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Program in Neuroscience (J.L.T., E.F.R) and Department of Biochemistry and Molecular Genetics (E.F.R.), University of Virginia Medical School, Charlottesville, Virginia 22908; and Medical Research Council Human Reproductive Sciences Unit (R.P.M.), Edinburgh EH3 9ET, Scotland, United Kingdom
Address all correspondence and requests for reprints to: Dr. E. F. Rissman, Department of Biochemistry and Molecular Genetics, University of Virginia Medical School, P.O. Box 800733, 1300 Jefferson Park, Charlottesville, Virginia 22908. E-mail: rissman{at}virginia.edu.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Almost 10 yr ago, GnRH-II was discovered in musk shrew (Suncus murinus) brain making this species the first placental mammal shown to have two forms of GnRH including GnRH-I and GnRH-II (13). Since that time, several other primitive mammals (tree shrews and golden moles), as well as several primates (humans, rhesus monkeys, and marmosets), have been examined either by in situ hybridization, HPLC, or immunocytochemistry, and GnRH-II has been located in brain tissues (1, 8, 14, 15, 16, 17). Although early studies indicated that GnRH-II was not present in rodent brain (18), recently it has been isolated in the midbrain of capybara and mouse (19, 20). These data remain controversial, however, as the GnRH-II peptide gene is not present in the completed mouse genome. When GnRH-II was first discovered, there was some speculation that this form of the peptide could have specific gonadotropin-releasing characteristics. However, few data support a primarily physiological role of GnRH-II in release of either FSH or LH (21). Once the anatomy of the neurons and their projections were defined, it was hypothesized that GnRH-II may be involved in regulation of behavior (1, 2, 12, 22). In several species of birds, sexual behavior in females is enhanced, to a modest degree, by infusion of GnRH-II (1, 23). In female musk shrews, however, intraventricular infusion of GnRH-II has no facilitatory effect on sexual receptivity (24).
In previous studies, we have shown that a mild 48-h food restriction blocks sexual receptivity in female musk shrews. However, 90 min of ad libitum (AL) food reinstates sexual behavior (25). Several lines of evidence lead us to hypothesize that this behavioral activation may be caused by one of the two GnRH peptides. Notably, others have shown that GnRH-I can stimulate sexual behavior in a variety of species from newts to rats (26, 27, 28, 29). To investigate this hypothesis, we administered saline, or GnRH-II to AL-fed and foodrestricted (FR) females. Next we asked whether the reinstatement of sexual receptivity we noted in FR females treated with GnRH-II was specific by repeating the study and administering GnRH-I, GnRH-II, or saline to FR females only. We found again that GnRH-II had a facilitatory effect, and GnRH-I had no effect on mating behavior. To ask whether endogenous concentrations of GnRH-II vary with nutritional condition, we used immunocytochemistry and analyzed both fiber density and cell numbers in brains of AL-fed and FR females. Finally, we used immunocytochemistry to determine whether the specific type II GnRH receptor was present in musk shrew brain. Our data show that GnRH-II, but not GnRH-I, activates mating in energetically challenged musk shrews, that immunoreactivity for GnRH-II cell bodies and fibers varies with nutritional status, and that this species has the GnRH-II selective type II GnRH receptors in brain.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Food restriction
Between the ages of 30 and 50 d, females with average body weights (between 19 and 27 g) were monitored for food intake daily for 4 d. To monitor food intake, the shrews received preweighed cat chow in clean small food cups between 1200 and 1400 h, at the same time body weights were recorded. Twenty-four hours later, the residual food was retrieved from the bowl as well as from the bedding on the floor of the cage and weighed. The average daily intake over the 4-d period was determined for each animal. Females were randomly assigned to one of two groups. The animals in the FR group received 60% of their average AL intake for the next 48 h. Females in the AL group continued to be fed as described above. These methods have been described previously (25). The University of Virginia Committee on Animal Care and use has approved all procedures in these experiments.
Surgical procedures
To implant intraventricular cannula, females between 35 and 55 d of age were anesthetized with sodium pentobarbital (4.5 mg/kg; 0.1 ml/10 g body weight). The animal was placed into the stereotaxic apparatus (Kopf Instruments, Tujunga, CA). The guide cannulae (33 gauge, 2.2 mm; Plastics One, Roanoke, VA) were aimed at the lateral ventricle by centering on the intersection of the sagittal suture and the most caudal portion of the olfactory bulb and moving -4.5 rostral-caudal and -1.0 medial-lateral. A hole was drilled and a cannula was lowered to a depth of 2.2 mm, aimed at the lateral ventricle. When the guide cannula was secure, a cannula insert was placed inside. For sham-operated animals, all procedures were the same as described above with the exception of the insertion of the guide cannulae.
The day after surgery, females were assigned to either the AL or FR feeding condition (described above). For 2 d, they were maintained on this feeding regimen. On the third day, each female was tested a single time for sexual behavior. All testing took place between 0800 and 1100 h. Before testing, females were briefly anesthetized with halothane and infused with saline (0.09%), GnRH-I (1 µg), or GnRH-II (1 µg), all peptides were dissolved in saline and given in a volume of 30 µl. The infusions were delivered by removing the cannula insert and replacing it with a longer cannulae that projected 0.1 mm further into the brain than the guide. Infusion was conducted by hand-delivery slowly over the course of 20–30 sec. Thirty seconds after the infusion, the temporary cannulae was removed, the insert replaced and the female was returned to her home cage. Fifteen to twenty minutes later, females were placed into a clean, clear Plexiglas test box with a sexually experienced male. Behavior was observed for 30 min in experiment 1 and 45 min in experiment 2 or until the male achieved ejaculation, whichever occurred first.
Experimental designs
In the first experiment, AL and FR females were infused with either saline (0.09%; AL, n = 10; FR, n = 11) or GnRH-II (1 µg; AL, n = 11, FR, n = 8). Doses for GnRH-I and -II were based on previous work done in our laboratory (24). Sham-operated females (AL, n = 8; FR, n = 9) did not receive infusions. In the second study, all the females were food restricted and each received saline (n = 8), GnRH-I (1 µg; n = 8) or GnRH-II (1 µg; n = 8). For GnRH-II immunocytochemistry, animals were maintained for 2 d in either the FR (n = 10) or the AL (n = 7) condition then killed and brains collected as described below. To validate the presence of the type II GnRH receptor in musk shrew brain, five AL females were used.
Behavioral testing
One notable aspect of sexual behavior in female musk shrews is the absence of a behavioral, vaginal, or ovarian estrous cycle (30). Thus, we can select females on any day and mating behavior ensures within a few minutes after introduction to the male. During a mating bout, sexually naive females are initially aggressive toward males. Sexual receptivity is marked by a significant reduction of aggression and the onset of rump presentations and tail wagging (31). At this time, the male begins to mount and intromit. Because female musk shrews do not have a reflexive posture such as lordosis, it is often difficult for the male to achieve a placed intromission (one in which the penis enters the vagina). Therefore, we score both missed and placed intromissions. Typically, a male will ejaculate after four to seven placed intromissions. During these tests, latencies for the female to rump present, tail wag, receive mounts, receive missed intromissions, receive placed intromissions, and receive five placed intromissions or an ejaculation were recorded by an experimenter blind to the treatment of the animals.
Confirmation of cannula placement and ovulation
Twenty to 24 h after behavioral testing, females were overdosed with halothane inhalant. India ink was injected into the cannulae and placement of the cannula was considered correct if the ventricles were filled with ink. Ovaries were examined for ovulation, which was scored if a corpus luteum was visible and at least one ovum was present in the oviduct. Female musk shrews exhibit mating-induced ovulation; however, typically few females ovulate after the first virgin mating (30).
Immunocytochemistry
Females were deeply anesthetized with sodium pentobarbital (10 mg/kg, ip) before perfusion between 0800 and 1200 h. Females were perfused with heparinized saline (100 U heparin/1 ml of 0.9% saline) followed immediately by Zamboni’s fixative (4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, containing 15% saturated picric acid). Brains were removed, cryoprotected overnight in 30% sucrose at 4 C, and quickly frozen in chilled 2-methyl butane. Brains were stored at -70 C until they were sectioned.
Brains were sectioned coronally at 30 µm and collected in a series of four wells. One well of tissue (one-quarter of the sections collected) was processed for immunocytochemistry using an antibody to GnRH-II (diluted 1:1000; antibody no. 741). This antiserum has no cross-reactivity with GnRH-I and has been previously validated for use in the musk shrew (32, 33). A subset of this same tissue was processed for type II GnRH receptor using an antibody made to the highly specific extracellular loop three domain of the human type II GnRH receptor (2). Because this antibody had not been tested in musk shrew, we also examined brain sections that were processed without primary antibody or secondary antibody. Immunoreactivity was visualized using the Vector Elite ABC method (Vector Laboratories, Inc., Burlingame, CA). All rinses and solutions were made in 0.02 M Tris-buffered saline (pH 7.8). Sections were pretreated in 1% sodium borohydride to remove residual aldehydes. Tissue was incubated in avidin and biotin blocking solutions (Vector Blocking Kit) to block endogenous biotin. Next, the tissue was incubated in the primary antiserum (a rabbit polyclonal used at a dilution of 1:1000) for 48 h at 4 C. After rinses, tissue was incubated in secondary antiserum (biotinylated goat antirabbit IgG from Vector Laboratories, Inc., 1:500) for 1 h. After incubation in secondary antiserum, the tissue was treated with avidin-biotin complex. Immunoreactivity was visualized with nickel intensified diaminobenzidine solution (0.25% nickel ammonium sulfate and 0.05% diaminobenzidine) and activated by 0.001% hydrogen peroxide. All tissue was processed at the same time for each study to eliminate inter-run variability and incubation and development times remained constant.
Quantification of GnRH-II-immunoreactive (ir) cell number and fiber density
Brains were processed as described and images were analyzed using the Metamorph Image analysis program for fiber density, cell bodies were counted manually. The majority of GnRH-II cell bodies are located in the midbrain just ventral to the periaqueductal gray. They form a cluster of cells that extend approximately 700–800 µm rostral to caudal. Cell bodies were counted throughout the extent of the midbrain. The midbrain was divided into anterior and posterior portions based on the presence or absence of the brachium pontis. The GnRH-II-ir fibers project to a number of areas, but the majority reside in the medial habenula (33). Immunoreactive fiber area was quantified in the preoptic area (POA), the medial habenula, and the median eminence. The habenula is a large structure and representative sections from the anterior, medial, and posterior portion of this nucleus were scored. The observer was blind to the feeding condition of the animals.
Statistical analyses
In the first experiment, latencies to perform various behaviors were analyzed using two-way ANOVA with feeding condition and drug treatment as the factors. In the second experiment, the effect of drug treatment was analyzed within each feeding condition using a one-way ANOVA. The numbers of females in each feeding/drug group displaying tail wagging and receiving mounts were analyzed using 
2 and Fisher’s exact tests. For ANOVA, all post hoc comparisons were made using Bonferroni’s multiple comparison tests. The number of GnRH-II cell bodies and fiber density were analyzed using a Student’s t test. Data were considered significantly different if P < 0.05.
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Results |
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]. Paired comparisons reveal that sham operated females took longer to rump present if they had been FR as opposed to fed AL (P < 0.05). Even more interestingly, FR females treated with GnRH-II were faster to perform the behavior than FR saline treated or sham operated females (P < 0.05).
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2 and Fisher exact tests. The numbers of FR females in each of the treatment groups that displayed tail wagging or received mounts from males did not differ [2(2) = 2.83, 4.71, respectively]. However, more selective Fisher exact tests between saline and GnRH-II-treated females revealed a trend whereby more GnRH-II-treated females performed tail wagging (P = 0.051) and a significant difference between numbers of saline vs. GnRH-II-treated females receiving mounts (P = 0.01; Fig. 1B
). Females treated with GnRH-II ovulated, and the numbers doing so did not vary with food condition (82% in the AL and 63% in the FR groups). In addition, one saline-treated animal that received an ejaculation ovulated.
GnRH-I has no effect on sexual receptivity in FR females
A one-way ANOVA performed on behavioral latency measures revealed a significant effect for tail wag, mounts and missed intromissions [F(2,24) = 7.14, 7.57, and 4.37 respectively; P < 0.026 at least; Fig. 2A]. Specifically for tail wag and mounts, administration of GnRH-II resulted in faster latencies than infusion of saline or GnRH-I (P < 0.05). The onset for males to perform a missed intromission was faster when test partners were GnRH-II-infused females as compared with GnRH-I-treated females (P < 0.05).
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2(2) = 6.39, P < 0.05]. More GnRH-II-treated females displayed each behavior as compared with GnRH-I-infused females (P = 0.005, Fig. 2B). A trend was noted when saline and GnRH-II-treated females were compared (P = 0.055). Both GnRH treatments promoted ovulation; 88% of the GnRH-II- and 76% of the GnRH-I-treated females ovulated as compared with none of the saline-treated females.
Food restriction significantly decreases the number of GnRH-II-ir cells
Food status had no effect on total numbers of GnRH-II-ir cell bodies [t(13) = 1.29]. When the anterior and posterior regions were analyzed separately, we did note a significant change in cell numbers in the anterior midbrain [t(13) = 1.88, P < 0.04; Fig. 3A] but not the posterior area [t(13) = 0.99]. More GnRH-II-ir cells were observed in brains of FR as compared with AL-fed females.
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]. There were no differences in fiber density among females in the two feeding conditions in the habenula [t(15) = 1.26] or the POA [t(15) = 0.11].
Type II GnRH receptor distribution in musk shrew brain
Type II GnRH receptor staining was present in many areas of the brain, including POA, cingulate cortex, arcuate nucleus, habenula, and infundibular stalk (Fig. 4A). The immunoreactivity was isolated to the membrane, which is similar to previously published results from other species (Ref. 2 ; Fig. 4B). No positive staining was observed in control tissue run without primary or secondary antibody (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been well documented that both peptide content and pulsatile release of GnRH-I is responsive to nutritional status. Food restriction inhibits LH and GnRH-I pulsatility, GnRH-I immunoreactivity, and GnRH-I content in brain (34, 35, 36, 37, 38). However, the effects of nutritional status on GnRH-II-ir cell number and fiber density reported here have not been observed previously. Thus, our data are consistent with data from other species showing, both directly and indirectly, that food restriction inhibits the release of GnRH peptide. However, our findings are novel because we have focused on the evolutionarily conserved GnRH-II, and we have revealed a direct relationship between food, mating behavior, and GnRH-II. The increased number of immunoreactive cell bodies and fiber dense area after food restriction suggests that an individual’s nutritional status affects either the production and/or the release of GnRH-II peptide. Furthermore, because administration of GnRH-II to FR females reinstates mating behavior, we hypothesize that food restriction inhibits release of GnRH-II, thus causing accumulation of the peptide in cell bodies and their terminals.
In addition to having different projection sites, GnRH-I and GnRH-II bind to different GnRH receptor subtypes with different affinities (2). GnRH-II has a 24-fold higher affinity for the type II, compared with the type I receptor, and GnRH-II has only 9–10% of the activity of GnRH-I at the type I receptor (2). Here we have mapped the distribution of immunoreactive type II GnRH receptors in the female musk shrew brain. The extracellular loop three antibody used for these studies has also been used in human, monkey, sheep, and mouse brains and in all cases membrane-like immunoreactivity was noted. In musk shrew, specific membrane-like staining was present in several brain regions including; olfactory forebrain, cingulate cortex, POA, arcuate nucleus, ventromedial nucleus of the hypothalamus (VMN), and medial habenula. This distribution overlaps with that reported in other species, with the exception of the cingulate cortex (2). In brains of AL-fed musk shrews, fibers immunoreactive for GnRH-II are located in all these regions. Of those regions mentioned above and assayed for GnRH-II peptide, we have noted its presence in POA, hypothalamus (that includes arcuate nucleus and VMN), and in the medial habenula (33). In fact, in musk shrew brain the highest concentration of GnRH-II peptide, outside of the region containing its cell bodies, is the medial habenula and electron microscopy reveals GnRH-II in presynaptic vesicles in this area (33). Because type II GnRH receptors are located in regions associated with mating behavior in the musk shrew, namely, POA and VMN (39), and we saw no behavioral effect with administration of GnRH-I, it is likely that the effects that we observed are mediated by actions of GnRH-II via the type II GnRH receptors in one or both locations.
Administration of GnRH-I can facilitate sexual receptivity in gonadectomized female rats (26, 27, 28). Work done on fragments of the GnRH-I peptide illustrate that specifically amino acids 5–10 are responsible for the behavioral actions of GnRH-I (28). Interestingly, only 3 of these 6 amino acids overlap with the GnRH-II peptide. More recently, a few studies suggest that GnRH-II is involved in the regulation of sexual behavior in birds. Administration of GnRH-II, but not chicken GnRH-I, to female sparrows facilitated solicitation behavior in response to male song (23). In addition, GnRH-II initiates courtship in female ring doves (1). To the best of our knowledge, ours is the first study showing any effect of GnRH-II on any behavior in a mammalian species.
The fact that the behavioral effect is only seen in energetically challenged females raises the possibility that GnRH-II evolved specifically to coordinate changes in nutrition with reproductive status. In the musk shrew, food restriction for 48 h inhibits mating behavior, and 90 min of refeeding reverses this effect (25). This rapid behavioral transition is not accompanied by changes in endogenous cortisol or testosterone concentrations in plasma (25). Estradiol levels are extremely low in plasma of mating females, and we have shown that estradiol is likely produced neurally for local actions at estrogen receptors which are essential for display of female sexual behavior in this species (39). Yet, we have also examined steroid receptors in brains of AL, FR, and refed females and found no differences in the POA or VMN in the numbers of cells expressing immunoreactive estrogen or androgen receptors (40). These results lead us to hypothesize that a neuropeptide was responsible for mediating these rapid behavioral effects.
Based on the current data, we hypothesize that GnRH-II acting at the type II GnRH receptor is responsible for rapid behavioral recovery in FR females after refeeding. The type II receptor has a carboxyl-terminal tail that the type I receptor lacks (2). This C-terminal tail in the GnRH-II receptor is implicated in rapid desensitization of G protein-coupled signaling and is present in all nonmammalian vertebrates (41). Because of the wide distribution of type II receptors, it has been hypothesized that GnRH-II may be involved in the regulation behavior including sexual arousal (2). In addition, because there is a high colocalization of type I and type II receptors in pituitary gonadotrophs (2), it is also possible that GnRH-II and GnRH-I operate in concert to control the release of FSH and LH. In birds, administration of either GnRH-II or chicken GnRH-I triggers the release of FSH and LH (42). Similarly, administration of either GnRH-I or GnRH-II in musk shrews stimulates ovulation (33). Considering that the hypothesis of specific roles for GnRH-I and GnRH-II have not been tested in FR animals, it may be the case that the function of GnRH-II has been somewhat masked.
Musk shrews live in tropical and semitropical geographical regions (43, 44) and, like early mammals that evolved in the tropics, their reproductive success depends on assessment of unpredictable nutritional resources. Under these conditions, a fast-acting peptide, such as GnRH-II, would be an ideal candidate to synchronize nutritional status and mating behavior. Our findings support much speculation that the conserved GnRH-II regulates behavioral aspects of reproduction, whereas the GnRH-I controls gonadotropin secretion. These two GnRH systems likely evolved together and act at present in a synergistic manner. In fact, in the musk shrew both GnRH systems are impacted by nutritional status (34). Nutrition and reproduction are inextricably linked, and we propose that over the course of evolution first the ancient GnRH (GnRH-II) served all functions that coordinated energy and reproduction. At some later point during evolution, a second gene evolved and usurped many of the nonbehavioral functions of the GnRH-II peptide. In mammals, this more recently evolved GnRH, GnRH-I, controls the interactions between the pituitary and gonads. Most studies of the HPG axis are conducted in domesticated, well-fed laboratory mammals, which may or may not even produce GnRH-II. Thus, the choice of animal models, up until now, may have hindered our ability to discover the behavioral role of GnRH-II. By manipulating nutritional status in musk shrews, we have uncovered one of the roles of GnRH-II and expect that this discovery will lead to more informed investigations of the function of this neuropeptide in other species.
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Footnotes |
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Present address for J.L.T.: National Institute of Neurological Disorders and Stroke, National Institutes of Health Building 36, Room 5A-21, Bethesda, Maryland 20892-4156.
Abbreviations: AL, Ad libitum; FR, food restricted; ir, immunoreactive; POA, preoptic area; VMN, ventromedial nucleus of the hypothalamus.
Received August 23, 2002.
Accepted for publication October 9, 2002.
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) represent females that underwent a sham surgery; the hatched bars (
) show data from females that received intracerebroventricular treatment with saline, and the black bars (
) represent data from females infused intracerebroventricular with GnRH-II (1 µg). A two-way ANOVA revealed a significant interaction between food condition and drug treatment (P < 0.01). Planned comparisons showed two significant (P < 0.05) effects. *, Significantly different from both other FR groups; **, significantly different from FR sham females. B, The percentage of females in each condition that received mounts. *, Significantly different from both of the FR groups.
