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.

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.

Electroejaculation is an accepted method of semen collection from nonhuman primates. Although both penile and rectal probe stimulation techniques have been used, there has been a general lack of consistency and detail regarding their application. This report describes the collection, processing, and evaluation of rhesus monkey semen contrasting two methods of penile electroejaculation: 1) a constant-voltage method where stimulus current is a variable and 2) a constant-current method where stimulus current is operator-controlled. The constant-current method was the more efficient procedure, requiring a lower stimulus current for successful electroejaculation. The influence on semen quality of potentially toxic agents used in the procedure, surgical glove powder and electrolyte cream, was tested; both were detrimental as measured by motility loss. No correlation was found between coagula volume and sperm numbers. The intra- and interanimal variability in semen samples from six monkeys was also evaluated. Penile electroejaculation, combined with control of stimulus current, provides a consistent, successful, and humane method for the collection of semen in the rhesus monkey.

It has been hypothesized that the secretion of gonadotropins, i.e. luteinizing hormone (LH) and follicle-stimulating hormone (FSH), is driven by a synchronized neural network (‘pulse generator’). This network, regulated in part by α-adrenergic activity, ultimately generates bursts of hypothalamic gonadotropin-releasing hormone (GnRH) release. In this study, we used the push-pull (PP) perfusion technique in ovariectomized rabbits to investigate three aspects of the (‘GnRH/gonadotropin pulse generator’) hypothesis. The objectives were to determine: (1) if plasma LH and FSH pulses occur concomitantly with mediobasal hypothalamic (MBH-) GnRH pulses, (2) changes in the patterns of pulsatile LH and FSH secretion when pulsatile MBH GnRH signals are interrupted by either local immunoneutralization of GnRH or intravenous infusion of the α-adrenergic antagonist phentolamine (PHEN, 4 mg/kg BW), and (3) whether third cerebroventricular (3VT-) GnRH patterns reflect neuronal GnRH release from the MBH. We found that while both plasma LH and FSH patterns were pulsatile, MBH GnRH pulses were significantly coupled only with LH pulses (94% coincidence). Both the local immunoneutralization of MBH GnRH pulses and the PHEN-induced suppression of MBH GnRH pulses obliterated the pulsatile secretion of LH, but not FSH. Neither MBH GnRH nor plasma LH or plasma FSH pulses were concurrent with 3VTGnRH pulses. However, the PP perfusion of the 3VT appeared to alter the pulsatile release of MBH GnRH and pituitary LH. The results support the hypothesis that in the absence of ovarian signals, the ‘pulse generator’ is maintained by tonic α-adrenergic input and that a ‘cellular unity’ of MBH GnRH release (GnRH pulses) drives the gonadotrophs to secrete LH in pulses. In contrast, the pulsatile release of FSH appears to involve additional nonovarian regulatory events to those controlling LH secretion.

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