic expression of gonadotropin-releasing hormone (GnRH) in human? Peptides 9:419–423[CrossRef][Medline]
  15. Fahien CM, Sower SA 1990 Relationship between brain gonadotropin-releasing hormone and final reproductive period of the adult male sea lamprey, Petromyzon marinus. Gen Comp Endocrinol 80:427–437[CrossRef][Medline]
  16. Robinson TC, Tobet SA, Chase C, Waldron T, Sower SA 2000 Gonadotropin-releasing hormones in the brain and pituitary of the teleost, the white sucker. Gen Comp Endocrinol 117:381–394[CrossRef][Medline]
  17. King JC, Ronsheim P, Liu E, Powers L, Slonimski M, Rubin BS 1998 Fos expression in luteinizing hormone-releasing hormone neurons of guinea pigs, with knife cuts separating the preoptic area and the hypothalamus, demonstrating luteinizing hormone surges. Biol Reprod 58:323–329[Abstract/Free Full Text]
  18. Rubin BS, Bridges RS 1989 Alterations in luteinizing hormone-releasing hormone release from the mediobasal hypothalamus of ovariectomized, steroid-primed middle-aged rats as measured by push-pull perfusion. Neuroendocrinology 49:225–232[Medline]
  19. Fallest PC, Hiatt ES, Schwartz NB 1989 Effects of gonadectomy on the in vitro and in vivo gonadotropin responses to gonadotropin-releasing hormone in male and female rats. Endocrinology 124:1370–1379[Abstract/Free Full Text]
  20. King JC, Liu E, Ronsheim PM, Slonimski, Rubin BS 1998 Expression of fos within luteinizing hormone-releasing hormone neurons, in relation to the steroid-induced luteinizing hormone surge in guinea pigs. Biol Reprod 58:316–322[Abstract/Free Full Text]
  21. Parlow AF 1964 Importance of differential quantitative bioassays for pituitary gonadotropins in the rat. Endocrinology 74:138–141
  22. Elskus AA, Phelps AF, Schwartz NB 1995 Acute sex differences in serum LH levels in gonadectomized rats: investigation of pituitary response to GnRH pulse frequency and prolactin secretion as etiological agents. Neuroendocrinology 61:301–309[Medline]
  23. Barry J, Dubois MP 1974 Immunofluorescence study of the preoptico-infundibular LH-RH neurosecretory pathway of the guinea pig during the estrous cycle. Neuroendocrinology 15:200–208[CrossRef][Medline]
  24. Silverman AJ 1976 Distribution of luteinizing hormone-releasing hormone (LHRH) in the guinea pig brain. Endocrinology 99:30–41[Abstract/Free Full Text]
  25. Kelly MJ, Ronnekleiv OK, Eskay RL 1984 Identification of estrogen-responsive LHRH neurons in the guinea pig hypothalamus. Brain Res Bull 12:399–407[CrossRef][Medline]
  26. Giri M, Kaufman JM 1995 Involvement of neuroexcitatory amino acids in the control of gonadotropin-releasing hormone release from the hypothalamus of the adult male guinea pig predominantly inhibitory action of N-methyl-D-aspartate-mediated neurotransmission and its reversal after orchidectomy. Endocrinology 136:2404–2411[Abstract]
  27. Kelsall R, Coe IR, Sherwood NM 1990 Phylogeny and ontogeny of gonadotropin-releasing hormone: comparison of guinea pig, rat, and a protochordate. Gen Comp Endocrinol 78:479–494[CrossRef][Medline]
  28. Gao CQ, Van den Saffele J, Giri M, Kaufman JM 2000 Guinea-pig gonadotropin releasing hormone: immunoreactivity and biological activity. J Neuroendocrinol 12:355–359[CrossRef][Medline]
  29. Ter Haar MB 1978 Luteinizing hormone responsiveness to LHRH in the adult guinea-pig: direct ovarian involvement. J Endocrinol 76:49–61[Abstract/Free Full Text]
  30. Terasawa E, Bridson WE, Weishaar DJ, Rubens LV 1980 Influence of ovarian steroids on pituitary sensitivity to LHRH in the ovariectomized guinea pig. Endocrinology 106:425–429[Abstract/Free Full Text]
  31. Donovan BT, Haar MB, Parvizi N 1977 Gonadotropin secretion in the ovariectomized guinea pig: effects of electrical stimulation of the hypothalamus and of LHRH. J Physiol 265:597–613[Abstract/Free Full Text]
  32. Nass TE, Terasawa E, Dierschke DJ, Goy RW 1984 Developmental changes in luteinizing hormone secretion in the female guinea pig. I. Effects of ovariectomy, estrogen, and luteinizing hormone-releasing hormone. Endocrinology 115:220–226[Abstract/Free Full Text]
  33. Gore AC, Terasawa E 2001 Neural circuits regulating pulsatile luteinizing hormone release in the female guinea-pig: opioid, adrenergic, and serotonergic interactions. J Neuroendocrinol 13:239–248[CrossRef][Medline]
  34. Gao CQ, Giri M, Van Hoecke MJ, Mertens K, Van den Saffele J, Kaufman JM 2001 Marked species specificity of guinea pig luteinizing hormone: validation of a bioassay. J Androl 22:226–234[Abstract]
  35. Flanagan CA, Millar RP, Illing N 1997 Advances in understanding gonadotropin-releasing hormone receptor structure and ligand interactions. Rev Reprod 2:113–120[Abstract]
  36. Sandow J, Konig W, Geiger R, Uhmann R, Rechenberg W 1978 Structure-activity relationship in the LH-RH molecule. In: Crighton DC, Haynes NB, Foxcroft GR, Lamming GE, eds. Control of ovulation. London: Butterworths; 49–70
  37. Yanaihara N, Tsuji K, Yanaihara C, Hashimoto T 1973 Synthesis and biological activities of analogs of luteinizing hormone-releasing hormone (LH-RH) substituted in position 1 or 2. Biochem Biophys Res Commun 51:165–173[CrossRef][Medline]
  38. Millar RP, Zhu YF, Chen C, Struthers RS 2000 Progress towards the development of non-peptide orally-active gonadotropin-releasing hormone (GnRH) antagonists: therapeutic implications. Br Med Bull 56:761–772[Abstract/Free Full Text]
  39. Fujino M, Kobayashi S, Obayashi M, Fukuda T, Shinagawa S 1972 Synthesis and biological activities of analogs of luteinizing hormone releasing hormone (LHRH). Biochem Biophys Res Commun 49:698–705[CrossRef][Medline]
  40. Yu WH, Karanth S, Walczewska A, Sower SA, McCann SM 1997 A hypothalamic follicle-stimulating hormone-releasing decapeptide in the rat. Proc Natl Acad Sci USA 94:9499–9503[Abstract/Free Full Text]
  41. Yu WH, Karanth S, Sower SA, Parlow AF, McCann SM 2000 The similarity of FSH-releasing factor to lamprey gonadotropin-releasing hormone III (l-GnRH-III). Proc Soc Exp Biol Med 224:87–92[Abstract/Free Full Text]
  42. Lescheid DW, Terasawa E, Abler LA, Urbanski HF, Warby CM, Millar RP, Sherwood NM 1997 A second form of gonadotropin-releasing hormone (GnRH) with characteristics of chicken GnRH-II is present in the primate brain. Endocrinology 138:5618–5629[Abstract/Free Full Text]
  43. Dellovade TL, King JA, Millar RP, Rissman EF 1993 Presence and differential distribution of distinct forms of immunoreactive gonadotropin-releasing hormone in the musk shrew brain. Neuroendocrinology 58:166–177[Medline]
  44. Kasten TL, White SA, Norton TT, Bond CT, Adelman JP, Fernald RD 1996 Characterization of two new preproGnRH mRNAs in the tree shrew: first direct evidence for mesencephalic GnRH gene expression in a placental mammal. Gen Comp Endocr 104:7–19
  45. White RB, Eisen JA, Kasten TL, Fernald RD 1998 Second gene for gonadotropin-releasing hormone in humans. Proc Natl Acad Sci USA 95:305–309[Abstract/Free Full Text]
  46. Chen A, Yahalom D, Ben-Aroya N, Kaganovsky E, Okon E, Koch Y 1998 A second isoform of gonadotropin-releasing hormone is present in the brain of human and rodents. FEBS Lett 435:199–203[CrossRef][Medline]
  47. Gestrin ED, White RB, Fernald RD 1999 Second form of gonadotropin-releasing hormone in mouse: immunocytochemistry reveals hippocampal and periventricular distribution. FEBS Lett 448:289–291[CrossRef][Medline]
  48. Urbanski HF, White RB, Fernald RD, Kohama SG, Garyfallou VT, Densmore VS 1999 Regional expression of mRNA encoding a second form of gonadotropin-releasing hormone in the macaque brain. Endocrinology 140:1945–1948[Abstract/Free Full Text]
  49. Lin XW, Otto CJ, Peter RE 1998 Evolution of neuroendocrine peptide systems: gonadotropin-releasing hormone and somatostatin. Comp Biochem Physiol C 119:375–388
  50. Montaner AD, Somoza GM, King JA, Bianchini JJ, Bolis CG, Affanni JM 1998 Chromatographic and immunological identification of GnRH (gonadotropin-releasing hormone) variants. Occurrence of mammalian and a salmon-like GnRH in the forebrain of an eutherian mammal: Hydrochaeris hydrochaeris (Mammalia, Rodentia). Regul Pept 73:197–204[CrossRef][Medline]
  51. Montaner AD, Affanni JM, King JA, Bianchini JJ, Tonarelli G, Somoza GM 1999 Differential distribution of gonadotropin-releasing hormone variants in the brain of Hydrochaeris hydrochaeris (Mammalia, Rodentia). Cell Mol Neurobiol 19:635–651[CrossRef][Medline]
  52. Keightley MC, Fuller PJ 1996 Anomalies in the endocrine axes of the guinea pig: relevance to human physiology and disease. Endocr Rev 17:30–44[Abstract/Free Full Text]
  53. Graur D, Hide WA, Zharkikh A, Li WH 1992 The biochemical phylogeny of guinea-pigs and gundis, and the paraphyly of the order rodentia. Comp Biochem Physiol B 101:495–498[CrossRef][Medline]
  54. Graur D, Hide WA, Li WH 1991 Is the guinea-pig a rodent? Nature 351:649–52[CrossRef][Medline]



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