Hypothalamic Sites of Action for Testosterone, Dihydrotestosterone ...

0013-7227/97/$03.00/0 Endocrinology Copyright © 1997 by The Endocrine Society

Vol. 138, No. 9 Printed in U.S.A.

Hypothalamic Sites of Action for Testosterone, Dihydrotestosterone, and Estrogen in the Regulation of Luteinizing Hormone Secretion in Male Sheep* CHRISTOPHER J. SCOTT†, DAVID E. KUEHL, SUZIE A. FERREIRA, GARY L. JACKSON

AND

Department of Veterinary Biosciences, University of Illinois, Urbana Illinois 61801 ABSTRACT Testosterone (T) inhibits LH secretion partly by acting at unknown sites within the brain to inhibit GnRH secretion. We tested the hypothesis that the preoptic area (POA) and arcuate-ventromedial region (ARC/VMR), areas rich in androgen and estrogen (E) receptors, are neural sites at which T and the T metabolites, dihydrotestosterone (DHT) and estrogen (E), act to suppress LH secretion. Bilateral guide cannulae were surgically implanted into either the POA or ARC/VMR of castrated male sheep. Experiments were conducted under a long day photoperiod to maximize the inhibitory effect of the steroids. In Exp 1, all sheep (n 5 6/site) sequentially received bilateral implants of cholesterol (CHOL), T, or E at each site. Jugular blood samples were taken at 10-min intervals for 4 h both immediately before implant insertion and 5 days later. In Exp 2, all sheep (n 5 6/site) sequentially received bilateral implants of CHOL, DHT, or E at each site according

to a latin square design. Blood samples were taken before and 7 days after implant insertion. In Exp 3, which followed the same design as Exp 2, implants of E, T, or DHT were placed only in the ARC/VMR. In the final experiment, the effects of T and CHOL implants in the ARC/VMR were compared. Neither T, DHT, nor CHOL implants at either site affected LH secretion. In contrast, E treatment in the ARC/VMR suppressed mean plasma LH levels (P , 0.01), primarily due to an increase in interpulse interval (P , 0.01). Estrogen implants in the POA caused a small, but nonsignificant (P . 0.05), decrease in mean LH levels in the first experiment and an increase in LH interpulse interval (P , 0.05) in the second experiment. These results suggest that the ARC/VMR and possibly the POA are sites at which E acts to reduce GnRH secretion in male sheep. (Endocrinology 138: 3686 –3694, 1997)

T

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.

ESTICULAR steroids inhibit LH secretion. Whether this is due to an action on GnRH secretion, responsiveness of the pituitary to GnRH, or both remains unclear in the rat (1–3). However, in the male sheep testicular steroids clearly reduce GnRH secretion (4 – 6). Whereas testosterone (T) is effective in suppressing GnRH secretion in castrated sheep (5, 6), it is not known whether this action is due to T itself or one of its metabolites. Testosterone may act directly on androgen receptors or, alternatively, it may be metabolized via 5a-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 Received March 26, 1997. 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: [email protected]. * Presented in part at the 39th Annual Meeting of the Endocrine Society of Australia, Sydney, Australia, 1996. This work was supported by USPH Grant HD-27543. † Present address: Department of Physiology, Monash University, Clayton, Victoria 3168, Australia.

Materials and Methods Yearling Suffolk and Hampshire rams were castrated at least 8 weeks before neurosurgery. With the exception of Exp 2, the sheep were housed outdoors before surgery and fed pasture grass. After surgery, the sheep were housed indoors under natural photoperiod and fed alfalfa hay or pellets and sheep chow (Ralston Purina Co., St. Louis, MO), with free access to water. All experiments were conducted under an inhibitory

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FEEDBACK SITES OF TESTICULAR STEROIDS ON LH SECRETION long day photoperiod to maximize the sensitivity to steroid feedback (19, 20). Exp 1, 3, and 4 were conducted under a natural photoperiod (February through May). Exp 2 was conducted during the normal breeding season; thus, controlled lighting was used to produce an inhibitory photoperiod. In Exp 2, the sheep were placed indoors 105 days after the summer solstice, by which time they would have been sensitive to the inhibitory influence of long days (21, 22). They then were kept on a lighting regimen of a constant long day photoperiod (16 h of light and 8 h of darkness/day). The first blood samples were taken 55 days later, by which time the animals were sensitive to the inhibitory effects of the steroid treatments (23). 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.

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Exp 1. In this pilot study, T, E, and CHOL (control) implants were placed bilaterally into either the POA or ARC/VMR (n 5 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.

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 mCi/mg). Unilateral implants were placed into the POA and ARC/VMR of castrated rams (n 5 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 mm) 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 mm. One hundredmicroliter 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 ml 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 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 5 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 5 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-mm paraffin sections were cut

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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 5 3.98, G2 5 2.4, G3 5 1.68, G4 5 1.24, and G5 5 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.

Results Diffusion studies

The total radioactivity recovered from around each implant in vivo after 5 days was 489 6 94 dpm. This corresponded to about 6 ng of total T present around the implant site. Ninety percent of the detected radioactivity was limited to within 0.5 mm of the implant site. Beyond 1 mm from the implant site, radioactivity always was at background levels (30 –50 dpm). No radioactivity was found in the samples of pituitary, cerebral cortex, or blood plasma. The release of [3H]T in vitro was 455 6 178 dpm/day into 10 ml water and 6209 6 2446 dpm/day into 1 ml water. This corresponded to approximately 6 and 78 ng/day steroid released, respectively. 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. ARC/VMR implants

Placements of guide cannulae into the ARC/VMR in Exp 1 and Exp 2– 4 are shown in Fig. 4. 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 de-

FIG. 1. Diagram showing the composite location of implant placement in sheep with guide cannulae in the POA in Exp 1 (upper panel) and Exp 2 (lower panel). Solid circles represent the site of the implant. Implants sites belonging to the same sheep are connected by a solid line. Only five sites are shown in the lower panel because placements were nearly identical in two animals. ac, Anterior commissure; db, diagonal band of Broca; ovlt, organum vasculosum of the lamina terminalis; son, supraoptic nucleus; ls, lateral septum.

scribed according to effect of steroid rather than to the order of the experiment. Although CHOL appeared to reduce LH pulse amplitude in Exp 1 (Table 3), 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 6 7.1; DHT, 77.1 6 6.0; E, 81.8 6 8.7 ng/ml). Discussion

These experiments showed that localized implants of T or DHT into either the POA or ARC/VMR of the castrated ram brain did not affect circulating LH secretory patterns. In contrast, implants of E into the POA marginally, but significantly, increased IPI, whereas implants into the ARC/VMR clearly and very significantly reduced mean LH concentrations whereas the effects on IPI varied with experiment. Collectively, these results suggest that the ARC/VMR and possibly the POA are sites at which E exerts negative feedback in the male sheep.


FIG. 2. Examples of LH secretory profiles in plasma of sheep with either stylets or steroid implants placed bilaterally into the POA in Exp 1. CON, Nonsteroid control. Sampling periods were 5 days apart. Peaks of LH pulses are represented by hollow circles. The number in the upper right refers to the animal number.

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

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FIG. 3. Examples of LH secretory profiles in plasma of sheep with either stylets or steroid implants placed bilaterally into the POA in Exp 2. CON, Nonsteroid control. Sampling periods were 7 days apart. Peaks of LH pulses are represented by hollow circles.

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

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TABLE 1. The effect of steroid implants into the preoptic area on mean (6SEM) plasma LH, pulse amplitude, and interpulse interval in castrated rams (n 5 6) in Exp 1

Mean LH (ng/ml) Pulse amplitude (ng/ml) Interpulse interval (min)

Control

Chol 1

T

Chol 2

E

Chol 3

18.3 6 2.1

18.4 6 0.4

18.9 6 2.3

20.3 6 2.6

16.9 6 2.3

18.9 6 3.1

7.6 6 1.0

7.9 6 0.6

7.6 6 0.9

8.2 6 0.9

6.9 6 0.9

6.3 6 0.6

47 6 2.1

44 6 3.2

43 6 2.0

46 6 1.7

51 6 3.6

43 6 1.4

Chol, Cholesterol; T, testosterone; E, estradiol. TABLE 2. The effect of steroid implants into the preoptic area on mean (6SEM) plasma LH, pulse amplitude, and interpulse interval in castrated rams (n 5 5– 6) in Exp 2


Control

Chol

Control

E

Control

DHT

13.9 6 1.4

12.6 6 1.1

11.7 6 0.8

12.1 6 0.7

10.8 6 0.6

11.2 6 0.7

8.7 6 1.1

5.9 6 1.3

5.2 6 0.8

6.6 6 1.5

4.3 6 0.4

5.8 6 1.1

44 6 2.7

42 6 1.9

41 6 2.3

48 6 2.4

42 6 1.9

42 6 3.1

a

Chol, Cholesterol; E, estradiol; DHT, dihydrotestosterone. In one animal the guide cannulae were damaged before the experiment was completed. a P , 0.05 compared with the respective control period.

FIG. 4. Diagram showing the composite location of implant placement in sheep with guide cannulae in the ARC/VMR in Exp 2 (upper panel) and Exp 3 and 4 (lower panel). Circles connected by solid lines represent the bilateral implant sites within an individual animal. arc, Arcuate nucleus; III, third ventricle; fx, fornix; me, median eminence; mt, mammillothalamic tract; ot, optic tract; dm, dorsomedial nucleus; vm, ventromedial nucleus.

(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 androgenresponsive 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 ex-

tensive 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


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FIG. 5. Examples of LH secretory profiles in plasma of sheep with either stylets or steroid implants placed bilaterally into the ARC/VMR in Exp 1. CON, Nonsteroid control. Sampling periods were 5 days apart. Peaks of LH pulses are represented by hollow circles.

FIG. 6. Examples of LH secretory profiles in plasma of sheep with either stylets or steroid implants placed bilaterally into the ARC/VMR in Exp 2 (no. 566 and 570) and Exp 3 (no. 602). CON, Nonsteroid control. Sampling periods were 7 days apart. Peaks of LH pulses are represented by hollow circles.

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

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TABLE 3. The effect of steroid implants into the ARC/VMR of the hypothalamus on mean (6SEM) plasma LH, pulse amplitude, and interpulse interval in castrated rams (n 5 3) in Exp 1


Control

Chol 1

T

Chol 2

E

Chol 3

15.8 6 0.83

14.6 6 1.42

14.8 6 1.6

15.2 6 0.8

9.3 6 2.5

12.5 6 2.2

8.7 6 0.9

5.1 6 0.3a

5.6 6 0.5

5.2 6 1.6

3.1 6 1.0

5.3 6 0.9

50 6 3.9

45 6 2.4

50 6 3.2

48 6 2.7

37 6 3.4

49 6 3.8

b

Chol, Cholesterol; T, testosterone; E, estradiol. a P , 0.05 compared with control. b P , 0.05 compared with Chol 2. TABLE 4. The effect of steroid implants into the ARC/VMR of the hypothalamus on mean (6SEM) plasma LH, pulse amplitude, and interpulse interval in castrated rams (n 5 8) in Exp 2 and 3


Control

Chol

Control

E

Control

DHT

12.2 6 1.3

12.1 6 1.1

11.7 6 1.0

7.8 6 0.8

11.5 6 1.4

12.0 6 1.2

6.3 6 0.7

6.1 6 0.7

6.1 6 0.5

3.8 6 0.7

5.4 6 0.8

5.4 6 0.8

43 6 1.8

44 6 3.3

44 6 3.3

94 6 23.4a

44 6 3.9

45 6 3.8

a

Chol, Cholesterol; E, estradiol; DHT, dihydrotestosterone. a P , 0.01 compared with control. TABLE 5. The effect of steroid implants into the ARC/VMR of the hypothalamus on mean (6SEM) plasma LH, pulse amplitude, and interpulse interval in castrated rams (n 5 5) in Exp 4


Cholesterol

Testosterone

Control

13.5 6 2.0

13.5 6 2.0

12.2 6 2.2

5.8 6 1.3

5.7 6 1.0

5.3 6 0.4

41 6 2.1

37 6 1.3

40 6 1.8

male rats elevated circulating LH concentrations (40). The previously mentioned observation that inhibition of 5a-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 g-aminobutyric acid (GABA), dopaminergic, and b-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 b-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

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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. Acknowledgments We thank Mrs. J. Thompson for histological preparations, Dr. David Schaeffer for statistical advice, Dr. J. Roser (University of CaliforniaDavis) for the LH antibody, Dr. S. Hileman for technical assistance and reviewing the manuscript, and the National Hormone and Pituitary Agency (University of Maryland-Baltimore) for the ovine LH.

References 1. Kalra SP, Kalra PS 1989 Do testosterone and estradiol-17b enforce inhibition or stimulation of luteinizing hormone-releasing hormone. Biol Reprod 41:559 –570 2. Levine JE, Bauer-Dantoin AC, Seck LM, Conaghan LA, Legan SJ, Meredith JM, Strobl FJ, Urban JH, Vogelsong KM, Wolf AM 1991 Neuroendocrine regulation of the luteinizing hormone pulse generator in the rat. Recent Prog Horm Res 47:97–153 3. Phelps CP, Kalra SP, Kalra PS 1992 In vivo pulsatile release into the anterior pituitary of the male rat-effects of castration. Brain Res 569:159 –163 4. Caraty A, Locatelli A 1988 Effect of time after castration on secretion of LHRH and LH in the ram. J Reprod Fertil 82:263–269 5. Jackson GL, Kuehl DE, Rhim TJ 1991 Testosterone inhibits gonadotropinreleasing hormone pulse frequency in the male sheep. Biol Reprod 45:188 –194 6. Tilbrook AJ, deKretser DM, Cummins JT, Clarke IJ 1991 The negative feedback effects of testicular steroids are predominantly at the hypothalamus in rams. Endocrinology 129:3080 –3092 7. Sanford LM, Palmer WM, Howland BE 1976 LH release in castrate-male sheep treated with testosterone and dihydrotestosterone. IRCS Medical Science: experimental animals. 4:408 8. Edgerton LA, Baile LA 1977 Serum LH suppression by estradiol but not by testosterone or progesterone in wethers. J Anim Sci 44:78 – 83 9. Huang X, Harlan RE 1993 Absence of androgen receptors in LHRH immunoreactive neurons. Brain Res 624:309 –311 10. Herbison AE, Skinner DC, Robinson JE, King IS 1996 Androgen receptorimmunoreactive cells in ram hypothalamus: distribution and co-localization patterns with gonadotropin-releasing hormone, somatostatin and tyrosine hydroxylase. Neuroendocrinology 63:120 –131 11. Shivers BD, Harlan RE, Morrell JI, Pfaff DW 1983 Absence of oestradiol concentration in cell nuclei of LHRH-immunoreactive neurons. Nature 304:345–347 12. Herbison AE, Robinson JE, Skinner DC 1993 Distribution of estrogen receptor-immunoreactive cells in the preoptic area of the ewe: co-localization with glutamic acid decarboxylase but not luteinizing hormone-releasing hormone. Neuroendocrinology 57:751–759 13. Lehman MN, Ebling FJP, Moenter SM, Karsch FJ 1993 Distribution of estrogen receptor-immunoreactive cells in the sheep brain. Endocrinology 133:876 – 886 14. Clarke IJ, Scott CJ 1993 Studies on the neuronal systems involved in the oestrogen-negative feedback effect on gonadotrophin releasing hormone neurons in the ewe. Hum Reprod [Suppl 2] 8:2– 6 15. Ferreira SA, Scott CJ, Kuehl DE, Jackson GL 1996 Differential regulation of luteinizing hormone release by g-aminobutyric acid receptor subtypes in the arcuate-ventromedial region of the castrated ram. Endocrinology 137:3453–3460 16. Lisk RD 1962 Testosterone-sensitive areas in the hypothalamus of the rat. Acta Endocrinol (Copenh) 41:195–204 17. Kalra PS, Kalra SP 1980 Modulation of hypothalamic luteinizing hormonereleasing hormone levels by intracranial and subcutaneous implants of gonadal steroids in castrated rats: effects of androgen and estrogen antagonists. Endocrinology 106:390 –397 18. McCann SM 1974 Regulation of secretion of follicle-stimulating hormone and luteinizing hormone. In: Knobil E, Sawyer WH. Handbook of Physiology, sect 7, vol 4, part 2. American Physiological Society, Washington DC, pp 489 –517 19. Pelletier J, Ortavant R 1975 Photoperiodic control of LH release in the ram. Acta Endocrinol (Copenh) 78:442– 450 20. Lubbers LS, Jackson GL 1993 Neuroendocrine mechanisms that control seasonal changes of luteinizing hormone secretion in sheep are sexually differentiated. Biol Reprod 49:1369 –1376 21. Jackson GL, Gibson M, Kuehl D 1988 Photoperiodic disruption of photorefractoriness in the ewe. Biol Reprod 38:127–134 22. Khalid M, Jackson GL 1991 Exposure of ewes to long-day photoperiods before the winter solstice can disrupt refractoriness to short days. Anim Reprod Sci 25:225–232 23. Jackson GL, Jansen HT, Kuehl DE, Shanks RD 1989 Time of the sidereal year

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24. 25. 26. 27. 28. 29. 30. 31.

32. 33. 34. 35. 36. 37. 38. 39. 40.

41. 42.


affects responsiveness to the phase-resetting effects of photoperiod in the ewe. J Reprod Fertil 85:221–227 Blache D, Fabre-Nys CJ, Venier G 1991 Ventromedial hypothalamus as a target for oestradiol action on proceptivity, receptivity and luteinizing hormone surge of the ewe. Brain Res 546:241–249 Lincoln GA, Maeda K-I 1992 Reproductive effects of placing micro-implants of melatonin in the mediobasal hypothalamus and preoptic area in rams. J Endocrinol 132:201–215 Rhim TJ, Jackson GL 1993 Comparison of pulsatile and constant testosterone on secretion of gonadotropins in the ram. Endocrinology 132:2399 –2406 Matteri RL, Roser JF, Baldwin DM, Lipovetsky V, Papkoff H 1987 Characterization of a monoclonal antibody which detects luteinizing hormone from diverse species. Dom Anim Endocrinol 4:157–165 Merriam GR, Wachter KW 1982 Algorithms for study of episodic hormone secretion. Am J Physiol 243:E310 –E318 Handa RJ, Kerr JE, Don Carlos LL, McGivern RF, Hejna G 1996 Hormonal regulation of androgen receptor messenger RNA in the medial preoptic area of the male rat. Mol Brain Res 39:57– 67 Schanbacher BD 1981 Testosterone regulation of LH secretion; effect of time after castration. J Androl 2:26 D’Occhio MJ, Galil KAA, Brooks DE, Setchell BP 1985 Differential effects of gonadectomy on sensitivity to testosterone of brain centres associated with gonadotrophin negative feedback and with mating behaviour in rams. J Endocrinol 104:69 –75 Madigou T, Tiffoche C, Lazennec G, Pelletier J, Thielant M-L 1996 The sheep estrogen receptor: cloning and regulation of expression in the hypothalamopituitary axis. Mol Cell Endocrinol 121:153–163 Hileman SM, Lubbers LS, Petersen SL, Kuehl DE, Scott CJ, Jackson GL 1996 Influence of testosterone on LHRH release, LHRH mRNA and pro-opiomelanocortin mRNA in male sheep. J Neuroendocrinol 8:113–121 Abdelgadir SE, Resko JA, Ojeda SR, Lephart ED, McPhaul MJ, Roselli CE 1994 Androgens regulate aromatase cytochrome P450 messenger ribonucleic acid in rat brain. Endocrinology 135:395– 401 D’Occhio MJ, Schanbacher BD, Kinder JE 1983 Androgenic and oestrogenic steroid participation in feedback control of luteinizing hormone secretion in male sheep. Acta Endocrinol (Copenh) 102:499 –544 Schanbacher DB 1985 Effects on intermittent pulsatile infusion of luteinizing hormone-releasing hormone on dihydrotestosterone-suppressed gonadotropin secretion in castrate rams. Biol Reprod 33:603– 611 Hileman SM, Lubbers LS, Kuehl DE, Schaeffer DJ, Rhodes L, Jackson GL 1994 Effect of inhibiting 5a-reductase activity on the ability of testosterone to inhibit luteinizing hormone release in male sheep. Biol Reprod 50:1244 –1250 Sanford LM 1987 Luteinizing hormone release in intact and castrate rams is altered with immunoneutralization of endogenous estradiol. Can J Physiol Pharmacol 65:1442–1447 Schanbacher BD 1984 Regulation of luteinizing hormone secretion in male sheep by endogenous estrogen. Endocrinology 115:944 –950 Grattan DR, Rocca MS, Sagrillo CA, McCarthy MM, Selmanoff M 1996 Antiandrogen microimplants into the rostral medial preoptic area decrease g-aminobutyric acidergic neuronal activity and increase luteinizing hormone secretion in the intact male rat. Endocrinology 137:4167– 4173 Selmanoff MK, Brodkin LD, Weiner RI, Siiteri PK 1977 Aromatization and 5a-reduction of androgens in discrete hypothalamic and limbic regions of the male and female rat. Endocrinology 101:841– 848 Simerly RB Chang C, Muramatsu M, Swanson LW 1990 Distribution of androgen and estrogen receptor mRNA containing cells in the rat brain: an in situ hybridization study. J Comp Neurol 294:76 –95

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43. Goodman RL, Robinson JE, Kendrick KM, Dyer RG 1995 Is the inhibitory action of estradiol on luteinizing hormone pulse frequency in anestrous ewes mediated by noradrenergic neurons in the preoptic area. Neuroendocrinology 61:284 –292 44. Herbison AE, Heavens RP, Dyer RG 1990 Oestrogen modulation of excitatory A1 noradrenergic input to rat medial preoptic gamma aminobutyric acid neurones demonstrated by microdialysis. Neuroendocrinology 52:161–168 45. Sar M, Stumpf WE 1981 Central noradrenergic neurons concentrate 3Hoestradiol. Nature 289:500 –502 46. Herbison AE, Simonian SX, Characterization of brainstem estrogen-receptive neurons projecting to the vicinity of the gonadotrophin-releasing hormone (GnRH) cell bodies in the rat. 26th Annual Meeting of the Society for Neuroscience, Washington DC, 1996, p 1589 (Abstract) 47. Blache D, Anderson ST, Blackberry MA, Curlewis JD, Martin GB, Effects of intracerebral implants of sex steroid on luteinizing hormone secretion in mature merino rams. 27th Annual Meeting of the Australian Society for Reproductive Biology, 1995, p 40 (Abstract) 48. Kalra PS, and McCann SM 1975 The stimulatory effects on gonadotropin release of implants of estradiol or progesterone in certain sites in the central nervous system. Neuroendocrinology 19:289 –302 49. Goodman RL 1978 The site of positive feedback action of estradiol in the rat. Endocrinology 102:151–159 50. Smith ER, Davidson JM 1974 Location of feedback receptors: effects of intracranially implanted steroids on plasma LH and LRF response. Endocrinology 95:1566 –1573 51. Ferin M, Carmel PW, Zimmerman EA, Warren M, Perez R, Vandewiele RL 1974 Location of intrahypothalamic estrogen-responsive sites influencing LH secretion in the female rhesus monkey. Endocrinology 95:1059 –1068 52. Watson RD Jr, Langub MC Jr, Landis JW 1993 Further evidence that most luteinizing hormone-releasing hormone neurons are not directly estrogenresponsive: simultaneous localization of luteinizing hormone releasinghormone and estrogen receptor immunoreactivity in the guinea pig brain. J Neuroendocrinol 4:311–317 53. Robinson JE, Kendrick KM, Lambart CE 1991 Changes in the release of gamma-aminobutyric acid and catecholamines in the preoptic/septal area prior to and during the ovulatory surge of luteinizing hormone in the ewe. J Neuroendocrinol 3:393– 400 54. Grattan DR, Selmanoff M 1993 Regional variation in g-aminobutyric acid turnover: effect of castration on g-aminobutyric acid turnover in microdissected brain regions of the male rat. J Neurochem 60:2254 –2264 55. Grattan DR, Selmanoff M 1994 Castration-induced decrease in the activity of medial preoptic and tuberoinfundibular GABAergic neurons is prevented by testosterone. Neuroendocrinology 60:141–149 56. Lehman MN, Karsch FJ 1993 Do GnRH, tyrosine hydroxylase-, and b-endorphin-immunoreactive neurons contain estradiol receptors? A double-label immunocytochemical study in the Suffolk ewe. Endocrinology 133:887– 895 57. Tortonese DJ, Lincoln GA 1994 Photoperiodic modulation of the dopaminergic control of pulsatile LH secretion in sheep. J Endocrinol 143:25–32 58. Havern RL, Whisnant CS, Goodman RL 1994 Dopaminergic structures in the ovine hypothalamus mediating estradiol negative feedback in anestrous ewes. Endocrinology 134:1905–1914 59. Ferreira SA, Lubbers LS, Jackson GL 1995 Temporal relationship of luteinizing hormone (LH), testosterone (T), and estradiol (E2) in the intact ram. Biol Reprod [Supp 1] 52:p147 (Abstract) 60. Glass JD, Amann RP, Nett TM 1986 Effects of season and sex on in vitro aromatase and 17b-oxireductase activities in the brain and anterior pituitary gland of the sheep. Dom Anim Endocrinol 3:227–236