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Endocrinology Vol. 144, No. 5 2034-2045
Copyright © 2003 by The Endocrine Society

Developmental Changes in Hypothalamic Insulin-Like Growth Factor-1: Relationship to Gonadotropin-Releasing Hormone Neurons

Shabrine S. Daftary and Andrea C. Gore

Kastor Neurobiology of Aging Laboratories, Fishberg Research Center for Neurobiology, Brookdale Department of Geriatrics, Mount Sinai School of Medicine, New York, New York 10029

Address all correspondence and requests for reprints to: Andrea C. Gore, Ph.D., The University of Texas at Austin, Division of Pharmacology/Toxicology, 1 University Station, A1915, Austin, Texas 78712. E-mail: andrea.gore{at}mail.utexas.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Reproductive development in vertebrates is controlled by changes in hypothalamic GnRH neurons and their inputs from other neurons and glia. One factor involved in the regulation of the GnRH system is the neurotrophic factor, IGF-1. To better understand the regulation of GnRH neurons by hypothalamic IGF-1, we quantified levels of IGF-1 mRNA in hypothalamic and preoptic regions containing GnRH cells, studied the effects of IGF-1 on GnRH gene expression, and examined the neuroanatomical relationship between GnRH neurons and hypothalamic IGF-1 in neonatal, peripubertal, and reproductively mature mice. Our results indicated that IGF-1 mRNA levels in the preoptic area and anterior hypothalamus peaked at postnatal day (P) 5, decreased through P20, and then increased through peripubertal and adult development. Second, IGF-1 had stimulatory effects on GnRH gene expression in explanted preoptic area-anterior hypothalamuses of P5 and peripubertal mice, with results varying by sex and duration of treatment. In contrast, IGF-1 had no effect or even inhibited GnRH gene expression in adult P60 mice. Third, GnRH perikarya coexpressed IGF-1, and this increased throughout sexual maturation. Taken together, the results suggest that IGF-1 can modulate GnRH neurons, that the sensitivity of GnRH neurons to IGF-1 changes developmentally, and that GnRH cells coexpress IGF-1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CONTROL OF reproductive function is driven by the GnRH neurons, located within the preoptic area and anterior hypothalamus (POA-AH) of mammals. The hypophysiotropic GnRH cells project to the median eminence (ME), where they release the decapeptide in a pulsatile manner into the portal capillary vasculature, to bring about the release of gonadotropins into the general circulation. The gonadotropins, in turn, affect the synthesis and secretion of the steroid hormones from the gonads. Although the GnRH system is anatomically established in late embryonic life and the cells are capable of producing GnRH, the juvenile period is marked by a long absence in the activity of the reproductive axis (1). Beginning at the onset of puberty, GnRH release and biosynthesis increase, resulting in the attainment of reproductive function. Although the exact mechanisms for the developmental fluctuations in GnRH levels are still unknown, it has been hypothesized that alterations in the intrinsic properties of GnRH neurons themselves, as well as the levels of neurotransmitters, hormones, and neurotrophic growth factors that regulate GnRH neurosecretion, result in the maturation/activation of GnRH release at the onset of puberty.

Neurotrophic factors, including IGF-1, have been implicated in the regulation of GnRH neurons and the hypothalamic-pituitary-gonadal axis (for review, see Ref. 2). Either peripheral or central IGF-1, or both, may play such roles, although relatively little is known about central IGF-1 in this process. In rodents and primates, circulating IGF-1 concentrations increase during the onset of puberty (3, 4, 5, 6). A premature elevation of IGF-1 levels in prepubertal rats and primates advances the onset of puberty (7, 8). In vitro, the administration of IGF-1 to hypothalamic explants causes the release of GnRH from neuroterminals (9), and IGF-1 can also up-regulate GnRH mRNA levels and release in GnRH-producing cell lines (10, 11, 12). To further implicate IGF-1 in regulating reproductive maturation, targeted mutagenesis of the IGF-1 or the IGF-1 receptor gene results in mice that are small and infertile (13, 14). Moreover, studies have indicated synergistic interactions between estrogen and IGF-1 in the hypothalamus (15). A recent report by Quesada and Etgen (16) indicated that intracerebroventricular infusion of an IGF-1 receptor antagonist blocked estrogen-induced GnRH release. Taken together, these studies suggest that IGF-1 plays a role in regulating reproductive function, possibly through the modulation of GnRH cells. Therefore, the present study evaluated changes in hypothalamic IGF-1, and its potential regulation of GnRH neurons, using molecular, physiological, and anatomical approaches.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male and female C57bl/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed at the Center for Laboratory Animal Science at Mount Sinai School of Medicine. All animal use was approved by the Institutional Animal Care and Use Committee, following Guidelines for the Care and Use of Laboratory Animals. To provide pups at postnatal days (P)0–P20, breeding pairs were established. Additional mice to be used at the peripubertal period and those for use at P60 were purchased directly from the vendor and were allowed to recover, for 1 wk, from the shipping process before use. In cases when mice from our breeding colonies were allowed to mature, they were weaned at P21. Postweanling mice were housed in same-sex groups of 1–2 per cage in a room with a controlled temperature (22 C) and light cycle (12 h light, 12 h dark, lights on at 0700 h) and were provided with water and food ad libitum. Starting on P25, male and female mice raised in the colony were monitored daily for preputial separation (PPS) and vaginal opening (VO) as an indication of attainment of puberty. PPS in males and VO in females reflect the peripubertal rise in testosterone and estradiol secretion, respectively, and have long been used as landmarks of the progression of reproductive maturity (17, 18). PPS and VO were similar in timing in our male and female mice, occurring between P28 and P33 in our colony.

Tissue preparation for molecular studies
Animals were killed by decapitation, and the brains were rapidly removed from the skulls and chilled on ice, and the POA-AH and mediobasal hypothalamus-ME (MBH-ME) were dissected out as described previously (19). The border of the dissection was made by a coronal cut just posterior to the entry point of the optic chiasm. The rostral border of the POA-AH was made by a coronal cut at the posterior third of the olfactory tubercle, whereas the caudal border of the MBH-ME was made by a coronal cut, 2 mm anterior to the entry point of the optic chiasm. This coronal section (3-mm thick for the POA-AH and 2-mm thick for the MBH-ME in adults, and adjusted accordingly by the boundaries of the tissue for younger animals) was laid rostral side up, and an isosceles-triangle-shaped cut was made. For the POA-AH, the apex of the triangle was just under the midline of the corpus callosum, with the two legs of the triangle passing through the anterior commissure. For the MBH-ME, the apex of the triangle was just below the thalamus, with the two legs of the triangle passing through the hypothalamic sulci. These dissections should include virtually all GnRH perikarya and terminals. A stainless steel brain slicer (model RBM-4000; Activational Systems, Warren, MI) was used for brains of peripubertal and P60 mice. For IGF-1 mRNA quantification, POA-AH and MBH-ME dissections were snap-frozen on dry ice and stored at -80 C until use. For perifusion experiments, fresh tissues were transferred directly into the perifusion chamber. The total dissection time was less than 3 min after death.

Tissue perifusion
The POA-AHs were transferred to an Endotronics, Inc. (Minneapolis, MN) chamber and perifused continuously with warmed (32–34 C), oxygenated (95% O2-5% CO2) Locke’s media (approximately 100 µl/min). The composition of the Locke’s media was (in mM): 154 NaCl, 5.6 KCl, 1 MgCl4, 6 NaHCO3, 1.25 CaCl2, 10 glucose, 2 HEPES, and 1 mg/ml BSA; pH was adjusted to 7.2–7.4.

Tissues were allowed to equilibrate for 2 h before treatment. IGF-1 (100 ng/ml, dissolved in Locke’s media) or glacial acetic acid (vehicle; 0.002 mM) was perifused for various set intervals of time (0, 1, 2, or 4 h). The 100-ng/ml IGF-1 dose was chosen based on previous work by Hiney et al. (9). After treatment, the POA-AHs were snap-frozen on dry ice and stored at -80 C for RNA extraction. Levels of total RNA and GnRH mRNA were measured in untreated tissues (0 h) and compared with levels in tissues perifused with the vehicle for 1, 2, or 4 h. RNA recovery was similar among these tissues, suggesting that there was no appreciable degradation caused by perifusion. In addition, we confirmed the integrity of the RNA by electrophoresis on a 0.8% agarose gel.

RNA extraction
Frozen tissue from individual POA-AH and MBH-ME dissections was fractionated into cytoplasmic and nuclear phases using a double-detergent lysis buffer, 1-cc syringes, and 22-gauge needles as homogenizers, as previously described (19). The nuclear fraction was subjected to a more extensive deoxyribonuclease (DNase) treatment for 30 min at 37 C with 60 U DNase-I. Then, both cytoplasmic and nuclear RNA were treated with proteinase K (200 µg/ml final concentration) at 45 C, followed by a phenol-chloroform-isoamyl alcohol extraction, chloroform extraction, and precipitation with ethanol (nuclear fraction) or isopropanol (cytoplasmic fraction) at -20 C. Both fractions were centrifuged and washed with 70% ethanol, and the pellets were resuspended in 20 µl hybridization buffer [0.1 M EDTA (pH 8) and 4 M guanidine thiocyanate (final pH 7.5)] dissolved in 20 µl hybridization solution [0.1 M EDTA (pH 8) and 4 M guanidine thiocyanate (pH 7.5)] for ribonuclease (RNase) protection assay (RPA). An aliquot (4 µl) from each cytoplasmic fraction was diluted in 200 µl of 10-mM Tris, 1 mM EDTA (pH 7.4), to determine the total RNA content, using absorbance at 260 nm.

Riboprobes and reference RNA
The following DNA subclones were used in this study: 1) to measure IGF-1 mRNA, a 386-bp cDNA subcloned into the EcoRI and HindIII restriction sites of a pGem2 vector [kindly provided by Drs. Derek LeRoith and Charles Roberts (20)]; 2) to measure GnRH mRNA in the cytoplasm, a murine GnRH cDNA, 443 bp in length, subcloned into the EcoO1091 and XbaI restriction sites of a Bluescript KS (+) vector (Stratagene, La Jolla, CA) (21); 3) cyclophilin mRNA, for use as an internal control for gel loading variability, measured in the above cytoplasmic fractions using a 111-bp clone, subcloned into the PstI and XmnI restriction sites of a Bluescript KS (+) vector (22); 4) to measure GnRH primary transcript levels in the nucleus, a 383-bp mouse GnRH fragment complementary to the intron A-exon 2-intron B (mA2B) region of the proGnRH gene, spanning the SpeI and HindIII restriction sites and subcloned into a Bluescript KS (+) vector (21).

Probe (antisense) and reference (sense) RNA were transcribed in vitro from template DNA, as described previously (19), using bacteriophage T3, T7, or Sp6 RNA polymerase; and transcription was terminated by digesting the template with RNase-free DNase-I. Free nucleotides were removed from the probe RNAs by ethanol precipitation in the presence of 2.5 M ammonium acetate. Reference RNAs were phenol-chloroform-isoamyl-alcohol extracted and purified by two ethanol precipitations. Reference RNAs were quantitated (using an absorbance reading at 260 nm), aliquoted, and stored at -80 C.

RPA
RPAs were performed separately on IGF-1 mRNA, GnRH mRNA in the cytoplasm, and GnRH primary transcript, an index of GnRH gene transcription in the nucleus. RPAs were performed as described previously (19). Briefly, GnRH mRNA, IGF-1 mRNA, and GnRH (mA2B) genomic probes were labeled with [{alpha}-32P] uridine 5'-triphosphate to high specific activity (~1,300,000 cpm/ng), and cyclophilin probe to low specific activity (~60,000 cpm/ng). Probes were added to samples at a final vol of 25 µl (20 µl RNA and 5 µl probe). Cytoplasmic samples were incubated with GnRH and cyclophilin probes or IGF-1 and cyclophilin probes in the same tubes. For standard curves, probes were mixed with increasing amounts of reference RNAs (GnRH cDNA, 0–10 pg; mA2B, 0–1 pg; IGF-1, 0–1 pg; cyclophilin, 0–50 pg). Samples and standards were allowed to hybridize for 16–18 h at 30 C. Samples were then treated with proteinase K for 15 min at 45 C, phenol-chloroform extracted, and precipitated with ethanol on dry ice. After centrifugation, samples were washed with 70% ethanol, and the dried pellets were resuspended in 5 µl 1.5x Ficoll loading buffer and then electrophoresed through 5% nondenaturing polyacrylamide gels. Gels were exposed to x-ray film for 18–24 h to produce an autoradiogram, and to a phosphor imaging screen (Molecular Dynamics, Inc., Sunnyvale, CA) for 2–7 d for quantification. Levels of cytoplasmic IGF-1 and GnRH mRNA levels were normalized to both the nanograms of cyclophilin mRNA and to the total micrograms of RNA, as determined by absorbance at 260 nm. Absolute mRNA amounts in samples were determined by comparison with the standard curve, using regression analysis.

Perfusions
Mice were deeply anesthetized with 0.05 ml each ketamine (100 mg/ml)/xylazine (20 mg/ml) and perfused transcardially with 1% paraformaldehyde for 1 min at a rate of 7.5–15 ml/min, followed by 4% paraformaldehyde for 10 min. Brains were immediately removed and postfixed in 4% paraformaldehyde for 4–6 h, followed by storage in PBS with 0.1% azide at 4 C. Brains were cut on a vibratome (Ted Pella, Inc., Redding, CA) at 40–50 µm and stored in PBS with 0.1% sodium azide at 4 C, before processing for immunohistochemistry.

Immunohistochemistry
Single- and double-label immunohistochemistry was performed on six tissue sections per animal, distributed evenly through the organum vasculosum of the lamina terminalis (OVLT)/POA-AH from each neonatal, peripubertal, and adult mouse. Anatomical boundaries of the POA-AH, OVLT and medial septum, and lateral septum (MS-LS) were determined relative to the anterior commissure, corpus callosum, and third ventricle, and sections were compared with a mouse brain atlas (23).

For single-labeling experiments, sections were pretreated with 2% normal goat serum (Vector Laboratories, Inc., Burlingame, CA) in PBS for 1 h, to reduce nonspecific staining, and then incubated with a rabbit polyclonal antibody to IGF-1 (IHC 7296; 1:200; Peninsula Laboratories, Inc., San Carlos, CA) for 96 h at 4 C. IHC 7296 is an affinity-purified rabbit polyclonal antibody raised against a recombinant human/bovine IGF-1 that differs from the corresponding mouse sequence by 4 amino acids distributed across the entire sequence (94% homology). This antibody shows no cross-reactivity with IGF-2, insulin, proinsulin, somatostatin, GH, and epidermal growth factor (Peninsula Laboratories, Inc., Manufacturer’s data sheet) and has been validated in GT-1 GnRH-producing cells (11) and rat tissue (24). The sections were then rinsed and incubated for 1 h with goat antirabbit antibody conjugated to fluorescein isothiocyanate (FITC; 1:200; Vector Laboratories, Inc.) and rinsed again in PBS. They were then mounted on gelatin-subbed slides, air-dried, and coverslipped with Vectashield (Vector Laboratories, Inc.). Controls for specificity of staining in mouse tissue included omission of the primary antibody and preabsorption of the primary antibody with human IGF-1 peptide (1 µg/ml; Sigma, St. Louis, MO). Both conditions resulted in complete elimination of signal.

To determine whether IGF-1 is colocalized in GnRH-expressing neurons, we used a double-labeling protocol that involved incubating the tissue in the rabbit polyclonal antibody to IGF-1 (IHC 7296) together with the mouse monoclonal antibody HU11B for GnRH-I (kindly provided by Dr. Henryk Urbanski, Oregon National Primate Research Center, Beaverton, OR). HU11B detects the amidated decapeptide corresponding to the sequence pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (25). Sections were pretreated with 2% normal goat serum and 2% normal horse serum (Vector Laboratories, Inc.) in PBS for 1 h. The polyclonal IGF-1 antibody (1:400) and monoclonal GnRH antibody (1:500) were applied together for 96 h at 4 C in PBS. Sections were rinsed and incubated for 1 h in a mixture of goat antirabbit antibody conjugated to FITC (1:200) and horse antimouse antibody conjugated to Texas Red (1:200, Vector Laboratories, Inc.) and rinsed in PBS. Sections were then mounted on gelatin-subbed slides, dried overnight, and coverslipped with Vectashield (Vector Laboratories, Inc.). The same controls were performed as described above for single-labeling studies.

Microscopy and image analysis
All sections were analyzed using both an Axioplan2 fluorescence microscope and an LSM410 inverted confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany), as reported previously (24). Sections were initially examined under the Axioplan2 microscope, using a FITC filter (excitation at 450–490 nm) to reveal IGF-1-expressing neurons, and a second Texas Red filter (excitation at 515–560 nm) to visualize GnRH neurons. We determined the percentage of GnRH neurons expressing IGF-1 (GnRH+/IGF-1+) and the percentage of GnRH neurons without IGF-1 (GnRH+/IGF-1-). The immunohistochemical results were confirmed and imaged using an LSM 410 inverted confocal microscope equipped with an ArKr 488/568 laser using various objectives [16x, numerical aperture (NA), 0.05; 25x, NA, 0.8; 63x, NA, 1.4; and 100x, NA, 1.4]. As described above, an FITC filter and a Texas Red filter were used to visualize IGF-1-expressing and GnRH neurons, respectively. The images obtained for each fluorophore were stored separately as 512 x 512 pixel images. By merging IGF-1 and GnRH images, using the Zeiss CLSM Imaging Software version 3.99 (Zeiss Microimaging Inc., Thornwood, NY), we confirmed our earlier conventional microscopy observations of the percentage of GnRH neurons expressing IGF-1 (GnRH+/IGF-1+). These cells were also mapped onto a representation of a mouse coronal brain section at the level of the POA-AH, as described previously (24). For presentation purposes, the illustrations for the manuscript were assembled using Adobe Photoshop 6.0 (Adobe Systems Inc., San Jose, CA).

Statistics
For molecular studies on IGF-1 gene expression, results were compared using two-way ANOVA, with age and sex as variables. For IGF-1 treatment of neonatal, peripubertal, and adult POA-AHs, GnRH transcript levels were analyzed by four-way ANOVA, with age, sex, treatment (IGF-1 vs. vehicle), and treatment time as variables. For colocalization of IGF-1 within GnRH perikarya, differences in double labeling were compared by two-way ANOVA, with age and sex as variables. In all cases, differences were considered significant at P < 0.05. Although the statistical analyses were performed on raw data, in the graphic presentation (see Figs. 3Go, 4Go, and 7Go), data are indicated as a percentage of the corresponding control levels.



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  		Figure 3. Effects of IGF-1 on GnRH mRNA levels. POA-AH tissues from neonatal (P5, A), peripubertal (~P30, B), and adult (P60, C) mice were perifused in vitro with IGF-1 (100 ng/ml) or vehicle (diluted acetic acid) for 1, 2, or 4 h. GnRH mRNA levels were measured by RPA. Data are expressed as percent of corresponding control, with the horizontal line indicating control values expressed as 100%. IGF-1 has significant stimulatory effects on GnRH mRNA in neonatal male (2 h) and peripubertal female mice (4 h). However, IGF-1 has no significant effects on GnRH mRNA levels in adult male or female mice at all times tested. *, P < 0.05 vs. vehicle control.

 


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  		Figure 4. Effects of IGF-1 on GnRH primary transcript levels. POA-AH tissues from neonatal (P5, A), peripubertal (~P30, B), and adult (P60, C) mice were perifused in vitro with IGF-1 or vehicle for 1, 2, or 4 h; and GnRH primary transcript levels were measured by RPA. Data are expressed as percent of corresponding control, with the horizontal line indicating control values expressed as 100%. IGF-1 significantly stimulates GnRH primary transcript levels in neonatal male mice at 2 h. In peripubertal mice, IGF-1 significantly stimulates GnRH primary transcript levels in peripubertal male (4 h) and female mice (2 h). In adults, IGF-1 exerts no (or has an inhibitory) effect (4 h in females). *, P < 0.05 vs. vehicle control.

 


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  		Figure 7. GnRH neurons express IGF-1, and this increases postnatally. The percentages of GnRH perikarya that colocalize IGF-1 in neonatal (n = 4 and 4), peripubertal (n = 4 and 4), and adult (n = 5 and 6) male and female mice are shown. The number of GnRH neurons coexpressing IGF-1 increases significantly with reproductive development. *, P < 0.001 vs. all other age groups. Data shown are mean ± SEM.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Experiment 1: Developmental changes in IGF-1 mRNA levels in the POA-AH and MBH-ME
Neonatal period.
We previously reported that GnRH gene transcription increases substantially during the neonatal period (19). Therefore, a detailed examination of IGF-1 gene expression was performed in neonatal mice to determine any temporal relationships between these systems. IGF-1 mRNA levels in the POA-AH and MBH-ME, regions that contain GnRH perikarya and terminals, were quantified in male and female mouse pups at P0, P5, P10, P15, and P20 (n = 7, 10, 9, 7, and 13 for males and n = 9, 10, 11, 12, and 9 for females, respectively) using the RPA. ANOVA indicated that there was a significant effect of age (P < 0.001) but no significant effect of sex, and no interaction of age and sex was observed. Post hoc analysis on changes in IGF-1 mRNA levels with age in POA-AH demonstrated that levels were significantly elevated at P5, compared with all other ages (P < 0.001; Fig. 1Go). In the MBH-ME, IGF-1 mRNA levels were significantly higher at P0, compared with all other ages (P < 0.0001; Fig. 1Go). Based on these results in the POA-AH, we chose the P5 age group for comparison with older developmental stages (peripubertal and adult periods) in subsequent experiments.



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  		Figure 1. IGF-1 mRNA levels in neonatal mice. IGF-1 mRNA levels in the POA-AH and MBH-ME were quantified using RPAs. Male and female mice were used at P0, 5, 10, 15, and 20. Mean IGF-1 mRNA levels [normalized to cyclophilin (cycl.) mRNA levels] ± SEM are indicated. IGF-1 mRNA levels in the POA-AH are significantly higher on P5 than all other early developmental ages tested, whereas IGF-1 mRNA levels in the MBH-ME peak at P0. *, P < 0.05 vs. all other ages.

 
Peripubertal period.
Based on previous reports that demonstrated a developmental increase in IGF-1 in the hypothalamus of developing rats (26) and significantly elevated IGF-1 levels at late proestrus in pubertal rats (7), we extended our study to other reproductively relevant time points, namely to peripubertal and reproductively mature adult mice. For the peripubertal period, we used the age at PPS in males and VO in females, because these are landmarks of peripubertal development (17, 18). In our colony, PPS and VO occurred at an average of P30 in both male and female mice.

IGF-1 mRNA levels were determined in the POA-AH and MBH-ME of neonatal (P5; n = 6 and 6), peripubertal (~P30; n = 8 and 5), and adult (P60; n = 7 and 8) male and female mice, respectively (Fig. 2Go). ANOVA indicated that there was a significant effect of age (P < 0.001), but no effect of sex or interaction of age and sex, in both brain regions. Post hoc analysis indicated that IGF-1 mRNA levels in the POA-AH were significantly higher in adult P60 mice, compared with the younger ages (P < 0.001; Fig. 2Go). Hence, in both the POA-AH and the MBH-ME regions, age-related increases in IGF-1 mRNA levels were observed.



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  		Figure 2. IGF-1 mRNA levels in neonatal, peripubertal, and adult mice. IGF-1 mRNA levels in the POA-AH and MBH-ME of neonatal (P5), peripubertal (~P30), and adult (P60) male and female mice were quantified using RPAs. IGF-1 mRNA (normalized to cycl. mRNA) levels in the POA-AH and MBH-ME of adult (P60) male and female mice are significantly higher than levels observed in neonatal (P5) and/or peripubertal mice. *, P < 0.01 vs. the other two ages; **, P < 0.01 vs. P5.

 
Experiment 2: IGF-1 effects on GnRH gene expression
IGF-1 regulation of GnRH mRNA levels in neonatal, peripubertal, and adult mice.
IGF-1 stimulates GnRH release in vivo (7) and GnRH gene expression in immortalized GnRH cell lines (11, 12). We tested whether IGF-1 may act directly in the hypothalamus to activate GnRH gene expression using explanted mouse POA-AHs in vitro. POA-AHs of neonatal (P5, n = 62 and 56), peripubertal (~P30, n = 48 and 41), and adult (P60, n = 39 and 50) male and female mice were perifused with IGF-1 or vehicle for 0, 1, 2, or 4 h, and GnRH mRNA levels were measured by RPA.

ANOVA indicated that there was a significant effect of age (P < 0.0001) and treatment (P < 0.0005), but no effect of sex (P = 0.16), on GnRH mRNA levels. There were several significant interactions, including that between age and treatment (P < 0.005), as well as significant interactions among all four variables (age, sex, treatment, and time; P < 0.01). Post hoc analysis showed that in P5 males, IGF-1 significantly stimulated GnRH mRNA levels at 2 h (P < 0.01; Fig. 3AGo). In peripubertal (~P30) female mice, IGF-1 significantly stimulated GnRH mRNA levels at 4 h (P < 0.01; Fig. 3BGo). In adult male and female mice (P60), IGF-1 had no significant effect on GnRH mRNA levels at all times tested (P = 0.7, Fig. 3CGo).

IGF-1 regulation of GnRH primary transcript levels in neonatal, peripubertal, and adult mice.
We reasoned that the rapid actions of IGF-1 on GnRH mRNA levels (seen at 2 h in P5 male mice and at 4 h in peripubertal female mice) may be attributable either to an enhancement of GnRH gene expression, an increase in GnRH mRNA stability, or both. Thus, we quantified GnRH primary transcript levels, because detection of the full-length primary transcript indicates the unprocessed GnRH pro-mRNA that is transcribed directly off the GnRH gene, before intron excision (21). The nuclear fractions of the POA-AH dissections from the same P5 (n = 62 and 40), ~P30 (n = 49 and 42), and P60 (n = 36 and 45) male and female mice used for measurements of GnRH mRNA in the cytoplasm, and which had been perifused with IGF-1 or vehicle for 0, 1, 2, or 4 h, were used to quantify GnRH primary transcript by RPA. Differences in n-values between cytoplasmic and nuclear fractions are attributable to the loss of some samples during RNA extraction or in the RPA.

ANOVA indicated that there was a significant effect of age (P < 0.0001), but no effect of IGF-1 treatment (P = 0.75) or sex (P = 0.09), on GnRH primary transcript levels. Several significant interactions were detected by ANOVA, including interactions of age and sex (P < 0.05), age and treatment (P < 0.05), and among all four variables (age, sex, treatment, and time; P < 0.01). Post hoc analysis indicated that IGF-1 significantly increased GnRH primary transcript levels in P5 males at 2 h (P < 0.02; Fig. 4AGo) and in both peripubertal males (at 4 h: P < 0.01; Fig. 4BGo) and females (at 2 h: P < 0.05; Fig. 4BGo). In adult (P60) mice, IGF-1 did not stimulate GnRH primary transcript significantly; and surprisingly, IGF-1 significantly decreased GnRH primary transcript at 4 h in P60 female mice (P < 0.001; Fig. 4CGo).

Experiment 3: GnRH neurons express IGF-1, and this increases developmentally
Immunofluorescence studies were performed to study the anatomical relationship between IGF-1 and GnRH in the POA-AH and to test the hypothesis that GnRH neurons express IGF-1 and that this is under developmental regulation. These studies were based on the observations that IGF-1 is widely expressed in the brain and that immortalized murine GnRH neurons (GT-7 cells) and GnRH neurons in adult rats express IGF-1 (11, 24).

Immunocytochemical experiments demonstrated that IGF-1 immunoreactivity is detectable throughout the OVLT, POA-AH, and MS-LS of neonatal (n = 4 and 4), peripubertal (n = 4 and 4), and adult (n = 5 and 6) male and female mice. Although we did not quantify signal intensity, in all the six sections examined per animal, we noted qualitative increases in IGF-1 immunostaining during development throughout the hypothalamus, POA, and septum (Fig. 5Go). In neonatal P5 mice, staining was light, rather diffuse, and mainly observed in the neuropil (Fig. 5Go, B and F). In peripubertal (~P30) mice, there seemed to be an increase in both the number of immunoreactive cells and the intensity of staining within these cells but a reduction of immunoreactivity in the neuropil (Fig. 5Go, C and G). In adult P60 mice, the intensity of immunoreactivity varied from light to very heavy within cells, with the most intensely labeled cells located within the MS and POA (Fig. 5Go, D and H). It is important to emphasize that these observations are purely descriptive but were consistently made in all sections from all of the animals.



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  		Figure 5. Single-label immunohistochemistry for IGF-1 in hypothalamic and preoptic regions. Low-power photomicrographs (A–D, 16x objective; and E–H, 25x objective on the confocal microscope) showing immunoreactivity for IGF-1 (labeled in green with FITC) in septal (A–D) and preoptic (E–H) regions of neonatal (P5), peripubertal (~P30), and adult (P60) mice. Control tissues incubated with the antigen to IGF-1 (1 µg/ml) are shown in A and E, demonstrating no specific staining. The intensity of staining, as well as the number of IGF-1 immunoreactive cells, increases during development. IGF-1 immunoreactivity in P5 mice is rather diffuse and mainly restricted to the neuropil (B and F). The intensity of staining and number of IGF-1-positive cells increase dramatically during the peripubertal period, and IGF-1 immunoreactivity is found within neurons and glia in peripubertal animals (C and G). IGF-1 immunostaining is abundant in the POA-AH and MS of adult mice (D and H). CC, Corpus callosum.

 
Double-label immunohistochemical studies, using antibodies to GnRH and IGF-1, were carried out in the same P5 (n = 4 and 4), peripubertal (n = 4 and 4), and P60 (n = 5 and 6) male and female mice. Six sections, distributed evenly through the POA-AH, OVLT, and septal regions, were counted per animal. Representative micrographs, showing double-labeling of GnRH perikarya with IGF-1 (A–I) and single-labeled GnRH cells that did not express IGF-1 (J–L), are shown in Fig. 6Go. Totals of 353, 70, and 153 GnRH neurons were identified in the P5, ~P30, and P60 male mice; and 251, 52, and 95 GnRH neurons were counted in the female mice of the same ages, respectively. We found that GnRH perikarya colocalize IGF-1, and ANOVA demonstrated that this increases developmentally. In the males, 16% (58 of 353), 31% (22 of 70), and 74% (113 of 153) of the GnRH perikarya colocalized IGF-1 at P5, ~P30, and P60, respectively (Fig. 7Go). In females, 20% (49 of 251), 52% (27 of 52), and 85% (81 of 95) of the GnRH perikarya coexpressed IGF-1 at P5, ~P30, and P60, respectively (Fig. 7Go). ANOVA indicated that there was a significant effect of age on the number of GnRH neurons that coexpressed IGF-1 per animal (P < 0.001), but there was no effect of sex (P = 0.12) and no significant interaction of age and sex (P = 0.69). Hence, GnRH neurons express IGF-1, and this increases with age.



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  		Figure 6. Dual-label immunohistochemistry for GnRH and IGF-1 (A–L) in hypothalamic and preoptic regions. High-power photomicrographs (60x objective, zoom 3 on the confocal microscope) of representative tissues showing double-label immunohistochemistry for GnRH (shown in A, D, G, and J, labeled with Texas Red), IGF-1 (shown in B, E, H, and K, labeled with FITC), and a double exposure (C, F, I, and L). A–C are from a neonatal mouse, D–F from a peripubertal mouse, and G–I from an adult mouse, showing a representative GnRH neuron that is double-labeled with IGF-1. J–L show a representative single-labeled GnRH neuron (that does not express IGF-1) from an adult mouse.

 
We also mapped the distribution of GnRH neurons that coexpressed IGF-1 in developing mice onto a representation of the OVLT/POA from a mouse brain atlas (23). Similar to a previous study in rats (24), most GnRH cells expressing IGF-1 in mice were found to lie in the more medial regions examined. Although we did not note any differences between males and females in the distribution of GnRH neurons that coexpressed IGF-1, we observed age-related shifts in the distribution, with P5 mice of both sexes having most double-labeled cells in dorsal regions, peripubertal mice having these cells in more ventral regions, and adult mice having these double-labeled cells distributed both dorsally and ventrally (Fig. 8Go). This suggests a developmental dorsal-ventral shift in the population of GnRH neurons that express IGF-1.



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  		Figure 8. Spatial distribution of GnRH neurons that coexpress IGF-1. Schematic representation of GnRH neurons that are IGF-1-positive in neonatal (A), peripubertal (B), and adult (C) mice. Data are combined for both males and females because no sex differences were observed. Cells are represented by circles on a mouse brain atlas coronal section at the level of the OVLT/POA and septum. GnRH neurons that coexpress IGF-1 are concentrated more medially throughout the POA-OVLT, and they exhibit a developmental shift in their dorsal-ventral distribution, being more dorsally concentrated in P5 mice and more evenly distributed across the rostral-caudal continuum in peripubertal and adult mice. Note: Not all cells may be visible, because of overlapping cells. AC, Anterior commissure.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results provide novel and fundamental information about how the neurotrophic factor IGF-1 influences the GnRH system during two key reproductive periods: the neonatal and peripubertal developmental stages. The data indicate that central IGF-1 gene expression changes before or during those periods when GnRH gene expression undergoes its greatest developmental changes; that IGF-1 can stimulate GnRH gene expression in neonatal and peripubertal, but not adult mice; and that GnRH neurons express IGF-1, and this is developmentally regulated. Moreover, these changes in hypothalamic IGF-1 gene and protein expression have implications for the development of other neuroendocrine and autonomic systems whose perikarya and/or neuroterminals may also be regulated by IGF-1.

Several studies have indicated a role for peripheral IGF-1 in the initiation and progression of rodent puberty (7). However, because IGF-1 crosses the blood-brain barrier (27), identification of the origin of IGF-1 (peripheral vs. central) regulating the GnRH/LH-releasing system has been difficult. Although several groups have produced knockout mice for IGF-1 or the IGF-1 receptor genes (13, 14), homozygous IGF-1 knockout mice almost always die at birth, and the few that survive have profound developmental defects (14) and are difficult to study with respect to reproductive development. Interestingly, Liu et al. (28) generated a conditional knockout mouse that lacks IGF-1 specifically in the liver, the primary site of peripheral IGF-1 production; and they found that although circulating and serum levels of IGF-1 were decreased by approximately 70% in these mice, these animals exhibited no developmental defects and were fertile. These results suggest that liver-derived IGF-1 may be relatively unimportant in regulating reproductive function. These observations led us to investigate the expression of IGF-1 in the hypothalamus and its regulation of GnRH neurons.

IGF-1 gene expression changes during reproductive development
In the first set of studies, we looked at the developmental expression of hypothalamic IGF-1 mRNA during the early postnatal period. We chose to study the neonatal period, because it is associated with substantial amounts of neurogenesis, synaptogenesis, and gliogenesis and is a critical period for sexual differentiation of the brain in rodents (29, 30). Furthermore, IGF-1 influences neuronal generation, survival, and/or differentiation in other regions of the rodent neonatal/developing brain (31). In the present study, IGF-1 mRNA levels were significantly elevated at P5 in the POA-AH and at P0 in the MBH-ME of neonatal male and female mice. This peak in IGF-1 gene expression in the POA-AH at P5 occurs just before the increase in GnRH gene expression in this same brain region (from P5-P10) reported by our laboratory and others (19, 32), and this suggests the possibility that the increase in IGF-1 mRNA levels at P5 may even be related to the increase in GnRH gene transcription from P5–P10 in mice (19, 32).

Based on previous reports showing a developmental increase in IGF-1 in the hypothalamus of developing rats (26) and significantly elevated IGF-1 levels at late proestrus in pubertal rats (7), we extended our study to peripubertal and reproductively mature, adult mice. In both POA-AH and MBH-ME of male and female mice, IGF-1 mRNA levels were higher in adults than in P5 or peripubertal mice. Taken together with our data on neonatal development, our results indicate that hypothalamic IGF-1 gene expression peaks transiently during the neonatal period, decreases through P20, and then increases again during puberty and through adulthood. This pattern of expression is remarkably similar to that of GnRH release, which undergoes a transient increase neonatally, decreases during the prepubertal hiatus, and then increases again during pubertal maturation. The developmental pattern of hypothalamic IGF-1 mRNA expression also resembles that of another neurotrophic factor (TGF{alpha}), which is also implicated in the sexual maturation of mammals. In rodents, TGF{alpha} mRNA expression demonstrates a neonatal (P12) and peripubertal peak (33). Therefore, it is likely that the increases in the expression of the neurotrophic factors IGF-1 and TGF{alpha} may be related to (or even contribute to) the maturation of GnRH gene expression and release during development.

IGF-1 stimulates GnRH gene expression in neonatal and peripubertal (but not adult) mice
In the second experiment, we tested whether IGF-1 could affect GnRH gene expression, and we observed some stimulatory effects of IGF-1 in neonatal and peripubertal mice depending on age, sex, and duration of treatment. At P5, IGF-1 stimulated both GnRH mRNA and primary transcript levels in male mice after 2 h of treatment. Thus, the increase in GnRH mRNA levels at this time is probably attributable to a concomitant activation of GnRH gene transcription (21). Female mice at P5 did not respond to IGF-1 with a change in GnRH gene expression, indicating a sex difference that may be caused by differences in sex steroid hormones or in the regulation of GnRH gene expression itself, which differs between male and female mice at P5 (19). In peripubertal female mice, IGF-1 treatment resulted in elevated GnRH mRNA levels (at 4 h) and GnRH primary transcript levels (at 2 h). Again, the increase in GnRH mRNA levels is likely attributable to a stimulation of GnRH gene transcription that precedes the increase in GnRH mRNA levels. In peripubertal males, IGF-1 stimulated GnRH gene transcription (at 4 h) but had no effect on GnRH mRNA; however, it is possible that elevated GnRH mRNA levels may have occurred at a later time point, which was not studied here. We feel that these sex and timing differences are relatively minor, our major conclusion being that IGF-1 can stimulate GnRH gene expression in neonatal and peripubertal male and female mice and that the mechanism for the increase in GnRH mRNA levels probably involves an activation of GnRH gene transcription. These results are also consistent with those from GT1–7 cells, in which we reported that IGF-1 can stimulate GnRH mRNA, probably through increased GnRH gene transcription (11).

In adult female mice, GnRH primary transcript levels were significantly lower in adult female mice exposed to IGF-1 for 4 h. We did not see such an effect in adult males, in which IGF-1 did not alter GnRH gene expression; and though we do not know the mechanism for the sex difference, it may involve differences in other hormones or sex differences in the regulation of the GnRH system. The age dependency of the IGF-1 effect on GnRH gene expression, which is stimulatory in neonatal and peripubertal mice, but is inhibitory or has no effect in adults, is similar to that reported previously in rats (34). Bourguignon et al. showed that IGF-1 inhibited GnRH secretion from hypothalamic explants of 50-d-old adult rats but not from immature 15-d-old explants. Moreover, a recent study showed that the direct action of {gamma}-aminobutyric acid (GABA) on GnRH neurons switches from depolarization to hyperpolarization around the time of puberty in female rats (35), presumably through a K+/Cl- cotransporter, which plays a role in neuronal maturation (36). Interestingly, in the hippocampus, a similar developmental switch of GABAergic responses to hyperpolarizing inhibition is accelerated by IGF-1, resulting in neuronal maturation (37). Hence, it is possible that IGF-1 causes the maturation of GnRH neurons through a similar mechanism, although this remains to be tested in future experiments.

Developmental changes in the coexpression of IGF-1 in GnRH neurons
In the third experiment, we analyzed the expression of IGF-1 immunoreactivity in the hypothalamus. IGF-1 immunoreactivity is robustly expressed throughout the POA-AH, OVLT, and septal regions. Although we did not quantify the intensity of IGF-1 immunostaining, qualitative observations consistently suggested that IGF-1 immunoreactivity increases developmentally. This increase in protein expression is consistent with our observation of maturational increases in IGF-1 mRNA levels, suggesting that IGF-1 gene transcription and translation occur roughly in parallel. However, in other areas of the brain, such as olfactory bulb, cortex, hippocampus, and amygdala, developmental decreases in IGF-1 have been reported (38, 39). Thus, there are regional differences in the developmental pattern of IGF-1 expression.

Double-labeling studies enabled us to address whether the IGF-1 is expressed specifically in GnRH neurons, and whether this is under developmental regulation. We previously reported that GnRH neurons of adult rats express IGF-1, but we did not investigate this in a developmental context (24). Here, we found that GnRH neurons of male and female mice express IGF-1 and that this is developmentally regulated, increasing from approximately 18% to 80% from P5 through adulthood. No sex differences in the coexpression of IGF-1 in GnRH neurons were found in mice of any age.

We also noted a developmental shift in the spatial distribution of the GnRH/IGF-1 double-labeled neurons. Although GnRH neurons were localized throughout the POA-AH, OVLT, and septum, those GnRH perikarya that coexpressed IGF-1 were concentrated more medially with a developmental change in the dorsal-ventral distribution of these cells. It has been suggested that different spatial populations of GnRH neurons may be heterogeneous in function; for example, their medial-lateral spatial distribution and activation change during reproductively relevant periods (40, 41). Although we do not know whether the dorsal-ventrally distributed cells represent functionally distinct subpopulations, this is certainly possible. Consistent with this, a recent study in the male hamster indicated that testosterone preferentially regulated GnRH gene expression in the most dorsal population of cells (42).

Although the role of IGF-1 in GnRH neurons is still unknown, its presence can be explained by two possibilities: first, GnRH neurons may retrogradely transport peripheral IGF-1 to their perikarya via their neuroterminals; alternatively, GnRH neurons may actively synthesize IGF-1. This is not unlikely, because although IGF-1 is produced peripherally, IGF-1 mRNA has been localized in the brain (26, 43, 44) and hypothalamus (45). Also, IGF-1 is colocalized in immortalized GnRH neurons (11) and in other hypothalamic neurosecretory neurons of the paraventricular and supraoptic nuclei (46). Moreover, preliminary studies, in our laboratory, using postembed immunogold electron microscopy, indicate the presence of the IGF-1 molecule in secretory vesicles in GnRH processes of female rats (unpublished observation). This would suggest that IGF-1 is synthesized, packaged, and released from GnRH neuroterminals.

If IGF-1 is indeed released by the GnRH neuroterminals, there are several possible mechanisms of action. We propose that, based on the developmental and reproductive state of the animal, IGF-1 may act locally within the ME as an autocrine or paracrine factor. This IGF-1 may even regulate the same GnRH neuroterminals from which it was released. This is supported by studies demonstrating that neurotrophic factors, including IGF-1, mediate synaptic remodeling in cycling rats (47). Alternatively, or in addition, IGF-1 released into the ME may act in a paracrine manner, acting locally on other neuroterminals in this region. Support for either of these two possibilities (autocrine or paracrine effects of IGF-1) is provided by reports that IGF-1 receptors are localized on neurons within the MBH-ME (48, 49, 50). Finally, IGF-1 could travel through the portal capillaries to the anterior pituitary gland, affecting gonadotropes and/or other cells within the pituitary; and indeed, the pituitary expresses IGF-1 receptors (51).

If IGF-1 acts as an autocrine regulator on GnRH cells, this may explain some of the developmental differences in effects of IGF-1 on GnRH gene expression and/or release. In adults, IGF-1 could exert negative feedback, whereby increased IGF-1 expression down-regulates the initial stimulation of GnRH gene expression and/or release. The inhibitory component of this regulation may occur through the actions of a cleavage product of IGF-1, the truncated N-terminal tripeptide (1, 2, 3) IGF-1, which can antagonize N-methyl D-aspartate receptors, the end result of which is a decrease in GnRH gene expression and/or release (34). In neonatal and peripubertal rats, in which IGF-1 gene and protein expression in the hypothalamus and coexpression of IGF-1 in GnRH cells is lower, IGF-1 may not have this negative effect and may even exert stimulatory effects. Future studies will attempt to elucidate whether GnRH neurons express IGF-1 receptors, and their specific localization on GnRH perikarya and/or nerve terminals, thereby providing a better understanding of the mechanisms that govern the IGF-1 regulation of GnRH neurons and the progression of reproductive maturation.


   Acknowledgments
 
We would like to acknowledge Bill Janssen, Twethida Oung, Daita Ciobanu, Sara Farrell, and Jonathan Steiglitz for technical assistance.


   Footnotes
 
This work was supported by funding from the National Science Foundation (IBN-0110047).

Abbreviations: AH, Anterior hypothalamus; DNase, deoxyribonuclease; FITC, fluorescein isothiocyanate; GABA, {gamma}-aminobutyric acid; LS, lateral septum; mA2B, mouse GnRH fragment complementary to the intron A-exon 2-intron B; MBH, mediobasal hypothalamus; ME, median eminence; MS, medial septum; NA, numerical aperture; OVLT, organum vasculosum of the lamina terminalis; P, postnatal day; POA, preoptic area; PPS, preputial separation; RNase, ribonuclease; RPA, RNase protection assay; VO, vaginal opening.

Received October 23, 2002.

Accepted for publication January 17, 2003.


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 Introduction
 Materials and Methods
 Results
 Discussion
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G. Bienvenu, D. Seurin, P. Grellier, P. Froment, M. Baudrimont, P. Monget, Y. Le Bouc, and S. Babajko
Insulin-Like Growth Factor Binding Protein-6 Transgenic Mice: Postnatal Growth, Brain Development, and Reproduction Abnormalities
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