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Neuroendocrinology |
a Department of Physiology and Pharmacology, West Virginia University, Morgantown, West Virginia 26506
b Department of Anatomy and Structural Biology, University of Otago Medical School, Dunedin, New Zealand
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Therefore, we tested the hypothesis that ß-estrogen receptors mediate the action of estradiol in the retrochiasmatic area by comparing the effects of estradiol and genistein, a selective ERß agonist. We also examined whether there are seasonal changes in response of the retrochiasmatic area to these agonists and if these effects are mediated by dopamine. To test these hypotheses, ovariectomized ewes were implanted with bilateral guide cannulae targeting the retrochiasmatic area. Crystalline agonists were administered via microimplants inserted down the cannulae. Blood samples taken before and 4 days after microimplant insertion were analyzed for LH concentrations, pulse frequency, and amplitude. Genistein treatment produced no significant change in LH levels in either season. Estradiol treatment decreased both mean LH concentrations and pulse frequency in anestrous but not breeding-season ewes. Administration of the dopamine antagonist sulpiride to ovariectomized ewes with estradiol microimplants in the retrochiasmatic area returned LH pulse frequency to levels indistinguishable from controls. From these data, we hypothesize that estradiol acts on local ER-containing neurons in this area to stimulate a dopaminergic pathway that inhibits LH secretion during anestrus.
estradiol, estradiol receptor, gonadotropin-releasing hormone, hypothalamus, luteinizing hormone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Luteinizing hormone suppression by E2 is believed to be mediated via the A15 DA neurons contained within the retrochiasmatic area (RCh). These neurons project to the median eminence, where they limit LH secretion, presumably through presynaptic inhibition of GnRH terminals [3, 4]. Destruction of these neurons by radiofrequency or chemical lesioning reduces the ability of E2 to inhibit LH secretion in anestrus but not during the breeding season [5, 6]. Furthermore, these neurons have been shown to be activated in anestrous ewes by systemic E2 exposure using both the early-immediate gene products Fos and Fos-related antigens [7] and amine metabolites as indices of activity [8, 9]. Administration of E2 into the RCh in OVX ewes resulted in a significant decrease in LH pulse frequency [10, 11] during anestrus, raising the possibility that E2 acts directly on A15 DA neurons. However, it is not clear whether this local response to E2 in the RCh varies with season. Moreover, no classical estrogen receptors (ER) have been found in A15 DA neurons [12, 13]. The discovery of a second estrogen receptor subtype (ERß) in the rat [14], human [15], and cow [16] raised the possibility that E2 exerts its actions via this novel ER subtype within the A15 nucleus. The recent reports that ERß message could be measured and differentially localized within the RCh area of sheep [17, 18] supports this possibility.
The purpose of this experiment was to determine if there are seasonal changes in the response of the RCh to E2 and to examine a role for ERß in the seasonal suppression of LH. We used E2 and genistein, an ERß-selective agonist [19], to differentiate ER versus ERß activity in anestrous and breeding season ewes. Additionally, we tested the hypothesis that the local effects of E2 in the RCh on LH secretion are conveyed via DA neurons by administering a specific DA-D2 receptor antagonist during E2 treatment.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Blackface ewes of mixed breeding were used for these studies. All ewes were housed outdoors until 3 days prior to surgery. Postsurgical animals remained indoors where the lighting condition for each experiment was adjusted twice per week to simulate the natural photoperiod. Each ewe received water ad libitum and a daily ration of silage and grain. All procedures involving animals were approved by the West Virginia University Animal Care and Use Committee.
Surgery
Bilateral ovariectomies were performed via midventral laparotomy under sodium pentobarbitol anesthesia, using sterile procedures. Following a 2-wk recovery period, chronic bilateral 18-gauge stainless steel guide cannulae were stereotaxically placed into the hypothalamus under halothane anesthesia as previously described [20]. Guide cannulae were lowered until 2 mm dorsal to the target area of the RCh (A15 DA nuclei: approximately 2.5–3.0 mm lateral to midline, 0.5 mm caudal to the posterior border of the optic chiasm, level in the dorsoventral plane with the floor of the third ventricle). Ewes were allowed at least 2 wk to recover before beginning experiments. Anestrous experiments were carried out between April and August, while breeding-season experiments were performed between October and January.
Drugs
Crystalline 17ß-estradiol or genistein (Sigma-Aldrich, St. Louis, MO) were tamped (approximately 40 times) into the lumen of sterile 22-gauge stainless steel microimplants. Genistein was chosen based on in vitro data that it has a 20-fold higher affinity for ERß than ER [19] and in vivo data demonstrating its effects on ERß-dependent gene expression, but not ER [21]. Within 30 min of tamping, microimplants were inserted down the guide cannulae so that they protruded 2 mm into the A15 cell group. The DA antagonist sulpiride (Sigma-Aldrich) was chosen for its specificity to D2 receptors [22] and absence of reported clinical side effects [20, 23, 24] and was injected i.m. in 0.1 M tartaric acid at a dose of 1.2 mg/kg.
Serum Sampling
Blood samples (3 cm3) were collected via jugular venipuncture at 12-min intervals. After clotting overnight at 4°C, serum was harvested and stored at -20°C until assayed for LH concentration.
Experimental Design
Experiment 1 In this experiment, we tested the effects of genistein and E2 delivered locally to the RCh area on LH suppression in anestrous ewes (n = 15). Jugular blood samples were collected at intervals of 12 min for a 5-h period for assessment of pretreatment LH pulse patterns and mean LH concentration in OVX ewes with chronically implanted guide cannulae. Immediately following sampling, bilateral microimplant pairs containing either E2 or genistein were placed in each animal. Frequent blood samples were again collected for 4 h on Day 4 after implantation; this time point was chosen based on previously published E2 data [20]. Microimplants were then removed and animals were allowed 1 wk to recover before the same protocol was repeated in a crossover design. Upon completion of the final blood sampling, brain tissue was collected for histological analysis of microimplant locations.
Experiment 2 Genistein and E2 microimplants were administered to OVX breeding season ewes (n = 8) to differentiate seasonal effects of these ligands within the RCh. Ewes were surgically prepared with guide cannulae and treated using the blood collection and treatment protocol as described above. Following completion, brain tissue was collected for histological analysis of microimplant locations.
Experiment 3 This experiment was designed to test the role of DA neurons in mediating the locally administered E2 effect on LH secretion during anestrus. The OVX ewes (n = 11) were surgically prepared with guide cannulae as described above. Following recovery, blood samples were collected every 12 min for 4 h and then either E2 or blank microimplant pairs were inserted into the RCh. Four days later, blood samples were collected for 4 h to determine the effect of E2 or blank implants. Sulpiride was then injected and sampling continued for an additional 4 h to determine if the DA antagonist could disrupt E2 actions. Microimplants were removed and all ewes were allowed a 1-wk recovery period before repeating the protocol in a crossover design. Histological analysis was used to exclude ewes with bilaterally misplaced microimplants as in experiments 1 and 2.
Tissue Analysis
Ewes were killed with an overdose of sodium pentobarbital (2 g) preceded by two heparin i.v. bolus injections (20 000 IU/injection, 10-min interval). Heads were removed and perfused with 6 L of 4% paraformaldehyde in 0.1 M phosphate buffer containing 2.5 IU heparin/ml and 0.1% NaNO3 prior to collecting the brain. Hypothalami were dissected and postfixed for 24 h and then infiltrated with 20% sucrose in phosphate buffer. Hypothalami were sectioned into 50-µm coronal slices using a freezing stage microtome for histological analysis of guide cannulae positions. Every fifth section was mounted onto gelatin-coated microscope slides and cover slipped after cresyl violet staining to visualize lesions created by the chronic guide cannulae. Sections rostral, through, and caudal to the lesion tracts were transcribed onto paper using a Bausch and Lomb overhead magnifying scope. Each microimplant placement was evaluated by comparing its location, measured 2 mm below the guide cannulae lesion and distance from midline (middle of third ventricle), to previously known locations of the A15 nucleus [11, 25]. Any ewes found to have microimplants completely outside these boundaries on both sides were considered misses.
Radioimmunoassay
Serum LH concentration was measured in 200-µl aliquots by RIA using a modification of a previously described method [26]. Values are expressed in terms of the ovine standard, NIH S24. Radioiodinated ovine LH (LER1374A; Dr. L.E. Reichert, Jr., Albany Medical College, Albany, NY) was used as tracer, and primary antiserum was CSU-204 (Dr. Gordon Niswender, Colorado State University, Fort Collins, CO; dilution 1:75 000). The sensitivity (95% confidence interval at 0 ng/ml) averaged 0.13 ng/tube over the 12 assays that contributed to the results. Intraassay coefficients of variation (CV) averaged 9.4% and 15%, respectively, for serum pools displacing radiolabeled LH to approximately 36% and 68% of the total bound, and interassay CVs were 12.5% and 25% for the same serum pools.
Statistical Analysis
The effects of treatments on LH pulse frequency in experiments 1 and 2, before and during treatments within seasons, were determined by the Wilcoxon-Mann-Whitney signed rank test. The effects on mean LH concentration were determined by ANOVA on repeated measures. Effects on LH pulse frequency among groups in experiment 3 were determined using Friedman repeated measures ANOVA on ranks. Mean LH concentrations were compared using two-way ANOVA for repeated measures. Amplitude data was analyzed using ANOVA. Results are presented ± SEM, and P < 0.05 was defined as statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Histological examination of implant lesions revealed accurate placements in 12 of 15 anestrous ewes (Fig. 1, Table 1). There was a significant decrease (P < 0.05) of LH pulse frequency (pulses/4 h) after 4 days of local RCh E2 microimplants (Fig. 2A). Eleven of 12 ewes exhibited a drop in pulse frequency of at least one pulse per 4 h during E2 microimplant treatment. In addition, the mean LH concentration was significantly reduced during treatment (Fig. 2B). This fall was evident in 11 ewes, with no change in concentration for the nonresponder (12.6 versus 12.9 ng/ml). An example LH pulse pattern is shown in Figure 3A. The location of the microimplants for the nonresponding ewe was observed to be at the lateral edge of the A15 area, in close proximity to the optic tracts (Fig. 1).
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In contrast, genistein microimplants produced no significant changes in either LH pulse frequency or mean concentrations (Fig. 2). An example LH pulse pattern is shown in Figure 3B. Furthermore, there were no significant effects of either E2 or genistein on pulse amplitudes (Table 2). The three misplaced microimplant animals exhibited no change in LH pulse frequency (Table 3), mean LH concentration, or amplitude (data not shown) following E2 or genistein microimplant administration.
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Experiment 2: Does E2 or Genistein Delivered to the RCh Affect LH Secretion During the Breeding Season?
Histological examination of implant lesions revealed accurate placements in five out of eight breeding-season ewes (Table 1). Average LH pulse frequency (Fig. 4A) during E2 microimplantation was slightly lower than control due to a drop in one ewe, and mean LH concentration (Fig. 4B) declined slightly during E2 microimplant treatment, but both were not significant changes (P > 0.05). Furthermore, there was a nonsignificant decrease in LH pulse amplitudes (P = 0.09, Table 2) which contributed to the nonsignificant decline in mean LH concentration in three of the five ewes. There was no apparent correlation between microimplant placement within the A15 area and the differences seen in LH measurements. Genistein microimplants did not significantly alter pulse frequency, mean LH concentrations (Fig. 4), or pulse amplitudes (Table 2). Luteinizing hormone pulse frequency in the three animals with misplaced microimplants was unaltered by either E2 or genistein (Table 3), as was mean LH concentration and pulse amplitude (data not shown).
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Experiment 3: Are E2 Actions in the RCh Mediated by DA Neurons?
Histological examination of microimplant lesions revealed accurate placements in 7 of 11 ewes (Table 1). As expected, E2 microimplants significantly reduced LH pulse frequency during treatment, an effect clearly evident in this group (Fig. 5A, P < 0.05 versus control). As with experiment 1, a single nonresponder had microimplants that were at the lateral borders of the A15 area. Sulpiride was effective in blocking E2 actions and returned pulse frequency to pretreatment levels (P < 0.05 versus E2). Mean LH concentrations showed similar trends (Fig. 5B), with E2 tending to reduce LH and sulpiride treatment tending to block this effect; however, these results did not attain significance (P = 0.20). Luteinizing hormone pulse amplitude was not significantly affected by E2 or sulpiride (22.2 ± 7.8 ng/ml before, 14.7 ± 4.4 during E2 treatment, and 15.8 ± 6.1 after sulpiride injection, P > 0.05). Blank implants had no effect on LH pulse frequency (Fig. 4A), mean LH concentration (Fig. 5B), or LH pulse amplitude (24.6 ± 9.2 ng/ml versus 29.0 ± 8.7 and 31.8 ± 9.6 after sulpiride injection, P > 0.05). In the four ewes with misplaced microimplants, no significant effects of treatment on LH pulse frequency, mean concentration, or amplitude were observed (Table 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because A15 DA cells in the RCh do not appear to contain ER [12], one simple explanation for these results is that E2 is acting via ERß in A15 cells. However, the lack of LH response to genistein microimplants does not support this hypothesis. It is possible that the estradiol was reaching a larger number of neurons than genistein, but based on their structures and relative solubility [27], it seems more likely that the volume of diffusion for genistein is greater than that of estradiol. Another explanation for the lack of effect of genistein is that it does not act at ERß sites in the ovine RCh. However, competitive binding was demonstrated between genistein and 17ß-estradiol in ovine pituitary and hypothalamic cytosol, indicating genistein may interfere with E2 negative feedback mechanisms [28]. Moreover, it has been shown to be an effective protein tyrosine kinase inhibitor [29, 30]. Also, in the rat hemipituitary, genistein was effective in reducing GnRH-induced LH secretion [31], while significantly affecting ERß-dependent gene expression in the ventromedial nucleus [21]. Given these data, we argue that ERß has no role in the seasonal suppression of GnRH by E2 negative feedback in the ewe. In further support of this conclusion, Lubbers et al. [32] failed to localize ERß in DA neurons of the A15. Furthermore, ERß knockout mice continue to exhibit normal reproductive behaviors and functions [33, 34], while ER knockout mice display abnormally high plasma gonadal steroid levels. Thus, ER appears to be the predominant ER involved in steroid negative feedback in this species [35]. The role of ERß, if any, has yet to be elucidated in the neuroendocrine regulation of reproduction.
The effectiveness of E2 microimplants targeting the A15 in suppressing LH via a DA pathway remains somewhat paradoxical because these DA neurons do not contain ER [12]. One possible explanation is that E2 is diffusing from the site of implantation to other DA perikarya, such as A14 neurons. Some A14 neurons rostral to the A15 contain ER [12]. But histological analysis revealed that E2 appears to act in an anatomically restricted zone of the RCh, encompassing the A15 nuclei. Specifically, microimplants that were misplaced more than 1 mm rostral or caudal to the A15, including those in the region of A14 perikarya, had no effect on LH secretion. These data are consistent with estimates that radiolabeled steroid diffusion in ovine neural tissue is limited to approximately 1 mm [36, 37]. Several other studies have also shown E2 microimplants to act in a site-specific manner [11, 24, 36–38]. Another possible explanation for these findings is that E2 may act via nongenomic pathways involving membrane-associated ER [39, 40]. However, effects of a nongenomic mode of action are usually seen within minutes [41]. Greater than 16 h are required before the negative feedback effects of E2 are observed, a time course more compatible with actions mediated by nuclear receptors [7, 10]. Therefore, we postulate that E2-receptive interneurons exist in the RCh that directly or indirectly stimulate DA release. Preliminary analysis of tissue following injection of a retrograde tract tracer into the A15 indicates the presence of ER-containing afferents in close proximity to the A15 area [42].
Recently, a group of ER-immunopositive neurons found in the ventromedial preoptic area (vmPOA) have also been implicated in E2 negative feedback [43]. These neurons are responsive to E2 [43] and also suppress LH pulse frequency during anestrus via a DA inhibitory pathway [20]. These data, together with the findings presented here and results from several preliminary neural tract-tracing studies [42, 44, 45], support the existence of a previously undescribed neural network involved with the seasonal inhibition of GnRH. Specifically, tract tracers, when injected into the vmPOA, are transported to nerve terminals in the A15 and the area just mediodorsal and caudal to the A15 nucleus [45]. These tract-tracing studies as well as the present E2-microimplant results support the existence of a neural network that extends from the vmPOA to the A15/RCh area, including the A15-associated cells, and then extends via A15 efferents on to GnRH terminals within the median eminence (ME). Furthermore, we have preliminary evidence that demonstrates an increase in synaptic connections associated with the A15 cells during anestrus [46]. This may account for the ability of the postulated vmPOA-RCh-ME system to suppress GnRH during anestrus, not the breeding season.
The findings of this study support prior research concerning seasonal changes in E2 negative feedback acting through an inhibitory DA pathway. The DA activity of A15 neurons is increased during anestrus following systemic E2 exposure [8, 9] in OVX ewes, and ablation of these neurons disrupts E2 inhibition of LH at this time [5, 9]. Furthermore, microimplants containing the DA antagonist pimozide and microdialysis of sulpiride or -methyl-para-tyrosine (but not D1-receptor analogs) into the ME disinhibit LH secretion in anestrous ewes [47, 48], suggesting a D2 receptor-dependent inhibition of GnRH secretion by DA. Our results, taken together with the sulpiride data from Anderson et al. [20], extends these findings and provides a developing, functional picture as to how the RCh and vmPOA E-responsive neurons interact to alter GnRH secretion. While local E2 in each area significantly reduced LH, microimplants in neither site were as effective as systemic E2 treatment [11, 20]. One explanation for this quantitative difference is that the E2 in the microimplants is not reaching all the E2-receptive neurons in the RCh or vmPOA. If this is the case, the two populations of E2-receptive neurons would be redundant such that one or the other population of cells is required for seasonal anestrus. Alternatively, these two areas might act in concert to establish the efficacy of E2 negative feedback seen in peripherally treated OVX+E ewes.
In summary, the results of this experiment lead to the hypothesis that a group of E2-responsive, A15-associated cells in the RCh contribute to seasonal changes in E2 negative feedback in the ewe. Microimplants delivering E2 to these neurons result in the stimulation of DA neurons, which in turn inhibit GnRH secretion. Further studies are needed to elucidate the location and identity of these A15-associated cells and to determine their role in E2 negative feedback.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Robert L. Goodman, Department of Physiology and Pharmacology, West Virginia University, P.O. Box 9229, Morgantown, WV 26506-9229. FAX: 304 293 3850; e-mail: bgoodman{at}hsc.wvu.edu
Received: 12 August 2002.
First decision: 28 August 2002.
Accepted: 16 September 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and ERß messenger ribonucleic acid in the preoptic area and hypothalamus of the sheep: comparison of males and females. Endocrinology 2000 141:2951-2962)-containing afferents to the hypothalamic A15 region of the ewe. In: Program of the 29th Annual Meeting of the Society for Neuroscience; 1999; Miami Beach, FL. Abstract 582.5 -containing neurons in the preoptic area of the ewe depends on reproductive status. Endocrinology 2000 141:190-196This article has been cited by other articles:
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  A. L. Bogusz, S. L. Hardy, M. N. Lehman, J. M. Connors, S. M. Hileman, J. H. Sliwowska, H. J. Billings, C. J. McManus, M. Valent, S. R. Singh, et al. Evidence that {gamma}-Aminobutyric Acid Is Part of the Neural Circuit Mediating Estradiol Negative Feedback in Anestrous Ewes Endocrinology, June 1, 2008; 149(6): 2762 - 2772. [Abstract] [Full Text] [PDF]
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 C. J McManus, M. Valent, S. L Hardy, and R. L Goodman Does nitric oxide act in the ventromedial preoptic area to mediate oestrogen negative feedback in the seasonally anoestrous ewe? Reproduction, July 1, 2007; 134(1): 137 - 145. [Abstract] [Full Text] [PDF]
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V. L. Adams, R. L. Goodman, A. K. Salm, L. M. Coolen, F. J. Karsch, and M. N. Lehman Morphological Plasticity in the Neural Circuitry Responsible for Seasonal Breeding in the Ewe Endocrinology, October 1, 2006; 147(10): 4843 - 4851. [Abstract] [Full Text] [PDF]
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G. M. Anderson, S. L. Hardy, M. Valent, H. J. Billings, J. M. Connors, and R. L. Goodman Evidence that Thyroid Hormones Act in the Ventromedial Preoptic Area and the Premammillary Region of the Brain to Allow the Termination of the Breeding Season in the Ewe Endocrinology, July 1, 2003; 144(7): 2892 - 2901. [Abstract] [Full Text] [PDF]
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