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. 2008 Dec 3;24(3):718–725. doi: 10.1093/humrep/den434

Effect of L-dopa on interleukin-1β-induced suppression of luteinizing hormone secretion in intact female rats

MP Sirivelu 1, AC Shin 2, GI Perez 1,3, PS MohanKumar 1,2,4, SMJ MohanKumar 1,2,5,6
PMCID: PMC2646791  PMID: 19054775

Abstract

BACKGROUND

The cytokine, interleukin-1β (IL-1β), increases during immune stress and is known to suppress the preovulatory luteinizing hormone (LH) surge in female rats by decreasing hypothalamic norepinephrine (NE). We hypothesized that IL-1β could produce this effect by decreasing NE biosynthesis.

METHODS

Female Sprague–Dawley rats were implanted with a push–pull cannula in the medial preoptic area (MPA) of the hypothalamus and a catheter in the jugular vein. They were treated i.p. with the vehicle or 5 µg of IL-1β, the NE precursor, L-dopa, or a combination of L-dopa and IL-1β at 1300 hours on the day of proestrus. They were subjected to push–pull perfusion and serial blood sampling. Perfusates were analyzed for NE levels and serum samples for LH.

RESULTS

IL-1β treatment blocked the increase in NE levels in the MPA and the LH surge. Treatment with L-dopa was able to partially restore both NE and LH levels during the afternoon of proestrus. IL-1β treatment caused failure of ovulation and this effect was also reversed by L-dopa.

CONCLUSIONS

These results suggest that IL-1β could decrease NE levels in the MPA to suppress reproductive functions and L-dopa can be used to counter this effect.

Keywords: IL-1β, hypothalamus, luteinizing hormone, L-dopa

Introduction

Stress produces a number of changes that involve the immune, endocrine and nervous systems (Licinio and Frost, 2000) that can lead to the suppression of the reproductive system. The cytokine, interleukin-1β (IL-1β), is an important mediator for these effects and inhibits the hypothalamo–pituitary–gonadal (HPG) axis, which results in loss of reproductive function (Rivier and Vale, 1989, 1990; Kalra et al., 1990a, b). This effect is marked by a reduction in gonadotrophin-releasing hormone (GnRH) and luteinizing hormone (LH) levels and failure of ovulation, which could translate into infertility. The mechanism by which IL-1β affects the HPG axis to suppress reproductive functions is still unclear. Since LH secretion is under the direct stimulatory control of GnRH neurons of the hypothalamus, this has been suggested to be a possible site of action (Rivier and Vale, 1990). The medial preoptic area (MPA) of the hypothalamus has a large number of GnRH perikarya (Ibata et al., 1979; Witkin et al., 1982) and plays a major role in the generation of the LH surge that is critical for ovulation. The terminals of these neurons extend to the median eminence. GnRH that is released from these terminals enters the portal circulation and reaches the anterior pituitary to release LH, which in turn causes the growth and eventually ovulation of the follicles in the ovary (Mueller and Nistico, 1989).

Regulation of GnRH secretion is highly complex and a large number of neurotransmitters and neuropeptides are believed to be involved in this process (Mueller and Nistico, 1989; Barraclough, 1992; Kalra and Crowley, 1992; Brann and Mahesh, 1995; Brann et al., 1997). The earliest to be identified were the catecholamines, and several studies support their role in LH regulation (Kalra and Kalra, 1977); reviewed in Barraclough and Wise (1982). Techniques such as measuring turnover, concentration and release have been used to demonstrate a stimulatory role for NE in the preovulatory LH surge (Wise, 1984; Mohankumar et al., 1994, 1995; Szawka et al., 2007). Moreover, lesioning of specific noradrenergic nuclei that innervate the hypothalamus, was also capable of suppressing LH levels (Anselmo-Franci et al., 1997) indicating a positive role for NE in the LH surge. Thus, it is possible that IL-1β-induced suppression of LH secretion could be mediated through a reduction in hypothalamic NE. In the present study, we examined the effect of IL-1β on all arms of the HPG axis. We measured NE release in the hypothalamus in conscious freely moving animals and concurrently monitored serum LH levels in these animals. We also observed changes in ovarian structures in response to IL-1β treatment. We attempted to reverse the effects of IL-1β by using a noradrenergic precursor, L-dopa to test our hypothesis that IL-1β probably decreases NE levels in the MPA by suppressing NE biosynthesis.

Materials and Methods

Animals

Three-month-old female Sprague–Dawley rats were obtained from Harlan Inc. (Indianapolis, IN, USA). They were housed in air-conditioned (23 ± 2°C) and light controlled (lights on from 0500 to 1900 hours) animal rooms and provided with rat chow and water ad libitum. The protocols were approved by the IACUC at Michigan State University.

Vaginal cytology

Vaginal lavage was performed daily between 0800 and 0900 hours and the smears obtained were dried at 60°C and stained with a methylene blue staining solution (0.5% methylene blue and 1.6% potassium oxalate in water). They were examined under a light microscope and the stage of the oestrous cycle was determined using previously described criteria (Mohankumar et al., 1994). Rats in proestrus typically had clusters of epithelial cells with few to no cornified cells and leukocytes.

Stereotaxic surgery

Intact cycling rats were implanted with push–pull cannulae in the MPA as previously described (Mohankumar et al., 1994; MohanKumar and MohanKumar, 2002). Briefly, the rats were given atropine sulphate (0.1 mg/kg BW) and anesthetized with a combination of xylazine (7 mg/kg BW) and ketamine (55 mg/kg BW) i.p. After placing them in a stereotaxic apparatus (Kopf, Tujunga, CA, USA), they were implanted with a push–pull cannula in the MPA using the following coordinates: 8.5 mm ventral, 0.3 mm posterior and 0.3 mm lateral to the bregma (Paxinos, 2004). Vaginal smears were obtained from these animals a week after surgery. They were implanted with a jugular catheter on the day of diestrus of their first regular cycle.

Implantation of jugular catheter

The rats were maintained under Isoflurane (2%) anesthesia and were implanted with a silastic catheter (0.64 mm ID × 1.19 mm OD; Dow Corning, Midland, MI) in the jugular vein as described before (Mohankumar et al., 1994). The surgical incision was secured with wound clips and the animals were allowed to recover.

Push–pull perfusion procedure

After a post-surgical rest period of 7–10 days, push–pull perfusion was performed on the day of proestrus in regularly cycling animals as previously described (Mohankumar et al., 1994; MohanKumar and MohanKumar, 2002, 2004). The stylet was replaced with an inner cannula assembly containing the push and pull tubes. The tubes were placed in opposite orientations within a peristaltic pump (Pharmacia-P3, Uppsala, Sweden) so that the pump pushes and pulls fluid at the same rate. Sterile artificial cerebrospinal fluid was used as the perfusion medium and its composition has been described previously (Mohankumar et al., 1994; MohanKumar and MohanKumar, 2002, 2004). Pump speed was adjusted to achieve a flow rate of 10 μl/min.

Treatment

On the day of proestrus, after collecting a pretreatment perfusate sample from the MPA and a blood sample, animals were randomly subjected to one of the following four treatments blinded to the investigators: control (PBS-1.0% BSA; n = 6), IL-1β (5 µg i.p; n = 6), L-dopa (50 mg/kg BW i.p; Sigma, St Louis, MO, USA; n = 6) or IL-1β+L-dopa (n = 9) at 1300 hours. Perfusate samples were collected every 30 min and blood samples at hourly intervals from 1300 to 1800 hours. Perfusates were stored at −70°C until HPLC-EC analysis for NE. Plasma was separated and stored at −20°C until analysis for LH levels by RIA. At the end of the experiment, the animals were sacrificed and the brains were removed, sectioned and stained with cresyl violet to verify cannula location. Only those animals that had a cannula in the MPA were included in the analysis.

Effects of L-dopa on IL-1β-induced suppression of ovulation in rats

Adult, cycling female rats were randomly subjected to one of the four treatments: control (PBS-1.0% BSA; n = 4), IL-1β (5 µg i.p; n = 4), L-dopa (50 mg/kg BW i.p; Sigma, St Louis, MO, USA; n = 4) or IL-1β+L-dopa (n = 4) at 1300 hours on proestrus. They were sacrificed between 1400 and 1500 hours on the following day (estrus) and the ovaries were collected in neutral buffered formalin and processed for sectioning. The sections were stained with hemotoxylin and eosin for detailed histological examination.

HPLC-EC

The HPLC-EC procedure for determination of NE has been described previously (Clark et al., 2008). At the time of analysis, the samples were thawed at room temperature and 120 µl of perfusate and 30 µl of the internal standard (0.05 M dihydroxy benzylamine; Sigma Chemical Co., St Louis, MO, USA) were loaded onto an autosampler (model SIL-10AF, Shimadzu, Columbia, MD, USA) and 125 µl of the mixture was injected into the HPLC system. NE concentrations in the chromatograms were determined using the Class VP software (version 7.3) in a double-blind fashion. The sensitivity of the system was <1 pg.

LH-radioimmunoassay

Double antibody radioimmunoassay was used to determine LH levels in the plasma samples as described before (Mohankumar et al., 1994; MohanKumar and MohanKumar, 2002). The reference preparation for LH was NIDDK rLH-RP-3. The primary antibody used was anti rLH-S11. LH standards, iodination quality LH protein and LH primary antibody were obtained from Dr A.F. Parlow, NIDDK. LH was iodinated by Peptide Radioiodination Service, University of Mississippi. Serum samples (50 µl) were assayed in duplicate. The assay had a sensitivity of <10 pg. The inter-assay variability was 10.4 ± 6.7% and the intra-assay variability was 3.8 ± 2.16%.

Statistical analysis

Changes in NE and LH profiles were analyzed by repeated measures ANOVA followed by Fisher's LSD. The average values of NE and LH were compared using one way ANOVA followed by post hoc Fisher's LSD. Changes in ovarian histology were analyzed by Kruskal–Wallis test followed by post hoc Bonferroni–Dunn test.

Results

NE release in the MPA

The location of the push–pull cannulae in animals subjected to perfusion are shown in Fig. 1. The profiles of NE release in the MPA in control rats and those treated with IL-1β, L-dopa or IL-1β in combination with L-dopa are shown in Fig. 2. The effect of treatment [F(3,93) = 43.33], time [F(11,53) = 3.46] as well as the interaction between treatment and time [F(33,53) = 1.73] were found to be statistically significant (P < 0.05). In control animals, NE levels (pg/min, mean ± SE) increased gradually from 1300 hours (7.63 ± 1.15), reached a peak at 1530 hours (21.53 ± 1.6; P < 0.05), were 16.94 ± 1.8 at 1600 hours and declined to 12.69 ± 1.28 at 1700 hours. In contrast, treatment with IL-1β suppressed the rise in NE levels. NE levels in this group were 2.34 ± 1.59 at 1300 hours and remained at that level for the remaining period of observation (Fig. 2A).

Figure 1.

Figure 1

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Push–pull cannula location.

Schematic representation of the sagittal section of a rat brain indicating the locations of the push–pull cannulae in control (PBS-1%BSA-treated group, n = 6), IL-1β treated (n = 6), L-dopa treated (n = 6) and the group treated with both IL-1β and L-dopa (n = 9). The numbers A1–P3 represent coronal plates extending 1 mm anterior (A1) to 3 mm posterior (P3) to the bregma (AP0). MPA, medial preoptic area; SCh, suprachiasmatic nucleus; AH, anterior hypothalamus; LA, lateroanterior hypothalamic nucleus; AVPO, anteroventral preoptic nucleus; MPO, median preoptic nucleus; StHy, striohypothalamic nucleus; VMH, ventromedial hypothalamus; OX, optic chiasm; SOX, supraoptic decussation. The location of the push–pull cannulae in individual animals was determined by examining stained serial brain sections under a light microscope.

Figure 2.

Figure 2

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Effects of L-dopa on IL-1β-induced decrease in NE release.

NE release profiles in intact female Sprague–Dawley rats (n = 6–7 per group) treated with PBS-1%BSA (Control) or 5 µg of IL-1β (A). (B) NE release profiles in the groups treated with 50 mg/kg BW of L-dopa or IL-1β in combination with L-dopa at 1300 hours on the afternoon of proestrus. NE profiles in the control and IL-1β-treated groups are provided in gray. Rats were subjected to push–pull perfusion of the MPA. Perfusates were collected at 30 min intervals and analyzed for NE levels by HPLC-EC. ‘a’ indicates significant difference (P < 0.05) from levels at 1300 hours and ‘b’ indicates difference (P < 0.05) from levels in IL-1β treated animals. (C) Average levels of NE during the entire period of observation in the different treatment groups. *P < 0.05 when compared to the rest of the groups.

In the group treated with L-dopa alone, NE levels increased from 6.77 ± 1.94 at 1300 hours, by more than 2-fold to 17.79 ± 1.16 at 1530 hours (P < 0.05), were 15.06 ± 3.34 and 15.15 ± 1.71 at 1600 and 1700 hours, respectively (Fig. 2B). They were not different from the levels in control group. However, treatment with L-dopa in combination with IL-1β partially reversed the suppressive effect of IL-1β on NE (Fig. 2B). The levels of NE in these animals increased significantly from 5.86 ± 1.99 at 1300 hours to 12.2 ± 2.6 at 1600 hours (P < 0.01). These levels were not different from the group treated with L-dopa alone at 1600 hours (15.06 ± 3.5) but was significantly higher compared to the IL-1β-treated group (3.6 ± 2.2; P < 0.05) at this time point. The average levels of NE over the entire period of observation are shown in Fig. 2C. NE levels were significantly higher in control animals (12.26 ± 1.06), in animals treated with L-dopa alone (10.24 ± 1.3) and in animals treated with IL-1β+L-dopa (7.67 ± 1.51) when compared with animals treated with IL-1β alone (3.01 ± 1.30; P < 0.05).

Plasma LH levels

Concurrent changes in plasma LH levels in the animals subjected to push–pull perfusion are shown in Fig. 3. The effects of treatment [F (3,58) = 8.57], and time [F (5,10) = 3.34] were found to be significant (P < 0.05). In control animals, LH levels (ng/ml, mean ± SE) were 0.5 ± 0.2 at 1300 hours and gradually increased to peak concentrations of 4.2 ± 0.8 at 1700 hours (P < 0.05), closely following the increase in NE levels in the MPA that occurred at 1530 and 1600 hours. In contrast, in IL-1β treated animals, LH levels were 0.05 ± 0.3 at 1300 hours and remained at that level during the rest of the observation period, parallel to the low levels of NE observed in the MPA (Fig. 3A).

Figure 3.

Figure 3

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Effects of L-dopa on IL-1β-induced decrease in LH levels.

Plasma LH levels in intact female Sprague–Dawley rats (n = 6–7 per group) treated with PBS-1%BSA (Control) or 5 µg of IL-1β are shown in (A). (B) LH levels in the groups treated with 50 mg/kg of BW of L-dopa or IL-1β in combination with L-dopa at 1300 hours on the afternoon of proestrus. Animals were implanted with jugular catheters for concurrent blood sampling at hourly intervals. Plasma samples were analyzed for LH levels by RIA. ‘a’ indicates significant difference (P < 0.05) from levels at 1300 hours and ‘b’ indicates difference (P < 0.05) from levels in IL-1β treated animals. (C) Average levels of LH over the entire observation period in the different treatment groups. *P < 0.05 when compared to the rest of the groups.

In the group treated with L-dopa alone, LH levels increased from 0.55 ± 0.31 at 1300 hours to 3.29 ± 0.93 at 1700 hours (P < 0.05). These levels were not different from those in the control animals. The suppressive effect of IL-1β on LH was partially reversed by co-treatment with L-dopa. In this group, LH levels at 1700 hours (1.99 ± 0.49) were significantly higher than the levels observed in the IL-1β-treated group (0.4 ± 0.7; P < 0.01) (Fig. 3B). Average LH levels over the entire period of observation in control animals and those treated with L-dopa alone were 1.91 ± 0.39 and 1.6 ± 0.4, respectively. IL-1β treatment decreased average LH levels significantly to 0.28 ± 0.31 (P < 0.05). However, the average LH levels in IL-1β+L-dopa-treated animals (0.97 ± 0.23) were in between the control and IL-1β treated groups and were not different from either group (Fig. 3C). This suggests that L-dopa partially reversed the IL-1β-induced suppression of the LH surge.

Ovarian histology

Representative sections of one ovary from each of the treatment groups are depicted in Fig. 4. The histological sections of the ovary were analyzed double-blind, for the presence of post-ovulatory fresh corpora lutea (CL), indicated by the presence of increased vascularity within the CL. The numbers of old CL as well as mature follicles were also enumerated (Fig. 5). The numbers of fresh CL (mean ± SE) in the control and L-dopa treated groups were 7.5 ± 0.96 and 5.75 ± 0.85, respectively. Treatment with IL-1β significantly decreased the fresh CL numbers to 1.25 ± 0.48 (P < 0.05). In contrast, treatment with IL-1β+L-dopa increased the number of fresh CL to 6.5 ± 0.5, which was significantly higher than that in the IL-1β treated group (P < 0.05).

Figure 4.

Figure 4

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Effects of L-dopa and IL-1β on ovarian structures.

Representative histological sections of ovaries (40× magnification) from female Sprague–Dawley rats (n = 4) treated with PBS-1%BSA (Control) (A), 5 µg of IL-1β (B), 50 mg/kg of BW of L-dopa (C) or IL-1β in combination with L-dopa (D) at 1300 hours on the afternoon of proestrus and sacrificed between 1400 and 1500 hours on the following day. Post-ovulatory, fresh corpora lutea (FCL) were identified by the histological presence of hemorrhage and the transition from a follicular appearance to luteal phenotype, old corpora lutea (OCL) by the presence of densely packed luteal cells, while the mature follicles (MF) were identified by the presence of prominent antral space and follicular fluid (E, 100× magnification).

Figure 5.

Figure 5

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Effects of L-dopa and IL-1β on the ovary.

Average numbers (mean ± SE) of post-ovulatory fresh corpora lutea (FCL), old corpora lutea (OCL) and mature follicles (MF) in the different treatment groups are shown. Female Sprague–Dawley rats (n = 4 per group) treated with PBS-1% BSA (Control), 5 µg of IL-1β, 50 mg/kg BW of L-dopa or IL-1β in combination with L-dopa at 1300 hours on the afternoon of proestrus and sacrificed between 1400 and 1500 hours on the next day. *P < 0.05 when compared to the rest of the groups.

Besides producing a significant reduction in fresh CL, IL-1β treatment was also associated with higher numbers of old CL (Fig. 5). The number of old CL in the IL-1β treated group (6.0 ± 0.57) was significantly higher (P < 0.05) compared with control (2.25 ± 0.63), L-dopa (2.25 ± 0.24) and IL-1β+L-dopa treated (2.23 ± 0.15) groups. The numbers of mature follicles were not significantly different between the different treatments.

Discussion

Results from this study indicate that systemic administration of IL-1β blocks the preovulatory LH surge in intact cycling animals and confirms earlier findings that IL-1β is capable of suppressing reproductive functions. The suppression of LH secretion was accompanied by a reduction in NE release in the MPA of the hypothalamus indicating a role for NE in this effect. Further, treatment with the noradrenergic precursor, L-dopa was able to block the effect of IL-1β and partially restore NE levels in the MPA. This suggests that the IL-1β most probably affects the biosynthesis of NE to produce its effect. The partial restoration of NE levels was accompanied by a partial reversal of the LH surge as well. Nevertheless, these LH levels were sufficient to cause ovulation in IL-1β-treated animals. L-dopa can therefore be considered as a viable treatment option in patients with reproductive failure that occurs as a result of stress, or other chronic diseases.

IL-1's effects on the HPG axis became apparent when it was first demonstrated that IL-1α could decrease LH secretion in male rats (Rivier and Vale, 1989). Subsequently this finding was extended to cycling female rats in which the more potent isoform, IL-1β not only blocked the preovulatory LH surge but also inhibited ovulation (Rivier and Vale, 1990). A similar reduction in LH levels was also observed after IL-1β treatment in ovariectomized, steroid-primed rats (Kalra et al., 1990b). Interestingly, these effects on LH secretion and ovulation were observed only when IL-1β was administered i.c.v and not when given systemically (Kalra et al., 1990b; Rivier and Vale, 1990). This could be attributed to the doses of IL-1β that were used in these studies. Rivier and Vale (1990) used 40 ng i.c.v and only 25 ng i.v., while Kalra et al. (1990a, b) used 30 ng i.c.v. and 1 µg i.p. Katsuura et al. (1988) demonstrated that lower i.c.v. doses of IL-1β (3 ng) can elicit neuroendocrine responses, but much higher doses (300 ng) are required when it is given i.v. We have previously shown that a dose-dependent change in neurotransmitter levels occurs in response to i.p. administration of 1, 2.5 and 5 µg of IL-1β (MohanKumar and Quadri, 1993; MohanKumar et al., 1998). Based on previous findings with iodinated IL-1 (Banks and Kastin, 1991), we estimate that only 4 ng of IL-1β (0.08%) would have crossed the blood brain barrier after administration of 5 µg i.p. The present study demonstrates that this dose could suppress the HPG axis effectively. This is supported by our previous study in ovariectomized steroid-primed rats in which treatment with 5 µg of IL-1β i.p. suppressed the LH surge (MohanKumar and MohanKumar, 2002).

The mechanism by which IL-1β produces its effect on the LH surge is not clear. IL-1β could act through any of the neurochemicals that are implicated in LH regulation (Smith and Jennes, 2001). Results from the present study indicate that IL-1β acts by decreasing NE levels in the MPA. There are several lines of evidence to indicate that NE is stimulatory to GnRH neurons and LH secretion. NE concentration, turnover and release are known to increase parallel to the LH surge (Wise, 1984; Mohankumar et al., 1994, 1995). Further, adrenergic antagonists (Herbison, 1997) and inhibition of NE synthesis (Hancke and Wuttke, 1979; Adler et al., 1983) both block the LH surge. Anatomical evidence suggests that the MPA is richly innervated by brainstem noradrenergic nuclei (Kumar et al., 2007) and lesioning these areas suppresses the preovulatory LH surge (Anselmo-Franci et al., 1997). Therefore, it is possible that IL-1β inhibits the LH surge by decreasing NE levels in the MPA. The decrease in NE levels in the MPA produced by IL-1β treatment in the present study is in agreement with this notion.

The mechanism by which IL-1β decreases NE levels in the MPA is not clear. This effect may be produced by decreasing NE synthesis in noradrenergic neurons. The rate-limiting step in NE biosynthesis is the conversion of L-tyrosine to L-dopa by tyrosine hydroxylase (Nagatsu et al., 1964). Therefore, IL-1β could inhibit tyrosine hydroxylase activity leading to a reduction in L-dopa production. In the present study, supplementation with L-dopa was able to partially reverse the decrease in both NE and LH levels produced by IL-1β suggesting that IL-1β probably affects tyrosine hydroxylase activity and thereby NE biosynthesis. The partial but not complete reversal suggests that IL-1β may also affect enzymes downstream of tyrosine hydroxylase such as dopamine β-hydroxylase that is involved in NE synthesis. On the other hand, IL-1β may act through other mechanisms to inhibit NE release from the terminals. This needs further investigation.

Another possibility is that IL-1β could produce its effect on HPG function by activating the hypothalamo–pituitary–adrenal (HPA) axis or the stress axis. Stress is known to inhibit both tonic and pulsatile LH secretion and in some instances, delay the LH surge (Dobson and Smith, 2000; Li et al., 2004). Corticotropin-releasing hormone (CRH) which is a key player in HPA activation is known to be directly involved in the inhibition of GnRH and LH secretion in female rats (Rivest and Rivier, 1993). CRH antagonists are effective in blocking fasting-induced suppression of LH secretion (Maeda et al., 1994); however, they were ineffective in blocking IL-induced inhibition of LH secretion (Rivest and Rivier, 1993). Moreover, it is not clear if CRH could produce its effects through NE. Mitchell et al. (2005) have demonstrated that infusion of CRH into a brainstem noradrenergic region can suppress LH secretion. The exact mechanisms by which this occurs is not clear and additional studies are needed to understand the underlying mechanisms.

The effect of IL-1β treatment on rat ovaries was examined to complete studying the effect of IL-1β on all three arms of the HPG axis. In the present study, control rats had large numbers of freshly formed CL the day following proestrus, suggesting that ovulation had occurred in this group (Figs 4 and 5). In contrast, IL-1-treated rats had fewer fresh CL suggesting decreased ovulation rate. This is supported by a previous study where ovulation failure was reported after central administration of IL-1β (Rivier and Vale, 1990). Moreover, in the present study, the numbers of old CL were higher in the IL-1β treated animals suggesting lack of luteolysis. Although the reasons for this are not clear, it is possible that systemic IL-1β could block prostaglandin F2α production which is necessary for luteolysis and progression of the oestrous cycle (Dauffenbach et al., 2003). Results from the present study also indicate that the effect of L-dopa treatment was also evident on the ovary. Animals that were treated with both L-dopa and IL-1 had CLs comparable to that seen in control rats. Since L-dopa increased hypothalamic NE and plasma LH levels, it is possible that these effects led to the increased ovulation rate. Previous studies have shown that the minimum levels of LH required for successful ovulation are only a fraction of the peak levels (Gosden et al., 1976). Therefore, it is likely that the L-dopa-induced increase in LH levels was sufficient to cause ovulation in IL-1β treated animals.

Several studies indicate that IL-1β can access central sites through different routes. Peripheral IL-1β can cross the blood brain barrier to affect the hypothalamus and other brain areas such as the area postrema that lie in close proximity to the brainstem NE neurons (Brady et al., 1994). IL-1β receptors have been identified in several parts of the brain and brain stem (Ericsson et al., 1995) and have also been localized in the para-abdominal ganglia of the vagus nerve (Goehler et al., 1997). Since the vagus projects to the brainstem nuclei, systemic IL-1β could act through the vagus to affect central noradrenergic neurons. Regardless of the route, the present study indicates that systemic IL-1β is capable of inhibiting the HPG axis most probably by decreasing NE levels in the hypothalamus which results in decreased circulating LH that causes failure of ovulation. These effects could only be partially reversed by treatment with L-dopa suggesting that other mechanisms could also play a role in this phenomenon.

Funding

This study was supported by National Science Foundation IBN0236385, partially by National Institute of Health AG 027697 and the Charles Cowham fund. Andrew Shin was supported by funds from the Biomedical Health Research Initiative, MSU.

Acknowledgements

We would like to thank Ms Katrina Linning for her technical assistance and Dr Robert Speth, Mississippi State University for providing the LH tracer.

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