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Endocrinology Vol. 138, No. 12 5618-5629
Copyright © 1997 by The Endocrine Society

ARTICLES

A Second Form of Gonadotropin-Releasing Hormone (GnRH) with Characteristics of Chicken GnRH-II Is Present in the Primate Brain1

David W. Lescheid, Ei Terasawa, Laurie A. Abler, Henryk F. Urbanski, Carol M. Warby, Robert P. Millar and Nancy M. Sherwood

Department of Biology, University of Victoria (D.W.L., C.M.W., N.M.S.), Victoria, British Columbia, Canada V8W 2Y2; Wisconsin Regional Primate Research Center (E.T., L.A.A.), Madison, Wisconsin 53715-1261; Oregon Regional Primate Research Center (H.F.U.), Beaverton, Oregon 97006; and the Department of Chemical Pathology, University of Cape Town Medical School (R.P.M.), Cape Town, South Africa

Address all correspondence and requests for reprints to: Dr. Nancy Sherwood, Department of Biology, University of Victoria, Victoria, British Columbia, Canada V8W 2Y2. E-mail: nsherwoo{at}uvic.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The primate brain was thought to contain only the GnRH known as mammalian GnRH (mGnRH). This study investigates whether a second form of GnRH exists within the primate brain. We found that brain extracts from adult stumptail and rhesus monkeys contained two forms of GnRH that were similar to mGnRH and chicken GnRH-II (cGnRH-II) based on the elution position of the peptides from HPLC and on cross-reactivity with antisera that are specific to mammalian or chicken GnRH-II in RIAs. The fetal brain of rhesus monkeys also contained mGnRH and a cGnRH-II-like peptide by the same criteria.

Immunocytochemistry with a cGnRH-II-specific antiserum in adult and fetal rhesus monkeys showed immunopositive neurons generally scattered in the periaqueductal region of the midbrain, with a few positive cells in the posterior basal hypothalamus. Neurons immunopositive for cGnRH-II were fewer in number and smaller in size, with less defined nuclei and thinner neurites compared with those for mGnRH. Administration of synthetic cGnRH-II to adult rhesus monkeys resulted in a significant increase in the plasma LH concentration during the luteal phase of the menstrual cycle, but not during the midfollicular phase. We conclude that the primate brain contains mGnRH and a cGnRH-II-like molecule, although the function of the latter is unknown.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH, THE KEY brain peptide involved in the initiation of vertebrate reproduction, was first isolated and sequenced from mammals. At present, there are 12 distinct forms of GnRH, including the mammalian form, that have been sequenced from vertebrates or protochordates. All known forms are 10 amino acids in length, with a pyro-glutamyl-modified amino-terminus, an amidated carboxy-terminus, and conserved amino acids in positions 1, 2, 4, 9, and 10 (1, 2). The most common structural variation among the different forms of GnRH resides in amino acids 5–8. There is also a hydroxylated derivative of mammalian GnRH (mGnRH) in frog and rat brains, suggesting that posttranslational modification may provide structural diversity (3). In fish, amphibians, reptiles, and birds, there are two or more forms of GnRH within the brain of single species. Usually each form has a unique location within the brain, suggesting a difference in developmental origin and/or adult function (4, 5, 6).

The diversity of GnRH peptides within the brain of early evolved vertebrates is well established. However, in mammals, only primitive taxa, such as monotremes, marsupials, and primitive eutherians, have been shown to contain more than one form of GnRH within the brain of a single species (7, 8, 9). The identity of the GnRHs present in the brain is best known in primitive placental mammals, such as the musk shrew and tree shrew, in which the brain has been shown to contain mGnRH and chicken GnRH-II (cGnRH-II)-like molecules by one or more of the following techniques: HPLC, RIA (9, 10), immunocytochemistry (11), or complementary DNA sequencing (12). Despite these reports showing two forms of GnRH within the brains of some primitive placental mammals, we did not find evidence of cGnRH-II in several rodents (Lescheid, D. W., unpublished data). Also, there was no clear evidence that more recently evolved mammals, such as primates, had an additional form of GnRH, such as cGnRH-II, within their brains.

In this report we determined whether primate brains contain another form of GnRH. HPLC, RIA, and immunocytochemistry are used to characterize mGnRH-like and cGnRH-II-like immunoreactivity in the brains of adult stumptail monkeys (Macaca speciosa) as well as in adult and fetal rhesus monkey (Macaca mulatta) brains. The in vivo effect of the synthetic cGnRH-II-like peptide on the release of LH at different times in the reproductive cycle is also reported.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and tissue preparation
Stumptail (Macaca speciosa) and rhesus monkeys (Macaca mulatta), were born and raised at the University of Wisconsin Regional Primate Research Center (for immunocytochemical or chromatographic studies) or at the University of Oregon Regional Primate Center (for physiological studies). The animal protocols of these studies were reviewed and approved by the institutional animal care committee at the University of Wisconsin or the University of Oregon. All experiments in this study were conducted under the guidelines established by the NIH and the USDA.

For chromatographic studies, whole brains (one male at 19 yr; two females at 16 and 14 yr of age) were removed at necropsy from three adult stumptail monkeys. The brains (total mass, 297 g) were quickly frozen and stored at -80 C. Later, the brains were crushed using a cold mortar and pestle, powdered in a Waring blender with liquid nitrogen, and then extracted with an acetone-HCl mixture as previously described (13). Soluble lipids were removed by five consecutive applications of petroleum ether (20%, vol/vol) (14). The resultant acetone-water soluble mixture was reduced in volume in a vacuum centrifuge to approximately 3 ml and filtered through a 45-µm filter.

Fetal brains were removed from five rhesus monkey fetuses on 72–125 embryonic (E) days (two females at E75 and E125; three males at E72, E76, and E77). Fetuses were delivered by cesarian section. The brains were removed, quickly frozen, and stored at -80 C. Four of the brains (total mass, 18.7 g) were pooled (E72, E75, E76, and E77), crushed with a cold mortar and pestle, powdered in a Waring blender with liquid nitrogen, and treated as described above for the adult whole brains. At a later date, the brain (35.0 g) from the E125 fetus was prepared and treated in the manner described above.

A dissected brain was prepared from one adult stumptail monkey (male, 19 yr of age). The brain was collected, quickly frozen, and later partially thawed to allow sectioning into three pieces using a razor knife. First, the brain stem region (14.0 g) was removed with a single cut. Second, the diencephalic region (7.7 g; including the thalamus, hypothalamus, and pituitary stalk) was cut from the telencephalon. Third, the remaining brain tissue (68.0 g), including the telencephalon with frontal cortex, parietal lobes, temporal lobes, occipital lobes, and cerebellum, was combined. Each region was treated as described above, with the same extraction, HPLC, and RIA procedures as those used for the whole brain studies.

For immunocytochemistry, two adult female rhesus monkeys kept under the housing conditions described previously (15) and four rhesus monkey fetuses at E34, E50, E70, and E85 from time-mated pregnancies (16) were used in this study. Adult monkeys were anesthetized with ketamine and pentobarbital sodium (50 mg/kg), perfused with 4% paraformaldehyde in PBS (pH 7.6), and then immersed in the same solution for several hours. The brain stem was removed and placed in 30% sucrose in PBS for 2–3 days (15). Meninges were carefully removed under a stereomicroscope, and frozen serial sections were cut at 50 µm in the frontal plane.

Fetuses were delivered by cesarian section under halothane anesthesia. The whole body (fetus at E34) or brain (fetuses at E50 and E70) was immersed in 4% paraformaldehyde in PBS (pH 7.6) for 1–5 h (time varying with tissue size). The oldest fetus (E85) was perfused with 4% paraformaldehyde, and the brain was then immersed in the same solution for 4 h. None of these monkeys was alive after delivery. After fixation, the tissue was placed in 30% sucrose in PBS (pH 7.6) until fully saturated, as evidenced by sinking. Tissue was then frozen and sectioned by cryostat at 15 µm in either the sagittal plane (E34) or the frontal plane (E50, E70, and E85) and thaw-mounted onto gelatin-coated slides. Slides were stored at -20 C until immunostaining.

Six adult female rhesus macaques (6–9 years of age) were used for the in vivo study.

HPLC
A Beckman model 166 HPLC System Gold (Beckman, Palo Alto, CA), which included a solvent Module 125 and a UV detector Module 166, was used for HPLC analysis. For a blank run, two consecutive 600-µl volumes of Milli-Q (Millipore, Bedford, MA) water were loaded at 2-min intervals onto a Supelco C18 column (25.0 cm x 4.6 mm; 5-µm particle size; Supelcosil LC-18, Supelco Canada, Oakville, Canada) with a guard column of the same material attached. Sixty fractions of 1 ml each were collected in polyallomer tubes; 500 µl were removed from each fraction, vacuum dried, reconstituted in PBS with 0.1% gelatin, and assayed for immunoreactive GnRH (irGnRH). A blank run was repeated between each application of brain extract to ensure that the column was free of any contaminating residual GnRH from previous HPLC analyses. GnRH standards had never been previously injected onto this column, also ensuring that results were not affected by any residual synthetic GnRH peptides.

The injection volumes were 3 ml or less and were applied as multiple injections of up to 700 µl each. These repeated injections were at 2-min intervals onto a 1-ml loop; the material in the loop was loaded onto the column using an isocratic gradient of 17% solution A (0.25 M triethylammonium formate, pH 6.5) and 83% solution B (100% acetonitrile) over a 10-min period at a flow rate of 1 ml/min. After 10 min, the percentage of solution A was elevated to 24% (1%/min) and maintained there for an additional 43 min. Sixty 1-ml fractions were collected. A 100-µl aliquot was removed from each fraction, dried, and assayed for GnRH immunoreactivity. The initial 15 fractions were brought to pH 6.0–6.5 using 5 M NaOH before assaying for GnRH immunoreactivity, because previous experiments had shown that the low pH of the early eluting fractions affects the binding of some of the antisera used.

Synthetic standards were applied to the column after brain extracts were completed. Eleven GnRH forms: mGnRH, hydroxyproline9 (hydroxy-Pro9)mGnRH, seabream GnRH (sbGnRH), cGnRH-I, salmon GnRH (sGnRH), cGnRH-II, dogfish GnRH (dfGnRH), catfish GnRH (cfGnRH), lamprey GnRH-I (lGnRH-I), tunicate GnRH-I (tGnRH-I), and tGnRH-II were applied to the HPLC system as described for the brain extracts. The elution positions of the standards on the chromatograph were confirmed by absorbance peaks (A = 280 nm) and GnRH-specific RIA.

Cross-reactivity
The cross-reactivity of antisera GF-4, B-6, 7CR-10, and Adams-100 with synthetic GnRH peptides [mGnRH, (hydroxy-Pro9)mGnRH, sbGnRH, cGnRH-I, sGnRH, cGnRH-II, dfGnRH, cfGnRH, lGnRH-III, and lGnRH-I] is shown in Fig. 1Go. Antisera GF-4, B-6, and 7CR-10 were raised in rabbits in the Sherwood laboratory against sGnRH (GF-4), mGnRH (B-6), and dfGnRH (7CR-10). Antiserum Adams-100 (a gift from Dr. T. Adams, University of California-Davis) was raised in rabbits against cGnRH-II. Briefly, RIAs for GF-4 and B-6 (which cross-react with mGnRH) were homologous, using 125I-labeled mGnRH tracer and mGnRH standard. The Adams-100 RIA was also homologous, using 125I-labeled cGnRH-II tracer and cGnRH-II standard. The 7CR-10 RIA was heterologous using a 125I-labeled lGnRH-I trace and a cGnRH-II standard. Five concentrations of each synthetic peptide (100, 500, 1,000, 5,000, and 10,000 pg) were tested in triplicate for their ability to displace the tracer from the respective antiserum. Relative cross-reactivity is expressed as picomoles of the reference peptide at 50% bound/free ratio (B/B0) divided by picomoles of the test peptide at 50% B/B0 multiplied by 100.



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  		Figure 1. A, Percent cross-reactivity between antiserum GF-4 or B-6 and 10 different synthetic GnRH peptides. mGnRH was used as the reference peptide for both antisera in the calculation of the percent cross-reactivity. Relative cross reactivity (percentage) is expressed as the number of picomoles of reference peptide at 50% B/B0 divided by the number of picomoles of the other peptide at 50% B/B0. 125I-Labeled mGnRH was used as the tracer for the assays. Final concentrations of GF-4 and B-6 were 1:25,000 and 1:5,000, respectively. B, Percent cross-reactivity between antiserum 7CR-10 or Adams-100 and 12 different synthetic GnRH peptides and between antiserum 675 and 5 GnRH peptides. cGnRH-II was used as the reference peptide for the 3 antisera in calculation of the percent cross-reactivity. Relative cross-reactivity (percentage) is expressed as the number of picomoles of reference peptide at 50% B/B0 divided by the number of picomoles of the other peptide at 50% B/B0. 125I-Labeled lGnRH-I was used for assays with 7CR-10, whereas 125I-labeled cGnRH-II was used as the tracer for assays with Adams-100 and 675. Final concentrations of 7CR-10, Adams-100, and 675 were 1:37,500, 1:25,000, and 1:5,000, respectively. nd., Not done.

 
RIA
The details of the RIA were described previously (17). Essentially, 100-µl aliquots of each semipure fraction from the different HPLC runs were assayed for GnRH immunoreactivity using various antisera and 125I-labeled synthetic GnRH tracers in a competitive RIA. For each antiserum, the final dilution used in the RIA, the percent binding, and the limits of detection (B/B0 = 80%) were: GF-4, 1:25,000, 37%, and 16.0 pg; B-6, 1:5,000, 52%, and 9.8 pg; 7CR-10, 1:37,500, 18%, and 19.6 pg; and Adams-100, 1:25,000, 7%, and 1.1 pg. In fractions in which tracer binding was less than B/B0 = 20%, 50-µl aliquots were diluted serially, and the value closest to 50% B/B0 was used to estimate the quantity of irGnRH present.

Immunocytochemistry
Adult brain sections were immunostained with a free float method (15), whereas fetal brain sections were stained on the slide, as described previously (18). Endogenous peroxidase was deactivated in sections by washing with PBS (pH 7.6) for 1 h (four times, 15 min each time) and treating with 0.03% hydrogen peroxide in methanol solution. To further remove nonspecific background staining, sections were washed with PBS for 1 h (four times, 15 min each time) and then blocked with 0.5% normal goat serum in PBS for 2 h (twice, 60 min each time). The sections were then exposed to antisera specific to chicken GnRH-II, antiserum 675 at a 2,500 to 5,000 dilution (J. A. King and R. Millar), or Adams-100 at a 5,000 to 10,000 dilution. A small number of sections were also exposed to antiserum GF-6 at a 6,000 dilution or antiserum LR-1 at a 10,000 dilution (a gift from R. Benoit, University of Montreal, Montreal, Canada). The sections were then incubated at 0–4 C for 40 h. Sections were washed with PBS for 1 h (four times, 15 min each time) and exposed to the second antibody (biotinylated goat antirabbit IgG, Vector Laboratories, Burlingame, CA) for 1.5 h at room temperature. This was followed by a PBS wash (twice, 15 min each time) and exposure to avidin-biotin-peroxidase complex solution (ABC, Vector Laboratories) for 1.5 h. After a final wash with a 0.05 M Tris-buffered saline solution (pH 7.6; twice, 15 min each time), the final reaction product was visualized with a 3,3'-diaminobenzidine solution (0.5% 3,3'-diaminobenzidine with 0.06% hydrogen peroxide in 0.1 M Tris-buffered saline at pH 7.6). Adult brain sections were mounted on gelatin-coated slides. All sections were coverslipped with glycerol jelly.

For specificity testing, each antiserum was preabsorbed with mammalian and chicken GnRH-II at concentrations ranging from 1–200 µg/ml for 24 h before application. In addition, to eliminate the possibility of nonspecific reactions with the second antibody, the primary antibody was omitted in a few cases.

Physiology
The effects of cGnRH-II on LH release were examined in six adult female rhesus monkeys. The synthetic peptide (Peninsula Laboratories, Belmont, CA) was infused iv, either during the midfollicular phase of the menstrual cycle (day 6, on the average) or during the luteal phase (i.e. after the midcycle preovulatory surge). Blood samples (1 ml) were collected from conscious unrestrained animals using a remote blood sampling system (19). The assay procedure has been previously reported (20). The same animals had been used to determine the effect of mGnRH (50 ng/kg BW, iv) on plasma LH concentrations during the midfollicular and luteal phases of the menstrual cycle; these findings have been reported previously (19). Plasma LH levels were measured using a mouse Leydig cell bioassay, and the results were expressed in terms of the cynomolgus LH RP-I standard. The assay detection limit was 3 ng/ml, the intraassay coefficient of variation was 12%, and the interassay coefficient of variation was 20%. Effects of synthetic mGnRH (50 ng/kg) on LH release were similarly examined in the same monkeys for comparative purposes. At a later date, the in vivo cGnRH-II experiments were repeated exactly as described above, but using a 2000 ng/kg dose. Mean plasma LH concentrations were analyzed by ANOVA, followed by the Student-Newman-Keuls test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cross-reactivity of antisera
The cross-reactivity with 10 different GnRH peptides is shown for GF-4 and B-6 in Fig. 1AGo. The cross-reactivity for 7CR-10, Adams-100, and 675 is shown in Fig. 1BGo. It is clear that B-6 is specific for mGnRH (Fig. 1AGo); Adams-100, 7CR-10, and 675 are nearly specific for cGnRH-II (Fig. 1BGo); and GF-4 cross-reacts with many forms of GnRH, such as mGnRH, (hydroxy-Pro9)mGnRH, sbGnRH, cGnRH-I, sGnRH, cGnRH-II, dfGnRH, cfGnRH, and lGnRH-III.

Adult whole brain
With antisera that detect mGnRH (GF-4 and B-6), 22.2 and 21.7 ng immunoreactive GnRH, respectively, were detected in fraction 22 (Table 1Go and Fig. 2Go). Synthetic mGnRH standard eluted in position 22 under the same HPLC conditions (Fig. 2Go). With cGnRH-II-specific antisera (7CR-10 and Adams-100), 7.9 and 2.6 ng irGnRH, respectively, were detected (Table 1Go). Both antisera detected this immunoreactivity in HPLC elution positions 26 and 27 (Fig. 3Go). Synthetic cGnRH-II eluted in fraction 26 under the same conditions. The most specific antisera, B-6 (for mGnRH) and Adams-100 (for cGnRH-II), detected 73.1 and 8.8 pg irGnRH/g whole brain tissue, respectively. Thus, there was 8 times as much mGnRH as cGnRH-II at the time of brain collection as measured by these antisera.


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  		Table 1. Immunoreactive GnRH in primate brain

 


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  		Figure 2. irGnRH in adult stumptail monkey (male, 19 yr old; females, 16 and 14 yr old) brain extracts showing HPLC elution positions (minutes) and the amount of immunoreactivity (nanograms per fraction) detected by antiserum GF-4 or B-6. Small arrows above HPLC elution profiles represent the different forms of GnRH detected by that specific antiserum as well as where those synthetic forms would elute under the same HPLC conditions. m-Hyp9, (Hydroxyproline-9)mammalian GnRH; m, mGnRH; c-I, cGnRH-I; sb, sbGnRH; c-II, cGnRH-II; s, sGnRH.

 


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  		Figure 3. irGnRH in adult stumptail monkey (male, 19 yr old; females, 16 and 14 yr old) brain extracts showing HPLC elution positions (minutes) and the amount of immunoreactivity (nanograms per fraction) detected by antisera 7CR-10 and Adams-100. Small arrows above HPLC elution profiles represent the different forms of GnRH detected by that specific antiserum as well as where those synthetic forms elute under the same HPLC conditions. l-I, lGnRH-I; c-II, cGnRH-II; df, dfGnRH; s, sGnRH.

 
Our mammalian-specific antiserum, B-6, also showed GnRH immunoreactivity primarily in fraction 22 (Fig. 2Go). This immunoreactivity does not appear to be (hydroxy-Pro9)mGnRH, because B-7 (the next bleed from the same rabbit and an antibody with the same characteristics as B-6) has only 1% cross-reactivity with synthetic (hydroxy-Pro9)mGnRH compared with mGnRH.

Fetal whole brain
irGnRH (4.3 ng) was detected in fractions 20 and 21 using antiserum GF-4 (Fig. 4Go). The antiserum (B-7) that is more specific for mGnRH than GF-4 detected 3 ng mGnRH and 160 pg/g whole brain tissue (pooled samples of E72, E75, E76, and E77) in the same fractions. There was 160 pg irGnRH/g fetal brain tissue compared with 73.1 pg/g adult brain tissue as detected by B-7 or B-6. Analysis of another fetal brain (E125) also showed immunoreactivity in elution positions 20 and 21 as detected by GF-4 and B-7, confirming the presence of mGnRH (data not shown). There was much more irGnRH detected in this fetal brain extract (657.1 pg/g) than in the previous pooled fetal brain extracts (160.0 pg/g) or adult brain extracts (73.1 pg/g). Also, the cGnRH-II-specific antisera, 7CR-10 and Adams-100, detected immunoreactivity in positions 25 and 26 (Fig. 5Go). Adams-100 detected 0.45 ng cGnRH-II-like immunoreactivity, whereas 7CR-10 detected 0.37 ng. Therefore, there was 12.9 pg cGnRH-II/g fetal (E125) brain tissue as detected by Adams-100 antiserum. Compared with adult rhesus brains (8.8 pg cGnRH-II/g tissue), there was a 1.5-fold increase in the concentration of cGnRH-II-like immunoreactivity in fetal rhesus monkeys.



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  		Figure 4. Fetal rhesus monkey (female, E75; males, E72, E76, and E77) mGnRH HPLC showing elution positions (minutes) and amount of immunoreactivity (nanograms per fraction) detected by antisera GF-4 and B-7.

 


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  		Figure 5. cGnRH-II in fetal rhesus monkey (female, E125) brain extracts showing HPLC elution positions (minutes) and amount of immunoreactivity (nanograms per fraction) detected by antisera 7CR-10 and Adams-100.

 
Dissected adult brain
All three dissected parts of the brain diencephalon, forebrain-cortex-cerebellum, and brain stem contained GnRH-like immunoreactivity primarily in fractions 21 (Fig. 6Go) and 26 (7CR-10) or 26 and 27 (Adams-100) in the diencephalon (Fig. 7Go), corresponding to the elution positions of synthetic mGnRH and cGnRH-II, respectively. RIA analysis with antiserum GF-4 detected 19.1 ng irGnRH in the diencephalon, 0.8 ng irGnRH in the cortex region, and 1.4 ng irGnRH in the brain stem. The more specific mammalian antiserum, B-6, detected 4.7, 0.9, and 2.2 ng irGnRH/fraction in the same three brain regions, respectively. Fraction 26 contained 0.9, 0.5, and 0.7 ng irGnRH, as detected by the 7CR-10 antiserum and 2.2, 0.3, and 0.7 ng/fraction immunoreactivity as detected by antiserum Adams-100. Quantitatively, B-6 detected more irGnRH/g brain tissue (610.4 pg/g) in the diencephalon than in the cortex (13.2 pg/g) or in the brain stem (157.1 pg/g). These results suggest a relative abundance of mGnRH in the diencephalon. With Adams-100 we detected 285.7, 4.4, and 50.0 pg/g immunoreactivity in the diencephalon, cortex, and brain stem, respectively. Hence, immunoreactive mGnRH and ir-cGnRH-II are more concentrated in the diencephalon than in the brain stem or cortical regions.



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  		Figure 6. mGnRH in adult stumptail monkey (male, 19 yr old) brain extracts showing HPLC elution positions (minutes) and amount of immunoreactivity (nanograms per fraction) detected in the diencephalon, cortex, and brain stem regions, respectively, by antisera GF-4 and B-6.

 


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  		Figure 7. cGnRH-II in adult stumptail monkey (male, 19 yr old) brain extracts showing HPLC elution positions (minutes) and amount of immunoreactivity (nanograms per fraction) detected in the diencephalon, cortex, and brain stem regions by antisera 7CR-10 and Adams-100.

 
Immunocytochemistry
In the fetuses at stages E50, E70, and E85, cGnRH-II-positive cells were seen in the basal region of the posterior hypothalamus, median eminence, pituitary stalk, and periaqueductal regions in the midbrain (Fig. 8Go, A, C, D, F, G, and H), and cGnRH-II-positive fibers were observed in the basal hypothalamus and pituitary stalk (Fig. 8Go, A and C). In the adult brain stem, cGnRH-II-positive cells were distributed in the periaqueductal regions (Fig. 8Go, I and J). cGnRH-II-positive cells were found in the periventricular region of the posterior hypothalamus in the E34 fetus (not shown). Generally, cGnRH-II-positive cells (~17 x 10 µm) were round in shape and smaller in size (Fig. 8Go, A, C, F, G, and H) than mGnRH-positive cells (~22 x 10 µm), which had a fusiform shape (Fig. 8BGo). However, in the adult midbrain there were some large cGnRH-II cells (Fig. 8Go, I and J). Although thick neurites were commonly observed for mGnRH-positive cells (Fig. 8BGo), generally cGnRH-II positive cells possessed fine short neurites (Fig. 8Go, A, C, and I).



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  		Figure 8. Examples of immunostained cells for cGnRH-II, indicated by arrowheads (A, C, D, F, G, H, I, and J), or mGnRH, indicated by arrows (B). In the medial basal hypothalamus (A) and the pituitary stalk (C) of the monkey fetus at E85, cGnRH-II-immunopositive cells stained with antiserum 675 are seen. In the medial basal hypothalamus (B) of the adjacent section in the same monkey fetus, mGnRH-positive cells stained with antiserum GF-6 are also seen. The shape of mGnRH-positive cells is different from that of cGnRH-II-positive cells. cGnRH-II-positive cells stained with antiserum 675 (D) or antiserum Adams-100 (F, G, and H) are seen in the midbrain periaqueductal region of fetuses on E85 (D), E50 (F and G), and E70 (H). In the adjacent section of the fetus on E85 (E), there are no immunopositive cells with antiserum 675 when preabsorbed with cGnRH-II peptide. cGnRH-II-positive cells with antiserum 675 are seen in the midbrain of an adult monkey (I and J). Two immunopositive cells shown in I were magnified in J. cGnRH-II-immunopositive fibers, indicated by long double arrows, are also seen in the pituitary stalk (C) and periaqueductal region (I). AQ, Aqueduct; STK, pituitary stalk. Scale bars: A, B, C, D, E, F, G, and J, 20 µm; I, 40 µm; and J, 10 µm.

 
Absorption tests indicated that tissues exposed to antiserum 675, which had been pretreated with cGnRH-II peptide (2 µg/ml for 24 h), had no immunostained cells (Fig. 8EGo). However, if the tissues were exposed to antiserum Adams-100, which had been pretreated with the cGnRH-II peptide (5- 200 µg/ml for 24 h), cGnRH-II-positive cells were still present (not shown). Preabsorption of both antisera 675 and Adams-100 with mGnRH did not alter the immunoreactivity.

Physiology
The iv administration of cGnRH-II (50 ng/kg BW) to rhesus macaques during the luteal phase of the menstrual cycle resulted in a marked increase (P < 0.01) in plasma LH concentrations, which was sustained (P < 0.05) for at least 40 min (Fig. 9Go). In contrast, the same dose of cGnRH-II failed to induce a significant increase (P > 0.05) in LH during the midfollicular phase of the cycle. This finding is similar to that previously reported for differential effects of mGnRH on LH during the macaque’s menstrual cycle (19). A higher dose of cGnRH-II (2 µg/kg BW) during the midfollicular phase resulted in a higher plasma LH concentration (P < 0.05) than that after administration of 50 ng/kg BW, although the LH levels were still significantly lower than those induced by 50 ng/kg BW cGnRH-II during the luteal phase (P < 0.01).



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  		Figure 9. Effect of iv administration of cGnRH-II (at time zero) on plasma LH concentrations in adult female rhesus macaques. Serial blood samples were collected from each animal during either the midfollicular or luteal phase of the menstrual cycle (lower and upper panels, respectively). A significant LH increase was detected 10 min after administration of cGnRH-II (50 ng/kg BW; solid symbols) during the luteal phase (P < 0.01), but not during the midfollicular phase (P > 0.05); in the latter case, however, a higher dose of cGnRH-II (2 µg/kg BW; open symbols) resulted in a significantly greater increase (P < 0.05). Each point represents the mean LH concentration from six animals, and the SEMs are shown as vertical lines.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is the first report that a second form of GnRH, with characteristics of cGnRH-II, is present within the primate brain. The HPLC elution position as well as cross-reactivity with specific cGnRH-II antiserum in RIA and immunocytochemistry suggest that the primate brain contains both mGnRH and a cGnRH-II-like molecule.

cGnRH-II is conserved from jawed fish to primates
The cGnRH-II form has been conserved in a variety of vertebrates, from the first jawed fish to primitive placental mammals. The primary structure for cGnRH-II has been obtained from chicken, alligator, frog, sea bream, tilapia, pacu, catfish, herring, dogfish, and ratfish (1, 2). In addition, the sequence of the complementary DNA that encodes cGnRH-II is reported for several fish (21, 22, 23) and the tree shrew (11). The only exception for jawed vertebrates to date is rodents; brain extracts from rats, hamsters, and guinea pigs did not have detectable cGnRH-II in our HPLC-RIA analysis (Lescheid, D. W., unpublished data). Thus, the evidence confirms the presence of cGnRH-II in representative members of the major vertebrate taxa, except for jawless fish (17, 24) and possibly rodents. Isolation of the cGnRH-II peptide from monkey brains and determination of the primary structure require more tissue than is available.

The combination of cGnRH-II and mGnRH in the brain of one species is not unique to primates, but has been detected for a variety of vertebrates. For example, mGnRH-like and cGnRH-II-like immunoreactivity in the brain has been shown in primitive bony fish, such as sturgeon (25, 26), reedfish, and alligator gar (27); teleosts, such as eels (28); tetrapod ancestors, such as lungfish (29); amphibians, such as Xenopus (30) and ranid frogs (31, 32); and primitive placental mammals, such as the musk shrew (10).

cGnRH-II neurons in primates are conserved in location
We have shown also, through HPLC with RIA, that cGnRH-II-like and mGnRH-like immunoreactivity exist throughout the brain of stumptail and rhesus monkeys. Thus, primates are like other vertebrates in that cGnRH-II is distributed throughout the brain due to the wide distribution of axons. Furthermore, through immunocytochemistry we have shown that there are some cGnRH-II-like immuno-positive cells and fibers in the basal hypothalamus, in close proximity to the hypophysial portal system. However, the relative scarcity of cGnRH-II-like immunopositive cells and fibers in the basal hypothalamus compared with mGnRH-immunopositive cells suggests that mGnRH functions as the primary gonadotropin releaser.

It is surprising that cGnRH-II has not been detected previously in vertebrates such as sheep and humans (33). Differences in methodology, such as specificity and sensitivity of cGnRH-II antisera or use of primate whole brain rather than just diencephalon, may have contributed to the detection of this form. Alternatively, cGnRH-II may have a relatively small window of maximal expression, and therefore, the timing of tissue collection may be crucial in its detection. Another technical problem is that our two cGnRH-II-specific antisera stained cells in the midbrain, but one (Adams-100) was not blocked by the cGnRH-II peptide, whereas the other (antiserum 675) was blocked. Specific cGnRH-II staining by antiserum 675 for the hypothalamus, pituitary stalk, and midbrain is shown in Fig. 8Go, A, C, D, E, I, and J.

The present study suggests that the GnRH systems in primates are not dissimilar from those in other vertebrates; i.e. there is 1) an anterior system of neurons that express one GnRH form (e.g. mGnRH, sGnRH, cfGnRH, sbGnRH, or cGnRH-I) with fibers projecting to the pituitary or median eminence for LH and FSH release, and 2) a posterior system of neurons that express cGnRH-II, with fibers projecting throughout the brain, including the hindbrain, posterior pituitary, and spinal cord. In the musk shrew, cGnRH-II neurons are localized in a discrete cluster within the midbrain. Most of the fibers appear to end in the medial habenula region, but some are widely scattered in the forebrain as well as in the median eminence, arcuate nucleus, and infundibular stem (11). In contrast, mGnRH is found primarily in the forebrain (10).

cGnRH-II is expressed early in development in primates
In the present study, cGnRH-II was detected as early as E125 by specific antisera with HPLC and as early as E34 by immunocytochemistry. There was insufficient material from the E72–E77 brains for testing with Adams-100 or 7CR-10 antisera. This timing of the origin of cGnRH-II-like cells is as early as that of the two populations of anterior mGnRH cells that migrate from the olfactory placode into the telencephalon and diencephalon in rhesus monkeys (18). The earliest migrating cells containing GnRH were detected in the telencephalon of the rhesus monkey as early as E30; GnRH in these cells is detected only by GF-6. A recent preliminary study indicates that these cells contain fragments of GnRH (34). These neurons ultimately settled in extrahypothalamic regions, such as the lateral septum, stria terminalis, amygdala, internal capsule, and claustrum. The second type of cells containing mGnRH were detected by several antisera, including GF-6; these cells originated at E32–36 and migrated into the forebrain at E38–42, about 1–2 weeks after the first type of cells. These late-migrating mGnRH neurons settle in the medial septum, preoptic area, and medial basal hypothalamus. It is clear that neither of these forebrain cells, which originate in the olfactory placode, contain cGnRH-II. At present, the origin of midbrain cells containing cGnRH-II is unknown. One possibility is that the cGnRH-II neurons originate in the ventricular wall of the posterior hypothalamus and the midhindbrain area, and migrate only a short distance.

The function of cGnRH-II in all vertebrates remains an enigma
The fact that cGnRH-II has been conserved in species separated by a long time in evolution suggests that it may have important functional significance. We have shown that a low dose of synthetic cGnRH-II stimulated LH release in adult rhesus monkeys when administered during the midluteal phase. There was no significant increase in plasma LH when the injection took place during the midfollicular phase, unless a high dose (40-fold) of cGnRH-II was used. This observation is, however, not different from the results with mGnRH. Administration of mGnRH at the same dose to the same monkeys showed a differential pattern of LH release between the luteal phase and the midfollicular phase of the menstrual cycle (19).

These data accord with high binding affinity of cGnRH-II for the GnRH receptor in humans (35) as well as in nonprimate mammals. Other studies also show that cGnRH-II is effective in releasing LH, FSH, or gonadotropins (1, 5, 36, 37, 38), but it is not clear whether the fibers containing cGnRH-II reach the portal vessels or pituitary in all vertebrates. Hence, cGnRH-II may have a different physiological function. Indeed, cGnRH-II administered to chickens was effective in releasing LH and FSH, but blockage of the peptide with antisera in vivo did not alter the reproductive cycle (36). The location of cGnRH-II in the midhindbrain is one of the few clues about its possible functions. Despite these studies showing that more than one form of GnRH can stimulate gonadotropin release from the mammalian pituitary, mGnRH is usually more effective (1, 5, 38, 39). A unique cGnRH-II receptor has not been isolated and sequenced in any vertebrate to date; the possibility remains that a single receptor type may exist that binds both mGnRH and cGnRH-II. King et al. (40) have presented evidence that a single receptor type binds both cGnRH-I and cGnRH-II in chicken pituitaries.

The presence of a few cGnRH-II neurons in the basal hypothalamus of the rhesus monkey suggests that cGnRH-II might play a minor hypophysiotropic role, aiding in the stimulus of LH synthesis and/or release during certain developmental periods or reproductive states. Alternatively, the presence of cGnRH-II in this brain area might not affect LH or FSH release, but may function in the control of reproductive behavior (5). Other vertebrate studies suggest that cGnRH-II is a neuromodulator. This hypothesis is strengthened by evidence that cGnRH-II binds to high affinity sites in bullfrog sympathetic ganglia membranes, resulting in altered potassium currents (41) or in late, slow postsynaptic excitatory potentials (42, 43). Behavioral studies show that injection of GnRH into the midbrain resulted in enhanced lordosis and female receptivity in rats (44, 45, 46, 47) and turtle doves. Intraperitoneal injection of cGnRH-II into a reptile, Iguana iguana, altered plasma steroid levels and female sexual receptivity (48). Furthermore, irGnRH cells in the midbrain of the sting ray, Urolophus halleri, project to motor neurons associated with the clasper appendage that is involved in mating behavior (49).

The question of the presence of multiple forms of GnRH within the vertebrate brain is important for our understanding of the control of reproductive physiology and behavior. The discovery of a cGnRH-II-like substance in the brain of adult and fetal monkeys suggests that an anterior and posterior GnRH system exists within the brains of recently diverged vertebrates, possibly even in humans. Each of the multiple forms of GnRH may be involved in the control of reproductive physiology and behavior, or some of the GnRH forms may have a function unrelated to reproduction.


   Acknowledgments
 
We thank Dr. Tom Adams (University of California-Davis) for his generous donation of antibody, as well as Drs. J. F. F. Powell and Cameron Quanbeck for performing some preliminary HPLC/RIA and immunocytochemistry work, respectively.


   Footnotes
 
1 This work was supported by the Canadian Medical Research Council (to N.M.S.); NIH Grants RR-00167, HD-15433, and HD-11355 (to E.T.); NIH Grants RR-00163, HD-29186, and HD-24312 (to H.F.U.); and the South African Medical Research Council, NIH Foundation for Research Development, and the University of Cape Town (to R.P.M.). Back

Received August 1, 1997.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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