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. 2010 Jan 22;151(3):1356–1366. doi: 10.1210/en.2009-1009

Hypothalamic Insulin-Like Growth Factor-I Receptors Are Necessary for Hormone-Dependent Luteinizing Hormone Surges: Implications for Female Reproductive Aging

Brigitte J Todd 1,a, Zaher O Merhi 1,a, Jun Shu 1, Anne M Etgen 1, Genevieve S Neal-Perry 1
PMCID: PMC2840696  PMID: 20097715

Abstract

Brain IGF-I receptors are required for maintenance of estrous cycles in young adult female rats. Circulating and hypothalamic IGF-I levels decrease with aging, suggesting a role for IGF-I in the onset of reproductive senescence. Therefore, the present study investigated potential mechanisms of action of brain IGF-I receptors in the regulation of LH surges in young adult and middle-aged rats. We continuously infused IGF-I, the selective IGF-I receptor antagonist JB-1, or vehicle into the third ventricle of ovariectomized young adult and middle-aged female rats primed with estradiol and progesterone. Pharmacological blockade of IGF-I receptors attenuated and delayed the LH surge in young adult rats, reminiscent of the LH surge pattern that heralds the onset of reproductive senescence in middle-aged female rats. Infusion of IGF-I alone had no effect on the LH surge but reversed JB-1 attenuation of the surge in young females. In middle-aged rats, infusion of low doses of IGF-I partially restored LH surge amplitude, and infusion of JB-1 completely obliterated the surge. Intraventricular infusion of IGF-I or JB-1 did not modify pituitary sensitivity to exogenous GnRH or GnRH peptide content in the anterior or mediobasal hypothalamus in either young or middle-aged rats. These findings support the hypothesis that brain IGF-I receptor signaling is necessary for GnRH neuron activation under estrogen-positive feedback conditions and that decreased brain IGF-I signaling in middle-aged females contributes, in part, to LH surge dysfunction by disrupting estradiol-sensitive processes that affect GnRH neuron activation and/or GnRH release.

Female reproduction and the generation of an appropriately timed pre-ovulatory luteinizing hormone surge depend upon estradiol mediated modulation of brain IGF1 synthesis, IGF receptor activation, and GnRH-LH release.

Estradiol (E2) and progesterone (P) act sequentially in the brain to coordinate female reproductive physiology and behavior. A growing body of evidence indicates that a number of E2-mediated processes in the brain require concomitant signaling by IGF-I receptors. Estrogen receptors and IGF-I receptors are colocalized in neurons and glia throughout the brain (1,2). Notably, hypothalamic GnRH neurons express both IGF-I and IGF-I receptors (3,4). Pharmacological antagonism of brain IGF-I receptors abolishes estrous cyclicity, and this action is not attributable to changes in food intake or metabolism (5). Brain IGF-I receptor blockade also impairs estrogen-positive feedback, reducing E2- and P-dependent LH release (6) and inhibiting hormone-dependent reproductive behavior in ovariectomized female rats (7). Intracerebroventricular (icv) infusion of an IGF-I receptor antagonist also prevents estrous cycle-associated fluctuations in synaptic structures in the arcuate nucleus (8,9). The site(s) and mechanism(s) of IGF-I action in the regulation of female reproduction remain unclear. However, recent findings from IGF-I null mice carrying a transgene that overexpresses IGF-I in the liver, thereby restoring circulating IGF-I, are informative (10). Restoration of circulating IGF-I rescued adult body weight and carbohydrate metabolism, but female reproductive function remained impaired. Thus, it is likely that local production of IGF-I in reproductive tissues, including the neuroendocrine brain, is critical for normal fertility in females.

Female reproductive senescence in rodents is typified by delayed and attenuated preovulatory LH surges (11). Central and peripheral IGF-I decline with aging (12,13), but IGF-I receptor levels in the hippocampus and cerebral cortex increase in senescent rats (14). Hence, it is likely that the aging brain retains responsiveness to IGF-I, and it is possible that LH surge dysfunction is related to reduced IGF-I availability in the brains of older female rats.

The purposes of the present experiments were to determine whether brain IGF-I signaling regulates the female reproductive axis in young adult and middle-aged rats at the level of the hypothalamus or the pituitary and to determine whether elevating brain IGF-I levels restores hormone-dependent LH surges in middle-aged rats. We used continuous icv infusion of IGF-I and JB-1, a specific IGF-I receptor antagonist (15), to amplify or interfere with brain IGF-I receptor signaling during steroid hormone priming in ovariectomized female rats. Our findings provide evidence that brain IGF-I signaling is required for E2 modulation of GnRH-LH release on the day of the LH surge and that reduced brain IGF-I signaling, in part, contributes to age-related LH surge dysfunction.

Materials and Methods

Animals

Young (3–4 months) and middle-aged (retired breeders, 9–11 months) adult female Sprague Dawley rats (Taconic Farms, Germantown, NY) were housed individually and maintained on a 14-h light, 10-h dark cycle with free access to chow and tap water. Daily vaginal smears were used to monitor estrous cycles. Only rats with at least two regular 4- to 5-d estrous cycles were ovariohysterectomized (OVX) and studied. All procedures followed the National Institutes of Health Guide for the Care and Use of Laboratory Rats and were approved by the Institutional Animal Care and Use Committee at the Albert Einstein College of Medicine.

Stereotaxic surgery and osmotic minipump placement

Rats anesthetized with a ketamine/xylazine mixture (80 and 4 mg/kg, respectively, im) were OVX and placed in a Kopf stereotaxic apparatus with the nosebar set at +5.0 mm. A 22-gauge guide cannula (Plastics One, Roanoake, VA) was placed into the third ventricle (A/P +0.2 mm; M/liter +0.0 mm; D/V −7.8 mm with respect to bregma) and anchored with dental acrylic. An osmotic minipump (Alzet model 2002, 14 d delivery; flow rate 0.5 μl/h; Durect Corp., Cupertino, CA) was implanted and used to deliver drugs or vehicle as previously described (5). Rats were allowed to recover for 5–7 d before further manipulations. Correct guide cannula placement was verified at the end of all experiments by injection of dye into the third ventricle.

Intracerebroventricular drug administration

Rats received continuous infusion of drugs or vehicle via osmotic minipumps. The vehicle used to dissolve drugs, and infused into all control rats, was artificial cerebrospinal fluid [aCSF: 140 mm NaCl, 3 mm KCl, 1.2 mm Na2HPO4, 1 mm MgCl2, 0.27 mm NaH2PO4, 1.2 mm CaCl2, and 7.2 mm dextrose (pH 7.4)]. IGF-I receptors were blocked by infusion of JB-1 (20 or 100 μg/ml, depending on experiment; Bachem, San Carlos, CA), a peptide analog corresponding to the recognition motif or D domain within the carboxy terminus of IGF-I that binds the IGF-I receptor. JB-1 effectively blocks IGF-I receptor-mediated autophosphorylation and cellular proliferation in cultured cells (15) and IGF-I potentiation of norepinephrine-stimulated cAMP formation in rat hypothalamic slices (6). Human IGF-I (0.5–10 μg/ml; Bachem or Gropep, Adelaide, Australia) was infused into the third ventricle alone or together with JB-1. The doses of JB-1 and IGF-I used were modified from regimens previously used in our laboratory in which IGF-I completely reversed JB-1 suppression of estrous cycles in ovary-intact female rats (5).

Jugular vein catheterization

In experiments in which blood sampling was performed, rats were anesthetized 5–7 d after OVX and stereotaxic surgery, and an indwelling catheter was placed into the right atrium via the right jugular vein for serial blood sampling (16). Catheters were kept patent by daily flushing with heparinized saline (50 U/ml). Blood collection was done 7–9 d after OVX.

Ovarian steroid administration

To induce LH surges, E2 benzoate (EB) and P (Steraloids Inc., Newport, RI) were dissolved in peanut oil and administered sc in a volume of 0.1 ml. At 0900 h on the day the jugular vein catheter was placed, rats received the first of two daily injections of 2 μg of EB. At 0900 h on the morning of blood collection (2 d after the first EB injection), rats were injected sc with 500 μg P. This hormone regimen reliably produces LH surges in OVX female rats (6,16). In experiments investigating pituitary responsiveness to GnRH or tissue GnRH content, EB (2 μg, sc) or peanut oil vehicle was injected at 48 and 24 h before the experiment.

Blood collection

On experimental days, blood (300 μl per sample) was collected from awake, freely moving rats starting at the time of P administration and every 1–2 h thereafter for a total of nine to 10 samples. Each sample volume was replaced with warm, sterile, heparinized saline (15 U/ml). Collected plasma was stored at −20 C until RIA. In the experiment assessing pituitary responsiveness to GnRH, blood was collected at 30-min intervals beginning at 0900 h, for a total of eight to none samples. GnRH acetate (25 and 100 ng; Sigma, St. Louis, MO) was administered iv after the third and sixth sample, respectively. These doses of GnRH have been used to test pituitary responsiveness to GnRH in other studies (17).

LH and prolactin

Plasma LH was determined in duplicate with rat double-antibody assays using reagents provided by the National Institute of Diabetes and Digestive and Kidney Diseases National Hormone and Pituitary Program (Torrance, CA; rLH-RP-3 and rPRL-RP-3 reference preparations). The lower limit of the LH assay, which was done by the Northwestern University Assay Core, was 0.2 ng/ml, and the intra- and interassay coefficients of variation were 2.85 and 8.65%, respectively. The lower limit of the prolactin assay was 1 ng/ml, and all samples were analyzed in a single assay by Dr. Arthur Parlow (National Hormone and Pituitary Program). An LH surge was defined as an increase in serum LH levels 1.5 times greater than baseline for a minimum of two consecutive samples, and surge onset was considered to occur at the first of these samples (16,18). Baseline LH levels were those at the time of P injection.

ELISA for plasma IGF-I

Plasma IGF-I levels were determined in duplicate using an ELISA for rat/mouse IGF-I from Immunodiagnostic Systems, Ltd. (Tyne & Wear, UK). The lower limit of the assay sensitivity is 63 ng/ml, and there is no cross-reactivity with IGF-II. The intra- and interassay coefficients of variation were 8.8 and 6.8%, respectively, at the midrange of the assay.

Determination of tissue GnRH content

For assessment of GnRH content in the hypothalamus, a tissue block containing the entire hypothalamus was rapidly dissected and then separated at the level of the optic chiasm into anterior hypothalamus [containing the suprachiasmatic nuclei and the preoptic area (POA)] and mediobasal hypothalamus (MBH; containing the ventromedial and lateral hypothalamus, arcuate and median eminence). Tissue was homogenized in 300 μl of ice-cold 0.1 n HCl using a glass homogenizer, followed by trituration. The homogenate was centrifuged for 15 min at 13,000 × g at 4 C and the supernatant recovered for GnRH determination. GnRH peptide content was determined by the Northwestern University Assay Core using an antibody supplied by Dr. Terry Nett (Colorado State University, CO). GnRH for standards and iodination was purchased from Sigma, and the tracer used was 125I-LHRH (GE Healthcare, Piscataway, NJ). The lower limit of the assay detection was 0.67 pg/ml, and the intraassay coefficients were 9.14, 4.91, and 3.10% for the low, medium, and high pools, respectively. The interassay coefficient of variation was 9.34%. The pellet was resuspended in saline and protein levels determined by Bradford assay.

Data analysis

All data are expressed as mean ± sem. LH data were analyzed using repeated-measures ANOVA with Fisher’s protected least-squares difference post hoc test when initial significant differences were found. Area under the curve (AUC) for total LH release was calculated using SigmaPlot (Systat Software Inc., San Jose, CA), and differences in AUC analyzed using group factorial ANOVA with Fisher’s protected least-squares difference post hoc test. All other data were analyzed using group factorial one-way or two-way ANOVA or Student’s t test, depending on numbers of groups and variables. The criterion for statistically significant differences was P < 0.05.

Results

Body weight and plasma IGF-I

As reported previously (5), young rats lost up to 15% of their body weight after OVX and minipump placement. However, their average body weights at the time of plasma or tissue collection were not significantly different from initial body weights in any treatment group. Middle-aged females lost up to 12% of their body weight after OVX and minipump placement. Body weights in middle-aged rats at the time of blood sampling or tissue collection had not completely recovered to presurgical baseline values, but the degree of weight loss did not correlate with treatment (control, IGF-I, or combined IGF-I + JB-1) or LH surge amplitude. Thus, in agreement with our work on young, ovary-intact females (5), it is unlikely that the IGF-I antagonist interfered with the reproductive axis by affecting food intake or body weight or that IGF-I at the doses infused in this study affects nutritional status in middle-aged rats.

Because we did not have sufficient plasma to measure circulating IGF-I levels as well as plasma LH, IGF-I was measured by ELISA in separate young and middle-aged animals that were OVX and treated with vehicle or the same EB plus P treatment used for LH surge induction (n = 6–10/group). There was a significant interaction between age and hormone treatment (F = 29.8, P < 0.0001). In young OVX females, E2 priming significantly increased circulating IGF-I levels from 781 ± 89 to 1116 ± 47 μg/ml (P < 0.05), whereas in middle-aged OVX rats, the same E2 treatment significantly decreased plasma IGF-I levels from 1309 ± 75 to 911 ± 52 μg/ml (P < 0.05). We noted similar age differences in hormonal modulation of plasma IGF-I in young and middle-aged females implanted with timed-release pellets of E2 for 2–3 wk before assay (data not shown).

Blockade of brain IGF-I receptors delays and attenuates the LH surge in young rats

When we infused young rats with 20 μg/ml of JB-1, a dose that suppresses estrous cycles in intact females, there was considerable variability in the LH surge induced by E2 and P (data not shown). When we raised the antagonist dose to 100 μg/ml, there was a main effect of brain IGF-I receptor blockade on the LH surge (F = 3.3, P < 0.05; Fig. 1A). The LH surge was significantly attenuated in rats receiving 100 μg/ml of JB-1 icv, and this attenuation of LH release was reversed by coadministration of 10 μg/ml of IGF-I. This is the same ratio of IGF-I to JB-1 (1:10) that reversed the effects of the antagonist on estrous cycles in gonadally intact rats in our previous work (5) and was selected based on the relative affinity of JB-1 and IGF-I for the IGF-I receptor (15). IGF-I alone had no effect on the LH surge in young rats, and JB-1 treatment did not affect baseline LH values at the time of P injection. Total LH released over the day, expressed as the integrated AUC, was also significantly lower in JB-1-treated rats compared with vehicle-infused controls or IGF-I-treated rats (F = 3.6, P < 0.05; Fig. 1B). Coadministration of IGF-I with JB-1 reversed the effects of JB-1 on total LH release. Time to the onset of the LH surge was significantly delayed in JB-1-treated rats compared with all other groups (F = 3.7, P < 0.05; Table 1), and mean peak amplitude of the LH surge was significantly lower in JB-1-treated rats compared with controls (F = 2.5, P < 0.05; Table 1).

Figure 1.

Figure 1

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IGF-I receptor antagonism attenuates the LH surge in young rats; exogenous IGF-I partially restores the LH surge in middle-aged rats. LH surges in young adult (A and B) and middle-aged (C and D) rats receiving continuous icv infusions of JB-1, IGF-I, JB-1 plus IGF-I, or aCSF. Rats were given two daily doses of EB (2 μg) followed by P (500 μg), and blood samples were collected beginning 48 h after the first dose of EB, just before P injection (arrow). A and C show the time course of the LH surge, and B and D show total LH secreted throughout the day. For young rats: aCSF controls, n = 5; JB-1 (100 μg/ml), n = 8; IGF-I (10 μg/ml), n = 5; JB-1 (100 μg/ml) plus IGF-I (10 μg/ml), n = 7. For middle-aged rats: aCSF, n = 6; IGF-I (0.5 μg/ml), n = 4; IGF-I (2 μg/ml), n = 5; IGF-I (5 μg/ml), n = 5; IGF-I (2 μg/ml) plus JB-1 (20 μg/ml), n = 6. Note that in C and D, results shown for young rats are the same as those young aCSF rats shown in A and B. Data represent mean ± sem. Different letters denote statistically significant differences (P < 0.05).

Table 1.

The LH surge is attenuated and delayed in young rats receiving continuous icv infusions of JB-1a

aCSF (n = 5) JB-1 (n = 8) IGF-I (n = 5) JB-1 + IGF-I (n = 7)
Peak LH release (ng/ml) 50.7 ± 5.8 21.8 ± 3.4b 41.0 ± 9.4 33.2 ± 8.8
LH surge onset (hours after P injection) 3.25 ± 0.7 5.25 ± 0.5c 3.2 ± 0.4 3.87 ± 0.3
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a

Data are reported as means ± sem and are from the same experiment shown in Fig. 1. 

b

Significantly different from aCSF and IGF-I (P < 0.05). 

c

Significantly different from all other groups (P < 0.05). 

To determine whether blockade of IGF-I receptors attenuated all ovarian hormone-induced pituitary hormone surges, we measured the prolactin surge in young JB-1-treated rats and controls. JB-1 did not modify E2 and P enhancement of prolactin release in OVX, hormone-treated female rats (Fig. 2).

Figure 2.

Figure 2

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Prolactin surges in young rats are unaffected by blockade of central IGF-I receptors. Prolactin surges in rats receiving continuous icv infusions of JB-1 (100 μg/ml; n = 6) and controls (n = 6). Rats were injected with EB and P as described in the legend to Fig. 1, and blood samples were collected beginning 48 h after the first dose of EB, just before P injection (arrow). Data represent mean ± sem.

Central IGF-I augments the LH surge in middle-aged rats

As previously reported (for review see Ref. 11), middle-aged rats generated delayed and attenuated LH surges (Fig. 1C and Table 2). When compared with middle-aged controls infused with aCSF, middle-aged rats receiving 0.5 and 2 μg/ml of IGF-I exhibited a 2- to 3-fold increase in peak (F = 23.6, P < 0.0001) and total (F = 9.8, P < 0.0001) LH release (Fig. 1, C and D, and Table 1). However, IGF-I did not restore LH surge amplitude to levels seen in young rats nor did it significantly alter the onset of the LH surge. In contrast to the lower doses, the highest dose of IGF-I (5 μg/ml) further delayed (P < 0.05) and attenuated peak LH release (P < 0.05) in middle-aged females compared with aCSF-treated controls.

Table 2.

The LH surge is augmented in middle-aged rats receiving continuous icv infusions of low doses of IGF-Ia

aCSF (n = 6) IGF-I (0.5 μg/ml) (n = 4) IGF-I (2 μg/ml) (n = 5) IGF-I (5 μg/ml) (n = 6)
Peak LH release (ng/ml) 11.5 ± 1.6 19.5 ± 1.9b 22.9 ± 0.7b 6.3 ± 1.0c
LH surge onset (hours after P injection) 6.7 ± 0.4 5.8 ± 0.6 5.6 ± 0.5 8.6 ± 0.5c
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a

Data are reported as means ± sem and are from the same experiment shown in Fig. 1. Note that no rats from the IGF-I + JB-1 group mounted a surge, and thus, we cannot report surge onset or peak data (for comparison with young rats, please refer to Table 1). 

b

Significantly different from aCSF (P < 0.05). 

c

Significantly different from all other groups (P < 0.05). 

To confirm that central infusion of IGF-I increased LH release by acting on IGF-I receptors rather than insulin receptors, additional middle-aged rats (n = 6) were infused with the IGF-I receptor antagonist JB-1 (20 μg/ml) in combination with IGF-I (2 μg/ml). The LH surge was blocked in 83% (five of six) of the rats that received both IGF-I and JB-1 (Fig. 1C). Total LH release was also significantly lower in rats infused with IGF-I plus JB-1 than in controls infused with aCSF (P < 0.05; Fig. 1D).

Blockade of IGF-I receptors does not affect pituitary responsiveness to GnRH in young rats

To ascertain whether central IGF-I receptor blockade affects pituitary sensitivity to GnRH, we performed GnRH challenge experiments (Fig. 3A) (17). We collected nine consecutive blood samples from OVX females treated with vehicle or JB-1. These rats received two injections of EB or oil vehicle 48 and 24 h before the first blood sample taken at 0900 h. Subsequent blood samples were collected every 30 min. Stepwise iv infusions of 25 and 100 ng GnRH acetate were administered after the third and sixth blood sample. In vehicle-treated rats, GnRH significantly increased serum LH in both JB-1-infused rats and controls (F = 6.2, P < 0.01), and there was no difference in the magnitude of LH response between groups. Likewise, in EB-treated rats, GnRH significantly increased serum LH (F = 10.3, P < 0.0001) in both control and JB-1-infused rats.

Figure 3.

Figure 3

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IGF-I regulation of LH release is at the level of the hypothalamus. A, Serum LH responses to iv GnRH in young adult rats receiving continuous icv infusions of JB-1 or aCSF (controls). Rats were infused with JB-1 or IGF-I and injected with EB or oil as described in the legend to Fig. 1. Nine blood samples were taken at 30-min intervals, and GnRH was administered after the third (25 ng) and sixth (100 ng) blood sample. Data represent mean ± sem (n = 4 rats in aCSF/oil group, n = 5 in JB-1/oil group, n = 6 in aCSF/EB group, and n = 6 in JB-1/EB group). *, P < 0.05 difference between hormone-treated rats and oil-treated controls. B, Serum LH responses to iv GnRH in middle-aged rats receiving continuous icv infusions of IGF-I or aCSF (controls). Rats were infused with 2 μg/ml of IGF-I and injected with EB as described in the legend to Fig. 1. Eight blood samples were taken at 30-min intervals, and GnRH was administered as described above. Data represent mean ± sem (n = 4 in both groups). C, LH surges in middle-aged rats receiving continuous icv infusions of IGF-I (2 μg/ml). LH surges were generated with EB and P as described in legend to Fig. 1. The GnRH receptor antagonist cetrorelix (100 μg) or vehicle was injected sc at 0900 h on the day before and the day of the LH surge. Blood samples were collected every 1–2 h. The arrow represents the time of P injection. Data represent mean ± sem (n = 4 rats per group).

Elevating central IGF-I does not affect pituitary responsiveness to GnRH in middle-aged rats

To determine whether icv infusion of IGF-I increased LH release in middle-aged rats by increasing pituitary sensitivity to GnRH, we performed a GnRH challenge experiment in OVX, middle-aged females primed with EB as described above (Fig. 3B). Similar to young rats, GnRH significantly increased serum LH (F = 21.2, P < 0.001) in middle-aged rats infused with the dose of IGF-I that produced the greatest increase in LH (2.0 μg/ml) and controls. There was no difference in the magnitude of LH response between groups.

GnRH receptor antagonism blocks the LH surge in IGF-I-treated middle-aged rats

To determine whether IGF-I facilitation of the LH surge in middle-aged females is mediated by GnRH release, we used pharmacological blockade of GnRH receptors. IGF-I-treated females were injected sc with the GnRH receptor antagonist cetrorelix or vehicle at 0900 h on the day before and the day of the LH surge. IGF-I-treated females injected with vehicle mounted an LH surge; however, none of the rats that received cetrorelix displayed an LH surge (F = 37.5, P < 0.0001; Fig. 3C).

Neither blockade of IGF-I receptors nor exogenous IGF-I affects tissue GnRH content

We measured GnRH peptide content in the anterior hypothalamus and MBH of young and middle-aged females, half of which were primed with EB 48 and 24 h before the experiment. In the MBH of young females (Fig. 4A), E2 significantly increased GnRH peptide content (F = 6.6, P < 0.05). This effect of E2 was not blunted in JB-1-treated rats (Table 3). In the anterior hypothalamus of young females, there were no effects of hormone treatment or IGF-I receptor antagonism on GnRH peptide content (Fig. 4A and Table 3). Similarly, E2 significantly increased GnRH content in the MBH (F = 5.8, P < 0.05) but not the anterior hypothalamus (Fig. 4B) of middle-aged females, and this was not affected by exogenous IGF-I or JB-1 (Table 3). Two-way ANOVA with age and hormone treatment as variables revealed a main effect of age on GnRH peptide content in both the anterior hypothalamus (F = 18.0, P < 0.0001) and MBH (F = 15.7, P < 0.0005), with older rats having significantly higher GnRH levels in both cases.

Figure 4.

Figure 4

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E2 increases GnRH content in the MBH in young and middle-aged females. GnRH content in the anterior hypothalamus and MBH of young (A) and middle-aged (B) rats receiving continuous icv infusions of JB-1 (in young rats, 100 μg/ml; in middle-aged rats, 20 μg/ml), IGF-I (2 μg/ml), or aCSF (controls) and injected with either EB or oil for 2 d. EB increased GnRH content in the MBH in both young and middle-aged rats. Further breakdown of groups treated with IGF-I and JB-1 is shown in Table 3. Data are expressed as picograms per milligram protein and represent mean ± sem (n = 10–12 rats per group in A, n = 16–18 rats per group in B). *, P < 0.05 vs. oil-treated controls.

Table 3.

Effects of E2 priming, exogenous IGF-I, and IGF-I receptor blockade on GnRH content in the anterior hypothalamus and MBH from young or middle-aged female ratsa

aCSF
JB-1
IGF-I
Oil EB Oil EB Oil EB
Young
 Anterior hypothalamus 1152.9 ± 688.6 890.9 ± 209.2 1615.0 ± 287.9 1265.4 ± 228.6 ND ND
 Mediobasal hypothalamus 3593.5 ± 919.9 4655.1 ± 1086.9b 2001.2 ± 242.9 5114.4 ± 742.9b ND ND
Middle agedc
 Anterior hypothalamus 2140.7 ± 621.9 6420.5 ± 2354.9 5480.5 ± 857.1 3717.7 ± 436.2 2248.4 ± 783.3 5105.6 ± 1198.9
 Mediobasal hypothalamus 4846.4 ± 836.0 16872.7 ± 4277.0b 8208.2 ± 2685.7 10559.5 ± 2449.3b 7233.3 ± 2361.4 8184.3 ± 1292.1b
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ND, Not done. 

a

Data are expressed as picograms per milligram protein and are reported as means ± sem (n = 5–6 rats/group). 

b

Main effect of E2 treatment compared with aged-matched controls in the same brain region (P < 0.05). 

c

Main effect of age compared with young rats in the same brain region (P < 0.05). 

Discussion

The present data clearly demonstrate that ongoing IGF-I signaling in the brain, most likely the hypothalamus, is necessary for ovarian steroids to produce a high-amplitude LH surge in female rats, regardless of age. Circulating IGF-I levels may play a minor role. E2 increased plasma IGF-I in young OVX females, but central infusion of the IGF-I antagonist delayed and attenuated the LH surge. Although E2 reduced circulating IGF-I in OVX middle-aged rats, these levels did not differ significantly from E2-treated young females. The conclusion that brain IGF-I levels are the key determinant of LH release is consistent with a recent report that restoration of circulating IGF-I in IGF-I knockout mice by overexpressing the growth factor in the liver rescued adult body weight and carbohydrate metabolism but not female reproductive function (10). Taken together, our data and the transgenic mouse findings provide strong evidence that local production of IGF-I in reproductive tissues is critical for normal fertility in females and suggest that the hypothalamus is one such tissue. We also found that exogenous IGF-I infused at low doses into the third ventricle, a route of administration that is unlikely to modify circulating IGF-I levels, partially restored LH surges in middle-aged rats but did not restore them to levels seen in young rats. Thus, the attenuated LH surge in middle-aged rats, in part, reflects age-related deficiency in hypothalamic IGF-I production, thereby resulting in reduced central IGF-I receptor signaling and dysregulation of E2-positive feedback.

Ongoing IGF-I brain receptor signaling is necessary for reproductive function in young females

We previously showed that icv infusions of JB-1, given at 12-h intervals throughout 2 d of EB priming, depressed LH release at the predicted time of the LH surge (6). Because this observation reflected a single time point, we could not determine whether peak and/or total LH release was attenuated or whether IGF-I receptor blockade simply delayed the onset of the LH surge. The present study measured serum LH concentrations throughout the day of the expected LH surge in E2- and P-primed OVX rats that received continuous infusions of JB-1 or aCSF. These new data reveal total LH secreted throughout the day is significantly reduced in JB-1-treated rats. Moreover, the LH surge amplitude in young adults with central IGF-I receptor blockade is significantly attenuated and the onset of the surge delayed. Furthermore, the inhibition of LH secretion by JB-1 is reversed by coinfusion of a 10-fold lower dose of IGF-I, confirming that the drug is selective for IGF-I receptors. Infusion of exogenous IGF-I does not alter estrous cycles (5) or modify LH surges in young adult rats, implying a permissive effect of brain IGF-I receptor signaling on hypothalamic responses to E2 that underlie generation of LH surges.

Increasing brain IGF-I augments LH surges in middle-aged females

The onset of reproductive senescence in rodents is characterized by a delayed and attenuated LH surge (19,20). These changes result from reduced excitatory drive to GnRH neurons rather than a reduction in GnRH neuron number (21,22,23). We have shown that GnRH neurons in middle-aged rats retain responsiveness to excitatory stimuli such as glutamate and kisspeptin (18,24). In both rodents and humans, central and peripheral IGF-I levels decline with aging (12,13,25). Because blockade of IGF-I signaling with JB-1 delayed and attenuated LH surges in young females, and because standard E2 priming doses actually decreased plasma IGF-I in middle-aged OVX rats rather than increasing IGF-I as observed in young OVX females, we hypothesized that LH surge dysfunction with aging may partly result from central IGF-I deficiency. Indeed, we observed that icv infusions of IGF (2 μg/ml) significantly increased LH surge amplitude in middle-aged females, albeit not to the levels of young controls.

JB-1 was more potent in reducing LH surge amplitude in middle-aged than in young rats. Reliable attenuation of the LH surge in young female rats required infusion of 100 μg/ml of JB-1. In middle-aged females, 20 μg/ml of JB-1 completely suppressed the LH surge, even in rats receiving exogenous IGF-I (2 μg/ml). This may be because endogenous hypothalamic IGF-I is lower in middle-aged than young adults, favoring the binding of the competitive antagonist JB-1 to hypothalamic IGF-I receptors in middle-aged rats (12). Alternatively, the reduced sensitivity of middle-aged rats to E2-positive feedback may render them more vulnerable to perturbation of central IGF-I receptor signaling. It is unclear why 5 μg/ml of IGF-I failed to amplify the LH surge in middle-aged rats. It is unlikely that this is attributable to activation of insulin receptors because icv infusions of insulin do not affect steroid-induced LH surges in young nondiabetic female rats (26). Moreover, a higher dose of 10 μg/ml of IGF-I had no effect on the LH surge in young, steroid-primed females.

IGF-I and the timing of the LH surge

Interestingly, although blockade of brain IGF-I receptors significantly delayed the LH surge in young rats, there was only a trend, albeit not statistically significant, for icv IGF-I infusion to advance the onset of the LH surge in middle-aged rats. Moreover, although the LH surge in young rats treated with IGF-I antagonist was delayed, it still occurred earlier than in middle-aged controls (5.25 ± 0.5 vs. 8.6 ± 0.5 h, respectively). Lastly, IGF-I-treated, middle-aged rats exhibited LH surge onsets (5.8 ± 0.6 h) similar to those of young JB-1-treated rats. How central IGF-I receptor antagonism delays the ovarian steroid-induced LH surge in young females is unclear. Young rats subjected to knockdown of vasoactive intestinal peptide (VIP) in the suprachiasmatic nucleus using antisense oligonucleotides exhibit a delayed and attenuated LH surge similar to that observed in middle-aged rats (22,27,28,29,30,31,32,33,34). IGF-I is hypothesized to be a downstream mediator of VIP activity, and VIP increases IGF-I mRNA levels (35). Thus, it is possible that JB-1 delays and attenuates LH surges in young rats by disrupting VIP-mediated effects on GnRH neurons. On the other hand, the inability of IGF-I to rescue the delayed onset of the LH surge in middle-aged rats is consistent with our observation that elevating kisspeptin or altering the balance of glutamate and γ-aminobutyric acid in the POA on the day of the LH surge reinstates amplitude without advancing the surge in middle-aged rats (18). Thus, it may be difficult to significantly advance the LH surge onset because female reproductive senescence is accompanied by the disruption of circadian rhythms and several neurotransmitter systems that independently contribute to a normally timed LH surge (11).

IGF-I and GnRH synthesis

GnRH neurons express IGF-I and IGF-I receptors (3,4), suggesting that IGF-I may regulate GnRH synthesis. Therefore, we explored the effects of IGF receptor manipulation on GnRH content in OVX control and E2-primed rats. In the MBH (site of GnRH terminals) of both age groups, E2 priming significantly increased GnRH content in all treatment groups, suggesting that estrogen-positive feedback increases the pool of GnRH peptide available for release. GnRH content in the anterior hypothalamus (major locus of GnRH somata) was not altered by E2 priming, JB-1 or exogenous IGF-I. Thus, our study suggests that activation of hypothalamic IGF-I receptors is not essential for GnRH peptide synthesis. This interpretation is consistent with the proposal that IGF-I effects on GnRH-LH release arise independent of de novo GnRH gene expression (12,36). Moreover, because total GnRH content was significantly higher in the hypothalamus of middle-aged than young rats, our data suggest that inadequate pools of readily releasable GnRH peptide do not underlie reduced LH secretion or the reduced LH surge amplitude in hormone-primed young or middle-aged rats treated with JB-1. Elevated GnRH content could suggest that impaired GnRH release from middle-aged rats causes GnRH accumulation. Consistent with this hypothesis, several studies (37,38) reported age-related increases in hypothalamic GnRH and reached similar conclusions. Because fewer GnRH neurons are activated on the day of the LH surge in middle-aged than young rats (39), it is likely that IGF-I modulates the activation of GnRH neurons and/or local factors that modulate GnRH release at the level of the median eminence (28) under estrogen-positive feedback conditions.

IGF-I and pituitary function

IGF-I receptors are expressed in the anterior pituitary (40,41), and exogenous IGF-I stimulates LH release from isolated pituitary glands (42). JB-1 or IGF-I infused into the third ventricle could reach the pituitary gland via circumventricular organs such as the median eminence and thus modulate LH secretion by activating IGF-I receptors on pituitary gonadotropes. Therefore, we explored the possibility that IGF-I directly affected pituitary LH release. However, in OVX young adult rats, icv administration of JB-1 had no effect on GnRH-induced LH release in either oil- or E2-treated females. Thus, pituitary sensitivity to GnRH is not altered by IGF-I receptor antagonism. Intracerebroventricular infusion of middle-aged rats with IGF-I likewise had no effect on pituitary sensitivity to GnRH. Moreover, the GnRH receptor antagonist cetrorelix blocked the LH surge in IGF-I-infused middle-aged rats, clearly showing that IGF-I enhancement of the steroid-induced LH surge requires GnRH receptor activation. These data suggest IGF-I regulates the reproductive axis in both young and middle-aged females at the level of the hypothalamus.

Female rats exhibit a preovulatory surge of prolactin concomitant with the LH surge (43). Similar to the LH surge, the prolactin surge is stimulated by positive feedback effects of E2 (44), primarily in the POA (45,46) and dopaminergic neurons in the arcuate nucleus (47). E2 also stimulates prolactin synthesis through direct actions on lactotrophs (48). If hypothalamic IGF-I receptors are necessary for all estrogen-dependent neuroendocrine processes, then JB-1 treatment should attenuate induction of the prolactin surge. However, neither the timing nor amplitude of the prolactin surge was modified in JB-1-treated rats. Thus, the permissive role of hypothalamic IGF-I receptors in E2-dependent neuroendocrine events seems to be specific to the female reproductive axis, including hypothalamic generation of the LH surge and female reproductive behaviors (6,7).

Potential mechanisms of IGF-I action

We hypothesize that IGF-I regulates the LH surge by modulating E2-regulated afferent systems that innervate GnRH neurons and/or axon terminals. One E2-regulated afferent system that affects GnRH neurons and might be affected by reduced IGF-I receptor signaling is the norepinephrine neurotransmitter pathway, more specifically E2 induction of α1B-adrenergic receptors (6,49). An alternate target may be kisspeptin. Kisspeptin, the product of the Kiss1 gene, is expressed in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (50,51). Kisspeptin neurons in the AVPV express estrogen receptor-α (52) and innervate GnRH neurons, which express kisspeptin receptors (53). Kisspeptin stimulates LH release on central or peripheral administration (54,55). Recently IGF-I given either systemically or centrally was shown to increase Kiss1 mRNA expression in both the AVPV and arcuate nucleus of prepubertal female rats, and this effect was blocked by JB-1 (56). Thus, kisspeptin is poised both mechanistically and spatially to be a potential downstream mediator of IGF-I regulation of the reproductive axis. We previously reported that Kiss1 mRNA expression in the arcuate nucleus and AVPV of gonadally intact young adult female rats was not modified by chronic icv infusion of JB-1. However, estrous cycles were suppressed in JB-1-treated females so that ovarian steroid levels were low, producing a steroid environment characterized by reduced hypothalamic kisspeptin expression. Likewise, the control rats that exhibited estrous cycles were in a low E2 environment (diestrus) when they were killed (5). Thus, it is not surprising that Kiss1 mRNA expression was similar in the two groups. We recently reported that the delayed and attenuated LH surge observed in middle-aged rats is associated with a reduced ability of E2 to increase kisspeptin expression in the AVPV (57). Thus, future studies will test the hypothesis that E2 regulation of Kiss1 mRNA expression in the AVPV requires brain IGF-I receptors.

The attenuated LH surge in middle-aged rats results in part from the failure of steroid positive feedback to increase the ratio of glutamate to γ-aminobutyric acid neurotransmission in the medial POA on the day of the LH surge (16,58). A 4-fold pharmacological increase of glutamate neurotransmission in the medial POA of middle-aged rats rescues LH surge amplitude (18,24). Expression of the N-methyl-d-aspartate receptor subunit NR2b is higher in GnRH neurons in middle-aged than young female rats (59). A selective antagonist of this subunit stimulates pulsatile LH release in the absence of changes in GnRH expression, suggesting a role for the NR2b subunit in the regulation of GnRH-induced LH release (60). Interestingly, NR2b expression in aged rats decreases in the AVPV, a major source of excitatory input to GnRH neurons (61). Thus, the composition of N-methyl-d-aspartate receptor subunits in reproductively relevant brain areas changes with age and may underlie some of the deficiencies in GnRH/LH release seen in middle-aged rats (62). IGF-I regulates NR2b and NR2a subunit levels and local glucose use in rat brain (63,64), suggesting another possible mechanism for IGF-I modulation of the LH surge.

Conclusions

The present findings provide evidence that endogenous IGF-I regulates the E2-dependent LH surge at the level of the hypothalamus. Moreover, they suggest that reduced levels of hypothalamic IGF-I in middle-aged female rats may contribute to the attenuated LH surge that heralds the onset of reproductive senescence. Brain IGF-I receptors are not required for the prolactin surge, suggesting that IGF-I specifically regulates the hypothalamic-pituitary-gonadal axis. Blockade of brain IGF-I receptors does not alter tissue GnRH content, indicating that IGF-I is not required for GnRH synthesis under E2-positive feedback conditions. Thus, it seems likely that generation of the LH surge involves actions of brain IGF-I receptors on GnRH release and/or on E2-sensitive hypothalamic neurons that provide excitatory afferent signals to GnRH neurons on the day of the LH surge.

Acknowledgments

The authors gratefully acknowledge the expert technical assistance of Zewei Jiang and Nina Mikkilineni. We thank Brigitte Mann and Arthur Parlow for performing hormone assays.

Footnotes

This work was supported by National Institutes of Health Grants T32 AG23475 and R01HD29856 and grants from the Resnick Gerontology Center, Robert Wood Johnson Foundation, American Federation for Aging Research, and the Skirball Foundation. This research was also supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development/National Institutes of Health through cooperative agreement HD058155 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research.

Disclosure Summary: The authors have nothing to disclose.

First Published Online January 22, 2010

Abbreviations: aCSF, Artificial cerebrospinal fluid; AUC, area under the curve; AVPV, anteroventral periventricular nucleus; E2, estradiol; EB, E2 benzoate; icv, intracerebroventricular; MBH, mediobasal hypothalamus; OVX, ovariohysterectomized; P, progesterone; POA, preoptic area; VIP, vasoactive intestinal peptide.

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