Episodic Gonadotropin Secretion: DISCUSSION
To our knowledge, this is the first study to clearly demonstrate that circulating FSH concentrations vary significantly over the course of a day. Furthermore, our data suggest that FSH is secreted in an episodic fashion, resulting in distinct pulses of FSH in the adult male fowl. As discussed below, it appears that FSH and LH pulses are asynchronous and are not derived from a common hypothalamic pulse generator.
The management program for the birds used in the present experiments was developed for elite male broiler breeders. Birds raised under this program were feed-restricted to prevent obesity and exposed to a long-day photostimulation after 20 wk of age to maintain reproductive performance throughout adult life. One consequence of this management system is the variability in testis size of sexually mature males.
Despite a slight decrease of the LH baseline on Day 0, concentrations were not significantly different from those on Day 1 and Day 2. Moreover, we did not observe any depressive effect on LH secretion over time, suggesting that frequent withdrawal of blood samples using the present cannulation procedure did not interfere with the normal secretion of LH in blood. Conversely, LH concentrations tended to decrease over time as a result of the handling associated with repeated blood sampling of cockerels in previous reports.
The pulsatile pattern of LH secretion has previously been documented in the male fowl. However, to our knowledge the present is the first to report pulsatile FSH secretion in avian species. Regardless of the statistical method used to identify hormone secretion, we found that LH pulses were more frequent and had greater amplitude than FSH pulses. However, disagreement was found between methods in the estimation of FSH pulse frequency. When six statistical procedures were used to evaluate gonadotropin secretion, a distinct number of pulses was estimated by different methods, suggesting that no single correct method exists to identify pulses. In the present experiment, Pulsar identified most of the pulses that were apparent by visual observation; however, some small amplitude pulses were not consistent with our visual observation.
Only 32% of the LH pulses were associated with FSH episodes in experiment 1, and only 23% of the LH pulses were associated with FSH episodes in experiment 2. Additionally, a significant rate of false-positive and false-negative episodes was observed in both experiments. Schally et al. postulated that one hypothalamic hormone, LH-releasing hormone/FSH-releasing hormone, or simply GnRH, controls the secretion of both LH and FSH from the pituitary gland. The terms LH-releasing hormone and GnRH have been widely adopted, and most journals allow the use of both names and abbreviations. It is well documented that LH-releasing hormone or GnRH regulates reproduction in all mammalians species. However, recent evidence strongly favors the existence of FSH-releasing factors.
Lesions to the median eminence of castrated male rats suppressed LH, but not FSH, pulses, whereas animals with posterior to mid-median eminence lesions had no FSH pulses but maintained LH pulses. Similarly, ablation of the dorsal anterior hypothalamus of ovariectomized rats suppressed FSH, but not LH, pulses. These results raise the possibility that another form of GnRH may contribute to the control of reproductive function or take an important neuroendocrine role.
The temporal relationship between GnRH and LH has been demonstrated in sheep, rodents, and monkeys. In birds, two forms of GnRH have been reported [4l-43], and only indirect measurements of the GnRH pulse generator are available by measuring plasma LH concentrations in frequent samples or in pituitary extracts. Both cGnRH-I and -II stimulate gonadotropin release in vivo and in vitro in the chicken. However, concentrations of FSH in small cockerels were not affected by cGnRH-I challenges, and intracere-broventricular infusion of cGnRH-II, but not of cGnRH-I, induced copulation solicitation in female sparrows, suggesting a behavioral role for GnRH-II that may be independent from GnRH-I. In the chicken, LH- and FSH-containing gonadotrophs reside in separate cells within the pituitary gland, suggesting that synthesis and secretion of LH and FSH may be differentially regulated. The distinct pulsatile pattern of FSH as well as the dissociation of LH and FSH pulses that we observed in the present experiments further suggest that FSH secretion may be uniquely regulated in male broiler breeders.
Although the existence of an FSH-releasing factor was proposed 38 yr ago, until fairly recently it was generally believed that mammals only express one form of GnRH. When male rats were administered GnRH antiserum and/or GnRH antagonists, pulsatile FSH release was maintained but that of LH was abolished, giving further credence to the view that reproductive function may be regulated by more that one GnRH neuronal system. Additionally, testosterone supplementation of intact rats can maintain the FSH content of the pituitary in GnRH antagonist-suppressed males. A possible autocrine-paracrine regulation of FSH release at the pituitary level by activins, inhibins, and follistatins also cannot be overlooked. It is possible that the concerted action of local pituitary factors and peripheral steroids could lead to a pulsatile FSH pattern. However, to our knowledge, the exact regulatory roles that these factors and their receptors might fill in avian species have not been investigated.
Testosterone was secreted in an episodic fashion, and testosterone pulses closely followed LH episodes in birds with testis weight greater than 10 g and relative high testosterone concentrations. Similarly, in male turkeys, testosterone pulses were preceded by an increase in LH concentration. The amplitude of the LH pulses that were associated with FSH episodes was 2.3-fold greater than that of the false-positive LH pulses. The small-amplitude LH pulses, as determined by Pulsar, were not consistent with our visual observation. These data suggest that in broiler males with normal testis size (testis weight, >10 g), a close association exists between LH and testosterone pulses. Testosterone is secreted by Leydig cells in response to LH challenges. However, in the bird with small testis size, testosterone pulse frequency, amplitude, and baseline concentrations were reduced, and sensitivity was only 23%. In contrast, LH pulse frequency and amplitude were similar to those of birds with normal testis size, and LH pulses that were associated with testosterone episodes were of approximately the same magnitude as the false-positive LH pulses (6.1 and 6.9 ng/ml, respectively). These data indicate that in the bird with small testis size, Leydig cells did not respond to LH challenges in the same manner as it did in birds with testis size greater than 10 g.
Birds used in the present study exhibited no outward appearance of distress throughout the experiment, indicating that the cannulation procedure minimized the disturbance of experimental animals. Corticosterone concentrations tended to increase during the first 2-4 h after can-nulation, and testosterone concentrations were significantly low on Day 0 compared to Day 2. We conclude that to maximize the effectiveness of experiments designed to evaluate acute hormonal changes, at least 4 h should lapse between cannulation and sampling. Furthermore, the lack of synchrony between the episodic release of LH and FSH in adult male fowl gives support to the view that LH and FSH secretion are regulated independently.