The hypothalamus is at the base of the brain just above the junction of the optic nerves. In order to influence the anterior pituitary gland, the brain requires a means of transmission or connection. A direct nervous connection does not exist. The blood supply of the anterior pituitary, however, originates in the capillaries that richly lace the median eminence area of the hypothalamus. The superior hypophyseal arteries form a dense network of capillaries within the median eminence, which then drain into the portal vessels that descend along the pituitary stalk to the anterior pituitary. The direction of the blood flow in this hypophyseal portal circulation is from the brain to the pituitary. Section of the neural stalk, which interrupts this portal circulation, leads to inactivity and atrophy of the gonads, along with a decrease in adrenal and thyroid activity to basal levels. With regeneration of the portal vessels, anterior pituitary function is restored. Thus, the anterior pituitary gland is under the influence of the hypothalamus by means of neurohormones released into this portal circulation. There also exists retrograde flow so that pituitary hormones can be delivered directly to the hypothalamus, creating the opportunity for pituitary feedback on the hypothalamus. An additional blood supply is provided by short vessels that originate in the posterior pituitary that in turn receives its arterial supply from the inferior hypophyseal arteries.

The influence of the pituitary by the hypothalamus is achieved by materials secreted in the cells of the hypothalamus and transported to the pituitary by the portal vessel system. Indeed, pituitary cell proliferation and gene expression are controlled by hypothalamic peptides and their receptors. In addition to the stalk section experiments cited previously, transplantation of the pituitary to ectopic sites (e.g., under the kidney capsule) results in failure of gonadal function. With retransplantation to an anatomic site under the median eminence, followed by regeneration of the portal system, normal pituitary function is regained. This retrieval of gonadotropic function is not accomplished if the pituitary is transplanted to other sites in the brain. Hence, there is something very special about the blood draining the basal hypothalamus. An exception to this overall pattern of positive influence is the control of prolactin secretion. Stalk secretion and transplantation cause release of prolactin from the anterior pituitary, implying a negative, inhibitory hypothalamic control. Furthermore, cultures of anterior pituitary tissue release prolactin in the absence of hypothalamic tissue or extracts.

Neuroendocrine agents originating in the hypothalamus have positive stimulatory effects on growth hormone, thyroid-stimulating hormone (TSH), adrenocorticotropic hormone (ACTH), as well as the gonadotropins, and represent the individual neurohormones of the hypothalamus. The neurohormone that controls gonadotropins is called gonadotropin-releasing hormone (GnRH). The neurohormone that controls prolactin is called prolactininhibiting hormone and is dopamine. Human corticotropin-releasing hormone (CRH) is a 41 amino acid peptide that is the principal regulator of ACTH secretion, and that also activates the sympathetic nervous system. As we shall see, CRH can suppress gonadotropin secretion, an action partly mediated by endorphin inhibition of GnRH.

In addition to their effects on the pituitary, behavioral effects within the brain have been demonstrated for several of the releasing hormones. Thyrotropin-releasing hormone (TRH) antagonizes the sedative action of a number of drugs and also has a direct antidepressant effect in humans. GnRH evokes mating behavior in male and female animals.¹

Initially, it was believed that there were two separate releasing hormones, one for folliclestimulating hormone (FSH) and another for luteinizing hormone (LH). It is now accepted that there is a single neurohormone (GnRH) for both gonadotropins. GnRH is a small peptide with 10 amino acids with some variation in the amino acid sequence among various mammals. Purified or synthesized GnRH stimulates both FSH and LH secretion. The divergent patterns of FSH and LH in response to a single GnRH are due to the modulating influences of the endocrine environment, specifically the feedback effects of steroids on the anterior pituitary gland.

The classic neurotransmitters are secreted at the nerve terminal. Brain peptides require gene transcription, translation, and posttranslational processing, all within the neuronal cell body. The final product is transported down the axon to the terminal for secretion. Small neuroendocrine peptides share common large precursor polypeptides, called polyproteins or polyfunctional peptides. These proteins can serve as precursors for more than one biologically active peptide.

The gene that encodes for the 92 amino acid precursor protein for GnRH, pro-GnRH, is located on the short arm of chromosome 8.² The precursor protein for GnRH contains (in the following order) a 23 amino acid signal sequence, the GnRH decapeptide, a 3 amino acid proteolytic processing site, and a 56 amino acid sequence called GAP (GnRH-associated peptide).³ GAP is a potent inhibitor of prolactin secretion as well as a stimulator of gonadotropins; however, a physiologic role for GAP has not been established.⁴ Its primary role may be to provide appropriate conformational support for GnRH.

It is now apparent that GnRH has autocrine-paracrine functions throughout the body. It is present in both neural and nonneural tissues, and receptors are present in many extrapituitary tissues (e.g., the ovarian follicle and the placenta). Although GnRH is identical in all mammals, other nonmammalian forms exist, indicating that the GnRH molecule has existed for at least 500 million years.^5,6 The central sequence, Tyr-Gly-Leu-Arg, is the nonconserved segment of GnRH, the segment with the most variability in other species. Accordingly, substitutions in this segment are well tolerated.

A second form of GnRH, known as GnRH-II, has been known to exist in many other species. GnRH-II consists of the following sequence: pGln-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly.
Prompted by its existence in other species, a search for its presence in humans was ultimately successful. A gene encoding GnRH-II is located on the human chromosome 20p13, obviously distinct from the GnRH-I gene on 8p11.2-p21.⁷ Both genes produce a peptide with a signal sequence, a GnRH decapeptide, a proteolytic site, and a GAP. Human GnRH-II expression is highest outside the brain. An analysis of the evolution of GnRH indicates three major forms: GnRH localized to the hypothalamus (GnRH-I), forms in midbrain nuclei and outside the brain (GnRH-II), and forms in several fish species (GnRH-III), thus indicating appearance of the various forms before the emergence of vertebrates.⁷ A total of 24 forms of GnRH have been identified in multiple species, but GnRH-I and GnRH-II are the primary GnRHs in mammals.⁸ GnRH-I is the main form found in the brain, whereas GnRH-II is widely distributed in other organs.

A hypothalamic 12-amino acid peptide that inhibits pituitary secretion of gonadotropins was isolated from the brain of the Japanese quail.⁹ This peptide was identified in mammals as well and labeled gonadotropin-inhibitory hormone (GnIH), but its presence and possible role in primate reproduction remain to be determined.¹⁰

Perhaps the notion that the pituitary is a master gland should not be discarded. Although it is highly regulated by input from other sites, its function is essential for sustaining life. The hormones from the pituitary gland regulate puberty, growth, reproduction, metabolism, osmotic balance, and responses to stress. Pituitary development and activity are under the control of the hypothalamus (with input from other central nervous system sites), and pituitary response is finely tuned by hormonal messages from tissues that are the targets of the pituitary trophic hormones. In addition, the pituitary has its own auto-crine-paracrine system for enhancement and suppression of growth and function. But the pituitary gland is the focus for all of this activity, and this central, coordinating role is critical for normal life.

Prolactin gene expression occurs in the lactotropes of the anterior pituitary gland, in decidualized endometrium, and in the myometrium. The prolactin secreted in these various sites is identical, but there are differences in mRNA indicating differences in prolactin gene regulation.

Transcription of the prolactin gene is regulated by a transcription factor (a protein named Pit-1) that binds to the 5′ promoter region and that is also necessary for growth hormone and TSH secretion.^11,12 The expression of Pit-1 is in turn regulated by a transcription factor, Prop1 (Prophet of Pit-1); mutations in the Prop1 binding site in the Pit-1 gene result in defi-ciencies in multiple pituitary hormones.^12,13 These mutations account for the majority of inherited cases of combined pituitary hormone deficiency. The remainder arise from mutations involving other transcription factors, specifically HESX1, LHX3, LHX4, TBX19, SOX2, AND SOX3.¹⁴

Prolactin gene transcription is regulated by the interaction of estrogen and glucocorticoid receptors with 5′ flanking sequences. Mutations in the sequences of these flanking regions or in the gene for the Pit-1 protein can result in the failure to secrete prolactin. The Pit-1 gene is also involved in differentiation and growth of anterior pituitary cells; therefore, mutations in this gene can lead not only to absent secretion of growth hormone, prolactin, and TSH but also to an absence of their trophic cells in the pituitary—the result is significant hypopituitarism.¹⁵ Molecular studies indicate that Pit-1 participates in mediating both stimulatory and inhibitory hormone signals for prolactin gene transcription. However, alterations in Pit-1 gene expression are not involved in pituitary tumor formation.¹⁶

The main function of prolactin in mammals is lactogenesis, whereas, in fish, prolactin is important for osmoregulation. The prolactin gene from the Chinook salmon contains coding sequences that are similar to those in mammals, and it is regulated similarly in the pituitary.¹⁷ Pit-1, the pituitary specific transcription factor, therefore, appears to be highly conserved among species. Sexual arousal and orgasm in men and women produce marked elevations of circulating levels of prolactin that persist for at least 1 hour, perhaps contributing to the suppression of sexuality immediately after arousal and orgasm.^18,19

Prolactin gene expression is further regulated by other species-specific factors. Prolactin gene transcription is stimulated by estrogen and mediated by estrogen receptor binding to estrogen-responsive elements. This activation by estrogen requires interaction with Pit-1, in a manner not yet determined. Proximal promoter sequences are also activated by peptide hormones binding to cell surface receptors, e.g., TRH and growth factors. In addition, various agents that control cyclic AMP and calcium channels can stimulate or inhibit prolactin promoter activity.

Pituitary secretion of prolactin is chiefly, if not totally, under the inhibitory control of hypo-thalamic dopamine released into the portal circulation, a tonic inhibition that requires a high output of dopamine.²⁰ The action of dopamine in the pituitary is mediated by receptors that are coupled to the inhibition of adenylate cyclase activity. There are 5 forms of the dopamine receptor divided into 2 functional groups, D₁ and D₂, encoded by a single gene on chromosome 5 near the growth hormone receptor gene.^21,22 The D₂ type is the predominant receptor in the anterior pituitary gland. The structure and function of the dopamine receptors are of the G protein system described in Chapter 2. Binding of dopamine to the receptor leads to suppression of adenylate cyclase and cyclic AMP maintenance of prolactin gene transcription and prolactin secretion. Other mechanisms are also activated, including suppression of intracellular calcium levels. Pit-1 binding sites are involved in this
dopamine response. In addition to direct inhibition of prolactin gene expression, dopamine binding to the D₂ receptor also inhibits lactotroph development and growth. These multiple effects of dopamine explain the ability of dopamine agonists to suppress prolactin secretion and the growth of prolactin-secreting pituitary adenomas. No activating or inactivating mutations of the dopamine receptors have been reported.

Several factors exert a stimulatory effect on prolactin secretion (prolactin-releasing factors), especially TRH, vasoactive intestinal peptide (VIP), epidermal growth factor, and perhaps GnRH. These factors interact with each other, affecting the overall lactotroph responsiveness; however, the only important clinical manifestation is the association of hyperprolactinemia with the elevation in TRH secretion that occurs with hypothyroidism. Prolactin homeostasis is regulated mainly by prolactin itself, feeding back on the dopamine-releasing neurons.

This dopaminergic mechanism is highly influenced by estrogen, either directly or through other neurotransmitters. Prolactin secretion can be understood by viewing dopamine received through the portal system as responsible for tonic inhibition. The dopaminergic system is stimulated by prolactin (decreasing secretion) and inhibited by estrogen (increasing secretion). Modulating influences include the inhibitory activity of endogenous opioids on dopamine release and stimulation by many substances, including serotonin and neuropeptide Y. Prolactin levels are highest during sleep.

Drugs used to treat psychological disorders are dopamine receptor antagonists. The inhibitory activity of dopamine on pituitary secretion of prolactin is blocked by these drugs, and prolactin levels increase in the circulation. Some of the newer drugs in this class do not affect prolactin secretion; these include clozapine, olanzapine, quetiapine, aripiprazole, and ziprasidone. Risperidone and amisulpride, however, act like the older drugs and increase prolactin secretion. The differences reflect the ability of each drug to cross the blood-brain barrier and the variations in affinity for the dopamine receptor.

The hypothalamus is the part of the diencephalon at the base of the brain that forms the floor of the third ventricle and part of its lateral walls. Within the hypothalamus are peptidergic neural cells that secrete the releasing and inhibiting hormones. These cells share the characteristics of both neurons and endocrine gland cells. They respond to signals in the bloodstream, as well as to neurotransmitters within the brain in a process known as neurosecretion. In neurosecretion, a neurohormone or neurotransmitter is synthesized on the ribosomes in the cytoplasm of the neuron, packaged into a granule in the Golgi apparatus, and then transported by active axonal flow to the neuronal terminal for secretion into a blood vessel or across a synapse.

The cells that produce GnRH originate from the olfactory area. By migration during embryogenesis, the cells move along cranial nerves connecting the nose and the forebrain to their primary location, where eventually 1,000-3,000 GnRH-producing cells can be found in the arcuate nucleus of the hypothalamus extending their axons to the median eminence.²³ The GnRH neurons appear in the medial olfactory placode (a thickened plate of ectoderm from which a sense organ develops) and enter the brain with the nervus terminalis, a cranial nerve that projects from the nose to the septal-preoptic nuclei in the brain.²⁴ This amazing journey accounts for Kallmann’s syndrome, an association between an absence of GnRH and a defect in smell (a failure of both olfactory axonal and GnRH neuronal migration from the olfactory placode). Three modes of transmission have been documented: X-linked,
autosomal-dominant, and autosomal-recessive.²⁵ The 5-7-fold increased frequency in males indicates that X-linked transmission is the most common. The mutations responsible for this syndrome result in the failure to produce proteins with functions that are necessary for neuronal migration, anosmin-1 in X-linked forms, a protein that is homologous to members of the fibronectin family and responsible for cell adhesion and protease inhibition, and the receptors for fibroblast growth factor and prokineticin in the autosomal forms.^26,27 and ²⁸ Normal GnRH neuron development and migration also depend on receptors that are tyrosine kinases, and abnormalites in these receptors might explain some clinical cases of GnRH deficiency.²⁹ Like olfactory epithelial cells in the nasal cavity, GnRH neurons have cilia.³⁰ The olfactory origin and the structural similarity of GnRH neurons and nasal epithelial cells suggest an evolution from reproduction controlled by pheromones.

Pheromones are airborne chemicals released by one individual that can affect other members of the same species. Odorless compounds obtained from the axillae of women in the late follicular phase of their cycles accelerated the LH surge and shortened the cycles of recipient women, and compounds from the luteal phase had the opposite effects.³¹ This may be one mechanism by which women who are together much of the time have been reported to exhibit a synchrony in menstrual cycle timing.^32,33,34,35 and ³⁶ However, the work on menstrual synchrony has been criticized, emphasizing that methodological problems have led to incorrect conclusions.³⁷

In primates, GnRH cell bodies are primarily located within the medial basal hypothalamus.^38,39 and ⁴⁰ Most of these cell bodies can be seen within the arcuate nucleus where GnRH is synthesized in GnRH neurons. The GnRH neurons exist in a complex network and are connected to each other and to many other neurons. This physical arrangement allows multiple interactions with neurotransmitters, hormones, and growth factors to modulate GnRH release. The delivery of GnRH to the portal circulation is via an axonal pathway, the GnRH tuberoinfundibular tract.

Fibers, identified with immunocytochemical techniques using antibodies to GnRH, can also be visualized in the posterior hypothalamus, descending into the posterior pituitary,
and in the anterior hypothalamic area projecting to sites within the limbic system.³⁸ Using hybridization techniques, messenger RNA for GnRH has been localized to the same sites previously identified by immunoreactivity. However, lesions that interrupt GnRH neurons projecting to regions other than the median eminence do not affect gonadotropin release. Only lesions of the arcuate nucleus in the monkey lead to gonadal atrophy and amenorrhea.⁴¹ Therefore, the arcuate nucleus can be viewed with the median eminence as a unit, the key locus within the hypothalamus for GnRH secretion into the portal circulation. The other GnRH neurons may be important for a variety of behavioral responses.

The gene for the a subunit of the gonadotropins is expressed in both the pituitary and placenta. The b subunit for human chorionic gonadotropin (hCG) is expressed in the placenta but only minimally (and with alterations in structure) in the pituitary, whereas the LH b subunit, as expected, is expressed in the pituitary but not significantly in the placenta.^66,67 Studies of gonadotropin gene expression confirm the relationships established by earlier studies. The sex steroids decrease and castration increases the rate of gonadotropin gene transcription as reflected by the levels of specific messenger RNAs. In addition, the sex steroids can act at the membrane level, affecting the interaction of GnRH with its receptor.⁶⁸

Both LH and FSH are secreted by the same cell, the gonadotrope, localized primarily in the lateral portions of the pituitary gland and responsive to the pulsatile stimulation by GnRH. GnRH is calcium dependent in its mechanism of action and utilizes inositol 1,4,5-triphosphate (IP₃) and 1,2-diacylglycerol (1,2-DG) as second messengers to stimulate protein kinase and cyclic AMP activity (Chapter 2).⁶⁹ These responses require a G protein receptor and are associated with cyclical release of calcium ions from intracellular stores and the opening of cell membrane channels to allow entry of extracellular calcium. Thus, calmodulin, protein kinase, and cyclic AMP are mediators of GnRH action. Gonadotrope gene transcription is mediated by several transcription factors, providing a mechanism by which the various subunits of FSH and LH can be synthesized and secreted by the same cell.⁷⁰ The rate-limiting step for the secretion of FSH and LH is the synthesis of the beta-subunits of each gonadotropin.

The GnRH type I receptor, a member of the G protein family, is encoded by a gene on chromosome 14q13.1-q21.1.^71,72 The location of the type II receptor is uncertain; in the marmoset, it is on chromosome 1q.⁷³ The exact roles of the two GnRHs and GnRH receptors in the human are yet to be determined. GnRH receptors are regulated by many agents, including GnRH itself, inhibin, activin, and the sex steroids.⁷⁴ The number of GnRH receptors available is significantly regulated by the frequency of GnRH pulses. The signaling pathways include the induction and modification of stimulatory and inhibitory transcription factor proteins. No mutations in the GnRH gene have been described in patients with hypogonadotropic hypogonadism; however, multiple mutations in the GnRH receptor gene have been reported.

Synthesis of gonadotropins takes place on the rough endoplasmic reticulum. The hormones are packaged into secretory granules by the Golgi cisternae of the Golgi apparatus and then stored as secretory granules. Secretion requires migration (activation) of the mature secretory granules to the cell membrane where an alteration in membrane permeability results in extrusion of the secretory granules in response to GnRH. The rate-limiting step in gonadotropin synthesis is the GnRH-dependent availability of the beta subunits.

Binding of GnRH to its receptor in the pituitary activates multiple messengers and responses. The immediate event is a secretory release of gonadotropins, whereas delayed responses prepare for the next secretory release. One of these delayed responses is the self-priming action of GnRH that leads to even greater responses to subsequent GnRH pulses due to a complex series of biochemical and biophysical intracellular events. This self-priming action is important to achieve the large surge in secretion at midcycle; it requires estrogen exposure, and it can be augmented by progesterone. This important action of progesterone depends on estrogen exposure (for an increase in progesterone receptors) and activation of the progesterone receptor by GnRH-stimulated phosphorylation. This latter action is an example of cross-talk between peptide and steroid hormone receptors.

Five different types of secretory cells coexist within the anterior pituitary gland: gonadotropes, lactotropes, thyrotropes, somatotropes, and corticotropes. Autocrine and paracrine interactions combine to make anterior pituitary secretion subject to more complicated control than simply reaction to hypothalamic-releasing factors and modulation by feedback signals. Substantial experimental evidence exists to indicate stimulatory and inhibitory influences of various substances on the pituitary secretory cells. Although the GnRH system is a primary mechanism, other hypothalamic peptides can influence GnRH secretion. Peptides can interact with GnRH at the pituitary; peptides can be transported to the pituitary gland where they can directly affect the gonadotropes (e.g., oxytocin, CRH, and neuropeptide Y) or indirectly have an effect on FSH and LH secretion by stimulating the release of active substances within the pituitary (e.g., glalanin, interleukins). Autocrine-paracrine activities involve peptides synthesized by pituitary cells.

Intrapituitary cytokines and growth factors provide an autocrine-paracrine system for regulating pituitary cell development and replication as well as pituitary hormone synthesis and secretion. The pituitary contains the familiar cast of substances encountered in organs throughout the body, including the interleukins, epidermal growth factor, fibroblast growth factors, the insulin-like growth factors, nerve growth factor, activin, inhibin, endothelin, and many others.^75,76 and ⁷⁷ As in most tissues, the interaction among these substances is a complex story, but the activin-inhibin mechanism deserves emphasis.

The most fascinating peptide group is the endogenous opioid peptide family.⁹⁶ β-Lipotropin is a 91 amino acid molecule that was first isolated from the pituitary in 1964. Its function
remained a mystery for more than 10 years until receptors for opioid compounds were identified, and, by virtue of their existence, it was postulated that endogenous opioid compounds must exist and serve important physiologic roles. Endorphin was a word coined to denote morphine-like action and endogenous origin in the brain.

Opiate production is regulated by gene transcription and the synthesis of precursor peptides and at a posttranslational level where the precursors are processed into the various bioactive smaller peptides.⁹⁷ All opiates derive from one of three precursor peptides.

POMC was the first precursor peptide to be identified. It is made in the anterior and intermediate lobes of the pituitary; in the hypothalamus and other areas of the brain; in the sympathetic nervous system; and in other tissues including the gonads, the placenta, the gastrointestinal tract, and the lungs. The highest concentration is in the pituitary gland.

Proopiomelanocortin is split into two fragments, an ACTH intermediate fragment and β-lipotropin. β-Lipotropin has no opioid activity but is broken down in a series of steps to β-melanocyte-stimulating hormone (β-MSH), enkephalin; and α-, γ-, and β-endorphins. Melanocyte-stimulating hormone acts in lower animals to stimulate melanin granules within cells, causing darkening of the skin. In humans, there is no known function.

Enkephalin and the α- and γ-endorphins are as active as morphine on a molar basis, whereas β-endorphin is 5-10 times more potent. In the adult pituitary gland, the major products are ACTH and β-lipotropin, with only small amounts of endorphin. Thus, ACTH and β-lipotropin blood levels show similar courses, and they are major secretion products of the anterior pituitary in response to stress. In the intermediate lobe of the pituitary (which is prominent only during fetal life), ACTH is cleaved to CLIP (corticotropin-like intermediate lobe peptide) and β-MSH. In the placenta and adrenal medulla, POMC processing yields a-MSH-like and β-endorphin peptides. β-Endorphin has also been detected in the ovaries and in the testes.

In the brain, the major products are the opiates, with little ACTH. In the hypothalamus the major products are β-endorphin and a-MSH in the region of the arcuate nucleus and the ventromedial nucleus. The pituitary opiate system is a system for secretion into the circulation whereas the hypothalamic opiate system allows for distribution via axons to regulate other brain regions and the pituitary gland.

β-Endorphin is appropriately considered a neurotransmitter, a neurohormone, and a neuromodulator. β-Endorphin influences a variety of hypothalamic functions, including regulation of reproduction, temperature, and cardiovascular and respiratory function, as well as extrahypothalamic functions such as pain perception and mood. POMC gene expression in the anterior pituitary is controlled mainly by corticotropin-releasing hormone and influenced by the feedback effects of glucocorticoids. In the hypothalamus, regulation of POMC gene expression is via the sex steroids.⁹⁸ In the absence of sex steroids, little, if any, secretion occurs.

Proenkephalin A is produced in the adrenal medulla, the brain, the posterior pituitary, the spinal cord, and the gastrointestinal tract. It yields several enkephalins: methionineenkephalin, leucine-enkephalin, and other variants. The enkephalins are the most widely distributed endogenous opioid peptides in the brain and are probably mainly involved
as inhibitory neurotransmitters in the modulation of the autonomic nervous system. Prodynorphin, found in the brain (concentrated in the hypothalamus) and the gastrointestinal tract, yields dynorphin, an opioid peptide with high analgesic potency and behavioral effects, as well as α-neoendorphin, β-neoendorphin, and leumorphin. The last 13 amino acids of leumorphin constitute another opioid peptide, rimorphin. The prodynorphin products probably function in a fashion similar to endorphin.

Opioid peptides are able to act through different receptors, although specific opiates bind predominantly to one of the various receptor types. Naloxone, used in most human studies, does not bind exclusively to any one receptor type, and, thus, results with this antagonist are not totally specific. Localization of opioid receptors explains many of the pharmacologic actions of the opiates. Opioid receptors are found in the nerve endings of sensory neurons, in the limbic system (site of euphoric emotions), in brainstem centers for reflexes such as respiration, and widely distributed in the brain and the spinal cord.