6 Pulsatile Secretion
Let us roll all our strength and all
Our sweetness up into one ball,
And tear our pleasures with rough strife
Through the iron gates of life:
Thus, though we cannot make our sun
Stand still, yet we will make him run.
—Andrew Marvell (1621–1678), “To His Coy Mistress”
The findings of Wakerley and Lincoln marked a watershed for endocrinology. Before 1970, it had been assumed that hormonal concentrations changed only sluggishly, so a single blood sample would characterize the “state” of any particular endocrine system. Just a few years later, no hormone system could be considered as understood until the pattern of secretion was characterized. Of all the endocrine systems, the gonadotropins displayed the most vivid, dynamic behavior. In females of all mammals, LH and FSH are secreted in large intermittent pulses whose frequency and amplitudes vary over the course of the ovarian cycle. At the midpoint of the cycle, the pulses fade and a massive surge of LH and FSH secretion triggers ovulation, heralding a dramatic change in behavior. At this time, females of most mammal species are not just receptive to sexual advances from males, but actively seek them.
LH and FSH are both made in gonadotroph cells of the anterior pituitary, but they are packaged in separate vesicles and secreted semi-independently. Through their control of the ovaries, these two gonadotropins orchestrate all of the physiological, hormonal, and behavioral events that lead to ovulation, mating, and reproduction. In males, FSH is required for spermatogenesis and LH for the production of the male “sex hormone,” testosterone. In females, FSH controls the growth and recruitment of immature ovarian follicles, and LH controls the production of estrogen by those follicles, which acts on the brain to regulate sexual behavior.
Pulsatile secretion of LH and FSH is regulated by gonadotropin-releasing hormone (GnRH); this is synthesized in the hypothalamus and released in pulses that are carried by portal blood vessels to the anterior pituitary. That sentence captures decades of work by hundreds of scientists and some remarkable technical achievements.
Studies of the pattern of hormone secretion depended on sensitive means for measuring hormones, which depended on the development of radioimmunoassays, for which Rosalind Yalow was awarded a Nobel Prize in 1977.1 The principle is simple. You take a sample that contains an unknown amount of a given hormone, and add to it a known amount of the same hormone labeled with a radioactive isotope and a small amount of an antibody to that hormone. Some of the labeled hormone and some of the unlabeled hormone in the sample will bind to the antibody. The binding is competitive: molecules of antibody have a limited number of binding sites, so the hormone in the sample competes with the labeled hormone for binding to these sites: less of the labeled hormone can bind when there is more unlabeled hormone in the sample. The bound fraction is then precipitated, usually by adding another antibody that recognizes the original antibody, forming a large complex that can be spun down into a pellet using a centrifuge. The amount of labeled hormone in the pellet is then measured by counting the radioactivity, and the values are compared with a standard curve generated by adding known concentrations of unlabeled hormone to aliquots of assay buffer, which are processed in the same way as the samples. The same principle is used in enzyme-linked immunosorbent assays (ELISAs), which differ from radioimmunoassays in using nonradioactive labeled hormones and a color reaction, rather than radioactivity, to measure binding.
Radioimmunoassays could be extremely sensitive and specific, were simple to perform, and could handle large numbers of samples. Antibodies could be raised to many types of molecules, and by 1975, twenty years after the first publication of the approach, more than 4,000 laboratories in hospitals, universities, and research institutes in the United States alone were running radioimmunoassays to measure many hormones and other biological molecules.
In Pittsburgh, Ernst Knobil and his colleagues developed a radioimmunoassay for LH. In rhesus monkeys, they used it to measure secretion in blood samples taken every ten minutes, and showed that LH was secreted in pulses that occurred at about once an hour.2 Over the next few years, the full profile of LH and FSH secretion during the menstrual cycle of monkeys and women was revealed,3 along with the pattern of secretion in males, and then the profiles in rats, guinea pigs, pigs, sheep, goats, cows, and horses. During the menstrual cycle of the rhesus monkey and of women, pulsatile secretion of LH and FSH is interrupted every month by a massive “surge” of these hormones, which, in the rhesus monkey, precedes ovulation by about 37 hours. A similar surge occurs in most female mammals, and it is generally an absolute prerequisite for ovulation. In all species, secretion is pulsatile at other stages of the ovarian cycle, and is pulsatile at all times in males.
The next step required understanding how LH pulses arise. Geoffrey Harris had hypothesized that secretion of hormones from the anterior pituitary depends on the secretion of releasing factors into the blood vessels that link the hypothalamus and pituitary. What exactly were these factors, if they existed at all (which some still disputed)? Rosalind Yalow shared her Nobel Prize with Roger Guillemin and with Andrew Schally, who, more or less simultaneously, discovered two of the releasing factors: thyrotropin-releasing hormone and GnRH.4,5
In the early 1960s, investigators had found that the hypothalamus contained a substance that stimulated LH secretion, and they provisionally named it “LH-releasing factor” (LRF). To get enough of this substance to enable it to be identified was a massive undertaking. From 160,000 hypothalami, Schally isolated 800 µg of LRF, just enough to determine its structure by the techniques then available. The deduced structure was confirmed by synthesizing the peptide, a process that also provided abundant supplies for further research. The synthesized peptide explained all of the biological activity of the hypothalamic extract—and it stimulated the secretion of not just LH but also FSH. This resolved the question of whether there was a separate releasing factor for FSH, and the early name LRF gave way to the present name, GnRH. GnRH has since been identified in many animal species from sea squirts to mammals. The amino acid sequence is well conserved, suggesting that it is evolutionally ancient, and in every species studied it has an important role in reproduction.6
Given that LH is secreted in pulses, it was natural to expect that these might be the result of pulses of GnRH, but this was not necessarily so. Many oscillatory phenomena in biology arise from constant stimulation; for example, the uterus of a pregnant female will contract rhythmically even when exposed to a constant concentration of oxytocin. To test the hypothesis that pulses of LH are the result of pulses of GnRH, it would be necessary to simultaneously measure the secretion of both. Measuring LH was not difficult, but GnRH is secreted at tiny amounts into the vessels that connect the hypothalamus to the pituitary. GnRH is also much smaller than LH, just ten amino acids, and measuring such small peptides by radioimmunoassay is much more difficult than measuring LH: blood samples must be processed to eliminate plasma matrix molecules that can interfere with immunoassays for small molecules. Moreover, any attempt to measure GnRH secretion would have to be made in a conscious and unstressed animal, since both anesthetics and stress stop pulsatile LH secretion.
Iain Clarke and James Cummins in Melbourne set out to do the seemingly impossible.7 In sheep, they devised a way to collect blood samples from the median eminence, the specialized region at the base of the hypothalamus where small blood vessels collect the hypothalamic releasing factors. They implanted two needles into this region. Once the sheep had recovered, they used one needle to introduce a fine stylus to “nick” some of the vessels, and the other to collect the resulting blood, and they also collected blood samples from the jugular vein. The results were clear: every LH pulse was preceded by a GnRH pulse. The approach was refined by Alain Caraty in Nouzilly and Fred Karsch in Michigan, who revealed the relationship between LH and GnRH pulses in sheep in portal blood samples taken every 30 seconds. They also showed that the preovulatory LH surge is the consequence of a surge of GnRH.8
Knobil and colleagues showed just how very important the pulsatile pattern of GnRH is. In rhesus monkeys, lesions of the mediobasal hypothalamus abolish LH and FSH secretion by eliminating the supply of GnRH. Normal LH secretion could be restored by injections of GnRH, but their effectiveness depended more on the pattern of delivery than on the amount given. Continuous infusions failed to sustain LH secretion, which after an initial increase declined to undetectable levels. However, hourly injections, mimicking the physiological frequency of LH pulses, reproduced the physiological pattern of LH secretion. The explanation is that, when GnRH is infused continuously, the initial response of the pituitary is followed by a downregulation of LH and FSH secretion. As later summarized by Knobil, “the intermittency of the GnRH signal, within a relatively narrow window of frequencies, is an obligatory component of the neuroendocrine control system that governs normal gonadotropin secretion.”9 Knobil went on to note that “these fundamental physiologic observations in a nonhuman primate were transferred with remarkable rapidity to the clinical arena in the treatment of infertility that was attributable to hypothalamic dysfunction and in the suppression of inappropriate gonadotropin secretion (e.g., precocious puberty).”
The findings became important for treatments to enhance fertility, but also for new methods of contraception. The depression of LH secretion by continued administration of GnRH is so marked that GnRH agonists can be used as contraceptives for both males and females. Long-acting agonists are now used for this purpose in many domestic species, including dogs and cats.10
The pattern of LH secretion changes throughout life. Large amounts are secreted by the newborn infant, but then LH secretion drops to very low levels until it is reactivated at puberty. After puberty, the adult pattern of pulsatile secretion is established, followed by high levels of pulsatile secretion in late life as gonadal steroid levels decline. In the adult female, the pattern of LH secretion varies across the ovarian cycle in response to feedback from changing levels of estrogen and progesterone and other hormonal signals from the ovaries.
When GnRH binds to its receptors on gonadotrophs, intracellular calcium stores are mobilized, and this triggers exocytosis of vesicles. Every pulse of GnRH stimulates a pulse of LH, but FSH follows less consistently.11 FSH is secreted in response to GnRH, but it is also secreted constitutively, at a rate that depends on the rate at which it is synthesized. The pattern of GnRH pulses makes a difference; low-frequency pulses favor FSH secretion, and high-frequency pulses favor LH secretion. Gonadal steroid and peptide hormones also modulate the synthesis of both FSH and LH. A variety of signals are involved, including three other hormones: activin, inhibin, and follistatin. Activin and inhibin are produced by the gonads (and also by pituitary cells) and have opposite effects: activin stimulates FSH production and inhibin inhibits it. Follistatin (and a variety of other messengers) is released from folliculo-stellate cells in the pituitary, curious cells of which we know little, but which appear to be an endocrine version of interneurons. These cells have long processes entwined among the endocrine cells, and they are electrically coupled. Follistatin binds activin, preventing it from stimulating the gonadotrophs, and so it is a functional antagonist, modulating the hormonal feedback from the gonads to the pituitary.
The ovarian cycle comprises the processes by which a set of follicles develops in the ovary to the point of ovulation, when the ova that they contain are released into the fallopian tube to become available for fertilization. Each follicle consists of an oocyte (an immature ovum) enclosed in a cluster of other cells, including granulosa cells that produce estrogen. At puberty, a woman will have several hundred thousand follicles in her ovaries, but normally only one will develop fully in each cycle. In a rat, the cycle occupies four or five days in three stages: proestrus, estrus, and diestrus. At the end of proestrus, a set of ovarian follicles begins to grow in response to pulsatile secretion of FSH from the pituitary. Soon, only a few follicles will continue to develop—the “leading” follicles secrete factors that suppress the development of less mature follicles. As the follicles develop, the granulosa cells proliferate and develop receptors for LH, and pulsatile secretion of LH stimulates them to produce more and more estrogen. The estrogen inhibits GnRH release, and hence the LH pulses become attenuated. Despite the attenuation of LH secretion, the increasing sensitivity of the follicles to LH means that estrogen levels continue to rise, reaching a peak in the afternoon of proestrus.
This escalating production of estrogen has many consequences. At the pituitary, it stimulates the production of LH and of GnRH receptors, and in the brain it activates neurons in the rostral hypothalamus that trigger a surge of GnRH secretion. This triggers the preovulatory LH surge, a surge that over a few hours depletes most of the large pituitary content of LH that has previously expanded in response to estrogen. The LH surge triggers ovulation in the afternoon of estrus: the follicles rupture, releasing ova into the fallopian tube, and their remnants form the corpus luteum. The corpus luteum produces a surge of progesterone coincident with the LH surge, and throughout diestrus it continues to produce progesterone until it is destroyed in a process called luteolysis, the mechanisms of which vary considerably between species. The high level of progesterone also acts at both the pituitary and the hypothalamus, suppressing the secretion of GnRH, LH, and FSH.
While continuing high levels of estrogen are needed for an LH surge, the mechanisms that drive the surge are present only in females—the hypothalamus is sexually dimorphic. The exact timing of the surge depends on other signals. The cat is a reflex ovulator: the LH surge in cats is triggered by coitus. Rats are spontaneous ovulators: the LH surge occurs at a fixed time of day, and its timing is governed by the suprachiasmatic nucleus of the hypothalamus. Many species are seasonal breeders: in them, a prolonged period of infertility is terminated by environmental cues, such as day length and ambient temperature, to ensure that young are born at a time when food is abundant. In many of these species, pheromonal signals from urine, feces, vaginal secretions, saliva, and specialized scent glands help to synchronize reproductive cycles. This ensures that the young are all born at about the same time; a greater proportion of them will survive when their predators are saturated with prey.
The actions of estrogen and progesterone in the brain are not confined to the control of GnRH secretion. The term estrus, from the Greek word for a gadfly, was coined in 1900 to describe the “special period of sexual desire of the female.”12 The human is unusual in being potentially willing to engage in sexual activity at any time of the cycle: in most mammals, the female is receptive to males only at estrus, just after ovulation, and this receptivity is governed by the actions of estrogen and progesterone on the rostral hypothalamus.
This is a very simplified account. The details of the ovarian cycle differ between species, but all involve reciprocal interactions between the brain, the pituitary, and the ovary, each of which has a pattern-generating machinery of its own, involving many cell types and many signals. When three complex systems interact reciprocally to generate a cycle, it makes little sense to call any one a master and any other a slave. The gonads control the brain and pituitary as much as the brain controls the others.
Endocrine cells are not passive; they form an organized community using multiple messaging systems to generate complex patterns of secretion, and the regulation of synthesis is as important as the regulation of secretion. The intrinsic properties of the gonadotrophs amplify and smooth the pulsatile signals from GnRH neurons to generate large and orderly LH pulses, but their signal-generating abilities really come into their own during the preovulatory LH surge.
The LH surge is triggered by a surge of GnRH, but it is far from a passive response. The extraordinary process that underlies this surge was unmasked by George Fink, who called it the “self-priming” actions of GnRH.13 Although it is the actions of steroids on the brain that ultimately result in the LH surge, steroid actions at the pituitary are also important. By increasing the synthesis of LH, estrogen increases the amount of LH that can be secreted, and by increasing the synthesis of GnRH receptors it enhances the sensitivity of gonadotrophs to GnRH. Also, and most importantly, gonadotrophs that have been exposed to high levels of estrogen for long enough change how they respond to GnRH (figure 6.1).
Self-priming. In gonadotrophs, LH and FSH are packaged in large dense-core vesicles. (a) shows an electron microscope image of these vesicles, which appear as small, dense, spherical objects that are scattered throughout the cell.14 (b) shows a reconstructed cross section of a gonadotroph, showing the nucleus (N) and the endoplasmic reticulum. In a proestrus rat, exposure of the pituitary to GnRH causes an increase in intracellular calcium that comes from the stores in the endoplasmic reticulum. This causes some LH to be secreted, but it also causes the remaining vesicles to be trafficked to sites close to the cell membrane. (c) shows the appearance of such a gonadotroph, and (d) shows a schematic reconstruction of the whole gonadotroph. Now, more of the vesicles are available to be released, so when a second GnRH challenge is given, much more LH is secreted. The graph (e) shows the resulting secretion. Redrawn from Scullion et al.15
To understand this, we might ask a simple question, a question that can be asked both about release of neurotransmitters at synapses and about hormone secretion from endocrine cells. In both cases, secretion results from exocytosis of vesicles, either the small synaptic vesicles that contain neurotransmitter or the large dense-core vesicles that contain peptides. In both cases, exocytosis is triggered by an increase in intracellular calcium. At synapses, exocytosis is the result of calcium entry through channels opened by depolarization. In endocrine cells, exocytosis can result from chemical signals that activate signaling pathways leading to mobilization of intracellular calcium stores. In both cases we can ask why only some vesicles are released. At synapses, the answer is that, at any given time, only a few synaptic vesicles are available for release. Exocytosis of synaptic vesicles can occur only at specialized sites, close to clusters of calcium channels. To be available for release, a vesicle must be “docked” at one of those sites, where spikes cause a local rise in calcium and where particular proteins enable the synaptic vesicle to fuse with the cell membrane. As one vesicle is released from one of these sites, another takes its place, awaiting the next signal. In endocrine cells, however, calcium signals from intracellular stores spread throughout the cell and are relatively prolonged. The secretory response must be carefully rationed, and when some vesicles are secreted others must be moved around from deep within the cells.
It’s complicated: you might expect it to be like a tube of marbles in which, when the marble at the bottom is released, a new marble enters at the top, but it is not. Newly synthesized vesicles go first to the plasma membrane to be part of a “rapid-release” pool. If they are not released, they are shuttled into a “reserve pool” from which they can reenter the rapid-release pool if it becomes depleted. If they are still not called upon, they enter a “nonreleasable pool” where they will be dismantled. This flow is regulated by a “scaffold” of actin filaments. Cells are not mere bags of stuff, they have an internal cytoskeleton of contractile filaments that organize the internal compartments and enable cells to move.
The pituitary of an ovariectomized rat responds consistently to repeated challenges with GnRH, secreting similar amounts of LH each time. However, in a pituitary that has been exposed to estrogen, GnRH pulses result in progressively escalating responses. GnRH “primes” the response of gonadotrophs to GnRH—this involves a translocation of vesicles to docking sites at the plasma membrane, which makes more of them available for release (figure 6.1). As a consequence, in some species an LH surge can be evoked without any GnRH surge. In rhesus monkeys, after hypothalamic lesions that remove all GnRH, ovarian cycles can be restored by hourly pulses of GnRH, and no change in this pattern is needed to produce the monthly surge of LH and FSH that triggers ovulation. The feedback actions of sex steroids, by modulating how the pituitary responds to GnRH, are enough.
Thus secretion from endocrine cells depends on the signals that they receive, on their sensitivity to those signals, on paracrine interactions among the endocrine cells and with neighboring cell types, on the number of vesicles available for secretion, and on where those vesicles are located within the cell. The rates of synthesis of hormones and receptors, the intracellular disposition of vesicles, and intercellular organization and communication are all dynamically regulated.
Any notion that endocrine cells are merely slaves to the brain’s bidding is mistaken. The gonadotrophs are not exceptional in being organized as a community, or in being active partners in pattern generation.
Growth hormone, for example, is secreted in large pulses, the amplitude and frequency of which determine the rate of body growth. In species such as rats where there is a large disparity between the size of males and females, the pattern of secretion is sexually dimorphic. In male rats, a large pulse is secreted every three hours or so, and the importance of this pattern was revealed by Iain Robinson and his colleagues at the National Institute for Medical Research at Mill Hill, London. They studied rats deficient in growth hormone16 and measured their growth rates in response to injections of growth hormone. They showed that growth rates typical of normal male rats could be achieved in either male or female rats if growth hormone was injected at similar intervals to those seen in normal male rats, but not if the same amount was delivered by constant infusion or given as larger, less frequent injections.
Robinson went on to study how the pulses of growth hormone are generated. He found that male rats released more growth hormone in response to injections of GHRH than female rats, but whereas the responses in female rats were very consistent, the responses in male rats were erratic. When injections were given every 90 minutes, male rats responded strongly only to every other injection. Robinson concluded that another factor was intermittently suppressing the response of the somatotrophs to GHRH.
We now know, thanks in large part to Robinson’s studies, that pulses of growth hormone secretion arise from an interplay at the pituitary between GHRH, which stimulates growth hormone secretion, and a second hypothalamic peptide, somatostatin, which inhibits it. When GHRH triggers a pulse of growth hormone secretion, that pulse triggers the secretion of another hormone, insulin-like growth factor-1, from the liver, which enters the brain to act on the somatostatin neurons. The somatostatin that these neurons then release acts at the pituitary to prevent the somatotrophs from responding to GHRH. Only when the somatostatin signal has died away can GHRH trigger another pulse of growth hormone. These periventricular somatostatin neurons are one of the sexually dimorphic populations of the hypothalamus.
An even stronger role of peripheral endocrine mechanisms is apparent in the case of the “stress hormone” ACTH. ACTH is released from the corticotroph cells of the pituitary in pulses that stimulate pulsatile production of glucocorticoids that act back on the corticotrophs to inhibit them. As revealed by an elegant mathematical model, this partnership of endocrine cells can, by the exchange of hormonal signals, generate pulsatile secretion without any involvement of the hypothalamus.17 In this case, the hypothalamic releasing factors seem to modulate an autonomous endocrine pulse generator, rather than dictate the pattern of endocrine secretion.
Thus endocrine cells have a repertoire of properties that are not normally associated with neurons, but which nevertheless have sophisticated computational implications. These properties are also present in some hypothalamic neurons. Generally, the temporal patterning of peptide signals in the periphery is an important determinant of their biological efficacy. Given that peptides are commonly used signals within the brain, it seems likely that, there too, the temporal pattern of peptide secretion is important.
Notes