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Department of Veterinary Biosciences, University of Illinois, Urbana Illinois 61801
Address all correspondence and requests for reprints to: Dr. Gary L. Jackson, Department of Veterinary Biosciences, University of Illinois, 2001 South Lincoln Avenue, Urbana, Illinois 61802. E-mail: g-jackson{at}uiuc.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-reductase to the more potent androgen
dihydrotestosterone (DHT) or via aromatization to estradiol (E). Both
DHT and E suppress LH secretion in male sheep (7, 8). The specific neural site(s) of action of these steroids also remains unknown. As GnRH neurons do not contain either androgen (9, 10) or estrogen receptors (11, 12), it seems unlikely that testicular steroids act directly on GnRH neurons. More likely, steroid regulation of GnRH neurons is mediated via effects on other neurons that contain steroid receptors and contact GnRH neurons directly or via interneurons. Among sites that may be important are the preoptic area (POA) and arcuate/ventromedial region (ARC/VMR). In sheep, these areas are rich in both androgen and estrogen receptors (10, 12, 13). In addition, these sites are important in the regulation of GnRH secretion (14, 15).
One approach to locate specific sites of action of steroids in the brain has been the use of localized steroid implants. T implants in the ventromedial nucleus (VMN) of male rats reduced the size of accessory sexual organs (16), implying a decrease in LH secretion. In addition, implants of DHT or E into the POA or mediobasal hypothalamus (MBH) of male rats elevated GnRH content, and MBH implants reduced serum LH (17). Although the results of that study suggest a localized effect of DHT and E on GnRH secretion, the effects of MBH implants on LH secretion in the rat are hard to interpret due to the possibility of steroid diffusion to the pituitary gland, "the implantation paradox of Bogdanove" (18), where it can affect the responsiveness to GnRH. This difficulty is greatly reduced in larger animals because the large size of the brain reduces the likelihood of diffusion from steroid implants to other active sites. Thus, we used the sheep as a model with which to study the specific sites of action of steroids on GnRH secretion. The aim of this study was to determine the effect on LH secretion of implants of T, DHT, or E discretely placed into the POA or ARC/VMR.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All experiments were approved by the institutional committee on laboratory animal care and were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
Brain surgery
Bilateral stainless steel guide cannulae were aseptically
inserted into the POA or ARC/VMR as previously described (15). Briefly,
the head of the anesthetized sheep was fitted into a stereotaxic frame
(Kopf Instruments, Tujunga, CA). The dorsal cerebral cortex was
exposed, the sagittal sinus was ligated, a radioopaque dye was injected
into the third ventricle, and a lateral x-ray was taken. The
ventriculogram provided landmarks for cannula placement.
Eighteen-gauge, thin-walled, stainless steel guide cannulae fitted with
a protruding (2-mm) wire stylet then were inserted into previously
described coordinates (15) using an XYZ manipulator (Kopf Instruments,
Tunjunga, CA). The guide cannulae were secured in place with dental
acrylic set around screws fixed in the skull. The top part of a plastic
bottle was set in the acrylic to protect the tops of the guide
cannulae. Sheep were given at least 2 weeks to recover from surgery
before the experiment was started.
Implant manufacture
Implants were made using a modification to the methods of Blache
et al. (24) and Lincoln and Maeda (25). Implants for each
steroid were made on a separate day, and the area was thoroughly
cleaned to avoid cross-contamination of steroids. Test tubes containing
powdered steroid [T, DHT, E, or cholesterol (CHOL)] were placed in an
oil bath heated to just above the melting point of the appropriate
steroid in a muffle oven. Implants were made from 20-gauge,
thin-walled, stainless steel tubing, with the depth fixed by a collar
of 18-gauge, thin-walled tubing. The steroid was drawn into the tubing
via capillary action by preheating the tubing to the temperature of the
steroid and placing the tips into the molten steroid for 15 min. After
crystallization, the outer surface of the tubing was cleaned with a
razor blade and alcohol, and then checked with a dissecting microscope
for the presence of contaminating steroid crystals on the exterior and
a smooth surface of steroid in the cannula tip. Implants were
sterilized by formalin vapors before insertion into the guide cannulae.
Each implant was used only once.
To confirm that the implants would release steroid and to estimate the distance of diffusion, a separate set of T implants was manufactured that contained 0.01% tritiated T (279 µCi/µg). Unilateral implants were placed into the POA and ARC/VMR of castrated rams (n = 2 sheep/site) and left in place for 5 days. The sheep then were killed, and pituitary, hypothalamus, and brain cortex samples were collected and frozen on dry ice. Blood plasma samples also were obtained. The tissue blocks were sectioned (50 µm) on a cryostat and then groups of 10 whole sequential sections were collected into scintillation vials, where they were solubilized (2 ml Soluene 350, Packard Instruments, Meriden, CT). This pooling procedure limited resolution to 500 µm. One hundred-microliter plasma samples also were placed into scintillation vials. Six milliliters of scintillation fluid were added to each vial and counted for radioactivity. Estimates of radioactivity were corrected for quenching and background. Release also was evaluated by placing the tips of tritiated T implants into 10 µl or 1 ml distilled water for 2 days and then determining the amount of radioactivity in the water. Use of the two volumes provides estimates of release under conditions of limited and unlimited diffusion gradients.
Experimental procedures
In each experiment, serial blood samples (3 ml) were taken via
jugular cannula every 10 min for 4 h during the morning.
Exp 1.
In this pilot study, T, E, and CHOL (control)
implants were placed bilaterally into either the POA or ARC/VMR (n
= 6/site). The implant cannulae containing steroids protruded 2 mm
beyond the end of the guide cannulae. Implants were left in place for 5
days, as previous work from our laboratory had shown that T infusion
into the jugular vein of long term castrated rams significantly
suppresses LH secretion within 2 days (26).
All sheep were treated according to the following schedule: day 0, serial blood samples, remove stylets, and insert CHOL implants; day 5, serial blood samples, remove CHOL implants, and insert T implants; day 10, serial blood samples, remove T implants and insert fresh CHOL implants; day 15, serial blood samples, remove CHOL implants, and insert E implants; day 20, serial blood samples, remove E implant, and insert fresh CHOL implant; and day 25, serial blood samples, remove CHOL implant, and insert stylets.
Exp 2.
This experiment was conducted under an artificial
inhibitory photoperiod (16 h of light, 8 h of darkness) as
described previously. In this experimental series, T implants were
replaced by DHT implants, and the treatment period was extended to 7
days. A new set of sheep was prepared, with bilateral guide cannulae
inserted into either the POA or ARC/VMR (n = 6/site). Within-site
sheep were allocated to treatments according to a balanced latin square
design such that each sheep received each treatment, CHOL, DHT, or E,
with a balance in order of treatment over time. Blood samples were
taken as described above, then stylets were removed, and the first
implants were inserted. These implants were left in place for 7 days
before blood samples were again taken. The implants then were removed,
and stylets were reinserted and left in place 7 days. Blood samples
were again taken, and the second steroid implants were inserted. This
sequence was repeated until all three steroid treatments had been
given. A final sampling was performed 7 days after removal of the final
steroid to determine whether the LH patterns returned to normal levels
for a castrated ram at the end of the experiment. Such a pattern
indicated that any suppression of LH secretion at the time of the final
steroid treatment was due to steroid treatment and not tissue damage
over time.
Exp 3.
A third experiment was performed on different animals
(n = 6) to further evaluate the effect of steroids placed into the
ARC/VMR. This experiment was conducted in an identical manner to Exp 2,
except that it was carried out under a natural photoperiod (increasing
inhibitory day length). In addition, we determined whether the observed
effects might be due to diffusion of steroids to the pituitary. At the
end of the standard 4-h blood-sampling period, each sheep was given an
iv injection of 250 ng GnRH (5) and sampled at 10-min intervals for an
additional 50 min. The response, or peak height, was calculated as the
highest subsequent LH concentration minus the basal concentration at
the time of the GnRH injection.
Exp 4.
These same animals then were used in Exp 4 to further
evaluate the effect of T and CHOL implants in the ARC/VMR. Blood
samples were taken 7 days after insertion of the stylet at the end of
Exp 3. This was treated as the control sample. The sheep then were
randomly allocated to first receive either T or CHOL implants. Seven
days after insertion of T or CHOL implants, blood samples again were
taken. Then these implants were removed, and those containing the
alternate steroid were inserted. Blood samples were taken again 7 days
later.
LH assays
LH was measured in duplicate samples using a previously
described RIA (27) fully validated in our laboratory (5). The assay
sensitivity was 2 ng/ml of NIH LH S20 at 90% binding. The intraassay
coefficients of variation were 11.9% and 9.8%, and the interassay
coefficients of variation were 4.2% and 4.0% for low and high quality
control samples, respectively.
Histology
After the completion of each experiment, all sheep were killed,
and their heads were perfused with saline followed by 10% formaldehyde
in PBS (pH 7.4). The brains were collected, and the hypothalami were
postfixed in the same fixative. Serial 10-µm paraffin sections were
cut in the coronal plane and stained with Luxol fast blue for
histological verification of the implantation site.
Statistical analysis
Pulses of LH were determined using the Pulsar algorithm (28).
The G values were set at G1 = 3.98, G2 = 2.4, G3 = 1.68,
G4 = 1.24, and G5 = 0.93. Effects of treatment on mean LH, LH
pulse amplitude, and LH interpulse interval (IPI) were analyzed using
ANOVA for repeated measures and, where appropriate, the ANOVA was
followed by the Newman-Keuls test. Effects of POA implants
vs. ARC/VMR implants were not compared directly,
i.e. they were analyzed as separate experiments. Data from
that part of Exp 2 dealing with the effects of ARC/VMR implants were
combined with data from Exp 3 and analyzed by ANOVA for repeated
measures, treating experiment as the main plot.
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Results |
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POA implants
Placements of guide cannulae into the POA in Exp 1 and 2 are shown
in Fig. 1
. Although two sets of implants
clearly were in the anterior region of the POA, POA guide cannula
placement was accepted for all 12 animals. Examples of the effects of
POA implants on LH concentrations are shown in Figs. 2 and 3 for
Exp 1 and 2, respectively. Summary data are presented in Tables 1 and 2.
CHOL, T, and DHT implants did not detectably affect LH secretion
patterns. In both Exp 1 and 2, E implants suppressed LH secretion to a
small degree, although the nature of this suppression varied between
experiments. In Exp 1 there was a small, but nonsignificant, reduction
in mean LH. The IPI after E treatment was not significantly different
from that after the preceding CHOL treatment. In Exp 2, E did not
affect mean LH or LH pulse amplitude, but there was a small, yet
significant (P < 0.05), increase in IPI.
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. Data from three
of six sheep in Exp 1, three of six sheep in Exp 2, and one of six
sheep in Exp 3 were deleted due to misplaced or damaged guide cannulae
or ill health of the animal. Locations of cannula placement in those
animals are not included in the figures. Examples of the LH pulse
profiles in animals with ARC/VMR implants are shown in Figs. 5 and 6 for
Exp 1 and Exp 2–3, respectively, and summary data are presented in
Tables 3, 4, and 5.
To facilitate comparison, results are described according to effect of
steroid rather than to the order of the experiment.
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),
no effect on any parameter of LH secretion was detected in subsequent
experiments (Tables 4 and 5). Neither T (Tables 3 and 5) nor DHT (Table 4) had any detectable effect on LH secretion.
In contrast, E reduced LH secretion, although the effect on pulse
frequency (IPI) appeared to differ between Exp 1 and Exp 2–3. In Exp 1
(Table 3), E appeared to reduce IPI (P < 0.05), pulse
amplitude, and mean LH. However, the effects on amplitude and mean LH
were not statistically significant (P > 0.05). In the
larger Exp 2–3, E clearly increased IPI (P < 0.01)
and reduced mean LH (P < 0.01). In one animal, plasma
LH was reduced by E to below assay detectability.
The steroid treatments did not significantly (P > 0.05) affect LH release in response to exogenous GnRH (peak heights: CHOL, 65.8 ± 7.1; DHT, 77.1 ± 6.0; E, 81.8 ± 8.7 ng/ml).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The failure of T implants kept in place for up to 7 days to reduce LH was unexpected given that systemic infusion of T into long term castrates will significantly suppress LH within 48 h (26). The reason for the ineffectiveness of T is not clear, but there are several possibilities. One is that there may be down-regulation of steroid (androgen) receptors in the long term castrated animal. In the male rat, long term castration decreases androgen receptor messenger RNA (mRNA) levels in the POA (29), and in sheep the efficacy of peripheral T treatment decreases with time after castration (30, 31). Although, castration reportedly increases the number of E receptors in the hypothalamus of the ram (32), the effect of castration on hypothalamic androgen receptors in this species is not known. The fact that systemic treatment of castrated rams with physiological doses of T suppresses LH within 48–72 h (26, 33) does suggest that androgen-responsive tissues in castrates either remain responsive or regain responsiveness, through as yet undefined mechanisms, to T. The 5- or 7-day treatment period to which our animals were subjected surely was long enough for T to have acted, although the localized effects may not have been extensive enough to reverse the effects of castration and adequately restore responsiveness.
A second possibility is that the implants delivered too little T to exert an effect. This possibility cannot be discounted; however, evaluation of radiolabeled T release in vivo and in vitro suggested that at least several nanograms of T were reaching tissue around the implant. The fact that the E implants handled identically were effective also suggests that the implants were delivering potentially effective T doses even though T may be less potent than E. Also, it was noted that in other pilot studies in our laboratory 2-mm long cocoa butter T implants extruded from 20-gauge tubing into these same locations were without effect on LH (data not shown). However, these observations must be tempered by the caveats that the diffusion gradients in vitro vs. in vivo are probably very different, that the diffusion characteristics of E and T may differ, and that an effective local in vivo dose of T remains undefined.
A third possibility is that T acts at one more or more sites that were untested in these studies. This issue can be resolved only with additional studies; however, it should be noted that the chosen implant sites are rich in androgen receptors (10).
A fourth, and compelling, possibility is that T must be converted to either DHT or E to exert its negative influence on GnRH secretion and that there was insufficient aromatase and/or reductase at or around the implant sites to produce sufficient concentrations of DHT or E from T to detectably suppress LH. This possibility is attractive given that castration of male rats reduces aromatase activity and mRNA in the POA and MBH (34). The observation that systemic treatment with either DHT (35, 36) or E (6, 8) suppresses LH in the male sheep also suggests that both metabolites are mediators of T action. This suggestion is supported by observations that systemic treatment with a reductase inhibitor (37), antibodies to estrogen (38), or aromatase inhibitors (39) compromises the ability of T to suppress LH. In an attempt to deal with this possibility, we substituted DHT and E in the second and third experiments in this series.
The observation that DHT implants did not suppress LH secretion when
implanted into the POA or ARC/VMR was surprising given that systemic
injections of DHT are very effective in acutely castrated rams (35),
and that there are abundant androgen receptors in the POA and arcuate
and ventromedial nuclei in the male sheep (10). Also, implants of the
antiandrogen hydroxyflutamide placed into the POA of male rats elevated
circulating LH concentrations (40). The previously mentioned
observation that inhibition of 5-reductase compromises the ability
of T to suppress LH (37) in the ram strongly suggests that DHT is a
physiologically important mediator of T action in this species. Thus,
the failure of DHT implants to suppress LH may have been due to one or
more of the same possible factors that affected the action of T: lack
of receptors, inadequate delivery of DHT to sites around the implants,
or the fact that DHT acts at sites other than the POA and ARC/VMR to
suppress LH.
There is fragmentary evidence to support this last suggestion. In both male and female rats, the MBH has relatively low levels of reductase activity (41). That observation also suggests that the ARC/VMR may not be a significant site of DHT actions. Although androgen receptors have not been mapped in regions other than the hypothalamus in the ram, they are found at numerous other sites in other species. In the rat, high concentrations are found in the amygdala, bed nucleus of the stria terminalis, hippocampus, and brainstem (42). Of these, the brainstem is of interest due to considerable evidence of GnRH regulation by noradrenaline (43, 44). It is not known whether the noradrenaline cells in the brainstem contain androgen receptors, but they do contain estrogen receptors in female rats (45, 46). Possibly, the brainstem may be a significant site for DHT feedback on GnRH secretion.
E implants suppressed LH, particularly when placed into the ARC/VMR. A direct statistical comparison between the effects of E in the ARC/VMR and POA was not conducted, but it appeared that POA implants had a less robust effect than those in the ARC/VMR even though the POA contains abundant E receptors in both ewe and ram (10, 12, 13). Our results, although not identical, are consistent with those in a preliminary report by Blache et al. (47), who found that E implanted into the POA for 19 h did not alter LH in male sheep. Interestingly, E implants into the POA of ovariectomized ewes did not induce a LH surge, whereas implants into the ARC nucleus did (24). In this respect, sheep appear to differ from female rats, in which POA implants of E induced LH surges (48, 49). In total, these observations suggest that in sheep, both the POA and ARC/VMR are sites at which E regulates basal LH.
Although implanting E into the ARC/VMR appeared to increase LH pulse frequency (decrease IPI) in Exp 1, it clearly decreased the number of LH pulses in all animals in Exp 2 and 3. The reason for this difference is not obvious, but it may have reflected false positives detected by the pulsar algorithm when dealing with greatly suppressed LH in some animals. However, the overall suppressive effect of E was consistent in all experiments. Also, it should be noted that in one animal (no. 414) there was a residual suppressive effect of E after the implant was removed. The suppressive effect of ARC/VMR E implants on LH secretion is consistent with the report of Blache et al. (47). There are no reports of localized E treatment in males of other species; however, E implants into the arcuate or ventromedial nucleus of female sheep induced a LH surge (47) and inhibited LH secretion in female rats (50) and female monkeys (51).
Several observations argue against the interpretation that steroid from ARC/VMR implants diffused to the pituitary gland and acted there to suppress LH secretion. First, no evidence of radioactivity was detected in the pituitary gland after treatment with implants containing tritiated T. The small spread of radioactivity from these implants suggests that diffusion was limited to less than 1 mm, although it is acknowledged that unmeasurable amounts may have reached more distant sites. Although E might have diffused differently than T, our results for radiolabeled T are comparable to those reported previously for radiolabeled E (47). Second, E implants did not reduce LH release in response to a high physiological dose of exogenous GnRH. Third, the effect of E was on LH pulse frequency rather than on pulse amplitude, indicating actions on the hypothalamic pulse generator function. Collectively, these observations suggest that the action of E was exerted in the hypothalamus either at or very near the implant site. However, final verification of this suggestion will require measurement of the effects of such implants on GnRH release.
The cell types within the hypothalamus on which E acts to suppress GnRH
(LH) secretion are not known. GnRH neurons do not appear to possess E
receptors in the sheep (12, 13) or other species (52); thus, this
effect of E must be mediated by one or more other neuronal systems. The
-aminobutyric acid (GABA), dopaminergic, and ß-endorphin systems
are leading candidates. There is much evidence to suggest that
GABAergic neurons in the POA may be involved in the actions of T and E
in the sheep (12, 53) and of T in the male rat (40, 54, 55). In the
ARC/VMR of sheep, E receptors have been colocalized with dopamine and
ß-endorphin (56). Both of these neurotransmitters have been
implicated in the mediation of steroid feedback in male sheep (57, 58).
However, the specific roles of any of these transmitter systems in
regulating LH remain poorly defined.
Placing these observations into a broader concept of how T affects GnRH secretion is important, although challenging. First, it must be reiterated that T can be converted to E, probably by both peripheral and central neural aromatization. The extent of peripheral aromatization was demonstrated by Hileman et al. (37), who found that infusion of sufficient T into the circulation of wethers to achieve a high physiological circulating T concentration of 20 ng/ml resulted in circulating E concentrations of up to 10–15 pg/ml after 4 days. These E concentrations are somewhat higher than those found in rams (59). These observations are complemented by the observation that in rams, blockade of the conversion of T to E by systemic treatment with an aromatase inhibitor reduced circulating E concentrations by nearly half (39). Aromatase is present in the hypothalamus of the ewe (60), but its distribution in the brain of the sheep has not been reported. However, high levels of aromatase activity (41) and aromatase mRNA are present in both the POA and ARC/VMR of rats. Thus, it seems likely that both peripheral and local hypothalamic aromatizations of T have significant roles in regulating LH secretion.
In conclusion, the results of this study show that the ARC/VMR and the POA are important sites at which E acts to suppress LH in the male sheep. In the intact ram, this E is probably produced by both local neural and peripheral aromatization of T. The relative importance in the intact animal of these two E sources in regulating LH is not known. Although previous work indicates that reduction of T to DHT clearly is important in the mediating T feedback in male sheep, the site at which DHT acts remains unclear. It is possible that the effects of DHT are exerted at one or more sites other than the POA and ARC/VMR and that DHT and E act simultaneously at multiple sites to effect changes in LH secretion.
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Acknowledgments |
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Footnotes |
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2 Present address: Department of Physiology, Monash University,
Clayton, Victoria 3168, Australia.
Received March 26, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-aminobutyric acid receptor subtypes in the arcuate-ventromedial
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-reductase activity on
the ability of testosterone to inhibit luteinizing hormone release in
male sheep. Biol Reprod 50:1244–1250[Abstract]
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-aminobutyric acid turnover: effect of castration on
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male rat. J Neurochem 60:2254–2264[CrossRef][Medline]
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