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Original Studies |
Division of Neuroscience, Oregon Regional Primate Research Center (V.S.L., S.G.K., V.T.G., H.F.U.), Beaverton, Oregon 97006; and Department of Physiology and Pharmacology, Oregon Health Sciences University (H.F.U.), Portland, Oregon 97201
Address all correspondence and requests for reprints to: Dr. Henryk F. Urbanski, Division of Neuroscience, Oregon Regional Primate Research Center, 505 NW 185th Avenue, Beaverton, Oregon 97006. E-mail: urbanski{at}ohsu.edu
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Abstract |
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Introduction |
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It is already well established that GnRH-I plays a crucial role in controlling reproductive function in humans, as underscored by the observation that GnRH-I deficiency in Kallmann’s syndrome is associated with the absence of puberty and infertility (22, 23). Although the exact trigger for the onset of puberty is unknown, it is generally assumed that a change in the pulsatile release pattern of GnRH-I plays a major role. However, the neurons that produce GnRH-I have a diffuse distribution pattern (24), and so it is enigmatic how they coordinate their secretory activity to generate a well defined pulsatile GnRH release pattern. In addition, there is little evidence to support the view that puberty in the primate is associated with an increase in the expression of GnRH-I mRNA (20, 25, 26) or peptide content (27) in the hypothalamus.
Interestingly, GnRH-II mRNA and peptide are also expressed in the primate hypothalamus (18, 21), and like GnRH-I, GnRH-II has been shown to be a potent stimulator of LH release in vivo (17). Therefore, it is plausible that GnRH-II may somehow contribute to the neuroendocrine control of the reproductive axis of primates, especially during development. To help resolve this issue, the present study used in situ hybridization histochemistry to examine the expression of GnRH-II mRNA in the hypothalami of male and female rhesus monkeys either before or after the onset of puberty (at 0.6 or 10–15 yr of age, respectively). Preliminary findings from this study have been published in abstract form (28).
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Materials and Methods |
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This study was approved by the institutional animal care and use committee at the Oregon Regional Primate Research Center (ORPRC) and involved six male and six female rhesus macaques (Macaca mulatta); half of the animals from each sex were sexually immature (0.6 yr old), and half were adults (10–15 yr old). Based on detailed menstruation records, one of the three adult females was in the early follicular phase of the menstrual cycle, one was in the late follicular phase, and one was in the luteal phase. The immature animals had all been weaned several months before use. Animal care was provided by the ORPRC in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and the euthanasia was performed as part of the ORPRC Tissue Distribution Program to provide tissue for this and other studies.
Tissue preparation
The animals were deeply anesthetized using ketamine/pentobarbital, according to procedures established by the Panel on Euthanasia of the American Veterinary Society. Their brains were fixed by perfusing 1 L 0.9% saline through the ascending aorta followed by 6.5 L ice-cold 4% paraformaldehyde in 0.1 mol/L phosphate-buffered (pH 7.6) saline (0.9%, wt/vol). Hypothalami were blocked just rostral to the optic chiasm and rostral to the mammillary bodies. They were then immersed in fresh fixative for an additional 3 h (at 4 C) and cryoprotected, as previously described (29). This involved their immersion in 0.02 mol/L phosphate buffer (pH 7.4) containing glycerol (10%, vol/vol) and dimethylsulfoxide (2% vol/vol) for 24 h, followed by immersion in a more concentrated glycerol (20%) phosphate/dimethylsulfoxide solution for an additional 72 h. The tissue blocks were rapidly frozen in 2-methyl butane (precooled in an ethanol/dry-ice bath) and stored at -85 C. Subsequently, they were sectioned (25 µm) using a sliding microtome and mounted on glass microscope slides (Fisherbrand SuperFrost/Plus, Fisher Scientific, Auburn, WA). After being air-dried for 30 min and vacuum-dried overnight, the mounted sections were stored at -85 C for later use.
Complementary RNA probe synthesis and in situ hybridization histochemistry
A 35S-labeled 430-nucleotide antisense riboprobe was transcribed from rhesus monkey complementary DNA that encodes the GnRH-II/GnRH-associated-peptide precursor. Because the GnRH-associated-peptide-coding regions of the GnRH-I and GnRH-II precursors are unique (18, 20), this probe specifically identifies only those cells that express GnRH-II mRNA (18).
In situ hybridization was performed on a series of coronal hypothalamic sections from each animal, collected at approximately 200-µm intervals. First, the sections were postfixed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4) for 15 min, rinsed in Tris-ethylenediamine tetraacetate (EDTA), and then digested with proteinase K (10 µg/mL) in Tris-EDTA buffer (pH 8.0; 100 mmol/L Tris and 50 mmol/L EDTA) for 30 min. Next, they were acetylated, dehydrated with ascending concentrations of ethanol, and dried under vacuum for 2 h. They were then hybridized for 18 h at 65 C with 100 µL 35S-labeled antisense riboprobe diluted to 1 x 107 cpm/mL hybridization buffer [50 mmol/L dithiothreitol, 250 µg/ml transfer RNA, 50% formamide, 0.3 mol/L sodium chloride, 1 x Denhardt’s solution, 20 mmol/L Tris (pH 8.0), 1 mmol/L EDTA, and 10% dextran sulfate]. For the hybridization, glass coverslips were affixed to the slides using DPX mounting medium (BDH Laboratory Supplies, Poole, UK). The posthybridization procedure involved removing the coverslips, after two 30-min soakings in 4 x SSC (saline-sodium citrate buffer; the 20 x stock SSC solution comprised 175.3 g sodium chloride and 88.2 g sodium citrate/L, pH 7.0) containing 20 mmol/L dithiothreitol. The sections were then incubated in Tris-EDTA buffer (pH 8.0; 10 mmol/L Tris, 1 mmol/L EDTA, and 0.5 mol/L sodium chloride) containing ribonuclease A (10 µg/mL) for 30 min at 37 C, followed by two 30-min washes at room temperature with 2 x SSC containing 1 mmol/L dithiothreitol. After a final 30-min wash at 70 C with 0.1 x SSC containing 1 mmol/L dithiothreitol, they were dehydrated through ascending concentrations of ethanol containing 0.3 mol/L ammonium acetate and then air-dried for 30 min. To visualize the hybridization pattern the sections were apposed to Hyperfilm ß-max (Amersham Pharmacia Biotech, Piscataway, NJ) for 6 days (i.e. an exposure period that maintained the hybridization signal in the linear response range of the film).
The regional hybridization pattern of the monkey GnRH-II riboprobe has previously been shown to be distinct from that of the monkey GnRH-I riboprobe (18, 21), thus emphasizing its specificity. As a negative control, in situ hybridization was also performed on a few sections using a 35S-labeled sense riboprobe.
For quantitation, the autoradiographs were uniformly transilluminated (Northern Light, Imaging Research, Inc., St. Catherines, Canada), and the images were captured using a Sony CCD camera (model XC-77, Sony Corp. of America, Cypress, CA) equipped with a 50-mm macro-lens. They were then digitized using a frame grabber (Data Translation, Marlboro, MA) installed in a Macintosh Power PC computer (Apple Computer Inc., Cupertino, CA) and analyzed using the NIH Image program. Specific hypothalamic areas were defined using the freehand outlining tool, and the mean optical density was measured after correcting for background noise. For each animal, the mean optical densities from different autoradiographs were averaged and then combined according to age and sex to give an overall group mean (±SEM; n = 3). Two-way ANOVA was used to assess statistical differences in regional GnRH-II mRNA expression between the sexes and also between the immature and adult animals. Because no sex differences were detected, the data from the males and females were pooled (i.e. n = 6/age group) and further analyzed by one-way ANOVA.
To further quantitate the expression of GnRH-II mRNA in the different groups, the hypothalamic sections were subsequently dehydrated using increasing concentrations of ethanol, defatted in xylenes for 1 h, and dipped in photographic emulsion (NTB-2, Eastman Kodak Co., Rochester, NY). They were exposed at 4 C in a light-tight box for 12 days and then processed with Kodak developer (D-19) and fixer, dehydrated with ethanol, cleared with xylenes, and finally coverslipped using DPX mounting medium. Silver grain density was examined under a microscope using a x40 objective lens. Only cells that had an obvious round or fusiform silver grain deposition pattern were counted and analyzed. The images were digitized, as described above, and the NIH Image program was then used to determine the silver grain density per cell (expressed as pixels per cell) and to determine the total number of positive cells per section. Again, for each animal the mean silver grain densities were averaged and then combined according to age and sex to give an overall group mean (±SEM; n = 3). Two-way ANOVA was used to assess statistical differences in regional GnRH-II mRNA expression between the sexes and also between the immature and adult animals. Because no sex differences were detected, the data from the males and females were pooled (i.e. n = 6/age group) and further analyzed by one-way ANOVA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The distribution of GnRH-II mRNA in specific regions of the rhesus
macaque hypothalamus is shown in Fig. 1
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High levels of expression were detected in the supraoptic nucleus
(SON), suprachiasmatic nucleus (SCN), paraventricular nucleus (PVN),
and also the mediobasal hypothalamus (MBH). The hybridization signal
showed tight clustering in the SON, SCN, and PVN, but was more diffuse
in the MBH. No hybridization was seen when in situ
hybridization was performed using a GnRH-II sense probe (data not
shown).
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To analyze sex and age differences in GnRH-II gene expression in
these hypothalamic regions the autoradiographic images were digitized,
and mean optical densities were determined for the SON, PVN, and MBH
(note, the SCN was not analyzed because only one or two sections from
each animal included this nucleus). In all of the hypothalamic regions
examined the level of GnRH-II mRNA expression was similar in the males
and females (P > 0.05), so the data from the two sexes
were pooled for the developmental analysis. In adults, the overall
level of GnRH-II mRNA expression appeared to be greater than that in
the immature animals, but the difference was statistically significant
(P < 0.05) only in the MBH (Fig. 2).
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Based on the autoradiographs alone it was unclear whether the
intense hybridization in the MBH of adults reflected an increase in
GnRH-II mRNA expression per cell or an increase in the number of cells
expressing GnRH-II mRNA. To resolve this issue, the same hybridized
sections were dipped in photographic emulsion, and the resulting silver
grain deposition patterns were analyzed microscopically (Fig. 3). A total of 1656 GnRH-II cells were
identified in hypothalamic sections from the 6 males, and 948 were
identified in hypothalamic sections from the six females. Regardless of
the animal’s age, the number of silver grains per cell was found to be
similar in each of the hypothalamic regions examined (P
> 0.05; Fig. 4A). In contrast, the
number of MBH cells expressing detectable quantities of GnRH-II mRNA
was significantly (P < 0.001) greater in the adults
than in the immature animals (Fig. 4B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the pivotal involvement of GnRH-I in the control of the primate reproductive axis is well established, it is unclear whether GnRH-II also plays a modulatory role. On the one hand, GnRH-II mRNA is highly expressed in the primate hypothalamus (18), and GnRH-II has been shown to be a potent stimulator of LH release in vivo (17). On the other hand, it is currently unknown whether GnRH-II neurons project to the median eminence to secrete the decapaptide into the hypothalamo-hypophyseal portal blood vessels. Such a humoral route to the pituitary gonadotropes (30) is certainly plausible given the high level of GnRH-II mRNA expression observed in the adult. However, it is also possible that the posterior lobe of the pituitary gland secretes some GnRH-II directly into the peripheral circulation. Although speculative, this view gains support from the observation that very high levels of GnRH-II mRNA were detected in the SON and PVN, two hypothalamic nuclei that have major projections to the posterior pituitary gland (31, 32). The primary neuropeptides produced by these nuclei include arginine vasopressin and oxytocin, which resemble GnRH-II in many ways. For example, the biosynthesis of each of these peptides involves amidation of the terminal glycine residue at the carboxyl end of the molecule by the donation of an amide group from an adjacent glycine residue. Furthermore, each peptide’s precursor contains an associated peptide that is cleaved off at a lysine-arginine processing site (19, 31). Taken together, these peptide similarities emphasize that some of the neurons in the SON and PVN share the same biochemical machinery to produce arginine vasopressin, oxytocin, and GnRH-II; these magnocellular neurons provide the main axonal projections from the SON and PVN to the posterior pituitary gland (32). Although this issue requires further investigation, the current findings raise the possibility that GnRH-II may contribute nontraditionally to the control of reproductive function or may play an important nonreproductive neuroendocrine role.
Although the level of GnRH-II mRNA expression in the SON and PVN was similar in immature and adult animals, it showed a marked developmental increase in the MBH. This increase appeared to stem from an increase in the total number of cells expressing GnRH-II mRNA rather than from an increase in the level of expression per cell. Although we cannot rule out the possibility that the number of GnRH-II cells in the MBH actually increases during postnatal development, it is more likely that the induction of the GnRH gene within these cells is developmentally regulated.
Possibly, the earliest manifestations of pubertal onset in primates is
a change in the pulsatile release pattern of LH, which is highlighted
by high amplitude nocturnal peaks (33). To date, the
underlying cause of this neuroendocrine activation is unclear, although
a pubertal change in the pulsatile release pattern of GnRH almost
certainly plays a pivotal role (34). Moreover, there is
evidence to indicate that the pubertal activation of LH secretion
following an extended juvenile hiatus is associated with increased
glutamatergic stimulation (33, 35) and/or reduced
-aminobutyric acidergic inhibition (36, 37, 38) of the GnRH
neuronal circuitry. It is possible that developmental changes in the
expression of neuropeptide Y within the MBH are also involved
(25, 39, 40, 41). Because enhanced GnRH release is maintained
after puberty, one might expect an underlying increase in GnRH gene
expression to also become prominent during prepubertal development.
Surprisingly, however, there is little evidence to support a
developmental increase in the expression of GnRH-I mRNA (20, 26) or GnRH-I peptide (27) in the hypothalamus of
primates, although a developmental increase in GnRH-I mRNA expression
has been observed recently in the MBH of gonadectomized monkeys
(25). In contrast, the present findings show that a
developmental increase in GnRH-II mRNA levels is prominent in the MBH
even in gonad-intact animals in both males and females. More
comprehensive studies are needed to determine whether a developmental
increase in GnRH-II expression is associated with the central mechanism
that triggers the onset of puberty, or whether it simply reflects a
consequence of the maturational change in the sex steroid
environment.
The reproductive significance of highly elevated GnRH-II mRNA expression in the hypothalamus of adult primates is unclear, because many human reproductive disorders, such as Kallmann’s syndrome (22, 23), can be attributed to specific perturbation of the GnRH-I system and can be effectively treated by intermittent stimulation of the pituitary-gonadal axis with GnRH-I alone. Nevertheless, such findings do not exclude the possibility that GnRH-II also plays a crucial role in normal reproductive function. The present findings are consistent with the idea that GnRH-II may contribute to the physiological development of the primate reproductive axis, possibly by helping to synchronize or modulate the pulsatile release of GnRH-I; gap junctions are known to exist between cells within the SON and PVN, suggesting that the secretory activity of GnRH-II cells with perikarya in these nuclei may be intrinsically more coordinated than that of the more diffuse GnRH-I cells, and that these GnRH-II cells represent an important component of the hypothesized GnRH pulse generator. It is also possible that GnRH-II neurons play an important role in mediating feedback from the developing gonads to the hypothalamus and in augmenting the central stimulus for the preovulatory gonadotropin surge. To substantiate these possible reproductive roles for GnRH-II, additional experiments need to be performed in which either the synthesis of GnRH-II is suppressed (e.g. using antisense GnRH-II oligodeoxnucleotides) or its action is blocked (e.g. by immunoneutralization).
In summary, the results from this study show that the hypothalamus of both male and female rhesus macaques highly expresses GnRH-II mRNA in discrete regions, including the SON, SCN, PVN, and MBH. Furthermore, this expression appears to be developmentally regulated within the MBH. Although GnRH-II is expressed by a population of neurons completely distinct from that which expresses GnRH-I (21), it has been shown that GnRH-II can stimulate LH release in vivo (17). Taken together, therefore, these findings give credence to the view that reproductive function in higher primates may be regulated by more than one GnRH neuronal system. Moreover, because the reproductive axis of the rhesus macaque closely resembles that of the human, the data shed new light on the possible etiology of idiopathic human reproductive disorders.
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Footnotes |
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Received November 5, 1999.
Revised March 13, 2000.
Revised June 7, 2000.
Accepted September 10, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and its receptor in the hypothalamus of female rhesus
macaques. Neuroendocrinology. 60:346–359.[Medline]
-amino butyric acid (GABA) and glutamate in control of
puberty in female rhesus monkeys: effect of an antisense
oligodeoxynucleotide for GAD67 messenger ribonucleic acid and MK801 on
luteinizing hormone-releasing hormone release. Endocrinology. 140:705–712.-Aminobutyric acid is an inhibitory neurotransmitter restricting the
release of luteinizing hormone-releasing hormone before the onset of
puberty. Proc Natl Acad Sci USA. 91:395–399.This article has been cited by other articles:
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 J. D. Veldhuis, J. N. Roemmich, E. J. Richmond, and C. Y. Bowers Somatotropic and Gonadotropic Axes Linkages in Infancy, Childhood, and the Puberty-Adult Transition Endocr. Rev., April 1, 2006; 27(2): 101 - 140. [Abstract] [Full Text] [PDF]
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 T. M. Plant and M. L. Barker-Gibb Neurobiological mechanisms of puberty in higher primates Hum. Reprod. Update, January 1, 2004; 10(1): 67 - 77. [Abstract] [Full Text] [PDF]
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K. Morgan, D. Conklin, A. J. Pawson, R. Sellar, T. R. Ott, and R. P. Millar A Transcriptionally Active Human Type II Gonadotropin-Releasing Hormone Receptor Gene Homolog Overlaps Two Genes in the Antisense Orientation on Chromosome 1q.12 Endocrinology, February 1, 2003; 144(2): 423 - 436. [Abstract] [Full Text] [PDF]
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