Gonadotropin-Releasing Hormone-II Messenger Ribonucleic Acid and ...

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Endocrinology 147(11):5069 –5077 Copyright © 2006 by The Endocrine Society doi: 10.1210/en.2006-0615

Gonadotropin-Releasing Hormone-II Messenger Ribonucleic Acid and Protein Content in the Mammalian Brain Are Modulated by Food Intake Alexander S. Kauffman, Karolina Bojkowska, Aileen Wills, and Emilie F. Rissman Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908 GnRH-II is the most evolutionarily conserved member of the GnRH peptide family. In mammals, GnRH-II has been shown to regulate reproductive and feeding behaviors. In female musk shrews, GnRH-II treatment increases mating behaviors and decreases food intake. Although GnRH-II-containing neurons are known to reside in the midbrain, the neural sites of GnRH-II action are undetermined, as is the degree to which GnRH-II is regulated by energy availability. To determine whether GnRH-II function is affected by changes in food intake, we analyzed the levels of GnRH-II mRNA in the midbrain and GnRH-II protein in numerous target regions. Adult musk shrews were ad libitum fed, food restricted, or food restricted and refed for varying durations. Compared with ad libitum levels, food restriction decreased, and 90 min of refeeding reinstated, GnRH-II mRNA levels in midbrain and GnRH-II

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nRH-II IS A REGULATORY neuropeptide that is the most evolutionarily conserved structural variant of all 23 forms of GnRH (1, 2). GnRH-II has been identified in the brains of many mammalian species, including musk shrews, moles, rodents, sheep, tree shrews, nonhuman primates, and humans (3–14). In mammals, the most detailed description of the neuroanatomical localization of GnRH-II has been reported for musk shrews (Suncus murinus), in which GnRHII-containing cell bodies are present not in the forebrain but rather in a central cluster in the midbrain (ventral to the midbrain central gray) (5, 6). A similar distribution of GnRHII-containing cells predominantly in the midbrain has been reported for other mammals including humans (9, 11–13), although such a population may be absent in some rodents (15, 16). The brain region with the most GnRH-II-containing axonal fibers is the medial habenula, with additional GnRHII-containing fibers present in several other regions, including the olfactory bulbs, septum, amygdala, hypothalamus, and the periaquiductal gray (6). Unlike the well-studied forebrain variant (GnRH-I), the primary role of endogenous GnRH-II in mammals is not to regulate gonadotropin release (16 –19). Rather, evidence implicates First Published Online July 27, 2006 Abbreviations: ARC, Arcuate nucleus; GAPDH, gylceraldehyde-3phosphate dehydrogenase; Hipp, hippocampus; ME, median eminence; mHB, medial habenula; PAG, midbrain central gray; POA, preoptic area; POMC, proopiomelanocortin; PVN, paraventricular nucleus; VMN, ventromedial nucleus. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

peptide in several target areas including the medial habenula and ventromedial nucleus. Refeeding for 90 min also reinstated female sexual behavior in underfed shrews. In male shrews, abundant GnRH-II peptide was present in all sites assayed, including the preoptic area, a region with only low GnRH-II in females. In contrast to females, food restriction did not affect GnRH-II protein in male brains or inhibit their mating behavior. Our results further define the relationship between GnRH-II, energy balance, and reproduction, and suggest that food restriction may inhibit female reproduction by reducing GnRH-II output to several brain nuclei. We postulate that this highly conserved neuropeptide functions similarly in other mammals, including humans, to fine-tune reproductive efforts with periods of sufficient energy resources. (Endocrinology 147: 5069 –5077, 2006)

a role for mammalian GnRH-II in the regulation of reproductive and feeding behaviors. In many mammalian species, including humans, food-restricted females exhibit significant reductions in sexual behaviors (20 –23). In mice and musk shrews, central administration of GnRH-II, but not GnRH-I, significantly restores mating behavior in underfed females (17, 21, 24). However, GnRH-II infusions in ad libitum-fed females do not further enhance sexual receptivity above normal control levels (21, 25), implying that the function of GnRH-II in reproduction is permissive and not merely stimulatory. In addition to its effects on sexual behavior, acute administration of GnRH-II also decreases short-term food intake (26). GnRH-II therefore acts similar to galanin-like peptide, cholecystokinin, leptin, and ␣-MSH in that it promotes sexual behavior while concurrently reducing feeding (27). Thus, we recently proposed that GnRH-II be classified as an energy balance neurotransmitter that has a role in coordinating reproductive effort, feeding, and energy availability (1, 16). The effects of GnRH-II on both feeding and sexual behavior are mediated by the specific type-2 GnRH receptor (17), which is present in several mammalian brain sites associated with feeding and/or reproduction (10, 24, 28). However, the critical neural site(s) of GnRH-II-mediated regulation of behavior remain undetermined. Additionally, the degree to which the activity of GnRH-II is regulated by energy availability is unknown. To further elucidate the novel role of GnRH-II in coordinating energy balance and reproduction, we tested whether variations in energy availability modify neural GnRH-II mRNA or protein concentrations in various brain regions of male and female shrews, and compared this with alterations in male and female sexual behavior.

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Endocrinology, November 2006, 147(11):5069 –5077

Materials and Methods Animals All studies used adult (2–3 months old), sexually naive musk shrews (Suncus murinus). All musk shrews were born in our breeding colony at the University of Virginia. Upon weaning, at 21 d of age, shrews were housed individually with food (Purina Cat Chow) and water available ad libitum (unless noted differently in specific experiments). Males and females were housed in separate rooms, each maintained on a 14-h light, 10-h dark photoperiod (lights off at 1900 h) at a temperature of 24 ⫾ 2 C. All experiments were performed in compliance with regulations of the Animal Care and Use Committee of University of Virginia.

Kauffman et al. • GnRH-II Varies with Energy Status

individual experiments for specific sites) and stored at ⫺70 C until tissue extraction. For RNA extraction, total RNA was isolated from frozen tissue punches using a QIAGEN RNA isolation kit, following the supplier’s recommended protocol (RNeasy Lipid Tissue Mini Handbook; QIAGEN, Valencia, CA). For protein extraction, frozen tissue punches were thawed into 400 ␮l of ice-cold 0.2 m acetic acid: ethanol (1:1). The tissue was homogenized and centrifuged at 10,000 ⫻ g for 30 min at 4 C. The supernatant fractions were then removed and dried overnight at 46 – 48 C. Dried samples were then reconstituted in 0.1 m PBS, pH 7.4 (250 ␮l per tube), vortexed, and stored at ⫺20 C until the RIA was conducted.

Food restriction and food intake measurements

Reverse transcription and real-time PCR

For each experiment, daily food intake was measured over a 4-d period at approximately the same time each day. Female musk shrews do not have ovarian or behavioral estrous cycles; thus, food intake is not influenced by daily or hourly variations in ovarian hormone secretion. The average daily food intake over the 4-d period was determined for each individual; animals undergoing subsequent food restriction received 60% of their average daily intake for a consecutive 2 d. Control animals remained on ad libitum food intake during this same 48-h period. Animals that were refed were also food restricted (60%) for 2 d and then given ad libitum access to food for varying amounts of time (see specific experiments for refeeding durations).

RNA extracted from tissue punches (RNAseOut kit; Invitrogen, Carlsbad, CA) was reverse-transcribed using the SuperScript Reverse Transcriptase enzyme (Invitrogen). The presence of GnRH-II cDNA in the samples was confirmed via a PCR. The primer sequences used in the PCR and real-time PCRs were: GnRH II forward 5⬘-CAT CTC CCC AGC TCT GCT ATG-3⬘; GnRH II reverse 5⬘-TCC TGG TTG GGC TGT CAG TAG-3⬘; gylceraldehyde-3-phosphate dehydrogenase (GAPDH) forward 5⬘-ACC ACA GTC CAT GCC ATC AC-3⬘; GAPDH reverse 5⬘-TCC ACC ACC CTG TTG CTG TA-3⬘. The GnRH II primers amplify a fragment of 84 bp and correspond to GenBank sequence AF107315. The primers for GAPDH amplify a fragment of 452 bp and correspond to GenBank sequence BC064681. To validate the PCR primers mRNA was extracted from tissue punches taken from the midbrain GnRH-II-containing cells, for controls we also used mRNA from several regions that contain GnRH-II fibers, but do not contain cells. These areas included the paraventricular nucleus (PVN), arcuate nucleus (ARC), median eminence (ME), ventromedial nucleus (VMN), medial habenula (mHB), midbrain central gray (PAG), and hippocampus (Hipp) from several female shrews that were either food restricted or fed ad libitum (see Fig. 1). As illustrated in Fig.

Tissue collection and extraction Animals from different feeding regimens were deeply anesthetized with isoflurane and then quickly decapitated (refed animals were killed immediately at the end of the refeeding time period). Brains were rapidly removed, frozen on dry ice, and stored at ⫺70 C. Later, each brain was sectioned in a cryostat at 165 ␮m and sections mounted onto clean frost plus slides. Tissue punches were obtained from discrete brain areas (see

FIG. 1. A schematic illustration of the location of the musk shrew brain regions analyzed. Tissue was collected from the following sites: POA, PVN, VMN, ARC, Hipp, mHB, ME, midbrain GnRH-II cells, and PAG. Approximate sites of tissue punches are indicated by the dotted circles. Tissue punches were collected bilaterally for all sites except those regions that reside along the midline (GnRH-II cells, the PAG, and the ME). For each site, punches were collected from four consecutive 165-␮m brain sections. ac, Anterior commisure; cc, corpus collosum; f, fornix; LV, lateral ventricle; d3V, dorsal third ventricle; 3V, third ventricle (ventral).


2A, only the samples that included the GnRH-II cell bodies had reaction product. Real-time PCR was performed using an Applied Biosystems 7300 Real Time PCR System (PE Applied Biosystems, Foster City, CA). All samples were run in duplicate using the iTaq SYBR green Supermix with ROX (Bio-Rad Laboratories, Hercules, CA). The reference gene GAPDH was used for normalization of variations in total RNA quantity. The Ct value for each sample, as well as the standard curves and mRNA levels in samples, were calculated using the 7300 System Sequence Detection Software version 1.2.3 (Applied Biosystems). The level of expression of genes was calculated on basis of standard curve created from serial dilutions of shrew cDNA. The calculated value is a ratio between the level of GnRH II and level of GAPDH expression.

RIAs GnRH-II protein was assayed via RIA using a protocol previously validated for musk shrew tissue (6). The GnRH-II antibody, selective for this GnRH variant, was generously provided by Dr. James Millam (University of California, Davis) and was previously used and validated in the musk shrew (6, 29, 30). For each experiment, all samples were run in a single assay to eliminate interassay variability. Mean intraassay CV ranged from 6 –10% for the four assays conducted, with a minimum detection level of 0.75 pg/tube.


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Experiment 1: quantify GnRH-II mRNA levels in females exposed to different feeding conditions In this experiment, we asked whether changes in energy availability impact GnRH II at the level of message. Adult female musk shrews were either maintained on ad libitum feeding, food restricted (60% of baseline) for 48 h, or food restricted and then refed for either 90 min. Animals were then killed, and their brains collected and analyzed for GnRH-II mRNA content using real-time PCR. All shrews were killed at the same time of day (between 1300 and 1700 h). All feeding groups were matched for average body weight and baseline food intake, and each group contained nine to 14 animals (Table 1).

Experiment 2: determine whether GnRH-II concentrations in specific nuclei vary in response to alterations in food intake This experiment assessed the effects of alterations in energy availability on GnRH-II protein levels in various brain regions. Adult female musk shrews were either maintained on ad libitum feeding, food restricted (60% of baseline) for 48 h, or food restricted and then refed for 90 min. Animals were then killed, and their brains collected and analyzed for GnRH-II protein content using RIA. Tissue punches were collected from the following brain regions: preoptic area (POA), ME, mHB, Hipp, PAG, and midbrain GnRH-II-containing cells. In a second study, tissue punches from a separate cohort of females were collected from several hypothalamic nuclei including VMN, PVN, and ARC, as well as the mHB and GnRH II cells. See Fig. 1 for a diagram of the brain regions assayed. All shrews were killed at the same time of day (between 1300 and 1700 h). All groups were matched for average body weight and baseline food intake, and each group contained nine to 11 animals (Table 1).

Experiment 3: determine the time-course of changes in GnRH-II content in response to food restriction and refeeding Here we assessed the time-course of changes in GnRH-II concentrations after refeeding in several brain regions that were impacted by food availability in experiment 2. Adult female musk shrews were maintained on ad libitum feeding, food restricted for 48 h, or food restricted and then refed for either 30, 90, or 180 min. Brains were then collected and processed for GnRH-II protein content. Tissue punches for each brain were collected from the following nuclei: VMN, mHB, PAG, and GnRH II cells. Groups were matched for mean body weight and food intake, and each group had nine to 11 animals (see Table 1).

Experiment 4: assess the time course for reactivation of sexual behavior in underfed female shrews

FIG. 2. Presence of GnRH II mRNA in different brain regions of females maintained on different feeding regimens. A, Amplification of cDNA for GnRH II (top) and the reference housekeeping gene GAPDH (bottom) via PCR in various brain regions, including POA, PVN, ARC, VMN, ME, mHB, Hipp, midbrain GnRH II cells, and PAG (see Fig. 1 for definition of abbreviations). GnRH II mRNA was detected only in midbrain GnRH II cells. B, Mean (⫾SEM) level of GnRH II RNA expression in the midbrain GnRH II-containing cells (GnRH-II cells) and in the mHB of female shrews as determined via real-time PCR. Data are expressed as a ratio of GnRH II RNA to GAPDH RNA. Females were either fed ad libitum, food restricted (60%), or food restricted and then refed for 90 min before they were killed. *, Foodrestricted animals possessed decreased GnRH-II mRNA in midbrain cells (relative to ad libitum-fed controls), whereas 90 min refed animals were not different than ad libitum controls. No GnRH II mRNA was detected in the mHB under any feeding condition.

Previous studies have shown that 90 min of refeeding reverses the inhibition on mating imposed by food restriction in female shrews (31). To examine the relationship between protein concentrations and behavior in this experiment, we examined sexual behavior in underfed females that were acutely refed for various durations: 0, 30, or 90 min. An additional control group was maintained on ad libitum feeding throughout. Immediately after the refeeding period, the females were tested for sexual behavior with a sexually experienced male. Female musk shrews do not normally exhibit behavioral or ovarian estrous cycles; their sexual receptivity is induced with 10 –20 min of direct exposure to a courting male. For each sexual behavior test, males were placed in a clear Plexiglas testing cage 30 min before testing. Upon subsequent introduction of the female subject, each test lasted 40 min or until the male had ejaculated. The latencies for the female to begin tail wagging (a stereotyped receptive behavior) and for the male to mount and intromit were recorded, as well as the latency to ejaculate. All groups were matched for average body weight and baseline food intake; each group contained eight to 10 females (see Table 1).

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TABLE 1. Treatment groups, body weights, and food consumption for females in experiments 1– 4 Daily food intake (g)

Amount consumed during refeeding (g)

% Daily intake consumed during refeeding

⫺1% ⫺9%a ⫺7%a

3.2 ⫾ 0.1 3.0 ⫾ 0.1 2.9 ⫾ 0.1

n/a n/a 0.62 ⫾ 0.10

n/a n/a 21%

23.9 ⫾ 0.3 21.6 ⫾ 0.4a 22.0 ⫾ 0.4a

0% ⫺8%a ⫺7%a

2.9 ⫾ 0.1 3.0 ⫾ 0.1 3.0 ⫾ 0.1

n/a n/a 0.52 ⫾ 0.06

n/a n/a 17%

24.5 ⫾ 0.7 23.3 ⫾ 0.3 24.0 ⫾ 0.7

23.9 ⫾ 0.7 21.0 ⫾ 0.4a 22.0 ⫾ 0.6a

⫺2% ⫺10%a ⫺8%a

2.9 ⫾ 0.1 2.9 ⫾ 0.1 3.0 ⫾ 0.1

n/a n/a 0.48 ⫾ 0.06

n/a n/a 16%

10 10 11 10 9

24.0 ⫾ 0.6 23.9 ⫾ 0.5 24.3 ⫾ 0.8 23.0 ⫾ 0.7 23.4 ⫾ 0.6

23.8 ⫾ 0.6 21.7 ⫾ 0.5a 22.3 ⫾ 0.7a 21.4 ⫾ 0.6a 22.0 ⫾ 0.5a

⫺1% ⫺9%a ⫺8%a ⫺7%a ⫺6%a

3.1 ⫾ 0.1 3.1 ⫾ 0.1 3.1 ⫾ 0.1 3.0 ⫾ 0.1 3.1 ⫾ 0.1

n/a n/a 0.35 ⫾ 0.02b 0.51 ⫾ 0.04b 0.85 ⫾ 0.05b

n/a n/a 11% 17% 27%

9 10 10 8

24.9 ⫾ 0.5 23.9 ⫾ 0.4 24.0 ⫾ 0.7 24.4 ⫾ 0.5

24.8 ⫾ 0.6 21.8 ⫾ 0.5a 22.2 ⫾ 0.7a 23.0 ⫾ 0.4a

0% ⫺9%a ⫺8%a ⫺6%a

2.9 ⫾ 0.1 2.9 ⫾ 0.1 2.9 ⫾ 0.1 2.9 ⫾ 0.1

n/a n/a 0.29 ⫾ 0.03 0.52 ⫾ 0.04b

n/a n/a 10% 18%

Group

Experiment 1 Ad libitum fed Food restricted Refed 90 min Experiment 2A Ad libitum fed Food restricted Refed 90 min Experiment 2B Ad libitum fed Food restricted Refed 90 min Experiment 3 Ad-libitum fed Food-restricted Refed 30 min Refed 90 min Refed 180 min Experiment 4 Ad libitum fed Food restricted Refed 30 min Refed 90 min

n

BW 1 (g)

BW 2 (g)

9 14 12

24.8 ⫾ 0.7 24.3 ⫾ 0.4 23.3 ⫾ 0.6

24.3 ⫾ 0.7 22.2 ⫾ 0.5a 21.6 ⫾ 0.7a

9 10 10

23.9 ⫾ 0.7 23.6 ⫾ 0.4 23.7 ⫾ 0.4

11 10 10

% Change in BW

BW1, Mean initial body weight prior to the food-restriction period. BW2, Mean body weight at the end of the 2-d food-restriction period, measured after any refeeding just before killing (or for experiment 3, just before the time of behavior testing). % Change in BW, Change in body weight from BW1 to BW2; negative values denote a decrease in body weight. Daily food intake: mean 24-h food intake, measured over 4 – 6 d before the 48-h food-restriction period. n/a, Not applicable (no refeeding). aSignificantly different than that experiment’s ad libitum-fed controls. b Significantly different from all other groups in the same experiment.

Experiment 5: determine whether food restriction affects GnRH-II levels and/or sexual behavior in males

Statistics

The distribution of immunoreactive GnRH-II has been mapped in the male musk shrew brain, but no studies have quantified GnRH-II protein content in the brains of male mammals under ad libitum conditions or after energetic challenges. In experiment 5A, adult male musk shrews were food restricted to 60% of their baseline diet for 2 d (n ⫽ 6) or ad libitum fed (n ⫽ 7). Animals were then killed and their brains analyzed for GnRH-II protein content. Tissue punches were collected from the POA, VMN, mHB, GnRH II cells, and PAG. To determine whether food restriction impairs male sexual behavior as it does female mating, an additional cohort of sexually naive food-restricted (n ⫽ 10) and ad libitum-fed (n ⫽ 9) males were tested in experiment 5B for sexual behavior. Each sexual behavior test took place in the males’ home cages with a sexually experienced receptive female. Upon introduction of the stimulus female, each test lasted 60 min or until the male had ejaculated. The latencies for the female to begin tail wagging (a stereotyped receptive behavior) and for the male to mount and intromit were recorded, along with the number of mounts, intromissions, and occurrence of an ejaculation. All groups were matched for average body weight and baseline food intake (Table 2).

Each brain region/nucleus was analyzed separately for specific treatment group differences, with planned comparisons made between each food-restricted or refed treatment group and the ad libitum-fed control group using one-way ANOVAs and Fisher’s protected least significant difference test. For experiments 4 and 5, the proportion of animals in each group that displayed sexual behavior were compared using ␹2 test. Group differences in mean body weights, % change in body weight, baseline food intake, and parameters of sexual behavior were analyzed with one-way ANOVAs. For all statistical comparisons, significance level was set at P ⬍ 0.05.

Results Experiment 1: GnRH-II mRNA in the midbrain fluctuates with food availability

This experiment assessed levels of GnRH-II mRNA in the brain and determined whether such levels were modified by energetic conditions. RNA extraction and PCR amplification revealed that GnRH-II mRNA is present in only one brain

TABLE 2. Male sexual behavior from experiment 5 Group

BW 1 (g)

BW 2 (g)

% Change in BW

Baseline food intake

% Males mating

Latency to mount (min)

Latency to intromit (min)

Ad libitum fed (n ⫽ 9) Food restricted (n ⫽ 10)

38.4 ⫾ 1.0 38.2 ⫾ 1.3

37.6 ⫾ 0.9 34.4 ⫾ 1.1a

-2% ⫺9%a

4.3 ⫾ 0.3 4.4 ⫾ 0.2

56% 50%

24.5 ⫾ 5.6 32.3 ⫾ 6.4

28.7 ⫾ 6.0 38.5 ⫾ 7.0

Sexually naive male musk shrews were fed ad libitum or food restricted for 48 h before a 60-min sexual behavior test with a sexually experienced female. Both ad libitum-fed and food-restricted males exhibited sexual behavior to the same degree (no significant group differences in mating parameters). All data are expressed as means ⫾ SEM. BW1, Mean initial body weight prior to the food-restriction period. BW2, Mean body weight at the end of the 2-d food-restriction period, measured just before the mating test. % Change in BW, Change in body weight from BW1 to BW2 (negative values denote a decrease in body weight). % Males mating, Percentage of males in each group that exhibited mating behavior (mounting and thrusting) during the 60-min test. Mean latency data calculated from only those males in each group that exhibited that particular parameter of sexual behavior. a Significantly different than ad libitum-fed controls.


region: the midbrain cells (Fig. 2A). All other sites assayed, including the POA, habenula, and various hypothalamic nuclei possessed no detectable GnRH-II RNA (Fig. 2A). Using real-time PCR, we quantified GnRH II mRNA in the midbrain cells under varying food availability. One-way ANOVA revealed a trend for group differences between the three feeding conditions (P ⫽ 0.09). Planned comparison between ad libitum-fed and food-restricted females revealed that GnRH-II mRNA levels were significantly lower, by approximately 40%, in underfed females (P ⬍ 0.05; Fig. 2B). In contrast, GnRH-II mRNA levels in underfed females allowed to refeed for 90 min were not significantly lower than ad libitum-fed levels (P ⬎ 0.05; Fig. 2B). As a control, GnRH II mRNA levels were also measured in the habenula; no detectable mRNA was found in this site under any of the three feeding conditions (Fig. 2B). Experiment 2: GnRH-II peptide content in females varies in response to changes in food availability

In experiment 2A, significant levels of GnRH-II protein were found in ad libitum-fed females in three of the six brain regions assayed, with highest concentrations detected in the mHB and GnRH II cells (Fig. 3). Moderate levels of GnRH-II peptide were also observed in the PAG, whereas the ME, POA, and Hipp contained little to no detectable GnRH-II (Fig. 3). Food restriction significantly reduced the levels of GnRH-II protein in the mHB, PAG, and the GnRH II cells (P ⬍ 0.05 for each site, relative to ad libitum levels; Fig. 3). The degree of reduction in protein concentrations in these sites ranged from 48 – 60% decreases. In the mHB, underfed females that were refed for 90 min had GnRH-II levels that were not statistically different from those of ad libitum-fed females (P ⬎ 0.05), indicating a full restoration of GnRH-II content in this site (Fig. 3). In contrast, the GnRH-II protein content in the midbrain GnRH II cells and PAG of underfed females refed for 90 min remained low at this time and were significantly different from that of ad libitum-fed females (P ⬍ 0.05 for each site; Fig. 3). Experiment 2B assessed the protein levels of GnRH-II in several additional hypothalamic nuclei. As noted above, ad libitum-fed females had high levels of GnRH-II in the mHB and GnRH II cells, and in addition, in the VMN (Fig. 4). Little to no GnRH-II protein was detected in the ARC or PVN. As in experiment 2A, food restriction caused a significant decrease in the concentration of GnRH-II in all three of the peak sites; peptide content after restriction in each of these sites was sig-

FIG. 3. Mean (⫾SEM) GnRH-II protein content in various brain regions of female musk shrews as determined via RIA. Females were either fed ad libitum, food restricted (60%), or food restricted and then refed ad libitum for 90 min before they were killed. Significant GnRH-II levels were observed in the mHB, the midbrain GnRH-II containing cells (GnRH II cells), and the PAG, and food restriction caused significant deceases in GnRH-II content in each of these areas. Refeeding increased GnRH-II levels in the mHB. *, Significantly different than levels of ad libitum-fed females for the same region.


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FIG. 4. Mean (⫾SEM) GnRH-II protein content in several hypothalamic nuclei of female musk shrews as determined via RIA. Females were either fed ad libitum, food restricted (60%), or food restricted and then refed ad libitum for 90 min before animals were killed. Significant GnRH-II levels were observed in the VMN as well as the mHB and the midbrain GnRH-II containing cells. Food restriction caused significant deceases in GnRH-II content in each of these areas, whereas refeeding for 90 min increased GnRH-II levels in the VMN and mHB. *, Significantly different than levels of ad libitum-fed females for the same region.

nificantly different from that of ad libitum-fed females (P ⬍ 0.05 for each site; Fig. 4). The levels of GnRH-II in the VMN of food-restricted females were reversed with refeeding for 90 min; GnRH-II content in this site was not different between ad libitum-fed animals and females refed for 90 min (P ⬎ 0.05; Fig. 4). As in experiment 2A, 90 min refeeding also increased GnRH-II content to ad libitum levels in the mHB but not in the GnRH II cells (Fig. 4). Experiment 3: different durations of refeeding are necessary to restore GnRH-II content in different brain regions

In experiment 3 we examined the time-course of GnRH-II restoration after varying durations of refeeding. As in experiment 2, food availability significantly altered GnRH-II peptide level in several nuclei: underfed females had significantly less GnRH-II in the VMN, mHB, PAG, and GnRH II midbrain cells than did ad libitum-fed controls (P ⬍ 0.05 for each brain region; Fig. 5). Foodrestricted females allowed to refeed ate increasingly more food the longer the refeeding period: animals refed for 30, 90, and 180 min consumed approximately 11%, 17%, and 27%, respectively, of their baseline daily food intake (Table 1). However, refeeding females for 30 min did not increase levels of GnRH-II in any of the brain regions assayed (P ⬍ 0.05 for each site, relative to ad libitum fed; Fig. 5). In contrast, refeeding females for 90 min restored

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FIG. 5. Mean (⫾SEM) GnRH-II protein content in various brain regions of female musk shrews after refeeding. Females were either fed ad libitum, food restricted (60%), or food restricted and then refed for varying amounts of time (30, 90, and 180 min). Significant increases in GnRH-II levels after 90 min of refeeding were observed in the VMN and mHB; all regions showed elevated GnRH-II levels after 180 min refeeding. *, Significantly different than levels of ad libitum-fed females. GnRH-II cells refers to the region of the midbrain with GnRH-II-containing cells.

GnRH-II levels back to ad libitum levels in both the VMN and the mHB (Fig. 5) but not in the PAG or GnRH II cells (P ⬍ 0.05 for both sites relative to ad libitum levels). By comparison, refeeding for 180 min restored GnRH-II peptide levels in all sites, including the PAG and the GnRH II cells (P ⬎ 0.05 for each region relative to ad libitum controls; Fig. 5). Experiment 4: refeeding for more than 30 min is necessary to reactivate sexual behavior in underfed females

To compare the time-course of reactivation of female sexual behavior with that of restoration of GnRH-II levels in brain nuclei (as determined in experiment 3), underfed females were tested for sexual behavior after refeeding for various durations. All control females maintained on ad libitum feeding displayed some aspect of mating behavior: 100% of ad libitum females tail-wagged and 90% received mounts and intromissions from the male. In contrast, few of the underfed females engaged in sexual behavior. The vast majority of the underfed females refed for 0 min or 30 min failed to display sexual behavior (P ⬍ 0.05 relative to ad libitum-fed controls; Fig. 6A), and the few underfed females that did mate displayed trends for longer latencies to begin tail-wagging (relative to ad libitum-fed shrews; P ⫽ 0.10; Fig. 6B). In contrast, food-restricted females refed for 90 min showed high levels of sexual behavior, with nearly 90% of females displaying sexual receptivity. The percentage of females refed for 90 min that mated, as well as their mean latency to begin mating, was not significantly different from that of ad libitum control females (Fig. 6). Experiment 5: GnRH-II concentrations and sexual behavior in males are not modified by food restriction

Male shrews possess GnRH-II in the same brain regions as do females, including the mHB, the VMN, midbrain cells, and the PAG (Fig. 7). In addition, males have high levels of GnRH-II protein in the POA, a region that has only minimal GnRH-II in females (Fig. 7; compare with Fig. 3). Interestingly, low food availability did not significantly alter GnRH-II levels in any of the brain regions assayed in males: for each site, GnRH-II protein content did not differ statistically between food-restricted and ad libitum-fed males (Fig. 7). Like GnRH-II content, the display of sexual behavior in male shrews was also not inhibited by low food availability. Approximately half of the males in each of the ad libitum and underfed groups mated with a stimulus female (Table 2) and

latencies for males to begin mounting were not different between the two feeding groups (Table 2). Two males in each of the feeding groups ejaculated in the allotted testing time. As an indicator that 60% food restriction was sufficient to impair the males’ energy balance, mean body weights of food-restricted males were approximately 9% lower than their original baseline values (before food restriction) and differed significantly from those of ad libitum-fed controls at the time of testing (P ⬍ 0.05). This degree of body weight loss in underfed males was comparable to that of females given the same food restriction treatment in experiments 1–3 (8 – 10% decrease for each sex; see Tables 1 and 2). Body weight and food intake

None of the treatment groups differed significantly in mean body weight or baseline daily food intake before the 48-h food restriction period (Table 1). By the end of this restriction period, food-restricted females had lost 8 –10% of their initial body weight and weighed significantly less than ad libitum-fed females (Table 1). Underfed females that were refed also weighed significantly less than they did before food restriction; these females lost 6 – 8% of their initial body weight and weighed significantly less than ad libitum-fed controls (Table 1). In no case did the body weights of food-restricted and refed animals differ significantly at the time of testing/killing. Discussion

The present study is the first to directly measure GnRH-II protein and mRNA concentrations in animals on different levels of caloric intake. Our results illustrate a novel relationship between GnRH-II regulation, energy balance, and sexual behavior, and suggest that energetic and metabolic challenges associated with underfeeding may inhibit female reproductive behaviors by reducing both GnRH-II mRNA and protein transport/release in critical regulatory nuclei in the hypothalamus and habenula. We also report previously unidentified sex differences in GnRH-II content in the POA and in the responsiveness of GnRH-II to variations in energy availability. In ad libitum-fed female musk shrews, GnRH-II mRNA was only detected in the midbrain where significant GnRHII-immunoreactive cells have previously been observed using immunocytochemistry (5, 6, 9). No GnRH-II mRNA was detected elsewhere in the shrew brain, confirming that its production is restricted to midbrain neurons; this contrasts



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FIG. 7. Mean (⫾SEM) GnRH-II protein content in various brain regions of male musk shrews as measured with RIA. Males were either fed ad libitum or food restricted (60%) for 48 h. GnRH-II was present in all brain regions assayed; food restriction did not cause a significant decrease in GnRH-II content in any of the assayed brain regions. For comparison, in the same assay, GnRH-II levels were also measured in GnRH-II cells of eight ad libitum-fed females. GnRH-II cells, the region of the midbrain with GnRH-II-containing cells.

FIG. 6. Sexual behavior of female musk shrews exposed to variations in food availability. Sexually naive females were either maintained on ad libitum feeding, food restricted for 48 h, or food restricted and then refed for either 30 or 90 min. Females were then tested for sexual behavior with a sexually experienced male shrew. A, Percent of female shrews exhibiting tail-wagging (a receptive sexual behavior). B, Latency for the females to begin tail-wagging; only the females in each group that exhibited tail-wagging behavior were included in this latency data. *, Significantly different from ad libitum-fed control females.

with findings in rhesus macaques in which GnRH-II mRNA has been detected in the PVN, Hipp, and lateral hypothalamus, in addition to the midbrain (13, 32). It is unclear whether this dissimilarity reflects species differences or differences in techniques used. Although mRNA in the musk shrew was restricted to the midbrain, high levels of GnRH-II protein were noted in the mHB, VMN, and GnRH-II cells, with moderate concentrations in the PAG. We did not detect significant GnRH-II protein in any of the other sites assayed in females, such as the ARC, ME, PVN, POA, or Hipp. A previous study in female shrews similarly reported abundant GnRH-II protein in the mHB and GnRH-II cells and low levels in the ME (6) but did not assess protein levels in specific hypothalamic nuclei (nor under different feeding/ energetic conditions). It has been well documented that 48-h food restriction severely impairs female musk shrew reproduction, as it does for other female mammals (17, 22, 31). In the present study, 48-h food restriction of female shrews reduced GnRH-II mRNA levels in the midbrain by approximately 40% and decreased GnRH-II protein concentrations in several target areas, including the VMN, mHB, and PAG, by 48 – 60%.

These findings indicate that alterations in metabolic conditions and energy balance can regulate the GnRH-II system (and thus its activity) at the level of both the gene and translation/protein processing. The VMN, mHB, and PAG have each been implicated in the regulation of female reproduction (33– 40), and the VMN is also involved in energy balance/feeding (41– 43). Thus, we hypothesize that in energetically challenged females, decreased output of GnRH-II peptide from the midbrain to these target sites results in alterations in feeding and sexual behaviors. Specifically, reduced GnRH-II signaling in the VMN and/or mHB may play a role in the decreased sexual behavior and increased feeding/foraging of underfed females. This possibility is supported by previous findings that GnRH-II infusions into the lateral ventricles significantly reduce short-term food intake and promote sexual behavior in underfed female shrews and mice (17, 24, 26). Interestingly, a recent report in marmoset monkeys indicates that GnRH-II plays a role in promoting female sexual behavior in primates, suggesting that GnRHII’s reproductive functions may also extend to humans (44). If GnRH-II plays a critical role in permitting female reproduction, then GnRH-II levels in underfed females that are refed should correlate with reinstatement of sexual behavior. Refeeding food-restricted female shrews for just 90 min is sufficient to restore sexual behavior and receptivity (present study), as is a single GnRH-II infusion (17, 24). Complimenting this, refeeding underfed females for 90 min was sufficient to restore GnRH-II concentrations to ad libitum levels in both the mHB and VMN. Collectively, these data suggest that GnRH-II acting in the VMN and/or mHB may re-activate female mating behavior upon brief refeeding. Under this model, low food availability or negative energy balance reduce GnRH-II output to the VMN and/or mHb, thereby reducing display of sexual behavior, whereas subsequent refeeding reactivates the release of GnRH-II at these target regions, thus restoring receptivity. In support of this conjecture, both the VMN and mHB possess type-2 GnRH receptors (10, 24) that have been shown to mediate the behavioral effects of GnRH-II in both shrews and primates (17, 44). Interestingly, low GnRH-II levels in the PAG

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caused by food restriction were not increased by 90 min of refeeding, suggesting that GnRH-II release/action in the PAG is not a critical component of feeding’s reinstatement of sexual behavior. Although either the VMN and/or mHB may be involved in the GnRH-II-mediated regulation of behavior, we cannot rule out involvement of other additional sites that were not addressed in this study. Indeed, multiple brain regions have been implicated in regulating reproduction and energy balance, and numerous neuroregulatory factors besides GnRH-II influence mating and/or feeding, including CRF, galanin, leptin, proopiomelanocortin (POMC), AgRP, and NPY (23, 27, 45). Any of these factors could be interacting with the GnRH-II system to control feeding or sexual behavior. Although abundant evidence indicates that many neuropeptides’ mRNA/protein decreases (POMC, CART, CRF, galanin) or increases (NPY, AgRP) after short-term food restriction, few studies have examined the time-course of restoration of these levels after refeeding. In rodents, food restriction-induced changes in hypothalamic mRNA of NPY, AgRP, and POMC are fully or partially reversed after 5– 6 h refeeding (46, 47). However, apart from one study in mice showing reduced NPY peptide levels in the PVN after 1 h refeeding (48), we are aware of no other rapid changes (ⱕ2 h) in mRNA/protein concentrations of energy balance neuropeptides in refed mammals. Our present findings of 90min alterations of both GnRH-II mRNA and protein in several brain sites after refeeding suggest that the GnRH-II system is rapidly responsive to acute changes in energy availability and may be an initial signal in the brain’s coordination of energy and behavior. Although many nonfeeding neural systems are also compromised by food restriction [ex: decreased GnRH I in underfed animals (49)], the direct modulation by GnRH-II of food intake (26) indicates that it is a critical component of energy balance regulation and that its changes are not simply part of a global neural response to energy challenges. The vast majority of studies regarding GnRH-II neuroanatomy and function in mammals have been restricted to females. Although Rissman and Li (50) previously used immunocytochemistry to identify GnRH-II-containing cells in male shrew midbrains, no mammalian studies have measured GnRH-II protein content in male brains or looked for GnRH-II in potential target regions of the male brain under varying caloric conditions. We found high GnRH-II peptide content in all brain regions assayed in males, including the VMN, mHB, and POA. Interestingly, male musk shrews possessed approximately three times higher concentrations of GnRH-II peptide in the POA as compared with females. These data reveal a novel sex difference in the POA, a region well known for sexual dimorphism in mammals, including sex differences in aromatase enzyme (51), estrogen receptors (52, 53), and the volume of the sexually dimorphic nucleusPOA nucleus (54). The function of the sex difference in POA GnRH-II is not immediately clear. Although there is abundant evidence that the POA regulates male sexual behavior in a numerous mammalian species, this site has also been implicated in female sexual behavior in several species, including musk shrews (34). In contrast to females, GnRH-II content in male brains did


not drop after food restriction, nor was male sexual behavior inhibited by food restriction. These findings reveal a possible sex difference in the role of GnRH-II in female vs. male reproduction. Similar to our present findings, studies in rodents have found male reproduction to be less susceptible to low energy availability than that of females (55–58), indicating this may be a generalized response of male mammals to moderate energetic challenges [likely owing to dissimilarities between the sexes in energetic resources devoted to gamete production, pregnancy, and lactation (59, 60)]. It is unlikely that the degree of food restriction in our study was too mild to affect the males’ energy balance because body weight of underfed males was markedly reduced (by 9 –10%). However, because reproduction of male mammals is not completely immune to the effects of large energetic challenges, it is unknown whether greater food restriction paradigms would inhibit male shrew sexual behavior, and if so, whether GnRH-II in critical nuclei would also be reduced. It also remains to be determined whether GnRH-II infusions would augment male sexual behavior in various mammalian species. In summary, our findings define a critical relationship between GnRH-II regulation, energy balance, and sexual behavior, and suggest that metabolic and caloric cues associated with underfeeding and refeeding may regulate female reproductive behaviors by modifying GnRH-II mRNA production and protein release in the hypothalamus and/or habenula. We suggest that this highly conserved neuropeptide has a similar regulatory function in other mammals, including humans, to fine-tune reproductive efforts and synchronize female mating behavior with periods of sufficient energetic resources. Acknowledgments The authors thank Dr. James Millam (University of California at Davis) for generously providing the GnRH-II antibody, Dr. Kevin Morgan (Medical Research Council, Edinburgh, Scotland, UK) for designing the GnRH-II RNA primers, and Jessie Gatewood for technical assistance. We also thank Aliesha Schoenfelder and Dr. Dan Haisenleder of the University of Virginia Ligand Assay Core Laboratory for helpful RIA assistance. Received May 8, 2006. Accepted July 14, 2006. Address all correspondence and requests for reprints to: Dr. Alexander S. Kauffman, Department of Physiology and Biophysics, Health Sciences Building, Box 357290, University of Washington, Seattle, Washington 98195. E-mail: [email protected]. This work supported by National Institute of Mental Health Grants F32 MH070084, K02 MH01349, and RO1 MH57759. The University of Virginia Ligand Assay Core Laboratory is supported through National Institute of Child Health and Human Development Grant U54 HD28934. Disclosure statement: The authors have nothing to disclose.

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