Effect of microsomal cytochrome P-450 isozyme induction on the mutagenic activation of 2-aminoanthracene.

Two rabbit microsomal cytochrome P-450 isozymes. Forms 4 and 6, which are differentially induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the liver in an age-dependent fashion, catalyze the activation of 2-aminoanthracene to a mutagen in the Ames Salmonella mutagenesis assay. We have shown previously that in the presence of saturating concentrations of 2-aminoanthracene, Form 4 is 15-fold more active than Form 6 in the activation of this mutagen. Similar differences in the activation of 2-aminoanthracene are observed between liver microsomes isolated from TCDD-treated adult rabbits, in which Form 4 predominates, and microsomes from rabbit neonates exposed transplacentally to TCDD prior to birth, in which Form 6 predominates. However, when the extent of mutagenesis is limited by the amount of 2-aminoanthracene and is independent of the rate of activation, the number of revertants produced is similar for microsomes isolated from either newborn or adult TCDD-treated rabbits. Under these conditions, the extent of mutagenesis obtained for a given amount of 2-aminoanthracene will depend on the balance between activation and competing reaction pathways leading to detoxication. Thus, differences in the rate of activation between adult and newborn microsomes are probably offset by similar differences in the rates of competing pathways of metabolism. This is consistent with the finding that the overall rate of 2-aminoanthracene metabolism by the adult microsomes is greater than that of the neonate. In order for the extent of mutagenesis to be independent of rate, the half-life of 2-aminoanthracene was seen to be less than approximately 12 min.

Temporal dissociation in the exposure times required for maximal induction of cytotoxicity, mutation, and transformation by N-methyl-N'-nitro-N-nitrosoguanidine in the BALB/3T3 ClA31-1-1 cell line.

Cytotoxicity, alkali-labile DNA lesions, ouabain resistance mutations, and neoplastic transformation were analyzed concurrently in the BALB/3T3 ClA31 -1-1 cell line treated with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for different exposure times (15, 30, 60, 90, 120, and 240 min; 24, 48, and 72 hr). The half-life of MNNG in complete medium was approximately 70 min, both without cells and with cell numbers as used in the assays for cytotoxicity (2 X 10(2) cells/60-mm dish), transformation (1 X 10(4) cells/dish), and mutation (1 X 10(5) cells/dish). The cytotoxic effect of MNNG (0.5 or 2 micrograms/ml) appeared to be completed after an exposure time between 100 and 200 min. Maximal frequency of ouabain resistance mutations, however, was reached after a much shorter treatment time (30 to 60 min). Detection of DNA damage by alkaline elution analysis showed maximal increase in single-strand breaks already after treatment for 30 min. Exposures for 30 min followed by posttreatment incubation for 30 or 90 min showed active repair of single-strand breaks during these periods, indicative of an even balance between the additional MNNG-induced damage and its repair. Morphological transformation assays, at the same treatment times and concentrations used in the mutation assays, yielded frequency curves that reached their maxima 1 to 3 hr later than did the mutation frequencies. The ratio of transformation to ouabain resistance mutation frequencies was 3.7 for short treatment times (30 to 60 min), while it increased to more than 20 for exposure times of 240 min or longer. The temporal dissociation in the exposure times for maximal induction of mutation and transformation, observed with MNNG in this cell line, supports the hypothesis that a single gene mutational event is not sufficient to account for the full expression of neoplastic transformation.

Sex differences in the neuroendocrine response to short-term fasting in rhesus macaques.

When energy intake is restricted in mammals, there are neuroendocrine adjustments in the secretion of reproductive and metabolic hormones to reallocate energy for vital functions. In the present study, we investigated whether there were differences in the luteinising hormone (LH), growth hormone (GH) and cortisol responses to a 48-h fast in adult gonad-intact male and female rhesus macaques. In both male and female macaques, blood glucose levels were significantly lower in fasted than in control studies, and levels were higher in males than in females. Male rhesus monkeys had significantly lower (P < 0.01) mean serum LH levels after a 48-h fast than under fed conditions and this was attributable primarily to a decrease in the amount of LH released during each secretory episode. In fasted females, serum LH levels were significantly greater (P < 0.05) than during the fed conditions but no differences were found in pulse amplitude or in the number of pulses. Almost twice as many GH pulses were observed in both males and females during fasting but there was no difference in either mean serum GH levels or pulse amplitude between control and fasted studies. A typical diurnal profile in cortisol levels was observed in both sexes and both experimental conditions. Under control conditions, male macaques released less cortisol than females, and although fasting increased mean cortisol levels in both males and females, only the males shown a significant rise over levels observed in control studies. The changes in plasma LH and cortisol levels in fasted rhesus macaques are similar to those observed in humans and suggest that gonadotrophin and corticotrophin secretion are more resistant to short-term energy deprivation in female than in male primates.

Brain lesions in infant female rhesus monkeys: effects on menarche and first ovulation and on diurnal rhythms of prolactin and cortisol.

Sexual maturation in female rhesus macaques was studied after surgical isolation of the medial basal hypothalamus [complete hypothalamic disconnection (CHD); n = 4] or after creation of amygdaloid lesions (AMYG; n = 6). In four animals, CHD at 8 months did not affect the age when menarche occurred (30 months) but did result in a significant (P less than 0.01) advancement in age at first ovulation (35.8 +/- 1.1 vs. 43.7 +/- 1.1 months for the five controls). Body weights in CHD and AMYG animals were not different from weights of controls at either menarche or first ovulation, but CHD animals gained weight faster than controls. Although there was no overall difference in age at menarche or first ovulation between AMYG animals and controls, the three AMYG animals that sustained damage to the corticomedial amygdaloid area ovulated later than three other AMYG animals without damage to this area. Daily serum levels of LH, FSH, and 17 beta-estradiol were measured in the second ovulatory cycle of each animal and found to be similar among the three groups. Serum progesterone levels revealed that three of the five controls and one of six AMYG animals had short luteal phases typical of pubertal monkeys, whereas all four CHD animals showed luteal phases typical of sexually mature adult animals. Serum cortisol and PRL showed significant diurnal changes in all three groups. These data indicate that in infant rhesus females, intact neural connections to the medial basal hypothalamus are not obligatory for sexual maturation or for the propagation of entrained diurnal rhythms in cortisol and PRL. That isolation of the medial basal hypothalamus resulted in an increase in the rate of weight gain and favored early sexual maturation may indicate that the main effect of the higher brain centers is inhibitory on hypothalamic mechanisms which control these processes.

Cyclic ovarian function in a male macaque: additional evidence for a lack of sexual differentiation in the physiological mechanisms that regulate the cyclic release of gonadotropins in primates.

Ovarian tissue from adult female rhesus macaques was transplanted into sc abdominal pouches of 4 male rhesus macaques that had been castrated after reaching sexual maturity. The animals were treated daily with cyclosporin A to prevent rejection of the ovarian transplants. Two males in which the transplants were successful showed preovulatory-like gonadotropin surges in response to increasing levels of estradiol. In one of these males (7082), circulating levels of gonadotropins and steroids indicated that cyclic ovarian function had been established. This male showed 5 successive ovarian cycles that averaged 28 days in length. Comparison of the changes in reproductive hormones between 7082 and females with normal menstrual cycles support the hypothesis that the neuroendocrine mechanisms that regulate cyclic release of gonadotropins in primates are not sexually different.

Circadian periodicity in circulating cortisol is absent after orchidectomy in rhesus macaques.

Circadian/ultradian patterns of plasma cortisol were assessed in four intact and four gonadectomized male rhesus macaques. Concomitant measures of testosterone (T) were analyzed in the intact animals. Blood samples were drawn every 15 min for 28 h via an indwelling catheter. Plasma concentrations of cortisol and T were determined by RIA. Inverse diurnal patterns of cortisol and T secretion were documented in the control group. The circadian pattern of plasma cortisol was characterized by a progressive rise during the early morning hours (0200-0800 h), followed by a gradual decline until lowest levels were reached at 1600-1900 h. T levels were lowest from 0800-1200 h (2-4 ng/ml) and reached a zenith from 2200-0200 h (8-10 ng/ml). Surprisingly, circadian fluctuations in cortisol were absent after orchidectomy. Cortisol levels in castrates were less (P less than 0.05) than those during peak levels of cortisol secretion in the intact animals (6.25 vs. 10.1 micrograms/100 ml) and elevated (P less than 0.01) compared to concentrations in the intact animals at their nadir (6.4 vs. 2.5 micrograms/100 ml). The loss of circadian fluctuation was not associated with a significant change in frequency of pulsatile cortisol release or a change in the daily mean level. These results are the first in nonhuman primates to demonstrate that the testes modulate circadian activity in the hypothalamic-pituitary-adrenocortical axis. The data differ from previous findings in rodents suggesting that castration increases the synthesis and release of ACTH/corticosterone.

Influence of the gonads on cortisol secretion in female rhesus macaques.

Circadian and ultradian patterns of plasma cortisol were assessed in four intact female rhesus macaques during both the late follicular (days 9-10) and midluteal (days 21-22) phases of the menstrual cycle and compared to patterns in four ovariectomized macaques. Blood samples were drawn from a remote site at 15-min intervals for 24 h via an indwelling catheter. Measures of estrogen and progesterone were obtained for each subject. For purposes of data analyses, group cortisol measurements were collapsed across hourly intervals and submitted to analysis of variance. Pulsatile characteristics of cortisol release were determined for each subject using the PULSAR computer program. Circadian cortisol patterns, present in all three groups, were characterized by a progressive rise during early morning hours (0300-0600 h), followed by a decline of short duration. All groups then displayed an unexpected midday peak (0900-1400 h), at which time cortisol levels reached their daily zenith. In each of the three groups, cortisol levels reached a nadir during late afternoon hours shortly after the light phase ended. The amplitude of circadian changes and daily mean levels of cortisol were significantly reduced by ovariectomy, without alterations in pulsatile characteristics of cortisol secretion. Daily mean cortisol levels decreased from approximately 8 micrograms/100 ml in intact subjects to 4.5 micrograms/100 ml after ovariectomy. No significant differences in the circadian/ultradian periodicity of cortisol secretion were detected between the follicular and luteal groups. When data in the intact female groups were combined and compared to those previously obtained from gonadally intact adult male macaques, similar 24-h patterns of cortisol secretion were detected. Surprisingly, amplitude changes in cortisol concentrations after ovariectomy were temporally and quantitatively similar to those in orchidectomized males. In both male and female animals, circadian patterns of cortisol secretion were reduced by gonadectomy. These results are discussed in terms of the activational influence of gonadal steroids on hypothalamic-pituitary-adrenocortical function.

Interaction of estradiol and LHRH on LH release in rhesus females: evidence for a neural site of action.

The effects on LH release of infusing luteinizing hormone-releasing hormone (LHRH 80 mug/20 min) into the third ventricle, the pituitary, and the peripheral circulation were compared in spayed rhesus monkeys. Within 30 min after iv administration, serum LH concentrations increased to twice to preinfusion levels, and by 120 min declined to original values. Intraventricular or intrapituitary infusions of LHRH resulted in similar LH increments, but the peaks occurred somewhat later (70 to 90 min) and the elevations persisted beyond 200 min. Estradiol-17beta (E2) administered by a sc silastic capsule caused a 5-fold increase in serum E2 within 1 h and reduced serum LH levels by 65% within 4 h. The LH release caused by intrapituitary LHRH was significantly suppressed by maintaining for 72 h E2 concentrations near 100 pg/ml, a level inadequate for stimulating an LH surge. A comparable E2 treatment before intraventricular infusion of LHRH, however, did not inhibit LH release. This difference between the effects of intrapituitary and intraventricular LHRH was demonstrable only in E2-treated monkeys. Moreover, the release of LH after intraventricular infusion of LHRH in E2-treated females was blocked (P less than 0.001) by a single iv injection (90 min before LHRH) of haloperidol (1 mg/kg BW) or phentolamine (5 mg/kg), but was not altered by phenoxybenzamine (3 mg/kg) or propranolol (5 mg/kg). Without E2 pretreatment, LH release after intraventricular LHRH was enhanced by each drug. Phentolamine, injected into both E2- and non-E2-treated monkeys 90 min before an intrapituitary infusion of LHRH had no demonstrable effects on the patterns of serum LH. Our interpretation of these data is that E2 at a concentration below the level that triggers an LH surge has a dual action on LHRH-induced LH release in monkeys: an inhibitory effect exerted directly on the pituitary and a stimulatory effect on the brain. Furthermore, the paradoxical effects of the drugs with and without E2 are due to the involvement of two distinct neuronal systems. The postulated neural effects of both E2 and these drugs can be explained either by an increase in the quantity of injected or secreted LHRH which ultimately binds to LH-secreting cells or by the release of additional endogenous LH-stimulating agents together with ventricular LHRH.

Adrenocorticotropin stimulates natural killer cell activity.

Central release of CRH has recently been implicated in modulating natural killer cell (NK) activity independent of its role in activation of the hypothalamic-pituitary-adrenal axis. In the present study, NK- and interleukin-2 (IL-2)-activated NK cytotoxicity against K-562 target cells was examined in porcine lymphocytes cultured in vitro with ACTH or pig lymphocytes after iv or im ACTH administration. Physiological concentrations of porcine ACTH (10(-8)-10(-11) M) added to the culture medium had no direct influence on NK- or IL-2-stimulated NK cytotoxicity. In a second experiment four unrestrained pigs with indwelling catheters given an iv bolus of vehicle or ACTH (1 IU/kg BW) at 0800 h showed significantly elevated cortisol levels for 3 h after ACTH. Although serum cortisol had returned to baseline by 4 h after ACTH treatment, NK- and IL-2-stimulated NK cytotoxicity was dramatically elevated (P less than 0.01) compared to that in saline-injected controls. NK cytotoxicity in control pigs followed a diurnal pattern, with low morning and high evening cytotoxicity. Exogenous ACTH, given by bolus in the morning, prevented the normal morning decline in NK cytotoxicity. Because of this unexpected dramatic increase in NK- and IL-2-stimulated NK cytotoxicity in animals given ACTH, the experiment was replicated in two subsequent studies using 16 pigs (8 controls and 8 experimental) in each. Pigs were injected im with either ACTH (1 IU/kg BW) or an equivalent volume of saline at 0600 h. Two hours later, blood was collected by venipuncture to determine NK cytotoxicity and measure the cortisol response. As was observed in the previous study, NK- and IL-2-stimulated NK cytotoxicity was significantly greater (P less than 0.01) than that in saline-injected controls. In the final experiment pigs were given either ACTH (1 IU/kg BW) or an equivalent volume of saline at 1800 h. Two hours later, blood was collected by venipuncture to determine NK cytotoxicity and cortisol response. ACTH administered in the evening increased NK cytotoxicity, but the effect was only marginally significant and far less dramatic than in previous studies. Because ACTH had little effect on NK- and IL-2-stimulated NK activity in vitro, we hypothesize that the stimulatory effect of exogenous ACTH is mediated through an indirect mechanism, possibly through the suppression of central CRH as a result of elevated cortisol. This effect is more pronounced when the stimulatory dose of ACTH is given at a time in the circadian cycle when NK cytotoxicity is normally low.

The anterior hypothalamus: how it affects gonadotropin secretion in the rhesus monkey.

The effects of hypothalamic lesions on spontaneous and estrogen-induced LH release were studied in 17 female rhesus monkeys with regular menstrual cycles. In the cycle before surgery, all of the animals experienced 3- to 10-fold increases in serum LH and elevated (above 3 ng/ml) serum concentrations of progesterone. Three to 6 days after the onset of menstruation, lesions were made in the preoptic-anterior hypothalamic area (POA-AHA) in 14 monkeys by radiofrequency thermocoagulation (RF) or by the 180 degrees rotation of a modified "Halász" knife. About 35 days after surgery, the circulating levels of estradiol-17 beta (E2) increased to more than 200 pg/ml in each of the 14 monkeys. Three of the animals with RF lesions and 3 with knife lesions did not release LH or have elevated serum progesterone levels, an indication that they had not ovulated (effective). In 8 animals, 5 with RF and 3 with knife lesions, an LH surge and elevations in serum progesterone were observed (ineffective). After a 90-day postoperative period, the effective and ineffective lesioned groups and an additional group of 6 intact controls were given E2 to test further the ability of the hypothalamic-hypophyseal axis to release LH. The animals with effective lesions did not respond to increased E2 titers (200-400 pg/ml), but those in the ineffective and control groups showed an LH surge. Six to 11 months after surgery, histological examination of the brains from the animals with effective lesions revealed extensive bilateral destruction of the ventral POA-AHA. The suprachiasmatic nuclei or connections between these nuclei and the medial basal hypothalamus (MBH) were destroyed. In 2 animals, the supraoptic and ventromedial nuclei were partially damaged. In no instance was there damage to the paraventricular, dorsomedial, or arcuate nuclei. In animals with ineffective lesions, bilateral destruction of the POA-AHA was less extensive and most of the lesions were unilateral. Ovaries from animals with effective lesions contained small to medium follicles but luteal tissue was conspicuously absent. Spontaneous LH surges and elevated serum P occurred in 2 of 3 additional animals that had 270 degrees cuts around the MBH which left one anterior quadrant intact. Damage to the median eminence region was evident in the one animal that did not ovulate. These results suggest that in rhesus monkeys bilateral destruction of the ventral POA-AHA blocks spontaneous ovulation and compromises the ability of the hypothalamic-hypophyseal axis to release LH in response to estrogen.

Prolactin release following electrical stimulation of the brain in ovarectomized and ovariectomized estrogen-treated rhesus monkeys.

Selected areas in the medial basal (MBH) and rostral (RH) hypothalamus and in the amygdala (AMYG) of long-term ovariectomized rhesus monkeys were electrically stimulated for 30 min through permanently implanted bilateral stainless steel electrodes. Stimulation of an area in the MBH extending from the dorsal part of the ventromedial nucleus through the arcurate nucleus to the upper median eminence resulted in a 200 to 400% increase within 5 min in 8 monkeys. In one monkey the elevated serum prolactin levels persisted after termination of stimulation and in 2 monkeys prolactin remained unchanged during the 30-min stimulation but increased after stimulation was discontinued. Stimulation of the paraventricular-dorsomedial nuclear area in one monkey had no effect on prolactin release. Prolactin responses to stimulation in the RH varied. In 2 monkeys the electrode tips extended into the optic chiasm but part of the uninsulated tips remained in contact with the RH; only one of these monkeys released prolactin in response to stimulation. In 4 monkeys the electrode tips were located in the suprachiasmatic-anterior hypothalamus area. Serum prolactin increased by 200 to 300% in response to stimulation in 2 of these monkeys but increased only slightly in the remaining 2 monkeys. Prolactin responses to stimulation of the AMYG varied with the location of the electrodes. Stimulation in the corticomedial region produced no change in serum prolactin but stimulation in the basal or basolateral area produced marked elevations. An increase in circulating levels of estradiol-17beta (E2) to 100 pg/ml by SC implantation of E2 capsules 72 h before stimulation had no significant effect on basal prolactin levels, but markedly enhanced the prolactin release induced by stimulation in both the MBH and RH. Sham-stimulation did not affect serum prolactin. We conclude that prolactin release in rhesus monkeys can be triggered by electrical stimulation of selected hypothalamic and amygdaloid areas and that stimulation-induced prolactin release in the RH and MBH can be enhanced by E2 pretreatment.

Regulation of the second (estrous) release of follicle-stimulating hormone in hamsters by the medial basal hypothalamus1,2.

In femal proestrous hamsters (1800 h), bilateral electrocoagulative lesions in the arcuate-median eminence (Arc-ME) region blocked the pituitary FSH release that normally would have begun in late proestrus and continued until the afternoon of estrus. This second (estrous) FSH elevation, which is not accompanied by LH release, was unaffected by neural disconnection of the medial preoptic area (MPOA) from the medial basal hypothalamus (MBH) or by production of MBH-pituitary islands. After gonadotropin-releasing hormone was injected into Arc-ME-lesioned hamsters, LH was released. These results suggest that the Arc-ME does not require the MPOA for initiation of the estrous release of FSH.

Pulsatile secretion of luteinizing hormone during the menstrual cycle of rhesus macaques.

The secretion of LH, PRL, and cortisol was investigated in 4 sexually mature female rhesus macaques with cardiac catheters protected by tethers. Based on endocrine parameters, all 4 of the animals ovulated within 2 months from the time they were tethered, and regular menstrual cycles of 24-34 days were observed. The catheters remained patent for 6-12 months without reposition or repair. Plasma levels of 2 stress-labile hormones, PRL and cortisol, showed diurnal fluctuations comparable to those observed in untethered animals. The frequency of LH secretory episodes was determined by measuring bioactive LH in blood samples collected at 10-min intervals in the follicular phase and at 15-min intervals in the luteal phase of the menstrual cycle. In 10 trials during the follicular phase, we estimated that an average of between 14 and 15 LH pulses occurred every 12 h. The interpulse interval ranged between 20-80 min and averaged 50 min. No change in pulse frequency was observed across the follicular phase. The number of LH pulses decreased after ovulation, and by the end of the luteal phase, the interpulse interval was 4-6 h. One example during the preovulatory LH surge revealed the high frequency, high amplitude nature of LH secretion at that time. Our experience indicates that tethered animals with cardiac catheters show no hormonal indications of stress and represent the best available model for studies requiring frequent and prolonged access to the vascular system. Our data suggest that peripheral LH fluctuations in rhesus monkeys, as in other mammals, are pulsatile, and the frequency of these pulsatile episodes changes with different phases of the menstrual cycle, presumedly in response to varying stimuli to the pituitary from the brain.

Effects of estradiol-17beta on the induction of gonadotropin release by electrical stimulation of the hypothalamus in rhesus monkeys.

Serum LH and FSH were measured at 60, 30, and 0 min before, at 5, 15, and 30 min during, and at 10, 45, and 90 min after bilateral electrical stimulation (ES) of various hypothalamic regions in 12 unanesthetized ovariectomized rhesus monkeys. ES of the arcuate-ventromedial nuclei (medial basal hypothalamus; MBH) induced a prompt increase in serum LH that persisted throughout stimulation and returned to basal levels within 90 min thereafter. FSH was also released, but the release was slower and less dramatic than that of LH. Sham stimulation (0muA) caused no change in serum gonadotropins. The amount of LH released after MBH-ES depended upon current strength (1.0 mA greater than 0,5 or 0.7 mA). Three sequential 30-min MBH-ES trials at 90-min intervals induced comparable LH responses and 3 h of continuous MBH-ES maintained elevated serum LH levels throughout the stimulation period, suggesting that these stimulation period, suggesting that these stimulation parameters did not completely deplete pituitary stores of releasable LH. The character of the LH response was similar in individual monkeys through 3 to 24 trials during 4 to 18 months. Comparisons were made of the effects of estradiol-17beta (E2) treatment at different doses and for different intervals of time before MBH-ES. ES-induced LH release was not affected by low levels (25 and 55 pg/ml) ofE2 for 48 h, but was reduced by higher E2 concentrations (100 or 230 pg/ml). E2 concentrations of 100 pg/ml had no effect at 24 h, but reduced MBH-ES-activated LH release at 48 to 96 h; the degree of depression was time-related (48 h less than 72 h less than 96 h). ES of the preoptic-suprachiasmatic region (rostral hypothalamus; RH) in non-E2-treated monkeys also released LH, but this increase was less than after MBH-ES. FSH release was not measurable after RH-ES. In contrast to the depressed LH response to MBH-ES after 48 h of E2 (100 pg/ml), the response to RH-ES was not inhibited by this E2 regimen. These data suggest that ES of an area extending caudally from the rostral hypothalamus to the arcurate-median eminence region will evoke LH release in rhesus monkeys. This electrically induced gonadotropin release was affected by administration of physiological levels of E2 but the nature of effect depended on the specific region stimulated: distinct inhibition of the gonadotropic response to MBH-ES and slight facilitation of the response to RH-ES.

Hormonal control of nuclear estradiol receptor content and the luminal epithelium in the uterus of the golden hamster.

Uteri were removed and blood was drawn from hamsters during the estrous cycle, on the eighth day of pregnancy, and after different hormonal treatments. Serum estradiol (E2) and progesterone (P) levels were determined by RIA. Portions of the uteri were prepared for light and electron microscopy. Endogenous uterine nuclear E2 receptor was measured by exchange assay. Large increments of nuclear E2 receptor occurred when the level of serum E2 was rising and that of serum P was falling (diestrus day 2 through proestrus morning); decrements occurred when the serum E2 level was falling and the serum P level was rising (proestrus evening through diestrus day 1). Ovariectomy on proestrus morning led to a decline in the amount of nuclear E2 receptor, and this decline was prevented by administration of E2 at the time of ovariectomy. P depleted nuclear E2 receptor in the presence of high levels of serum E2. The uterine luminal epithelium passed through a phase of mitosis and hypertrophy (diestrus day 2 through proestrus morning), a brief phase suggestive of secretion (proestrus evening), a phase of degeneration (estrus), and a phase of quiescence (diestrus day I). Ovariectomy on proestrus morning led within 24 h to degenerative changes identical to those seen during estrus, and these changes were prevented by treatment with E2 at the time of ovariectomy. P induced these degenerative changes in the presence of high levels of serum E2. After 8 days of pregnancy, the epithelium resembled that seen during diestrus day 1. Elevated uterine nuclear E2 receptor levels were associated with growth and hypertrophy of the luminal epithelium, and severely depressed levels were associated with the degenerative and quiescent phases of the epithelium.

In vivo gonadotropin-releasing hormone release and serum luteinizing hormone measurements in ovariectomized, estrogen-treated rhesus macaques.

The push-pull perfusion technique was used to measure GnRH release in unanesthetized female rhesus macaques (Macaca mulatta) and to examine the dynamic relationship between GnRH release and LH levels during the estrogen-induced LH surge. Each ovariectomized macaque was anesthetized and stereotaxically fitted with a push-pull cannula directed into the median eminence (ME). After at least 1 week of recovery, each animal received an estradiol benzoate (E2B) injection (42 micrograms/kg BW) or an oil (OIL) injection and underwent push-pull perfusion of the ME and blood sampling for at least 5 h between 28 and 56 h postinjection. Continuous 10-min push-pull perfusates were collected and prepared for GnRH RIA. Peripheral venous blood samples were obtained either hourly or every 10 min, and serum LH levels were determined by Leydig cell bioassay. GnRH release was detectable and pulsatile in areas in or adjacent to the ME or arcuate nucleus. In eight OIL monkeys, GnRH pulses were regular (approximately one pulse every 60 min) and of low amplitude (14.7 +/- 12.0 pg), with a mean GnRH release rate of 4.0 +/- 1.7 pg/10 min. In five E2B-treated monkeys, GnRH release during the rising phase of the LH surge occurred as an apparent burst of high amplitude GnRH pulses. The mean GnRH release rate (37.5 +/- 17.9 pg/10 min) and mean GnRH pulse amplitude (170.0 +/- 90.0 pg) during the 5 h before the peak LH level in E2B-treated monkeys were greater than OIL values (P less than 0.025, mean release; P less than 0.05, mean amplitude). Within individual E2B-treated monkeys, hourly mean GnRH release rates were significantly correlated with LH levels during the ascending limb of the LH surge (r = 0.75 +/- 0.11; P less than 0.025). We have concluded that an increase in GnRH neurosecretion occurs in E2B-treated monkeys and that it is associated with generation of the LH surge. On the basis of our observations, we hypothesize that the primate hypothalamus, through changes in GnRH secretion, actively participates in the E2B-induced LH surge.

Different prolactin, thyrotropin, and thyroxine responses after prolonged intermittent or continuous infusions of thyrotropin-releasing hormone in rhesus monkeys.

Serum PRL, TSH, and T4 secretion during prolonged continuous or intermittent iv infusions of TRH were studied in 14 adult ovariectomized rhesus monkeys (Macaca mulatta). For 9 days, TRH was administered intermittently at 0.33 or 3.3 micrograms/min for 6 of every 60 min and continuously at 0.33 micrograms/min. With both modes, the PRL levels and responsiveness to TRH simulation peaked on day 1 and then fell to levels that were still higher than the preinfusion values; levels for the intermittently treated group on days 3-9 were 2- to 4-fold above prestimulation levels and significantly (P less than 0.01) higher than levels for the continuously treated group. Elevated basal levels and PRL responses to TRH pulses were similar during the 0.33 and 3.3 micrograms/min pulses of the 9-day treatment period. For both TRH modes, TSH levels were elevated significantly (P less than 0.001) on day 1 [this increase was higher with continuous infusion (P less than 0.001)] and then fell to preinfusion levels by day 3. Serum T4 also increased during both continuous and intermittent TRH stimulations. However, serum T4 levels were significantly lower (P less than 0.01) after intermittent TRH (both 0.33 and 3.3 micrograms/min) than after continuous (0.33 micrograms) TRH (8 +/- 1.1 and 10 +/- 1.8 micrograms T4/dl vs. 18 +/- 3.1 micrograms, respectively). These PRL and T4 responses were replicated when the mode of administering 0.33 micrograms/min TRH was reversed after 9 days. An iv bolus of TRH (20 micrograms) after 9 days of continuous or intermittent TRH infusion caused significant release of PRL and TSH, an indication that neither mode of administration resulted in pituitary depletion of releasable hormone. We have concluded that intermittent TRH is more effective in elevating serum PRL, and continuous TRH is more effective in raising TSH and T4 levels. Thus, the manner of TRH secretion by the hypothalamus may determine its relative physiological importance in the stimulation of lactotropes and thyrotropes.

Differential sensitivity of prolactin release to dopamine and thyrotrophin-releasing hormone in intact and pituitary stalk-sectioned rhesus monkeys.

The effects of dopamine and thyrotrophin-releasing hormone (TRH) on prolactin release was studied in 14 intact and six pituitary stalk-sectioned (SS) female rhesus monkeys (Macaca mulatta). Baseline prolactin values were ninefold higher in SS animals (149+/-16 ng/ml) than in intact animals (16+/-1 ng/ml). Prolactin release after intravenous administration of TRH in doses of 0, 125, 250, 500 and 1000 ng revealed that SS monkeys were more sensitive to the prolactin-releasing activity of this tripeptide than were intact animals. A significant (P less than 0.05) increment in serum prolactin was observed in SS animals after injection of 125 ng TRH whereas 250 ng was required to raise prolactin levels in the circulation of intact animals significantly (P less than 0.05). Furthermore, at each comparable dose level of TRH, the increment in serum prolactin was distinctly greater in SS animals than in intact monkeys. Infusion of dopamine at the rate of 10 microgram/kg body weight per min significantly (P less than 0.05) lowered prolactin levels within 60 min in intact animals and no further decline was observed with 20 or 40 microgram dopamine. Serum prolactin concentrations were not affected by saline infusion or by 5 microgram dopamine. Infusion of dopamine at the rate of 10 microgram/kg body wt per min also resulted in significant (P less than 0.01) suppression of serum prolactin in SS animals. This prolactin decrease was apparent within 40 min. Prolactin release after 500 ng TRH was less in these dopamine-treated SS monkeys than after an infusion of saline. Higher doses of dopamine (20 and 40 microgram) did not cause a further decrease in basal serum prolactin concentrations, but these two dopamine treatments blocked the increase in prolactin elicited by 500 ng TRH. The results suggest that the removal of hypothalamic influence, possibly related to the effects of dopamine, renders the pituitary gland more sensitive to the prolactin-releasing action of TRH.

Effect of leptin on ACTH-stimulated secretion of cortisol in rhesus macaques and on human adrenal carcinoma cells.

OBJECTIVE: Because glucocorticoids stimulate leptin release and, at least in vitro, leptin inhibits cortisol secretion, a feedback system between glucocorticoids and leptin has been proposed. However, in humans and non-human primates there are no in vivo studies to support any role for leptin in the control of the hypothalamic-pituitary-adrenal axis. In this study, we investigated the effect of leptin on (i) ACTH-stimulated secretion of cortisol in six male rhesus monkeys and (ii) basal and forskolin (FSK)-stimulated cortisol secretion by the human adrenal carcinoma cell H295R in vitro. DESIGN AND METHODS: In vivo studies: after suppression of endogenous ACTH with either dexamethasone (n=6) or a corticotropin-releasing factor (CRF) antagonist (d-Phe CRF(12-41)) (n=3), 1 microg bolus of human ACTH(1-24) was administered to stimulate adrenal cortisol release. Blood samples were collected every 15 min for 3 h. Leptin (1 mg) was infused over 4 h, starting 1 h before ACTH bolus. IN VITRO STUDIES: NCI-H295R cells were incubated for 6, 12, 24 and 48 h in the absence or presence of 20 micromol/l FSK in combination with leptin (100 ng/ml medium). Cortisol levels in serum and medium were measured by solid phase radioimmunoassay. RESULTS: Acute leptin infusion to rhesus monkeys did not change basal cortisol levels, peak cortisol levels after ACTH(1-24) or the area under the curve when compared with studies in which leptin was not given. FSK increased cortisol levels in medium at 24 and 48 h, but leptin did not change cortisol release in either control or FSK-stimulated cells. CONCLUSIONS: Short-term leptin infusion affected neither the cortisol response to ACTH in non-human primates in vivo nor cortisol release (basal or FSK stimulated) by H295R cells, in vitro. These data suggest that leptin may not be an acute regulator of primate adrenal cortisol secretion.

Consequences of restraint stress on natural killer cell activity, behavior, and hormone levels in rhesus macaques (Macaca mulatta).

Three experiments were performed to determine the effect of stress on the neuroendocrine-immune system in nonhuman primates. In Experiment 1 the diurnal variation in cell and hormone levels was determined. The percentages of neutrophils, monocytes, and eosinophils fluctuated throughout the 24-hr period, while white blood cell (WBC), neutrophil-to-lymphocyte ratio (N:L), hemoglobin (Hgb), natural killer cell cytotoxicity (NK activity) and beta-endorphin levels did not. Experiment 2 investigated the effects of ketamine and restraint on behavior. Scratching was increased in control monkeys and animals receiving ketamine, whereas passivity was increased in chair-restrained animals. In Experiment 3, eight adult male rhesus monkeys were restrained in primate chairs at 0600h. Behavior was filmed for 3 hr and blood samples were collected at 0700, 0800, and 0900. Whole blood was analyzed for total WBC and percentage of each leukocyte type. NK activity was also measured. Plasma levels of cortisol and beta-endorphin were determined and behavior was quantitated from video-records. WBC and the percentage of neutrophils increased during the restraint period, while the percent lymphocytes and monocytes decreased. NK activity also decreased over time after restraint whereas plasma cortisol and beta-endorphin levels increased significantly. Although after the 3 hr of restraint stress, changes were found in hormone levels, behavior, and NK activity, there were no significant correlations between the parameters measured. Thus, our results indicate that there is not a common neuroendocrine response or single neuroendocrine mediator that results in predictable behavioral changes and immune suppression following stress.