Complement activation and hemodynamic changes following intravenous administration of phosphorothioate oligonucleotides in the monkey.

Rapid intravenous infusion of GEM 91, a 25-mer phosphorothioate oligonucleotide complementary to the gag site of HIV, in the monkey produces transient decreases in peripheral total WBC and neutrophil counts, hemoconcentration, and a brief increase followed by a prolonged decrease in arterial blood pressure. These changes are preceded by and are likely mediated by activation of C5 complement. These effects are dose and infusion rate dependent and can be avoided by administering GEM 91 by slow intravenous infusion. Similar hemodynamic effects are produced with rapid intravenous infusion of other phosphorothioate oligonucleotides varying in length from 20- to 33-mer, and are, therefore, not sequence specific but a property of this chemical structure.

Role of endogenous opiates in the modulation of gonadotropin secretion in infant monkeys (Macaca mulatta): effects of naltrexone.

A potential inhibitory role of endogenous opioids in the gonadotropin decline from infancy to the prepubertal period in primates was assessed by examining the effect of the specific opioid antagonist naltrexone on gonadotropin levels in infant rhesus monkeys. Paradoxically, both chronic administration of naltrexone to neonatally castrate males as well as acute administration of graded doses to intact infant females resulted in gonadotropin suppression compared to appropriate vehicle-treated controls. Thus, naltrexone behaves as a gonadotropin secretory antagonist in infant monkeys and cannot be used to unmask a putative inhibitory mechanism involving endogenous opiates.

Gonadotropin responsiveness to estrogens and low-level progesterone in the ovariectomized rhesus monkey.

When estrogen is administered to gonadectomized rhesus monkeys in sufficient quantity, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels are initially suppressed and gonadotropin secretion is subsequently augmented. This study was designed to examine the ability of various orally administered estrogens to elicit biphasic pituitary responses in adult castrate rhesus monkeys and to investigate the role of low-level progesterone in altering these responses. In an incomplete block design, 13 treatments were constructed: (E2): 0.5, 5.0, 50 micrograms/kg; (DES): 0.05, 0.5, 5.0 micrograms/kg; zearalenone (Z): 10, 100, 1000 micrograms/kg; or oil vehicle, each combined with progesterone. The remaining treatments (5.0, 0.5, and 100 micrograms/kg of E2, DES, and Z, respectively) did not include progesterone. During six intervals, E2, DES, or Z was administered orally on Days 1, 2, and 3 and blood was sampled on Days 0, 3, and 8 of each period. A main effect of estrogen treatment was observed for LH and FSH secretion on Day 3 but not Day 8. There was no main effect of progesterone alone on release of either LH or FSH on days 3 or 8, nor were significant carry-over effects of progesterone, E2, DES, or Z on LH or FSH concentration apparent by Day 8 of any treatment. However, progesterone synergized with E2 to suppress Day 3 LH levels. Conversely, DES and Z interacted with progesterone to facilitate LH secretion at this time.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of long-term testosterone exposure on ovarian function and morphology in the rhesus monkey.

This study was aimed at developing a model in the rhesus monkey for the human gynecologic disorder termed the polycystic ovary syndrome (PCOS). The effects of chronic constant androgen exposure upon quantitative ovarian morphology and ovulatory function were examined. Twenty-five normally cycling females, aged 4-12 yr and weighing 3.3-8.2 kg, were enrolled in the study in random fashion. Seventeen animals were implanted subcutaneously (s.c.) with 10 or 25 mg testosterone-filled silastic tubing so as to maintain steady serum levels of testosterone averaging 80 ng/dl (low-dose group, n = 8) and 115 ng/dl (high-dose group, n = 9) for 13-16 months. Eight animals served as controls (sham implants); in these, mean serum testosterone levels averaged 24 ng/dl. No effect of androgen treatment was observed on ovulatory function as gauged by periodic luteal phase progesterone determinations and the presence of a fresh corpus luteum at laparoscopy. Menstrual cycle frequency (number of cycles over number of months of observation) was, however, slightly but significantly (P less than 0.05) reduced in the high-dose (88.9%) vs. the control (96.7%) and low-dose (95.0%) groups. Quantitative morphology, performed by light microscopy on a single ovary obtained from 16 of the 25 animals and read in a blinded fashion, revealed no differences in ovarian weight, capsular width and numbers, size, or proportion of healthy and atretic follicles among the three groups.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of gonadal steroids in feedback regulation of gonadotropin secretion at different stages of primate development.

The serum gonadotropin response to castration was assessed in 8 foetal, 2 neonatal, 30 juvenile, and 2 adult rhesus monkeys (M. mulatta). In the 30 castrated juvenile monkeys and 8 sham-operated controls, concentrations of oestrone, oestradiol, androstenedione, dihydrotestosterone, testosterone and 17OH-progesterone were measured in 10 ml serum pools before, one month after, and one year after the surgical procedure. Castration during foetal life (83-137 days gestation) was followed within 48-72 h by a significant rise in serum FSH levels in males, but had no effect on the already high levels in females. Similarly, castration of males during the first post-natal month raised serum FSH and LH into the adult castrate range; however, after 3 months of age serum gonadotropin levels again declined to the normal juvenile range in spite of the open feedback loop. Orchiectomy of prepubertal juvenile monkeys (age 3 month-2 8/12 years) had no immediate effect on serum gonadotropins, but was followed by a delayed rise in FSH (at age 2 3/12-4 3/12 years) and LH (at age 2 7/12-4 4/12 years) to adult castrate levels. Orchiectomy of older prepubertal (by serum testosterone) or adult males resulted within a few days in a progressive and sustained rise in serum FSH and a more gradual rise in LH. Prepubertal gonadotropin regulation appeared to be sexually dimorphic, since ovariectomy in juvenile females (age 3 months-1 5/12 years) was followed by generally elevated, if somewhat erratic, serum FSH values, with a secondary rise in both FSH and LH levels at 2-2 1/12 years. In both sexes, prepubertal castration caused a significant and sustained decline in serum concentrations of oestradiol; castrated males also showed a decline in serum testosterone levels. Although prepubertal castration also caused in both sexes a slight decline in serum oestrone, and ovariectomy a decline in serum androstenedione and dihydrotestosterone, these effects were not sustained one year later, and values were not significantly different from sham-operated controls. Taken together, these data lend support to a model of primate sexual maturation in which the primary regulator of gonadotropin secretion in both sexes during the prolonged juvenile phase is central inhibition of the hypothalamic GnRH regulator. However, during foetal and neonatal life, and again following the onset of puberty, the major modulator of gonadotropin secretion becomes sex steroid-mediated feedback inhibition.

Dissociation of cortisol and adrenal androgen secretion in the hypophysectomized, adrenocorticotropin-replaced chimpanzee.

We tested the hypothesis that adrenal androgen production is supported by a pituitary factor distinct from ACTH. Six adult male chimpanzees who had completed adrenal maturation (adrenarche) were castrated and either hypophysectomized or sham hypophysectomized. Hypophysectomized animals received synthetic ACTH-(1-24) and T4 to prevent adrenal insufficiency and hypothyroidism. Adrenal function was evaluated with a 3-h ACTH infusion before and 7, 21, 40, 120, and 180 days after hypophysectomy. Plasma cortisol (F), dehydroepiandrosterone (DHA), DHA sulfate (DHAS), and androstenedione (delta 4A) were measured at six time points during the ACTH infusions. The mean ratios of DHA to F, DHAS to F and delta 4A to F during ACTH infusion were calculated as indices of the relative activity of the androgen pathway compared to that of the cortisol pathway. The DHA to F ratio during ACTH infusion was 31% of the pretreatment level 40 days after hypophysectomy (P less than 0.01 compared to the sham-hypophysectomized controls). The DHAS to F ratio during ACTH infusion, which paralleled the DHA to F ratio, also fell significantly (P less than 0.025). Hypophysectomy did not alter the delta 4A to F ratio. None of the ratios was altered by sham hypophysectomy. MCRs for F and DHA, which were measured before and 180 days after hypophysectomy or sham hypophysectomy, did not change significantly. Additionally, plasma corticosteroid-binding globulin levels remained unchanged throughout the study for both groups of chimpanzees. Thus, the changes in the DHA to F ratio cannot be explained by alterations in the MCRs of DHA or F or in the plasma transport protein for F. These data suggest that ACTH maintained normal F and delta 4A secretion after hypophysectomy but failed to maintain normal DHA and DHAS secretion. This is consistent with the hypothesis that normal delta 5-adrenal androgen secretion is dependent upon a non-ACTH pituitary factor or with the hypothesis of different ACTH requirements for the maintenance of F and delta 5-adrenal androgen secretion.

Influence of restraint and ketamine anesthesia on adrenal steroids, progesterone, and gonadotropins in rhesus monkeys.

Changes in gonadotropins, progesterone, cortisol, DHA, and DHAS were monitored in 10 female rhesus monkeys (Days 20-23 of the menstrual cycle) subjected to cage restraint with or without ketamine anesthesia for successive venipunctures. All animals were bled without sedation for 2 hr at 30-min intervals. Then 4 of the animals were anesthetized with ketamine-HCl and bleedings in all animals were continued for an additional 2.5 hr. FSH and progesterone were not appreciably affected by either restraint technique. LH declined steadily for the duration of the bleedings (P less than 0.05). Serum levels of cortisol and the adrenal androgens increased twofold (P less than 0.05). Anesthesia with ketamine had no effect on any of the six variables when compared with saline controls. Cortisol and dehydroepiandrosterone (DHA) levels tended to plateau (P less than 0.01) after 2 hr in both treated and control groups. In contrast, dehydroepiandrosterone sulfate (DHAS) levels increased continuously throughout the entire study period. These data indicate that ketamine anesthesia does not alter endocrine responses to venipuncture when administered following cage restraint of conscious animals. These findings further confirm the difficulties in obtaining estimates of basal levels of hormones which are responsive to stress and suggest that the first sample may provide the best estimate.

Effects of naloxone and an enkephalin analog on serum prolactin, cortisol, and gonadotropins in the chimpanzee.

To assess a possible regulatory influence of opioids upon anterior pituitary function in the chimpanzee, we evaluated the effects of the specific opiate receptor antagonist naloxone and the agonistic enkephalin analog [D-Ala2, MePhe4,Met(o)-ol]enkephalin (FK 33-824; Sandoz) on serum levels of PRL, cortisol, FSH, and LH. Under ketamine anesthesia, the following were administered by iv injection during the early follicular phase of successive menstrual cycles in nine female chimpanzees: naloxone (10 mg; n = 7) or saline vehicle (n = 7) randomly assigned in the first two cycles, FK 33-824 0.25 mg (n = 3) in the third cycle, FK 33-824 0.50 mg (n = 4) in the fourth cycle, and FK 33-824 (0.50 mg) immediately preceded by naloxone (10 mg; n = 4) in the last cycle. Five pretreatment and 12 posttreatment serum samples were obtained at 10- to 15-min intervals for subsequent RIA. Naloxone caused a significant reduction in PRL levels from a pretreatment mean of 29.3 ng/ml to a mean of 11.1 ng/ml at 180 min. Values from 60-180 min were significantly below the saline control group at comparable times. A dose-related increment in PRL levels was seen after FK 33-824 administration, with mean peak values at 30 min of 61.0 and 92.3 ng/ml after the low and high doses, respectively. Naloxone pretreatment markedly attenuated the response to high dose FK 33-824. Cortisol levels rose in all groups throughout the study period, a presumed effect of the ketamine anesthesia. Compared to the saline group, no effects of FK 33-824 were observed. Naloxone, given alone or with FK 33-824, had a small, but significant, stimulatory effect on cortisol from 60-120 min posttreatment compared to the control group. Naloxone caused a significant increment in LH levels from a pretreatment mean of 11.7 micrograms/dl to a peak of 19.1 micrograms/dl at 30 min and in FSH level from 33.2 micrograms/dl before therapy to 40.0 micrograms/dl at 45 min. There was no influence of FK 33-824 on gonadotropin levels, although the high dose did blunt the response to naloxone. Taken together, these effects suggest that opiate agonists and endogenous opioid pathways may modulate anterior pituitary function in the chimpanzee, as in man.

Further studies on adrenarche in nonhuman primates.

Serum concentrations of dehydroepiandrosterone (DHA), DHA sulfate, and cortisol were measured in 52 chimpanzees (aged 0.5--10 yr), 76 Macaca mulatta (aged 0.25--5 yr), and 80 Macaca nemestrina (aged 0.5--9 yr). Sexual maturation was assessed by age and by the presence of menarche or the appearance of perineal turgescence in the females and by measurement of serum testosterone in the males. In an additional group of 10 young adult female M. mulatta, four repeated determinations of these same steroids at 30-min intervals demonstrated that the stress of capture and venipuncture caused a significant rise in serum levels of not only cortisol but also of DHA and DHA sulfate. The chimpanzees demonstrated an age-related rise in serum concentrations of DHA and DHA sulfate relative to cortisol which began before the onset of puberty and thus closely resembled human adrenarche. In M. mulatta, serum DHA levels showed no change with age, while DHA sulfate values decreased progressively both before and during puberty. The pattern in M. nemestrina was similar, with stable DHA and declining DHA sulfate levels before and during puberty. However, in the oldest group (aged 6--9 yr) of mature M. nemestrina, there was a significant postpubertal rise of both DHA and DHA sulfate with no change in serum cortisol. These data suggest that monkeys, just as higher primates, may show increasing adrenal secretion of C19 steroids at around 6--9 yr. This adrenarchal process appears to be completely independent of sexual maturation and probably merely reflects the influence of progressive adrenal growth and the resulting impact of changing intraadrenal steroid concentrations upon steroidogenesis in the zona reticularis.

Biotransformation of diethylstilbestrol in the rhesus monkey and the chimpanzee.

Diethylstilbestrol (DES) is considered a teratogen and a transplacental carcinogen in humans. In order to compare its biotransformation in nonhuman primates to that in humans, a metabolic study was carried out in rhesus monkeys and chimpanzees. After an oral dose of 1 mg/kg [14C]DES, approximately 59% of the ingested radioactivity was found in the urine and 28% in the feces of two female rhesus monkeys after 4 days, while in male rhesus monkeys urinary radioactivity accounted for 43% and fecal radioactivity for 35%. In chimpanzees, 63% of a 0.5 mg/kg dose was excreted with the urine in a female and 47% in a male animal. In both species, urinary radioactivity was predominantly (greater than 70%) associated with glucuronides. Besides DES, three metabolites were found in the urinary glucuronide fraction of rhesus monkeys and chimpanzees by radio gas chromatography and were identified as dienestrol and omega-hydroxy derivatives of DES and dienestrol. Fecal radioactivity in rhesus monkeys was shown to consist exclusively of DES. Glucuronidation of DES appears to occur in neonatal and fetal rhesus monkeys.

Radioimmunoassay for rhesus monkey gonadotropins.

Heterologous double-antibody radioimmunoassay methods are described for the measurement of circulating levels of rhesus monkey (Macaca mulatta) FSH and LH; the latter assay is also applicable to rhesus chorionic gonadotropin (CG) estimations. The FSH assay utilizes purified rat FSH for trace, either of two anti-human FSH antisera and a semipurified rhesus pituitary standard. The LH assay utilizes purified ovine LH for trace, an anti-human CG antiserum and the same rhesus pituitary standard. The use of these systems obviates the necessity of purifying rhesus gonadotropins which are required for the development of homologous radioimmunoassay systems.