Effects of Testosterone Implants and Hypothalamic Lesions on Luteinizing Hormone Regulation in the Castrated Male Rat CECILIA Y. CHEUNG1 AND JULIAN M. DAVIDSON Stanford University, Department of Physiology, Stanford, California 94305 suppression of endogenous LHRH release, the median eminence-arcuate region was destroyed to remove the source of LHRH. In these animals, the suppression of plasma LH was evident on the first day after the lesion, but pituitary responsiveness to LHRH was unaffected until after one week. When Silastic capsules were implanted SC into lesioned animals, a more rapid (less than 1 week)

ABSTRACT. The effects of intrahypothalamic and subcutaneous implants of testosterone (T) and those of hypothalamic lesions on resting levels of circulating LH and pituitary responsiveness to exogenous LHRH were studied in castrated male rats to elucidate hypothalamic and pituitary regulation of LH secretion. Two hundred /ig implants of testosterone propionate (TP) in the median eminence region suppressed plasma LH titers before evidence of direct inhibition of pituitary function (as indicated by testing with LHRH) was found. Such implants release appreciable amounts of T into the peripheral circulation in the immediate postoperative period, and SC Silastic (constant release) capsules containing T have similar effects. The findings suggest that, regardless of the site of implant, the initial negative feedback inhibition of LH by T is not dependent on direct action at the pituitary level but rather appears to be a hypothalamic effect. In the days following exposure to hypothalamic or peripheral implantation of T, however, a progressively developing decline in the response to exogenous LHRH was observed. In order to determine whether this effect results from

T

HE SITE(S) of the feedback actions of steroid hormones constitute a perennial problem in neuroendocrinology. For androgen, as for other classes of steroids, convincing evidence exists for feedback receptors located in the hypothalamus and pituitary (1-4), but there is no agreement as to specific sites of action. The present study uses the experimental approach of measuring basal (resting) circulating LH levels (to define negative feedback effects) coupled with determination of responses to LHRH (to identify direct pituitary effects) Received January 12, 1976. Correspondence to: Dr. Julian M. Davidson, Stanford University, Department of Physiology, Stanford, California 94305. Supported by NIH Grant HD 00778. 1 Present address: Department of Obstetrics and Gynecology, University of California, School of Medicine, San Francisco, California 94143.

inhibition

of pituitary

responsivity

ensued.

Suprachiasmatic lesions did not affect basal LH secretion or pituitary responses to LHRH. The data provide evidence for a dual feedback action of T on LH in castrated male rats: an initial inhibitory effect presumably due to hypothalamic inhibition (commencing at around 6 h after hypothalamic or SC implantation of T), and a subsequent suppression of pituitary responsiveness (after one day) presumably due to direct action of T on the pituitary. In addition to these phenomena, findings in rats bearing median eminence-arcuate lesions suggest that the removal of endogenous LHRH by itself leads to an eventual decline in pituitary responsiveness (>one week postoperative]y). (Endocrinology 100: 292, 1977)

at different times following the intrahypothalamic implantation of crystalline hormones. With this approach, evidence for hypothalamic actions of estradiol and progesterone has been obtained (5). In preliminary experiments (6) intrahypothalamic TP-implants suppressed plasma LH levels before any change in pituitary responsiveness could be detected. Subsequently, pituitary responsiveness did decline. However, since we have found that such implants release significant amounts of T into the systemic circulation during the initial postoperative period (7), the extent to which these findings reflected a hypothalamic action of T was not altogether clear. It was similarly not clear whether the eventual decline in LHRH response resulted from a direct pituitary action of T or was secondary to suppression of endogenous LHRH secretion.

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TESTOSTERONE, HYPOTHALAMUS AND LH IN RAT The present study was performed to delineate the pattern of change in resting LH level and pituitary responsiveness to exogenous LHRH following intrahypothalamic and SC implantation of T, and to correlate these with levels of T in peripheral blood. The findings were compared with those resulting from medial basal hypothalamic lesions, which presumably deprive the pituitary of endogenous LHRH, in an attempt to define the extent to which LHRH deprivation, alone or in combination with T treatment, can influence pituitary responsiveness. Some of the data have been presented in preliminary form (8,9). Materials and Methods Adult male Long-Evans rats were castrated at 60 days of age and maintained on a 14 h light: 10 h dark schedule, with water and Wayne Lab Blox supplied ad libitum. Operations involving steroid implantation or lesions in the medial basal hypothalamus (median eminence region) were performed 5-6 weeks after castration. At various times postoperatively, blood samples were taken from the jugular vein, before and 10 minutes after an iv injection of 10 ng synthetic LHRH in 0.1 ml saline. The responsiveness to LHRH was expressed as the increment in plasma LH after LHRH injection. Sodium pentobarbital in an ip dose of 50 mg/kg body weight was used for bleeding, implantation and lesion procedures. Although the anesthesia produced a rapid drop in plasma LH, the levels were stabilized by 30 min after pentobarbital administration (unpublished experiments), and no blood samples were taken before this time had elapsed. Intrahypothalamic TP implantation was performed by ejecting a 200 £ig pellet of TP or cholesterol which had been tamped into one end of a length of 20 gauge stainless steel tubing into the median eminence region, and removing the carrier tube. The location of the implant was always visible at autopsy under the dissecting microscope. Medial basal hypothalamic lesions were produced using a radiofrequency lesion maker and double electrodes made from 24 gauge stainless steel wire insulated with glass capillary tubing except at the tip and joined together with dental cement. When sagittally oriented, this electrode facilitates de-

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struction of the entire median eminence arcuate region. Sham lesions were made by positioning the electrode at the appropriate site without passage of current. Suprachiasmatic lesions were produced bilaterally using single electrodes. The locations of all lesions were verified histologically. In a separate group of animals, Silastic capsules (Silastic medical grade tubing, Dow Corning 0.062 in id and 0.125 in od) were implanted SC. These were either empty (controls) or were filled to a length of 25 mm with T (free alcohol), and the ends closed with wooden plugs and sealed with Silastic Medical Adhesive. Before use, the Silastic capsules were pre-incubated for 48 h in phosphate-buffered saline (PBS) with three changes per day. LH concentration in plasma was determined by the double antibody radioimmunoassay of Niswender et al. (10), using NIH-LH-RPl as the standard. For plasma testosterone the immunoassay method of Frankel et al. (11) was used. Assay coefficients of variations were: LHinterassay, 9.5%, intraassay, 9.0%; T-interassay, 9.9%, intraassay, 8.3%. For pituitary LH determinations, the quick-frozen adenohypophyses were thawed, homogenized in 4 ml of distilled water, and diluted with 0.1% gelatin-PBS. The plasma LH and LHRH response data were analyzed by two-factor (time and group) analysis of variance with repeated measures on one factor (time). Pairs of group means at each time point were compared using the NewmanKeuls test (12). Results Intrahypothalamic propionate (TP)

implants of testosterone

The results of two experiments are shown in Fig. 1 (A and B). The resting level of plasma LH was generally suppressed by 6 h following implantation, however, the variance in LH values was such that a statistically significant decrease was not always seen at this time (Fig. 1A, P < .05 vs. controls; Fig. IB, P > .05). By 24 h, plasma LH was significantly suppressed in both experiments when compared to controls (P < .01 in each case), while at 1 week it had dropped to within the range found in intact adult male rats in this laboratory. One ng LHRH had no significant effect on plasma LH (Fig.

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.--ir •-«CHOI •



IM LEVEL 10' AFTER Ing LHRH

Endo • 1977 Vol 100 • No 2

release was inhibited during the first day following hypothalamic TP implantation, in the face of apparently normal pituitary responses. Subcutaneous implantation of testosterone

A s

Effects of testosterone implants and hypothalamic lesions on luteinizing hormone regulation in the castrated male rat.

Effects of Testosterone Implants and Hypothalamic Lesions on Luteinizing Hormone Regulation in the Castrated Male Rat CECILIA Y. CHEUNG1 AND JULIAN M...
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