Effects of hyperandrogenemia and increased adiposity on reproductive and metabolic parameters in young adult female monkeys.

Many patients with hyperandrogenemia are overweight or obese, which exacerbates morbidities associated with polycystic ovary syndrome (PCOS). To examine the ability of testosterone (T) to generate PCOS-like symptoms, monkeys received T or cholesterol (control) implants (n = 6/group) beginning prepubertally. As previously reported, T-treated animals had increased neuroendocrine drive to the reproductive axis [increased luteinizing hormone (LH) pulse frequency] at 5 yr, without remarkable changes in ovarian or metabolic features. To examine the combined effects of T and obesity, at 5.5 yr (human equivalent age: 17 yr), monkeys were placed on a high-calorie, high-fat diet typical of Western cultures [Western style diet (WSD)], which increased body fat from <2% (pre-WSD) to 15-19% (14 mo WSD). By 6 mo on WSD, LH pulse frequency in the controls increased to that of T-treated animals, whereas LH pulse amplitude decreased in both groups and remained low. The numbers of antral follicles present during the early follicular phase increased in both groups on the WSD, but maximal follicular size decreased by 50%. During the late follicular phase, T-treated females had greater numbers of small antral follicles than controls. T-treated monkeys also had lower progesterone during the luteal phase of the menstrual cycle. Although fasting insulin did not vary between groups, T-treated animals had decreased insulin sensitivity after 1 yr on WSD. Thus, while WSD consumption alone led to some features characteristic of PCOS, T + WSD caused a more severe phenotype with regard to insulin insensitivity, increased numbers of antral follicles at midcycle, and decreased circulating luteal phase progesterone levels.

Elevated androgens during puberty in female rhesus monkeys lead to increased neuronal drive to the reproductive axis: a possible component of polycystic ovary syndrome.

BACKGROUND: Hyperandrogenemia is associated with several clinical disorders in which both reproductive dysfunction and metabolic changes may coexist [i.e. polycystic ovary syndrome (PCOS), obesity and congenital adrenal hyperplasia]. Moreover, there is growing evidence that the elevated levels of circulating androgens in obese girls may lead to an increased neuroendocrine drive to the reproductive axis, similar to that associated with PCOS. METHODS: To test whether androgen exposure in the childhood and adolescent period could lead to pubertal alterations in LH secretory patterns, female rhesus monkeys received subcutaneous testosterone implants prepubertally beginning at 1 year of age, maintaining a 3.7-fold increase (P = 0.001) in circulating testosterone levels over cholesterol-implant controls (n = 6/group) into the post-pubertal period. In early adulthood, pulsatile secretion of LH was measured over 12 h during the early follicular phase of a menstrual cycle, and responsiveness of the pituitary to gonadotrophin-releasing hormone was determined. In addition, ultrasounds were performed to assess ovarian morphology and glucose tolerance testing was performed to assess insulin sensitivity. RESULTS: The timing of menarche was similar between groups. Testosterone-treated animals had a significantly greater LH pulse frequency during the early follicular phase compared with controls (P = 0.039) when measured at 5 years of age. There was a larger LH response to GnRH when testosterone-treated animals were 4 years of age (P = 0.042), but not when the animals were 5 years old (P = 0.57). No differences were seen in insulin sensitivity or ovarian morphology, and the groups showed similar rates of ovulation in early adulthood. CONCLUSIONS: Exposure to increased levels of androgens over the course of pubertal development appears to trigger physiological changes in the neural drive to the reproductive axis that resemble those of obese hyperandrogenemic girls in early adulthood and are characteristic of PCOS.

Treatment with a CRH-R1 antagonist prevents stress-induced suppression of the central neural drive to the reproductive axis in female macaques.

In response to everyday life stress, some individuals readily develop reproductive dysfunction (i.e., they are stress sensitive), whereas others are more stress resilient. When exposed to mild combined psychosocial plus metabolic stress (change in social environment plus reduced diet), female cynomolgus monkeys can be categorized as stress sensitive (SS; they rapidly become anovulatory in response to stress), medium stress resilient (MSR; they slowly become anovulatory in response to prolonged stress), or highly stress resilient (HSR; they maintain normal menstrual cycles in response to stress). Previously, we reported that monkeys that develop abnormal menstrual cycles following exposure to mild combined stress (MSR + SS) have increased plasma cortisol levels the day they move to a novel room and start a reduced diet compared with HSR monkeys. In this study, we examined whether there is a similar acute effect of mild combined stress on the reproductive axis specifically in the combined group of MSR + SS animals by measuring LH pulse frequency and whether treatment with a CRH-R1 antagonist can prevent a stress-induced suppression of LH pulse frequency presumably by inhibiting activity of the HPA axis. Animals that developed abnormal menstrual cycles in response to stress (MSR + SS monkeys) suppressed LH pulse frequency in response to stress exposure. Pretreatment with 10 mg/kg iv antalarmin prevented the stress-induced suppression of LH secretion in these animals without the stress-induced increase in cortisol secretion being blocked. We conclude that CRH, acting via nonneuroendocrine mechanisms to regulate neurotransmitter systems other than the HPA axis, plays a role in causing stress-induced reproductive impairment in stress-sensitive individuals.

A selective monotropic elevation of FSH, but not that of LH, amplifies the proliferation and differentiation of spermatogonia in the adult rhesus monkey (Macaca mulatta).

BACKGROUND: Unilateral orchidectomy in monkeys increases spermatogenesis in the remaining testis in association with elevated follicle-stimulating hormone (FSH) secretion and testicular testosterone. The present study examined the relative importance of FSH and testosterone in driving the primate testis toward its spermatogenic ceiling. METHODS: Adult male rhesus monkeys were treated with a gonadotropin-releasing hormone receptor antagonist to inhibit endogenous FSH and luteinizing hormone (LH) secretion. The gonadotrophin drive to the testis was replaced with a pulsatile recombinant human FSH and LH infusion to maintain testicular volume and circulating testosterone and inhibin B at physiological levels. A selective monotropic elevation of FSH or LH that doubled the concentrations of inhibin B or testosterone, respectively, was then imposed for 4 weeks, each in a group of four monkeys. In a third group (n = 4), the gonadotrophin drive remained clamped at physiological levels. Bromo-deoxyuridine was administered 3 h prior to castration, and the effects of the monotropic hormone increments on germ cell number, S-phase labeling and degeneration were determined. RESULTS: Increased FSH, but not LH, produced increases in testicular volume (P < 0.05), the proportion of A pale spermatogonia entering the cell cycle and the numbers of differentiated spermatogonia and more advanced germ cells (P < 0.05). Indexes for spermatogonia labeling and germ cell degeneration were not affected. CONCLUSIONS: Under physiological conditions, circulating concentrations of FSH directly dictate sperm output of the primate testis by regulating the proportion of Ap spermatogonia in the growth fraction. An effect of FSH on survival of the first generation of differentiated B spermatogonia is not excluded.

Effect of transient hypothyroidism during infancy on the postnatal ontogeny of luteinising hormone release in the agonadal male rhesus monkey (Macaca mulatta): implications for the timing of puberty in higher primates.

The present study examined whether a transient thyroid hormone (T(4)) deficit during infancy in male monkeys would compromise the arrest of luteinising hormone (LH) secretion during the infant-juvenile transition, and/or interfere with the pubertal resurgence of LH. Animals were orchidectomized and thyroidectomized (n = 3; Tx) or sham Tx (n = 3) within 5 days of birth. T(4) replacement was initiated in two Tx monkeys at age 19 weeks to reestablish a euthyroid condition. Blood samples were drawn weekly for hormone assay. Body weight, crown-rump length, and bone age were assessed throughout the study. Within a week of Tx, plasma T(4) declined to undetectable levels and, by 6-8 weeks of age, signs of hypothyroidism were evident. Transient hypothyroidism during infancy failed to prevent either arrest of LH secretion during the infant-juvenile transition or the pubertal resurgence of LH secretion, both of which occurred at similar ages to sham Tx animals. Although body weight exhibited complete catch-up with T(4) replacement, crown-rump length and bone age did not. Thus, bone age at the time of the pubertal LH resurgence in Tx animals was less advanced than that in shams. Although Tx did not influence qualitatively the pattern of gonadotrophin secretion, LH levels during infancy and after pubertal LH resurgence were elevated in Tx monkeys. This was not associated with changes in LH pulse frequency and amplitude, but half-life (53 versus 65 min) of the slow second phase of LH clearance was greater in Tx animals. These results indicate that hypothalamic mechanisms dictating the pattern of gonadotrophin-releasing hormone release from birth to puberty are not dependent on T(4) action during infancy, and fail to support the notion that onset of puberty is causally coupled to skeletal maturation. They also indicate that LH renal clearance mechanisms may be programmed in a T(4) dependent manner during infancy.

Induction of a hypothyroid state during juvenile development delays pubertal reactivation of the neuroendocrine system governing luteinising hormone secretion in the male rhesus monkey (Macaca mulatta).

The present study aimed to determine the influence of thyroid status on the timing of the pubertal resurgence in gonadotrophin-releasing hormone pulse generator activity [tracked by circulating luteinising hormone (LH) levels] in male rhesus monkeys. Six juvenile monkeys were orchidectomised and then treated with the antithyroid drug, methimazole, from 15-19 months until 36 months of age, at which time thyroxine (T(4)) replacement was initiated. Four additional agonadal monkeys served as controls. Blood samples were drawn weekly for hormonal assessments. Body weight, crown-rump length and bone age were monitored at regular intervals. By 8 weeks of methimazole treatment, plasma T(4) had fallen sharply, and the decline was associated with a plasma thyroid-stimulating hormone increase. In controls, plasma LH levels remained undetectable until the pubertal rise occurred at 29.3 +/- 0.2 months of age. This developmental event occurred in only half of the methimazole-treated animals before 36 months of age when T(4) replacement was initiated. The hypothyroid state was associated with a profound arrest of growth and bone maturation, but increased body mass indices and plasma leptin levels. T(4) replacement in methimazole-treated monkeys was associated with the pubertal rise in LH in the remaining three animals and accelerated somatic development in all six animals. Although pubertal resurgence in LH secretion occurred at a later chronological age in methimazole-treated animals compared to controls, bone age, crown-rump length and body weight at that time did not differ between groups. There were no long-term differences in plasma prolactin between groups. We conclude that juvenile hypothyroidism in male primates causes a marked delay in the pubertal resurgence of LH secretion, probably occasioned at the hypothalamic level. Whether this effect is meditated by an action of thyroid hormone directly on the hypothalamus or indirectly as a result of the concomitant deficit in somatic development remains to be determined.

A switch from continuous to episodic testicular testosterone release in response to pulsatile LH stimulation in juvenile rhesus monkeys (Macaca mulatta).

This study examined the ontogeny of the testicular testosterone response to precocious pulsatile LH stimulation in the juvenile rhesus monkey. LH stimulation was achieved with an i.v. infusion (one pulse every 3 h) of either single-chain human (sch)LH, administered alone or in combination with recombinant human (rh)FSH, or recombinant monkey (rm)LH in combination with rmFSH. Homologous gonadotropin treatment resulted in an adult profile of circulating mLH concentrations. The schLH infusions produced a similar pulsatile pattern in circulating LH with peak concentrations of approximately 5 IU/l. Although a robust testicular testosterone response was observed after 24 h of intermittent LH stimulation, surprisingly testosterone release at this time was continuous. The apulsatile mode of testosterone secretion, however, did not persist, and a switch to an unequivocal episodic mode of secretion, comparable to that observed in adult monkeys, occurred by day 4 of LH stimulation. FSH did not influence the pattern of the testosterone response. We conclude from these findings that progenitor Leydig cells in the primate testis are able to respond rapidly to a physiological LH stimulus. While the cell biology underlying the switch from a continuous to a pulsatile mode of testosterone secretion remains unclear, we suggest that this phenomenon may be related to the hypothesis that episodic testosterone secretion is required for the operation of the neuroendocrine axis governing testicular function.

Elevating circulating leptin in prepubertal male rhesus monkeys (Macaca mulatta) does not elicit precocious gonadotropin-releasing hormone release, assessed indirectly.

The purpose of this study was to examine the hypothesis that the pubertal reaugmentation of pulsatile GnRH release in male primates is triggered by a rise in circulating leptin concentrations. Agonadal juvenile male rhesus monkeys (n = 7) were implanted with indwelling venous catheters and housed in specialized cages that allow continuous access to the venous circulation. GnRH release was monitored indirectly using LH secretion from the in situ pituitary sensitized to the LH releasing action of GnRH as a bioassay for the hypothalamic peptide. Infusion of recombinant human leptin (5 micro g/kg body weight.h for 16 d resulted in a marked square wave increment in circulating leptin concentration from approximately 2-20 ng/ml but did not elicit precocious GnRH release. GH secretion, however, was stimulated confirming that the heterologous leptin preparation was bioactive in the monkey. Parenthetically, recombinant human leptin was found to be immunogenic in the monkey and circulating antileptin IgG was demonstrable 22-35 d after the initial exposure to the human protein. These findings further support the view that circulating leptin is unlikely to provide the signal that triggers the onset of puberty in male primates.

Circulating concentrations of nocturnal leptin, growth hormone, and insulin-like growth factor-I increase before the onset of puberty in agonadal male monkeys: potential signals for the initiation of puberty.

The factor(s) responsible for initiating the developmental increase in nocturnal gonadotropin-releasing hormone secretion, defining the onset of puberty, are not known. Although signals regulating prepubertal growth seem to be obvious candidates to control such a process, it is unclear whether prepubertal alterations occur in these growth-related factors such that they might provide the brain information on changing body size. Using samples analyzed previously describing the initiation of nocturnal pulsatile LH secretion in agonadal male monkeys (Endocrinology 139: 2774-2783, 1998), developmental changes in plasma concentrations of leptin, GH, and insulin-like growth factor I (IGF-I) were determined to test the hypothesis that an increase in circulating levels of one or all of these growth-derived signals precedes the onset of puberty. Hormone concentrations were determined in five juvenile males at 10-day intervals from approximately 60 days before and 50 days after the initiation of pulsatile nocturnal LH secretion. Leptin concentrations were determined in samples obtained at 1000 and 2200 h, 36 and 48 h before the nocturnal assessment of pulsatile LH. Mean nocturnal GH concentrations were determined from the sequential samples collected at night. IGF-I was determined in the 1000- or 2200-h presequential samples. Although daytime leptin concentrations did not increase developmentally, nocturnal leptin levels increased significantly during the 30 days before the onset of puberty. Furthermore, both nocturnal GH and IGF-I concentrations showed a significant sustained increase from the early prepubertal period to the 30 days preceding the onset of puberty. These data are the first to demonstrate an increase in nocturnal leptin and GH-induced IGF-I secretion prior to the onset of puberty in the agonadal male monkey and that these developmental changes occur independent of the gonadal influences. These findings provide justification for empirical investigation of the role of leptin and the GH axis, in particular IGF-I, in regulating developmental increases in pulsatile nocturnal gonadotropin-releasing hormone secretion initiating puberty in primates.

Application of an enzyme immunoassay for urinary follicle-stimulating hormone to describe the effects of an acute stressor at different stages of the menstrual cycle in female laboratory macaques.

An enzyme-linked immunosorbent assay (ELISA) for human urinary beta follicle-stimulating hormone (FSH) subunit was validated for use in the laboratory macaque (Macaca mulatta and Macaca fasicularis). This ELISA is based on the dissociation of the FSH heterodimer in urine and the subsequent measurement of the beta subunit as a representation of total urinary FSH. This assay was then used to describe the gonadotropin escape following ovarian senescence in post-menopausal macaques. In addition, the assay was used to observe the impact of an acute stressor on the pituitary-gonadal axis and how the impact of this stressor varies when experienced at different stages of the menstrual cycle. The study design involved the measurement of ovarian steroids and FSH in urine collected daily during a period of time when animals experienced a well-defined event on two occasions consisting of capture, restraint, and anesthesia. This unique study design was made possible by the ability to monitor both ovarian and pituitary function in the absence of confounding daily captures and restraint for blood collection. There was a high correlation between urinary FSH measured in macaques with the beta FSH subunit ELISA and serum FSH measured in paired blood samples by radioimmunoassay (n=39, r2=0.878, P<0.001) and the composite urinary FSH profile obtained from normal, premenopausal macaques exhibited the expected dynamics with a transient rise of FSH during the luteal-follicular transition as well as an acute rise of FSH at mid-cycle. This pattern was lost in castrate and post-menopausal monkeys in which FSH levels were significantly increased (P<0.0001) above those of intact males and young females, respectively. In the stress study, we found that stressors occurring during the luteal-follicular transition not only resulted in acute perturbations of FSH but also led to abnormalities in the subsequent menstrual cycle in 50% of the cases.

Intrathecal administration of an anti-ganglioside antibody results in specific accumulation within meningeal neoplastic xenografts in nude rats.

Intrathecal (i.t.) administration of monoclonal antibodies (MAbs) represents a new therapeutic approach for the treatment of leptomeningeal (LM) cancer, which is presently rapidly fatal. In this study, we quantitated the accumulation of an intrathecally administered anti-ganglioside GD2 MAb (3F8) within leptomeningeal neoplastic xenografts of GD2 positive melanoma and neuroblastoma in nude rats by measuring concentrations of radiolabeled and unmodified MAbs and by immunohistochemistry. Intrathecal administration of 125I-3F8 resulted in area under the tissue concentration versus time curve (AUC) values in SK-MEL-1 melanoma xenografts (53.1 microCi*h/g) that were 14-fold greater than in corresponding blood (3.9 microCi*h/g), whereas i.t. administration of a control nonspecific MAb resulted in AUC values in tumors (7.1 microCi*h/g) that were less than those in blood (9.5 microCi*h/g). Administration of acetazolamide and furosemide, which slow the clearance of IgG MAb from rat cerebrospinal fluid resulted in a fivefold increase in AUC of 125I-3F8 in melanoma (262.9 microCi*h/g). The highest concentration of 125I-MAb in tumor after i.t. administration was seen at the first sampling time of 2 h, and this fell to 50% of maximum values at 8-16 h. Pharmacokinetic analysis of unmodified MAb demonstrated retention of MAb within the LM space of animals with tumor. The concentration of MAb 3F8 appearing in serum after i.t. administration was 10-fold lower in animals with melanoma xenografts than in those without tumor implants. Radiation dose estimates after intraventricular administration of radiolabeled MAb indicated delivery to tumor of 1,870 rad/mCi of 125I-3F8 but only 40 rad/mCi of 125I-labeled control MAb. These results indicate that anti-ganglioside MAbs and other MAbs directed to tumor-associated antigens are excellent candidates for i.t. treatment of appropriate leptomeningeal cancers in humans.

Indirect assessment of pulsatile gonadotropin-releasing hormone release in agonadal prepubertal rhesus monkeys (Macaca mulatta).

The major purpose of this study was to characterize the open-loop frequency of pulsatile GnRH release in the female rhesus monkey at an age (15-20 months) when the prepubertal restraint on the hypothalamic-pituitary axis is maximally imposed. Additionally, evidence for pulsatile GnRH release in agonadal males of comparable age was also sought. Episodic LH secretion from the pituitary was used as an indirect index of GnRH discharges. In order to maximize the sensitivity of this in situ bioassay, the responsiveness of the pituitary gonadotrophs was usually first heightened by an i.v. intermittent infusion of the synthetic peptide. Monkeys (five females, three males) were castrated between 9 and 14 months of age, implanted with indwelling venous catheters, fitted with nylon jackets and housed in specialized cages that permitted remote access to the venous circulation with minimal restraint and without interruption of the light-darkness cycle. In females, LH secretion was generally assessed at 20-day intervals during alternate nighttime (1900-0200 h) and daytime (0700-1400 h) windows. In males, LH was assessed less frequently and only at night. The mean frequency of pulsatile LH release in agonadal prepubertal females was 4 pulses/7 h during the night and 2 pulses/7 h during the day. These findings indicate that, prior to puberty in the female monkey, the GnRH pulse generator operates at a relatively slow frequency and is subjected to diurnal modulation. In males, evidence for robust pulsatile GnRH release was not observed. The striking difference in activity of the GnRH pulse generator in agonadal prepubertal male and female monkeys reinforces the view that the ontogeny of the hypothalamic drive to the pituitary-gonadal axis in higher primates, including man, is sexually differentiated.

The time course of follicle-stimulating hormone suppression by recombinant human inhibin A in the adult male rhesus monkey (Macaca mulatta).

In higher primates, FSH secretion appears to be regulated by a control system consistent with that described by the classical inhibin hypothesis. The purpose of the present experiment was to examine the time course of inhibin's action to suppress FSH secretion in the intact adult male rhesus monkey. To this end, five adult males implanted with indwelling venous catheters and exhibiting typical episodic patterns of LH and testosterone (T) secretion received a 4-day i.v. infusion of recombinant human (rh) inhibin A (832 ng/h x kg) followed, after a 4-week interval, by vehicle infusion of similar duration. Changes in circulating FSH concentrations during the inhibin and vehicle infusions were determined using a sensitive homologous macaque RIA, whereas enzyme-linked immunosorbent assays were employed to track inhibin A, inhibin B, and inhibin pro-alpha-C levels during the experiment. Normal pulsatile activity in the hypothalamic-pituitary-Leydig cell axis was confirmed by monitoring changes in circulating concentrations of LH and T in 12-h windows of sequential blood collection (1200-2400 h; every 20 min) before, during, and after the rh inhibin A and vehicle infusions. Although infusion of rh inhibin A, which led to a 12 ng/ml square wave increment in circulating levels of this inhibin dimer, produced a marked decline in circulating FSH concentrations, significant suppression of the secretion of this gonadotropin was not manifest until 54 h after initiation of the infusion. Despite the marked decline in FSH secretion during the last 24 h of the 4-day infusion of recombinant hormone, circulating inhibin B and pro-alpha-C concentrations were maintained at preinfusion control levels (1 ng/ml). The finding that imposition of an exaggerated circulating inhibin signal led to suppression of FSH secretion in the male monkey only after 2 days of exposure to the hormone indicates that in this species the feedback action of testicular inhibin on FSH secretion is heavily lagged. Moreover, as the decrease in FSH did not lead to changes in native inhibin secretion, it seems reasonable to propose that the FSH-inhibin feedback loop that governs testicular function in higher primates operates with considerable hysteresis at both the pituitary and gonadal levels. The failure of dramatically elevated inhibin A levels to influence the pulsatile secretion of LH in the monkey reinforces the idea that in this species the pituitary action of testicular inhibin is specific for FSH and does not involve modulation of GnRH receptor levels.

The pattern and tempo of the pubertal reaugmentation of open-loop pulsatile gonadotropin-releasing hormone release assessed indirectly in the male rhesus monkey (Macaca mulatta).

The purpose of this study was to determine the pattern and tempo of the open-loop reaugmentation of pulsatile GnRH release at the time of puberty in the male rhesus monkey. Episodic LH secretion from the in situ pituitary, in which responsiveness to GnRH was first heightened and subsequently sustained by priming with an i.v. intermittent infusion of the synthetic peptide, was used as an index of GnRH discharges. Ten male monkeys were castrated between 12 and 20 months of age, implanted with indwelling venous catheters, and housed in specialized cages that permitted remote access to the venous circulation with minimal restraint and without interfering with the light-dark cycle. Endogenous GnRH release was assessed by examining moment-to-moment changes in circulating LH concentrations measured at 12-min intervals for 7 h while GnRH priming was temporarily interrupted. A discharge of GnRH was inferred whenever a pulse of LH secretion was identified by a pulse detection program. Examination of nocturnal pulsatile GnRH release (1900-0200 h) was initiated as early as 14 months of age. GnRH release was assessed at 40-day intervals before 20 months of age and at 10-day intervals whenever possible thereafter. A simple algorithm was developed to identify the age at which a developmental increase in hypophysiotropic drive to the gonadotroph occurred. This was termed day zero and was considered to represent the age at which a pubertal mode of GnRH release was initiated. After the initiation of pubertal GnRH release was established, alternate nighttime and daytime (1100-1800 h) assessments of GnRH were performed. Before day zero, which was observed between 24 and 29 months of age, a stable, low frequency (<1 pulse/7 h), low amplitude pattern of pulsatile GnRH release was observed. Termination of the prepubertal mode of GnRH pulse generator activity was manifest as a relatively rapid nocturnal shift to a robust high-frequency pattern of activity. In some animals, the nocturnal acceleration to an adult GnRH pulse frequency (6-7 pulses/7 h) was attained within an epoch of only 30 days. Although initiation of the pubertal acceleration in nocturnal GnRH pulse generator activity seemed to be associated with an increase in GnRH pulse amplitude, it was not possible to decipher the subsequent developmental changes in this parameter. In some animals, the pattern of pulsatile GnRH release after the initiation of the pubertal acceleration was punctuated by periods of diminished activity, which seemed to be unrelated to the state of the pituitary-adrenal axis. These findings demonstrate that the neurobiological mechanisms that lead to the termination of the prepubertal mode of diminished GnRH release, and that therefore initiate the insidious process of puberty, have the potential to unfold with a surprisingly rapid time course. The extent to which the intrinsic tempo of the pubertal acceleration of pulsatile GnRH release in the agonadal situation is dampened by testicular feedback in the intact monkey remains to be established.

Pharmacokinetics of IgG and IgM anti-ganglioside antibodies in rats and monkeys after intrathecal administration.

Intrathecal (i.t.) administration of monoclonal antibodies (mAbs) represents a new therapeutic approach for the treatment of leptomeningeal cancer, which is rapidly fatal. This study describes the pharmacokinetics of intrathecally administered mAbs in rats and monkeys to optimize their use for regional antineoplastic therapy. We hypothesized that mAbs, which are high-molecular-weight, polar compounds, would be eliminated from the cerebrospinal fluid (CSF) at the same rate as bulk flow of CSF. We found that an IgM mAb was cleared from rat CSF at the rate of CSF bulk flow (0.0041 ml/min), but an IgG mAb was cleared at a faster rate (0.011 ml/min). We attempted to reduce the CSF clearance of an IgG mAb by administration of acetazolamide and furosemide, which inhibit the rate of CSF production and CSF bulk flow. We demonstrated that the administration of acetazolamide and furosemide reduced the clearance of IgG mAb from rat CSF by 58%. These results establish that bulk flow of CSF determines a minimum rate of elimination from the CSF for IgM mAbs and that additional mechanisms operate to clear IgG mAbs from the CSF. Inhibition of CSF production by acetazolamide and furosemide increased the area under the CSF concentration vs. time curve of IgG mAbs in the CSF. The increased area under the CSF concentration vs. time curve is likely to improve the therapeutic index of these agents for i.t. therapy.

Effects of continuous inhibin administration on gonadotropin secretion and subunit gene expression in immature and adult male rats.

To begin to examine inhibin as a male contraceptive, recombinant human (rh) inhibin-A was administered for 3 days via osmotic minipumps to male rats. Doses of inhibin of 0-150 mg/kg per day did not produce a concentration-dependent suppression of FSH secretion or pituitary FSH beta mRNA levels in adult rats. Treatment of immature rats at a dose of 145 micrograms/kg per day, which was without effect in adults, reduced plasma FSH levels by 49% (p < 0.01), and FSH beta mRNA levels to 47 +/- 11% of control (p < 0.01). Inhibin also decreased levels of LH beta mRNA (63 +/- 8% of control; p < 0.01), alpha-subunit mRNA (86 +/- 10% of control; p < 0.05), and GnRH-receptor mRNA (77 +/- 17% of control; p <0.01) in immature rats. Rh inhibin-A was more effective in immature than in adult animals; plasma inhibin levels were increased (p = 0.03) by rh inhibin-A treatment only in immature rats. Pharmacokinetic studies revealed that the weight-adjusted clearance was greater (p < 0.01), and the elimination half-life of rh inhibin A was shorter (p < 0.01) in adult than in juvenile rats. These data indicate that partial suppression of FSH beta mRNA inhibin is associated with a decline in GnRH receptor gene expression, suggesting that the notion that inhibin can act as a male contraceptive through selective and complete inhibition of FSH production, without effect on LH and Leydig cell function, may be mistaken. In addition, increased inhibin clearance appears to contribute to the fall in plasma inhibin levels with maturation in the male rat.

Gonadal and nongonadal mechanisms contribute to the prepubertal hiatus in gonadotropin secretion in the female rhesus monkey (Macaca mulatta).

The present study reexamined the role of the ovary in determining the prepubertal hiatus of gonadotropin secretion in the rhesus monkey. Day- and nighttime blood samples were obtained weekly from neonatally (7-10 days of age) ovariectomized and intact monkeys from birth until 3 yr of age. In the intact monkeys, plasma FSH levels increased during the first month of life, remained elevated until approximately 3 months of age, and then decreased to become undetectable by 7 months of age. Thereafter, plasma FSH remained undetectable until approximately 19 months of age, at which time it again increased to detectable concentrations. In animals ovariectomized as neonates, the developmental pattern in FSH secretion was similar to that in intact animals, but, quantitatively, mean plasma FSH concentrations in the agonadal females were greater than those in the intact control group at all times. Circulating daytime LH concentrations in intact animals were generally below the sensitivity of the assay during the neonatal and prepubertal phases of development, but after 27 months of age, this plasma hormone was measurable on occasion. In neonatally ovariectomized monkeys, daytime LH was elevated during the first month of life, undetectable between 2-20 months of age, and then rose into the adult range by the end of the study. Nocturnal plasma FSH and LH concentrations in agonadal monkeys were generally greater than those during the day at all stages of development. Of particular note was the finding that during the prepubertal hiatus in gonadotropin secretion, when daytime LH levels were mostly immeasurable, nighttime levels of this gonadotropin were consistently elevated. The hypersecretion of gonadotropin during prepubertal development in agonadal animals also occurred when ovariectomy was performed at 61-62 weeks of age. These findings demonstrate that in the female monkey, the open loop activity of the GnRH pulse generator during juvenile development is only partially suppressed, and the ovary contributes significantly to the prepubertal restraint on gonadotropin secretion. We also report the serendipitous finding that a precipitous, albeit transient, decline in circulating gonadotropin concentrations occurred in juvenile monkeys after separation from their mothers and relocation to individual cages. This suppression, which was accompanied by elevated plasma cortisol levels, was apparently not related to any impairment in growth.

Studies of the role of the N-methyl-D-aspartate (NMDA) receptor in the hypothalamic control of prolactin secretion.

To further examine the role of excitatory amino acids in the control of prolactin (PRL) secretion, the effects of administering a specific agonist and an antagonist of the N-methyl-D-aspartate (NMDA) receptor on plasma PRL concentrations were examined in the adult male rat. Animals of the Sprague-Dawley strain weighing 250-300 g were implanted with an indwelling cardiac catheter via the right jugular vein. Blood samples were collected through the catheter at 5 min intervals for 40 min, beginning 5 min before the iv administration of drug or the saline vehicle (V). Plasma PRL and luteinizing hormone (LH) concentrations were estimated using RIAs. Groups of animals (n = 5-7) received N-methyl-D,L-aspartate (NMA), D,L-2-amino-5-phosphonopentanoic acid (AP5), AP5 and NMA, norvaline (NOR), or V. The effects of administering the NMDA receptor antagonist alone were studied on two separate occasions. Injection of NMA (4.5 mg/rat) resulted in unambiguous PRL and LH discharges. Treatment with AP5 (9 mg/rat) 1 min prior to NMA administration completely blocked the LH releasing action of NMA, but did not significantly alter the discharge of PRL. Injection of AP5, alone, generally elicited a distinct and robust discharge of PRL, although plasma LH levels in these animals remained unchanged. NOR, an amino acid structurally related to AP5, administered at a dose (5.3 mg/animal) isomolar to that of AP5, was without effect on PRL and LH secretion, as was injection of V alone. These findings suggest that neuroexcitatory amino acids acting at the NMDA receptor may play a role in modulating the activity of neuronal systems that govern the release of both PRL releasing factor (PRF) and PRL inhibiting factor (PIF) into hypophysial portal blood.

The hypogonadotropic state of the prepubertal male rhesus monkey (Macaca mulatta) is not associated with a decrease in hypothalamic gonadotropin-releasing hormone content.

Hypothalamic contents of gonadotropin-releasing hormone (GnRH) in neonatally orchidectomized infant, juvenile, and adult monkeys were measured by a radioimmunoassay (RIA) and by an in vivo bioassay that utilized luteinizing hormone (LH) secretion in estrogen- and progesterone-treated ovariectomized rats. The results of the bioassay provided no evidence to suggest that hypothalamic GnRH content in juvenile monkeys (mean = 83 ng/hypothalamus; n = 3) was less than that in infants (mean = 54 ng/hypothalamus; n = 4) and adults (mean = 36 ng/hypothalamus; n = 3). A similar developmental pattern in hypothalamic GnRH content was also observed when the decapeptide was measured by RIA. In striking contrast to the maintenance of hypothalamic GnRH content throughout postnatal development, pituitary gonadotropin contents and serum gonadotropin concentrations were markedly reduced in juvenile monkeys.