Reproductive Endocrinology PDF

4 Reproductive Endocrinology Neuroendocrinology, Gonadotropins, Sex Steroids, Prostaglandins, Ovulation, Menstruation, and Hormone Assay Nataki C. Douglas, Roger A. Lobo KEY POINTS Gonadotropin-releasing hormone (GnRH) an...

4 Reproductive Endocrinology Neuroendocrinology, Gonadotropins, Sex Steroids, Prostaglandins, Ovulation, Menstruation, and Hormone Assay Nataki C. Douglas, Roger A. Lobo KEY POINTS Gonadotropin-releasing hormone (GnRH) analogs are Kisspeptin (KISS1) plays a key role in the regulation of GnRH synthesized by substitution of amino acids in the parent release. molecule at the 6 and 10 positions for GnRH agonists and at Ovulation occurs about 24 hours after the estradiol peak and the 2 or 3 position for GnRH antagonists. The various analogs 32 hours after the initial rise in LH, as well as about 12 to have greater potencies and longer half-lives than the parent 16 hours after the peak of LH levels in serum. Serum GnRH and are used clinically for suppression. progesterone is less than 1 ng/mL before ovulation and LH and FSH have the same ! subunit as thyroid-stimulating reaches midluteal levels of 10 to 20 ng/mL. hormone (TSH) and human chorionic gonadotropin (HCG). Four characteristics of hormone assays establish their The " subunits of all these hormones have different amino reliability: sensitivity, specificity, accuracy, and precision. acids and carbohydrates, which provide specific biologic activity. The endocrine regulation of the reproductive system is GnRH-associated peptide (GAP). (Several forms of the GnRH very complex. This chapter presents only the basic information decapeptide have been identified, the principal of which is required to understand this complex subject. More detailed and GnRH-2, which differs from GnRH by 3 amino acids. It is found in-depth information is found in the several books that have been in several areas of the body, where it may subserve functions un- dedicated to this subject. related to those of GnRH.) Its role in fertility, if any, remains to Successful function of the reproductive system requires the be determined. involvement of several organs, none of which acts independently. In this chapter we discuss the physiology of the hypothalamic- Anatomy pituitary-ovarian (HPO) axis. For ease of understanding, each organ will be discussed first as an individual unit; information The Relationship of the Olfactory and GnRH will include the central nervous system control of gonadotropin- releasing hormone (GnRH), the primary neurohormone con- Systems in Early Fetal Life trolling the whole reproductive endocrine axis; the GnRH Surprisingly, GnRH-synthesizing neurons do not originate action on the anterior pituitary and the resultant secretion within the brain, like the majority of all neurons. Rather, GnRH of the gonadotropins; the gonadotropins’ action on the ovaries neurons derive from progenitor cells in the embryonic olfactory and the release of gonadal steroids; and finally the action of placode where they develop. In a particular journey unique for a these sex steroids on the uterus and cervix. Although it is fair neuron, GnRH neurons migrate toward the brain during to state that the HPO axis is driven by the hypothalamus early fetal life to reach the locations that they will occupy and its release of GnRH, it is important to point out that during adult life. This migration of GnRH neurons over long normal function of the HPO-endocrine axis requires a re- distances and through changing molecular environments sug- markable information flow and coordination between each gests that numerous factors, local and possibly external, influence of these organs, as exemplified by the existing inhibitory this process at its different stages. Such factors play critical roles, and stimulatory feedback loops. Their relevance will become such as mediating the adhesion of GnRH neurons to changing obvious in the discussion of the menstrual cycle, which closes surfaces along their voyage, promoting cytoskeleton remodeling, the chapter. and modulating axonal guidance. Functional connections between GnRH neurons and the hy- pophyseal portal system that will transport GnRH to the anterior THE HYPOTHALAMUS AND GNRH pituitary gland are established by about 16 weeks of fetal life. The reproductive process starts in the brain, through the activa- Migration failure of GnRH neurons and the resultant lack tion of the initial hormonal signal that will release the gonado- of the establishment of functional connections are charac- tropins from the pituitary gland. This hormone released by teristic of patients with the Kallmann syndrome, who show the hypothalamus is GnRH, a decapeptide (10 amino acids) hypogonadotropic hypogonadism accompanied by anosmia (Fig. 4.1). The GnRH gene (GNRH1, situated on the short (Tsai, 2006). In the 19-week-old fetus with X-linked Kallmann arm of chromosome 8) encodes for a 92 amino acids precur- syndrome, the GnRH neurons accompanying the olfactory sor molecule, composed of a signal peptide sequence, the nerves have been shown to be arrested in their voyage within the GnRH sequence itself, a posttranscriptional processing signal meninges, and therefore contact with the brain and the hypophy- (3 amino acids long), and a 56-amino acid peptide known as seal portal system is not established. 76  CHAPTER 4 Reproductive Endocrinology 77 4 Fig. 4.1 The 10-amino acid sequence of gonadotropin-releasing hormone (GnRH). (From Klerzky OA, Lobo RA: Reproductive neuroendocrinology. In Mishell DR, Davajan V, Lobo RA, eds. Infertility, Contraception and Reproductive Endocrinology. 3rd ed. Cambridge, MA: Blackwell Scientific; 1991.) The GnRH Neuronal System portal vessels, which descend along the pituitary stalk to termi- In adults, neurons producing GnRH are present in several hypo- nate within another capillary plexus (hence the term portal) thalamic nuclei and other parts of the brain. However, the major- within the anterior lobe of the pituitary (see Fig. 4.2A). (Unlike ity of GnRH neurons controlling the HPO axis are located the posterior lobe of the pituitary, also referred to as the within the anterior hypothalamus and primarily within the me- neurohypophysis, the anterior lobe has no direct blood supply dial basal hypothalamus, with the greatest number in the primate and receives all of its vascularization from this portal system.) within the arcuate nucleus. The 92 amino acid GnRH precursor The vascular arrangement whereby GnRH and other neuro- is released into the axons of these neurons and cleaved during hormones reach the anterior pituitary is very important to the transport to yield GnRH and GAP. (The biologic function of proper function of the endocrine system: It allows for the rapid GAP or fragments thereof remains to be clarified.) (within minutes) and undiluted transport of relatively small A substantial number of GnRH axons terminate within the amounts of neurohormones to the pituitary. This is especially external zone of the median eminence (infundibulum) where crucial to GnRH because this neurohormone has a short GnRH is released. This area is the site of an important capillary half-life of about 2 to 4 minutes (it is rapidly degraded by plexus, with a fenestrated epithelium similar to that of peripheral peptidases in blood; as a consequence, GnRH is not measur- capillaries, which allows passage of large molecules. (These able in peripheral blood) and because of its pulsatile mode of capillaries differ from brain capillaries, which are not fenestrated. release (discussed later). Thus the median eminence is viewed as an area outside the blood- brain barrier.) This pathway is the most relevant one in regard to the control of the pituitary-ovarian axis (Fig. 4.2, A). Another Physiology substantial projection of GnRH axons is through circumventricu- The GnRH Pulse Generator lar organs, the major of which is the organum vasculosum of the lamina terminalis (OVLT). These areas are also outside the Studies have shown that GnRH is characteristically released blood-brain barrier. (The function of GnRH release into these intermittently, in a pulsatile fashion. From this comes the concept areas remains somewhat unknown. One role may be to enable the of the “GnRH pulse generator” responsible for the pulsatile release of GnRH into cerebrospinal fluid [CSF], perhaps to facili- release of the hormone (Herbison, 2018). GnRH pulses occur at tate actions of GnRH in other areas of the brain. GnRH levels about hourly intervals (Fig. 4.3). The rising edge of each GnRH have been found to be elevated in CSF as opposed to being mini- pulse is abrupt, such that GnRH can increase by a factor of 50 mal in peripheral blood.) Another possible route of GnRH release within 1 minute. Each GnRH pulse is preceded by an increase in may involve specialized ependymal cells, referred to as tanycytes. multiunit activity within the area of the arcuate nucleus. These have been found to extend from the lumen of the third ventricle to the external zone of the median eminence. Mechanisms Responsible for GnRH Pulsatility The cellular basis and the mechanisms that determine the timing Transport of GnRH to the Anterior Pituitary of the increase in multiunit activity resulting in pulsatile GnRH The capillary plexus of the external median eminence, into activity are still under study. First, there is a growing consensus which GnRH is released, collects into several hypophyseal that pulsatile activity originates from an inherent pace-making 78 PART I Basic Science Dorsal hypothalamic area Dorsomedial nucleus Paraventricular nucleus Posterior hypothalamic nucleus Anterior hypothalamic area Premamillary nucleus Preoptic area Tanycyte Supraoptic nucleus Mamillary Suprachiasmatic body nucleus Tuberal area Optic chiasm Ventromedial nucleus Arcuate nucleus Median eminence Superior hypophyseal artery A Posterior lobe Anterior lobe B Fig. 4.2 A, Nuclear organization of the hypothalamus, shown diagrammatically in a sagittal plane as viewed from the third ventricle. Rostral area is to the left and caudal area is to the right. Fast transport of GnRH pulses released into the median eminence from axons originating from GnRH neurons in the arcuate nucleus occurs through the portal vessels derived from the capillary plexus in the median eminence. B, The pituitary stalk and several individual hypophyseal portal veins transporting hypothalamic neurohormones to the anterior pituitary in a nonhuman primate. GnRH, Gonadotropin-releasing hormone. (A, Redrawn from Moore RY: Neuroendocrine mechanisms: Cells and systems. In Yen SSC, Jaffe R, eds. Reproductive Endocrinology: Physiology, Pathophysiology and Clinical Management. Philadelphia, WB Saunders; 1986. B, Image courtesy Drs. Peter Carmel and Michel Ferin.) activity of the GnRH neuron itself; in vitro data have shown kisspeptin (KISS1), a product of the KISS1 gene, and its that individual neurons have the capacity of spontaneous oscilla- receptor (GPR54 or KISS1R) in the regulation of GnRH re- tions in activity. In this case such activity would also require a lease (Skorupskaite, 2014). KISS1 neurons have been found to synchronized action from enough neurons to provide a discrete directly innervate and stimulate GnRH neurons. In humans, mu- GnRH pulse. Intercommunication between GnRH neurons may tations or targeted deletions of KISS1 or of its receptor cause occur through gap junctions between such neurons, which have hypogonadotropic hypogonadism. Patients with these muta- been demonstrated, and through synaptic forms of interaction tions, however, do not have anosmia, unlike those with between cells. Second, evidence identifies a key role of Kallmann syndrome, suggesting that there are no major deficits in  CHAPTER 4 Reproductive Endocrinology 79 35 D2 D5 20 30 4 15 GnRH pg/mL GnRH pg/mL 25 20 10 15 10 5 5 0 0 180 180 160 160 140 140 LH (ng/mL) LH (ng/mL) 120 120 100 100 80 80 60 60 40 40 20 20 0 0 0 60 120 180 240 300 360 420 480 0 60 120 180 240 300 360 Time (min) Time (min) Fig. 4.3 GnRH release by the hypothalamus is pulsatile. Shown in the upper panel are hourly GnRH pulses over an 8-hour period in an ovariectomized monkey in the absence of ovarian steroid modulation. Note the concordance of LH pulses (lower panel). GnRH, Gonadotropin-releasing hormone; LH, luteinizing hormone. (From Xia L, Van Vugt D, Alston EJ, et al. A surge of gonadotropin-releasing hormone accompanies the estradiol-induced gonadotropin surge in the rhesus monkey. Endocrinology. 1992;131:2812.) the embryonic migration of olfactory or GnRH neurons. KISS1 Rostral hypothalamus neurons within the arcuate nucleus (rodents) and infundibu- lar nucleus (humans) express the estrogen, progesterone, and androgen receptors, and KISS1 signaling in the brain is impli- MnPO CNS centers controlling cated in mediating sex steroids feedback loops, especially during heat-defense effectors the preovulatory GnRH/luteinizing hormone (LH) surge. Kiss- peptin has been used to induce egg maturation in women undergo- Arcuate ing in vitro fertilization therapy, with subsequent fertilization of GnRH Tail skin the mature eggs, embryo transfer into the uterus and successful vasodilation KNDy human pregnancy (Jayasena, 2014). There is the potential for rou- ERα tine use of kisspeptin to induce ovulation during fertility treatment cycles. KISS1 has also been shown to play a role in the initia- OC tion of puberty. A subpopulation of kisspeptin neurons in the human infundibular nucleus coexpress neuropeptides neurokinin B and dynorphin (an opioid inhibitor) (Skorupskaite, 2014). These Thermosensory kisspeptin-neurokinin-dynorphin (KNDy) neurons express information from AP warm afferent neurokinin B and dynorphin receptors, as well as estrogen pathway and progesterone receptors. KNDy neurons coordinate inputs, = NK3R including sex steroid feedback, to regulate pulsatile kisspeptin se- LH E2 cretion. The interaction of KISS1 neurons with other neurotrans- mitter systems is currently being studied. Fig. 4.4 depicts the hypothalamic circuitry between the KNDy neurons and GnRH release, as well as the newly appreciated connections Ovary to the thermoregulatory areas in the hypothalamus (Mittle- man-Smith, 2012). Thus blockade of the neurokinin (NK3) receptor with a specific antagonist has been found to control Fig. 4.4 Diagrammatic depiction (in a rat model) of KNDy neurons in hot flushes in postmenopausal women by blocking the pathways of the hypothalamus, not only influencing GnRH pulses and gonadotropin the thermoregulatory center. secretion but interacting with thermoregulatory and heat regulatory centers in the brain. AP, Anterior pituitary; CNS central nervous system; E2, estradiol; ER!, estrogen receptor alpha; GnRH, gonadotropin- Modulatory Influences on GnRH Pulsatility releasing hormone; KNDy, kisspeptin, neurotensin, and dynorphin; The foremost modulatory influence on the frequency and amplitude LH, luteinizing hormone; MnPO, median preoptic nucleus; NK3R, of GnRH pulses is exerted by the ovarian steroid hormones through neurokinin receptor. (From Lobo RA: menopause and aging. In Strauss their feedback loop actions. In general, estradiol is known to decrease JF and Barbieri RL, eds. Yen and Jaffe’s Reproductive Endocrinology: GnRH pulse amplitude, whereas progesterone decreases GnRH Physiology, Pathophysiology and Clinical management. 8th ed. pulse frequency (see the discussion presented later for details). Philadelphia: Elsevier; 2019.) 80 PART I Basic Science Numerous other studies suggest that the spontaneous activity antagonist naloxone, as demonstrated in studies in nonhuman of the GnRH pulse generator may also be modulated by a variety primates and in patients with hypothalamic amenorrhea, many of of additional stimulatory and inhibitory afferent neural signals. whom have elevated levels of cortisol. Stimulatory inputs to GnRH release may originate from neurons using the biogenic amine neuroepinephrine (NE), the amino acid glutamate, and the peptide neuropeptide Y Metabolic Influences and GnRH Release (NPY). Inhibitory inputs may come from the amino acid There is good clinical evidence linking energy homeostasis and gamma-aminobutyric acid (GABA), the biogenic amine do- reproductive function in the human. A functional reproductive pamine (DA), the endogenous opioid !-endorphin, and the system requires an accurate integration of energy balance, and a neurosecretory peptide corticotropin-releasing hormone significant imbalance may lead to reproductive dysfunction and (CRH) neurons. These systems may affect the GnRH pulse amenorrhea. Nutritional deprivation and abnormal eating generator either tonally or conditionally. habits are known to interfere with the normal reproductive In the first category we find, for example, NE as a potential process. Anorexia nervosa is a well-known and extreme exam- tonal stimulator and GABA as a tonal inhibitor of GnRH release. ple of how alteration in food intake can result in the suppression Administration of alpha-adrenergic blockers has been shown to of the menstrual cycle. Obesity also may contribute to menstrual reduce pulse frequency in animals, in accord with the postulated disorders. tonal stimulatory role for NE. The role of GABA as a tonal in- Growing evidence indicates that complex and extensively hibitor may be more prominent during the prepubertal period, at integrated physiologic mechanisms connect an active reproduc- which time a diminishing inhibitory GABA tone may activate tive axis to the metabolic state. The brain, and in particular the puberty and the resumption of GnRH pulsatile release. Gluta- hypothalamus and the GnRH pulse generator, function as the mate’s role is more uncertain, although it also is suspected in the center for the integrative metabolic response process. The nature initiation of pulses at puberty. Dopamine infusions in women are of the afferent signals that provide information about energy associated with a decrease in circulating LH and prolactin (dopa- metabolism to the reproductive axis is presently under intense mine is also known as the prolactin-inhibitory neurohormone). study, and data have shown possible roles for several energy- The effect on LH is thought to be mediated through GnRH related proteins. because, in patients with hypothalamic amenorrhea in whom One such example is leptin, an anorexigenic protein that is there appears to be an excess of dopaminergic tone, administra- the product of the ob gene and that is primarily produced by tion of a dopamine blocker may return the LH pulse frequency adipocytes (Ahima, 2000). Leptin levels are reduced when body to normal. It should also be remembered that specific effects of fat stores are decreased by fasting. Besides conveying metabolic neurotransmitters on GnRH neurons may be altered by the ad- information to several parts of the brain through its own recep- ministration of certain drugs, which may interfere with the tors, leptin also appears to function as one of the metabolic cues proper synthesis, binding, storage, or receptor function of these regulating the GnRH pulse generator. High leptin levels are neurotransmitters. Thus on treatment with such drugs (e.g., interpreted as conducive to reproduction, and the adminis- methyldopa; reserpine; tricyclic antidepressants such as tration of leptin stimulates the secretion of GnRH and of the propranolol, phentolamine, haloperidol, and cyprohepta- gonadotropins, with the effects most pronounced in individuals dine; selective serotonin reuptake inhibitors [SSRIs]; and showing signs of reproductive impairment. Peripheral injections serotonin-norepinephrine reuptake inhibitors [SNRIs]), pa- of leptin can prevent the reduction in GnRH/gonadotropins and tients may develop disorders such as oligomenorrhea or the disturbances in cyclicity that accompany caloric reduction. galactorrhea, the result of alterations in GnRH secretion or Ob/ob mice, which are leptin deficient because of a mutated hyperprolactinemia. Other studies also suggest that hypotha- leptin gene, besides exhibiting a pronounced obesity, show a lamic prostaglandins may also modulate the release of GnRH; for complete failure to display normal estrous cycles because of instance, the midcycle surge of LH (see The Menstrual Cycle absent GnRH secretion. The latter can be reversed by leptin later in this chapter) can be abolished in animals by the adminis- administration. Evidence suggests a role for kisspeptin in modu- tration of aspirin or indomethacin, which blocks the synthesis of lating metabolic leptin signals on the hypothalamus and pituitary prostaglandins. (Skorupskaite, 2014). Forty percent of kisspeptin neurons in the In the second category, other systems may affect the GnRH mouse arcuate nucleus express leptin. Leptin-deficient mice show system only conditionally—that is, under specific hormonal or decreased expression of Kiss1 messenger RNA (mRNA). How- physiologic conditions. One example is the endogenous opioid ever, leptin administration only partially restores Kiss1 mRNA !-endorphin, which exerts an inhibitory action on GnRH pulsa- levels, indicating that other mediators are involved in inhibiting tile activity that depends largely on the endogenous endocrine kisspeptin signaling in leptin deficiency (Smith, 2006). Another milieu. This is related to the fact that ovarian hormones control example is the orexigenic peptide NPY, which is synthesized in the release of !-endorphin within the brain, which is lowest in the arcuate nucleus. During fasting, expression of NPY mRNA the absence of estradiol, such as in the ovariectomized nonhuman increases in this nucleus and intracerebroventricular injec- primate, and highest in the presence of both estradiol and pro- tion of NPY stimulates food intake. NPY has been shown to gesterone, such as during the luteal phase of the menstrual cycle. affect pulsatile GnRH/LH activity in the nonhuman primate, but Experimental administration of the opiate antagonist naloxone this occurs in two apparently contradictory modes, one excitatory during the luteal phase increases GnRH/LH pulse frequency, and one inhibitory. It was shown in the ovariectomized monkey significantly suggesting the reversal of a inhibitory action under and rodent that a pulsatile intracerebroventricular infusion of the endocrine milieu that characterizes the luteal phase. No ef- NPY stimulates GnRH release, whereas a continuous infusion fect on LH pulsatility follows naloxone injection in postmeno- clearly decreases the pulsatile electric activity of GnRH neurons, pausal or ovariectomized women, unless they are treated with an as well as pulsatile LH release. In accord with this observation of estrogen-progesterone therapy. Another example is CRH, the an inhibitory effect of NPY is that, whereas fasting decreases LH main neuropeptide controlling the adrenal endocrine axis, secretion in normal mice, fasting mice lacking NPY Y1 receptor which is released in greater amount during stress. In this have a higher pituitary LH content than wild-type ones. What condition, increased CRH release negatively affects the these data suggest is that although a supportive effect of NPY on GnRH pulse generator, which results in a decrease in GnRH the GnRH pulse generator may occur within a limited window of pulse frequency. This action is indirect through the release of normalcy (i.e., within a normal background of basic and pulsatile !-endorphin and is prevented by the administration of the opiate NPY release), in physiopathologic situations (i.e., in circumstances  CHAPTER 4 Reproductive Endocrinology 81 mimicking increased endogenous NPY activity such as in under- chapter), the GnRH-R lacks a carboxy-terminus located on the nutrition) an inhibitory effect of NPY on the GnRH pulse intracellular site. 4 generator can be observed. Evidence suggests that kisspeptin neurons can sense and convey information about energy Activation of the GnRH Receptor status to GnRH neurons. In rodents, hypothalamic levels GnRH activation of the receptor requires the release of con- of Kiss1 mRNA are reduced in metabolic conditions, such as straining intramolecular bonds, which maintain the receptor in undernutrition, uncontrolled diabetes, and immune/inflammatory an inactive configuration. Once activated, the GnRH-R stimu- challenge, which are associated with suppressed gonadotropins. lates cellular production of specific membrane-associated lipid- Administration of kisspeptin in rodent models with disrupted like diacylglycerols, which, acting as a second messenger, activate metabolism and energy reserves restores gonadotropin secretion, several cellular proteins. Among these are the enzyme protein suggesting a potentially important central role of KISS1 kinase C (PKC) and mitogen-activated protein kinase neurons in the regulation of reproduction by metabolic factors (ERK; also called MAPK). Phosphorylated ERK activates tran- (Skorupskaite, 2014). scription factors, the end result being gene transcription of Overall, in regard to the reproductive system, the GnRH gonadotropin subunits and the synthesis of both gonadotropins. pulse generator actually acts as the link between the envi- Binding of GnRH to its receptor also rapidly mobilizes ronment, the internal milieu, and the reproductive axis. Its transient intracellular calcium, which triggers a burst of overall activity most probably reflects the summation of simulta- exocytosis to rapidly release LH and FSH. It also provokes a neous stimulatory and inhibitory inputs. It is evident that events, rapid influx of Ca## into the cell from the extracellular pool, disorders, or drug administration may tip the physiologic bal- which in turn activates calmodulin, a calcium-binding protein, ance, cause disruption or cessation of GnRH pulse activity, and maintaining gonadotropin release. Diacylglycerols amplify the lead to disruptions of the menstrual cycle and to reproductive action of Ca##-calmodulin, thereby synergistically enhancing disorders such as oligomenorrhea and hypothalamic amenorrhea. the release of gonadotropins. Administration of a calmodulin antagonist has been shown to decrease GnRH-stimulated THE ANTERIOR PITUITARY GLAND gonadotropin release. AND THE GONADOTROPINS Estrogens and the GnRH Receptor Pulsatile GnRH increases GnRH-R gene expression and the Anatomy number of GnRH-Rs on the gonadotrope’s cell surface. The The anterior pituitary (also referred to as the adenohypophysis) number of GnRH-Rs also varies with the hormonal environ- derives from Rathke’s pouch, a depression in the roof of the devel- ment, with highest number of receptors expressed when high oping mouth in front of the buccopharyngeal membrane. concentrations of estrogens are present. This leads to an increase It originates at about the third week of life. Origin of the in the overall Ca2## response and a significantly amplified adenohypophysis contrasts with that of the posterior pituitary gonadotropin response to a GnRH pulse. This action explains (neurohypophysis), which develops as a direct extension of the variations in the gonadotropin response to GnRH at the brain. It should also be noted that whereas the neurohy- various times of the menstrual cycle: GnRH pulses of similar pophysis receives a direct arterial blood supply from the amplitude elicit greater gonadotropin responses during the late hypophyseal arteries, the only vascularization to the adenohy- follicular phase and luteal phase when estradiol levels are highest, pophysis is through the hypothalamic-hypophyseal portal system but the responses are lower during the early follicular phase when (into which GnRH and several other neuropeptides are secreted; estradiol levels are lowest (Fig. 4.5). discussed earlier). The gonadotropes are the specialized cells within the GnRH Pulse Frequency and Gonadotropin Release adenohypophysis that produce the gonadotropins. On stimu- It is also important to note that varying frequencies of the GnRH lation of the gonadotropes by GnRH, two gonadotropins are pulse signal regulate gonadotropin subunit gene transcription released into the general circulation and regulate endocrine differentially. Overall, a low GnRH pulse frequency favors function in the ovaries and testes. FSH synthesis, whereas a high GnRH pulse frequency favors LH synthesis. This is well demonstrated experimentally where changing a pulsatile infusion from a high to a low pulse frequency Physiology results in a matter of days in an increase in the FSH/LH ratio (Fig. 4.6). This phenomenon may play a role during the luteal The GnRH Receptor phase of the menstrual cycle and in the changing FSH/LH ratio Pulses of GnRH released by the GnRH neurons in the arcuate that occurs during the passage from one menstrual cycle to an- nucleus reach the gonadotropes in the anterior pituitary via the other (see The Luteal Phase, discussed later). It is also reflected hypophyseal portal circulation. These GnRH pulses then act on in patients known to have a high GnRH pulse frequency, GnRH receptors (GnRH-Rs) on the gonadotropes to stimulate such as in women with the polycystic ovary syndrome, a high both the synthesis and release of both gonadotropins, LH and proportion of whom have a characteristically elevated follicle-stimulating hormone (FSH). Women with GnRH-R LH:FSH ratio. mutations typically present with incomplete or absent pubertal development and primary amenorrhea. Although reproductive GnRH Receptor Desensitization function is compromised, conception may be successfully Gonadotropin release after a GnRH pulse is rapid: Within min- obtained after gonadotropin treatment. utes, both FSH and LH are released. It is important to recognize On the cell membranes of the gonadotrope, GnRH interacts that the pulsatile release mode of GnRH is essential for the main- with high-affinity GnRH-Rs. The gene encoding the GnRH-R is tenance and control of normal gonadotropin secretion. located on chromosome 4q13.2-13.3, spanning 18.9 kb. This In contrast to the response to the normal pulsatile mode of receptor belongs to a large family of G protein–coupled re- GnRH release, sustained exposure of the GnRH-R to constant ceptors. These contain seven transmembrane helices connected GnRH concentrations drastically reduces the response of the by six alternating intracellular and extracellular loops, with the gonadotrope to subsequent stimulation with GnRH. This phe- amino-terminus located on the extracellular side. In contrast to nomenon is referred to as homologous desensitization or down- other protein receptors (see Fig. 4.10, presented later in the regulation of the receptor, which denotes a reduction in the 82 PART I Basic Science Pulsatile Continuous Pulsatile 20 200 15 150 FSH (ng/mL) LH (ng/mL) 10 100 5 50 0 0 10 5 0 5 10 15 20 25 30 35 40 Days Fig. 4.7 Gonadotropin-releasing hormone (GnRH) release in a pulsatile mode is required for a normal pituitary gonadotropin response. An experiment was performed in a monkey lacking endogenous GnRH and infused with hourly pulses of GnRH (left and right panels) or with a continuous GnRH infusion (center panel). LH, Luteinizing hormone. (From Belchetz PE, Plant TM, Nakai Y, et al. hypophyseal responses to Fig. 4.5 GnRH pulses of similar amplitude elicit greater overall gonad- continuous and intermittent delivery of hypothalamic gonadotropin- otropin responses during the late follicular phase and luteal phase releasing hormone. Science. 1978;202:631-633.) when estradiol levels are highest, but they elicit lower responses during the early follicular phase when estradiol levels are lowest. Note also a greater early response in the late follicular phase, denoting greater LH reserves under the effect of estradiol. GnRH, Gonadotropin-releasing the pulsatile mode, a profound inhibition of LH concentrations hormone; LH, luteinizing hormone. (From Hoff JD, Lasley BL, Wang occurred, which reflects desensitization of the GnRH-R. This CF, Yen SSC. The two pools of pituitary gonadotropins: regulation phenomenon, which takes a few days to be established, may during the menstrual cycle. J Clin Endocrinol Metab. 1977;44:302.) reflect a loss of active cell surface receptors and be maintained by a loss of functional Ca## channels. However, the mechanism of desensitization is still under investigation, and additional intermediary changes remain to be characterized. 994 50 1 Pulse/hour 1 Pulse/ 1 Pulse/hour 500 GnRH Analogs and the GnRH Receptor 3 hours The GnRH half-life in the peripheral circulation is very short 45 450 40 400 because peptidases rapidly degrade naturally occurring GnRH by cleaving the decapeptide molecule at the Gly6 to Leu7 and at FSH (ng/mL) LH (ng/mL) 35 350 30 300 the Pro9 to Gly10 bonds. However, by substituting amino acid 25 250 6 in the natural GnRH molecule with a d-amino or replacing 20 200 amino acid 10 with an N-methylamide (Na-CH2-CH3) or 15 150 Aza-Gly (NHNHCO) moiety, GnRH analogs were synthesized 10 100 and shown to have acquired a greater resistance to enzymatic 5 50 proteolysis and hence a longer half-life (hours vs. 2 to 4 minutes). 0 0 After administration of these GnRH agonists, there is an 20 15 10 5 0 5 10 15 20 25 30 35 40 initial stimulation of gonadotropin release (flare), followed by the process of desensitization blocking the releasing Days effect on the gonadotropins. This observation has led to the Fig. 4.6 Increase in the FSH/LH ratio after a decrease in the gonadotropin- clinical application of the functional desensitization property of releasing hormone (GnRH) pulse frequency (from 1 pulse/hour; left GnRH:GnRH analogs; they have been used to induce a “medical and right panels) to 1 pulse/3 hour (center panel). Experiment was castration” state by shutting down the pituitary-gonadal axis in a performed in a monkey lacking endogenous GnRH and infused with variety of clinical conditions. In contrast to GnRH agonists, GnRH. FSH, Follicle-stimulating hormone; LH, luteinizing hormone. GnRH antagonists act by competing with GnRH for recep- (From Wildt L, Hausler A, Marshall G, et al. Frequency and amplitude tor sites and thereby never activating a stimulatory signal. of gonadotropin-releasing hormone stimulation and gonadotropin Many of these result from the substitution of amino acids at the secretion in the rhesus monkey. Endocrinology. 1981;109:376.) 2 or 3 position. Thus GnRH antagonists have the advantage over the GnRH agonists of a rapidly decreasing LH and FSH release, without the flare. Clinical applications for both GnRH agonists and antagonists are listed in Box 4.1. Although GnRH ability of GnRH to elicit gonadotropin release after prior agonists have been in use longer and have several Food and Drug continuous exposure to GnRH. This phenomenon is well illus- Administration (FDA)–approved indications, the GnRH antago- trated in a classic experiment in ovariectomized monkeys lacking nists, though theoretically useful for the same conditions, are endogenous GnRH secretion after lesion of the arcuate nucleus only approved for a few indications at present. An orally active (Fig. 4.7). As illustrated, 6-minute pulses administered once an GnRH antagonist, elagolix (Orilissa), is available in the United hour restored normal LH levels in these animals. In contrast, States and has been approved in two dosages, 150 and 200 mg when a continuous mode of GnRH infusion was substituted to twice daily, for the treatment of severe endometriosis.  CHAPTER 4 Reproductive Endocrinology 83 THE OVARIES BOX 4.1 Clinical Applications of GnRH Agonists and Antagonists 4 Anatomy Activation of pituitary–gonadal function (GnRH) Delayed puberty Ovarian Gametogenesis (Oogenesis) Cryptorchidism Oogenesis begins in fetal life when the primordial germ cells, or Functional hypothalamic amenorrhea oogonia, migrate to the genital ridge. The number of oogonia Hypogonadotropic hypogonadism (Kallmann syndrome) increases dramatically from about 600,000 by the second month Pituitary–gonadal inhibition (agonists) of fetal life to a maximum of about 7 million by the sixth to sev- Precocious puberty enth month. The oogonia then begin meiotic division (they are Hormone-dependent tumors now referred to as primary oocytes) until they reach the diplo- Endometriosis tene stage of the prophase (the germinal vesicular stage), in which Uterine leiomyoma they will remain until stimulation by gonadotropins in adulthood Breast cancer during the menstrual cycle (discussed later). However, by a pro- Prostatic cancer cess of apoptosis and atresia of the enveloping follicle, which Suppression of ovarian function in polycystic ovary syndrome and starts prenatally and persists throughout childhood, the number in vitro fertilization of primary oocytes declines drastically from about 2 to 4 million Premenstrual syndrome at birth to become 90% depleted by puberty. Further depletion Abnormal uterine bleeding including clotting disorders of the pool occurs throughout adulthood, so that by age 37 only Contraception about 25,000 and by age 50 only about 1000 oocytes remain. Suppression of spermatogenesis In recent years, the traditional dogma that mammals have fixed, Ovulation inhibition nonrenewable oocyte stores established before birth has been chal- lenged. Some studies suggest that adult mammalian ovaries possess GnRH, Gonadotropin-releasing hormone. pluripotent germline stem cells (GSCs) that can differentiate into oocytes, as well as other cell types. Nonmammalian organisms, such as Drosophila, do possess ovarian GSCs. Whereas the exis- tence of spermatogonial cells in the adult human testis that give rise to pluripotent GSCs is well accepted, there is considerable evidence that disputes the existence of mammalian adult ovarian GSCs. At this point, there is not sufficient evidence to prove that mammalian oogenesis occurs after birth (Hanna, 2014). The Gonadotropins There are two distinct gonadotropins: LH and FSH (Bousfield, 2006). (A third gonadotropin, human chorionic gonadotropin Ovarian Folliculogenesis [HCG], is produced in the primate by the placenta.) The primary oocyte is surrounded by a single layer of flattened granulosa cells in a unit referred to as the primordial follicle. Structure Even in the absence of stimulation by gonadotropins, some LH and FSH are glycoproteins of high molecular weight. They primordial follicles will develop into primary (or preantral) fol- are heterodimers, containing two monomeric units licles, at which stage multiple layers of cuboidal granulosa cells (subunits). Both LH and FSH have a similar !-subunit, the surround them. Development of follicles to this stage appears to structure (92 amino acids) of which is highly conserved. (The be relatively independent of pituitary control but is probably same "-subunit is also shared with HCG and thyroid- influenced by intraovarian, nonsteroidal processes that remain to stimulating hormone [TSH].) However, !-subunits have dif- be understood. Development to this stage occurs during the non- ferent structures consisting of different amino acids and carbo- ovulatory stages of childhood, pregnancy, and oral contraceptive hydrates. These LH and FSH subunits are each encoded by a use, as well as during ovulatory cycles. separate gene. (The HCG subunits are also different and With formation of an antrum (cavity), the follicle, now re- encoded by six genes.) ferred to as secondary or antral follicle, enters the final stages of The "- and !-subunits are joined by disulfide bonds, which folliculogenesis, characterized by the transition from intraovarian are essential to maintain biologic activity. Reducing agents regulation to a major control by the hypothalamic-pituitary unit. break the disulfides bounds and reduce or remove the biologic This requires the presence of the characteristic increase in FSH activity of the gonadotropin. Highly purified free subunits have that occurs in the early menstrual cycle (see The Menstrual little if any biologic activity relative to that of the intact Cycle, presented later). hormone. However, it is the !-subunit that confers the spe- The development process from primary follicle (preantral cific biologic activity of each hormone. For instance, LH has a follicle) to secondary or antral follicle and to a mature preovula- !-subunit of 121 amino acids, a structure that is responsible for tory follicle, the latter during the follicular phase of the cycle, the specificity of the interaction with the LH receptor. LH and takes about 1 year to complete (Fig. 4.8). Only about 400 follicles FSH also differ in the composition of their sugar moieties. The complete this process, whereas the majority of follicles undergo different composition of several different oligosaccharides af- programmed cell death. Although little is known about factors fects bioactivity and speed of degradation of each gonadotropin. controlling growth during the earlier stages, more is known For example, the biologic half-life of LH is 20 minutes, much about the final stage of folliculogenesis during the follicular shorter than that of FSH (3 to 4 hours). (The half-life of HCG phase of the menstrual cycle (discussed later). is 24 hours.) Although both gonadotropins act synergistically in women, FSH acts primarily on the granulosa cells of the ovarian follicles Physiology to stimulate follicular growth, whereas LH acts primarily on the theca cells of these follicles, as well as on the luteal cells to The Gonadotropin Receptors stimulate ovarian steroid hormone production (see the following Although the two gonadotropins act synergistically in the woman, discussion). FSH acts primarily on the granulosa cells of the maturing antral 84 PART I Basic Science Life history of ovarian follicles Initial recruitment Atresia Atretic Ovulation Cyclic recruitment Antral Maturation Secondary Endowment and maintenance Primary Exhaustion of follicles Primordial Continuing initial recruitment Depletion Fig. 4.8 Life history of ovarian follicles: endowment, maintenance, initial recruitment, maturation, atresia or cyclic recruitment, ovulation, and exhaustion. A fixed number of primordial follicles are endowed during early life, and most of them are maintained in a resting state. Growth of some of these dormant follicles is initiated before and throughout reproductive life (initial recruitment). Follicles develop through primordial, primary, and secondary stages before acquiring an antral cavity. At the antral stage, most follicles undergo atresia; however, under the optimal gonadotropin stimulation that occurs after puberty, a few of them are rescued (cyclic recruitment) to reach the preovulatory stage. Eventually, depletion of the pool of resting follicles leads to ovarian follicle exhaustion and senescence. (From McGee EA, Hsueh AJW. Initial and cyclic recruitment of ovarian follicles. Endocrine Rev. 2000;21:200-214.) follicle to stimulate follicular growth, whereas LH acts primarily Extracellular fluid NH3+ on the theca cells of these follicles to induce steroidogenesis. Binding to and activation of their respective receptors at the cell surface membrane is the necessary first step in the hormonal function of both FSH and LH. Gonadotropin receptors are transmembrane G protein– Plasma membrane coupled receptors that possess seven membrane-spanning do- mains (Fig. 4.9). It is believed that the receptor molecule exists in a conformational equilibrium between active and inactive states, which is shifted by binding of LH or FSH. On binding to the COO– 1 2 3 4 gonadotropin, the receptor shifts conformation and me- 6 7 Cytoplasm chanically activates the G protein, which detaches from the 5 receptor and activates cyclic adenosine monophosphate Fig. 4.9 The seven transmembrane !-helix structure of a G protein– (cAMP)–dependent protein kinases. These protein kinases are coupled receptor, such as that for LH or HCG. GnRH, Gonadotropin- present as tetramers with two regulatory units and two catalytic releasing hormone; HCG, human chorionic gonadotropin; LH, luteinizing units. On binding of cAMP to the regulatory units, the catalytic hormone. (The structure for the GnRH receptor is similar, except that units are released and initiate the phosphorylation of proteins, the GnRH receptor lacks a carboxy-terminus on the intracellular site; which bind to DNA in the cell nucleus, resulting in the activation see the preceding text “Physiology: The GnRH Receptor”.) (From of genes and leading to the physiologic action (Fig. 4.10). Wikipedia. Trans-membrane helix of G-protein coupled FSH receptor. https://commons.wikimedia.org/wiki/File:7TM_receptor.png) The Ovarian Steroids: Biosynthesis One primary function of the ovary is the secretion of ovarian and dehydroepiandrosterone (DHEA). Because of the lack of the steroids, which occurs after binding of both FSH and LH to their appropriate enzymes, the ovary does not synthesize mineralocor- respective receptors. The ovary secretes three primary hormones: ticoids or glucocorticoids. estradiol (the primary estrogen), progesterone, and andro- Steroids are lipids that have a basic chemical structure or nu- stenedione. These hormones are the chief secretory products of cleus (Fig. 4.11). The nucleus consists of three six-carbon rings the maturing follicle, the corpus luteum, and the ovarian stroma. (A, B, and C) joined to a five-carbon atom (D) ring. The carbon The ovary also secretes, in varying amounts, estrone (a less potent atoms are numbered as shown in Fig. 4.11. Functional groups estrogen), pregnenolone, 17-hydroxyprogesterone, testosterone, above the plane of the molecule are preceded by the ! symbol  CHAPTER 4 Reproductive Endocrinology 85 Hormone 4 NH2 Sites for N-linked glycosylation Receptor Adenylate cyclase COOH ATP CAMP Protein kinase Regulatory Catalytic subunit subunit CAMP ! Regulatory Catalytic subunit subunit CAMP Regulatory Catalytic subunit subunit CAMP Phosphorylation Regulatory ! of proteins Catalytic subunit subunit CAMP Physiologic events Phosphodiesterase 5' AMP Fig. 4.10 On binding to their receptor, the gonadotropins activate cAMP–dependent protein kinases (see text). ATP, Adenosine triphosphate; cAMP, cyclic adenosine monophosphate. (Adapted from Speroff L, Friz M, eds. Clinical Gynecologic Endocrinology and Infertility. New York: Lippincott Williams & Wilkins; 2005:71-72.) and shown in the structural formula by a solid line, whereas those pregnenolone, 17-hydroxypregnenolone, progesterone, and 17- below the plane are indicated by an ! symbol and a dotted line. hydroxyprogesterone), 19 carbon atoms (androgens such as All steroids, whether secreted by the ovary, testis, or adrenal, DHEA, androstenedione, and testosterone), and 18 carbon atoms are derived from acetate (a 2-carbon compound), which, in a (estrogens such as estradiol and estrone). In the first step, choles- series of complex reactions, is transformed into cholesterol terol is transferred from the outer mitochondrial membrane to (a 27-carbon steroid) (Fig. 4.12). the inner membrane where cytochrome P450 enzyme is located. The sex steroids (as well as the corticosteroids) are then The latter will split off the cholesterol side chain, which is the derived from a stepwise transformation of the cholesterol mole- first enzymatic step in steroid biosynthesis. Being lipophilic, cule into steroids with 21 carbon atoms (the corticosteroids, cholesterol is unable to cross the aqueous phase between these 86 PART I Basic Science other tissues, and has been found to play a significant role in endometriosis, where aromatase is expressed in endometriotic lesions. It is also particularly relevant to note that in humans, in contrast to other species, estrogens are also synthesized in adi- pose tissue, which in postmenopausal women becomes the major site of estrogen biosynthesis. The tissue-specific expression of the CYP19 aromatase gene is regulated by the use of different pro- moters. For instance, expression in the ovary uses a promoter element proximal to the start of translation, whereas expression in adipose tissue uses distal elements. Overall, the C18 estrogen produced in different tissue sites of biosynthesis is rather specific and dependent on the nature of the C19 steroid presented to the aromatase enzyme: In the ovary, the main androgen source is ovarian testosterone and thus the main estrogen product from the ovary is estradiol, whereas in adipose tissue the main andro- gen source is circulating androstenedione (produced by the adrenals) and hence the principal estrogen produced is estrone. (The greater the amount of fat present, the greater the amount Fig. 4.11 Phenanthrene (top left). Cyclopentanoperhydrophenanthrene of androstenedione that is converted to estrone.) nucleus (top right), which incorporates the three six-carbon rings of the Mutation of the CYP19 aromatase gene leads to the aroma- phenanthrene ring system (A, B, and C) and a five-carbon ring (D), tase deficiency syndrome, which is inherited in an autosomal which resembles cyclopentane. Cholesterol (bottom) is the common recessive way (Morishima, 1995). In these female patients, accu- biosynthetic precursor of steroid hormones. Numbers 1 to 27 indicate mulation of androgens during pregnancy may lead to virilization the conventional numbering system of the carbon atoms in steroids. at birth. Individuals of both sexes have abnormal pubertal matu- (From Stanczyk FZ. Steroid hormones. In Lobo RA, Mishell DR, ration and are tall because of the lack of estrogen to affect Paulson RJ, Shoupe D, eds. Mishell’s Textbook of Infertility, Contraception epiphyseal closure. Female patients have primary amenorrhea. and Reproductive Endocrinology. 4th ed. Malden, MA: Blackwell Aromatase inhibition evidently leads to profound hypoestrogen- Science; 1997.) ism. Aromatase inhibitors have become useful in the manage- ment of patients with estrogen receptor positive tumors— for example, in breast cancer. Interconversion between androstenedione and testosterone and estrone and estradiol can occur outside the ovaries. Oxida- tion of the latter to the former reduces biologic potency because two membranes unassisted. It is now believed that steroidogenic both androstenedione and estrone have weaker biologic activity. acute regular protein (StAR) plays that role. The next steps in Estrone is also converted to estrone sulfate, which has a longer steroid biosynthesis require participation of a variety of enzymes, half-life and is the largest component of the pool of circulating most of which are part of the cytochrome P450 superfamily of estrogens. Estrone sulfate is not biologically active; however, heme-based enzymes. First is the transformation of cholesterol sulfatases in various tissues (such as breast and endometrium) can into pregnenolone by hydroxylation of C-20 and C-22 and cleav- readily convert it to estrone, which in turn can be converted age between these two atoms, reducing the C-27 cholesterol to to the more biologically active estradiol. Steroids are, in general, the C-21 compound pregnenolone. At this point, ovarian steroid insoluble in water but dissolve readily in organic solvents. biosynthesis proceeds along two major pathways, controlled by In contrast, steroids that have a sulfate or glucuronide group specific enzymes at each step: (1) the $5 pathway through 17- attached (conjugated steroids)—such as, for example, estrone hydroxypregnenolone and DHEA to $5 androstenediol and sulfate, dehydroepiandrosterone sulfate (DHEAS), or pregnane- (2) the $4 pathway via progesterone and 17-hydroxyprogesterone diol glucuronide—are water soluble. to the androgens androstenedione and testosterone. The Ovarian Steroids: Blood Transport and Metabolism The Aromatase Enzyme After release into the circulation, sex steroids bind to a steroid- Androgens are converted to the estrogens estrone or estra- specific transport protein, sex hormone–binding globulin diol by the enzyme aromatase, through the loss of the C-19 (SHBG) (a !-globulin synthesized by the liver), to the non– methyl group and the transformation of the A-ring to an aro- steroid-specific albumin, or circulate in an unbound or “free” matic state (hence the enzyme’s name) through oxidation and form. (There is a separate steroid specific protein, corticosteroid- subsequent elimination of a methyl group. The aromatic (or binding protein [CBG; transcortin], which binds primarily adre- phenolic) ring is characteristic of the estrogens (Fig. 4.13). nal steroids and to a lesser degree progesterone.) Both SHBG Aromatase is a complex enzyme comprising two proteins. The and CBG have a high affinity (by definition) but low capacity for first, P450arom (also a member of the cytochrome P450 super- steroids. Albumin, in contrast, has a high capacity but binds with family of genes), catalyzes the series of reactions required for the low affinity; thus steroids can readily dissociate from its binding formation of the phenolic A ring. The second is reduced nicotin- and enter target cells. amide adenine dinucleotide phosphate (NADPH)–cytochrome The free and loosely albumin-bound steroids are believed to P450 reductase, a ubiquitous protein required for transferring be the most biologically important fractions because the steroid reducing equivalents from NADPH to any microsomal form is free to diffuse or be actively transported through the capillary of cytochrome P450 with which it comes into contact. (All wall and bind to its receptor. (There is also evidence, however, microsomal P450 enzymes require this reductase for catalysis. that uptake of protein-bound hormone may also play a role.) Disruption of this reductase has lethal consequences, as shown in SHBG binds primarily dihydrotestosterone, testosterone, and knockout mice.) estradiol, in order of decreasing affinity. Thus in premenopausal The aromatase enzyme is found in many tissues besides the women, 65% of testosterone is bound to SHBG, 30% to albu- gonads, such as the endometrium, brain, placenta, bone, skin, and min, and 5% is free, whereas 60% of estradiol is bound to SHBG,  CHAPTER 4 Reproductive Endocrinology 87 4 Fig. 4.12 Biosynthesis of androgens, estrogens, and corticosteroids. (From Stanczyk FZ. Steroid hormones. In Lobo RA, Mishell DR, Paulson RJ, Shoupe D, eds. Mishell’s Textbook of Infertility, Contraception and Reproductive Endocrinology. 4th ed. Malden, MA: Blackwell Science; 1997.) 88 PART I Basic Science responses of endogenous stimulators and inhibitors, such as ovar- ian stimulation by LH, which is modulated by prostaglandin F2! (PGF2!), which in turn regulates ovarian receptor availability. Prostaglandins play an important role in ovarian physiology. They help control early follicular growth by increasing blood supply to certain follicles and inducing FSH receptors in granu- losa cells of preovulatory follicles. Both PGF2! and PGE2 are concentrated in follicular fluid of preovulatory follicles and may assist in the process of follicular rupture by facilitating proteo- lytic enzyme activity in the follicular walls. Many prostanoids are produced in the endometrium. Concentrations of PGE2 and PGF2! increase progressively from the proliferative to the secre- tory phase of the cycle, with highest levels at menstruation. These prostaglandins may help regulate myometrial contractility and may also play a role in regulating the process of menstruation. COMMUNICATION WITHIN THE HYPOTHALAMIC- PITUITARY-OVARIAN ENDOCRINE AXIS Fig. 4.13 Interconversion of the three main circulating estrogens. (From Stanczyk FZ. Steroid hormones. In Lobo RA, Mishell DR, The Steroid Receptors Paulson RJ, Shoupe D, eds. Mishell’s Textbook of Infertility, Contraception Gonadal steroids are integrated into every aspect of reproduc- and Reproductive Endocrinology. 4th ed. Malden, MA, Blackwell tion, and disruption of their signaling pathways, which obviously Science, 1997.) require initial binding to their receptors, leads to reduced fecun- dity and aberrations in multiple organs systems. For the sex steroid feedback loops (discussed later) to be active, there must be steroid receptors in the appropriate regions of the hypothalamus 38% to albumin, and 2% to 3% is free. The metabolic clearance and pituitary gland to respond to the ovarian signals. rate of sex steroids is inversely related to their affinity to SHBG. As opposed to peptide or protein hormone receptors that It is thus important to remember that the level of SHBG, and reside on the cell membrane (discussed earlier), steroid recep- therefore the level of free active hormone, may be influenced by tors reside in the nucleus or in the cytoplasm, in between which various clinical conditions. For instance, circulating levels of they may shuttle in the absence of hormone (Fig. 4.14). The SHBG are increased by estrogens (oral contraceptives, preg- cytoplasmic receptor is sequestered (hence in an “inactive” nancy) and by thyroid hormone (hyperthyroidism) and are state) within a multiprotein inhibitory complex that includes lowered by androgens and in hypothyroidism. heat shock proteins. Hormone binding leads to dissociation The major sites of steroid metabolism are the liver and kidney. from the heat shock proteins. The lipophilic steroids freely dif- Steroids are mainly oxidized by cytochrome P450 oxidase en- fuse across the nuclear membrane to bind to their cognate re- zymes through reactions that introduce oxygen into the steroid ceptor. This binding leads to conformational changes that ring, allowing a breakdown by other enzymes to form bile acids transform the receptor into an “activated” state, which allows it as final products. These bile acids can then be eliminated through to bind to a hormone responsive element (HRE), the specific secretion from the liver. In another process, which involves DNA-binding site to which steroid receptors bind conferring conjugation, the steroids are transformed from lipophilic hormone sensitivity within target gene promoters. Nuclear re- compounds, which are only sparingly soluble in water, into me- ceptors can inhibit or enhance transcription by recruiting an tabolites that are readily water soluble and can be eliminated in array of coactivator or corepressor proteins to the transcription urine. Examples are estradiol-17 glucuronide, estrone sulfate, and complex (Fig. 4.15) (Ellmann, 2009). mRNA is then generated pregnanediol-3-glucuronide (the major urinary metabolite of from a segment of nuclear DNA in the process of transcription. progesterone). Transcription is the most important process regulated by steroid hormones. All genes share a common basic design composed of a structural region in which the DNA encodes the Prostaglandins specific amino acids of the protein and a regulatory region that Prostaglandins (a subclass of eicosanoids and prostanoids) are in interacts with various proteins to control the rate of transcrip- general mediators of inflammatory and anaphylactic reactions. tion. Coactivators and corepressors modify the chromatin state Their most abundant precursor is arachidonic acid, itself and recruit/activate or hinder the basal transcriptional machin- formed from linoleic acid supplied in the diet. Their biosynthesis ery. Members of the SRC family of coactivators, including can be inhibited by several groups of compounds, including the SRC-1, SRC-2, and SRC-3, and the nuclear receptor corepres- nonsteroidal antiinflammatory drugs (NSAIDs) type 1 (aspirin sor (NCoR1) interact with both the estrogen and progesterone and indomethacin), which inhibit endoperoxide formation (the receptors (see Fig. 4.15) (Ellmann, 2009; Horwitz, 1996). The immediate precursor of eicosanoids), and type 2 (phenylbuta- mRNA migrates into the cytoplasm, where it translates infor- zone), which inhibits the action of endoperoxidase isomerase mation to ribosomes to synthesize the required new protein. and reductase. Corticosteroids also can inhibit prostaglandins Several alternative receptor mechanisms besides the classic synthesis. one outlined previously appear to exist. Some are plasma mem- In contrast to steroid hormones, which are stored and act at brane steroid signaling events that are mediated through various targets distant from their source, prostaglandins are produced kinases and second messengers including cAMP. These are inde- intracellularly shortly before they are released and generally act pendent of nuclear interactions and do not involve direct steroid locally. Specific prostanoids can have variable effects on different activation of gene transcription (nongenomic). As opposed to the tissues and variable effects on the same organ, even when released longer time required by the genomic pathway (hours to days), at the same concentration, hence the difficulty of studying their these alternate mechanisms may be responsible for some of the actions. One important effect is their ability to modulate the very rapid effects of steroids—for instance, as activated by the  CHAPTER 4 Reproductive Endocrinology 89 Steroid 4 hormone 1 CYTOPLASM Binding of steroid hormone to receptor New protein mRNA 5 Translation 2 Steroid Translocation of receptor steroid-receptor complex to nucleus 3 Binding of complex NUCLEUS to DNA regulatory site 4 Transcription mRNA Regulatory DNA site Fig. 4.14 The steroid receptor activation process. As opposed to protein hormones receptors, which reside on the cell membrane, steroid receptors reside in the nucleus or in the cytoplasm. See the text for details. mRNA, Messenger RNA. negative steroid feedback loop (discussed later), which occurs folliculogenesis (i.e., the cyclic recruitment of a pool of follicles within minutes. to produce a mature follicle ready for ovulation) and the forma- Members of the steroid receptor superfamily share amino acid tion of a corpus luteum. These processes occur in sequence, homology and a common structure. They contain key structural conferring a monthly rhythm to the reproductive cycle. Granu- elements that enable them to bind to their respective ligands with losa and theca cells within the follicle and luteal cells respond to high affinity and specificity and to recognize and bind to discrete LH by synthesizing and releasing ovarian steroids, mainly estra- response elements within the DNA sequence of target genes with diol-17! and progesterone. The type and amount of hormone high affinity and specificity. For instance, estrogen receptors will released depend on the status of the follicle and the corpus bind natural and synthetic estrogens, but not androgens or pro- luteum (see The Menstrual Cycle, presented later). gestins. The affinity of a receptor for a steroid also correlates Feedback communication between the ovaries and the hypo- with steroid potency; for example, the estrogen receptor has a thalamic-pituitary unit is an essential component to the physiol- greater affinity for estradiol than for estrone and estriol, which ogy of the reproductive cycle. It is important for the brain and are much less potent than estradiol. Overall, the magnitude of the pituitary gland to modulate their secretion in response to the signal to the cell and of the cell response to the steroid depend minute-to-minute activity status of the ovary. Through their re- on the concentration of the hormone and of the receptors, as well ceptors, both in various areas of the hypothalamus and in the as on the affinity of the receptor to the hormone. anterior pituitary gland, the two ovarian steroids, estradiol and In humans there are actually two estrogen receptors, ER-! progesterone, play a major role in these feedback communica- and ER-", which are distinct receptor forms encoded by separate tions. Evidence now shows that several nonsteroidal compounds genes. There are also two forms of the progesterone receptor, but are also involved in these feedbacks. these are isoforms (differing only by minor structural differ- ences), which are encoded by the same gene. The Negative Steroid Feedback Loop The Ovarian-Hypothalamic-Pituitary As in other endocrine systems, the major ovarian to brain- pituitary feedback loop is inhibitory (the negative feedback Feedback Loops loop), whereby the steroid secreted by the target organ (the FSH and LH act on the ovaries to induce morphologic changes ovary) regulates the hypothalamic-hypophyseal unit to adjust and ovarian steroid secretion. Morphologic processes include GnRH and gonadotropin secretion appropriately (Fig. 4.16). 90 PART I Basic Science Estradiol-17! is a potent physiologic inhibitor of GnRH and HDACS SHARP of gonadotropin secretion. The threshold for the negative feed- back action of estradiol is such that even small increases in the levels of the hormone induce a decrease in gonadotropins. RTA Sin3 SRA Levels of LH and FSH during the follicular phase vary in accord BRCA1 with the changes in estradiol concentrations that accompany NCoR maturation of the follicle. Thus, as circulating estradiol levels SMRT hPR-A increase during the follicular phase, gonadotropin concentrations decrease. In postmenopausal women or women who have under- REA p68 gone ovariectomy or have aromatase enzyme deficiency, all of ERβ whom lack estradiol secretion, sustained increases in LH and FSH release occur because of the absence of an active negative – feedback loop. In these conditions, administration of physio- SRC1 SHP TIF2 logic doses of estradiol results in a rapid and sustained decrease ERα AIB1 in LH and FSH to levels equivalent to those seen during the menstrual cycle. The estradiol negative feedback loop acts to decrease LH secretion rapidly, mainly by controlling the ampli- TRAP tude of each LH pulse. Most evidence suggests that this action DAX1 220 is secondary to inhibitory effects on the GnRH pulse, most + probably relayed by estrogen-receptive kisspeptin and possibly GABA neurons. Effects on the pituitary gonadotrope, whereby estradiol decreases the gonadotropin response to GnRH, may Cyclin RIP PELP1 also take place. D1 PGC1 140 Progesterone, at high concentrations such as those observed during the luteal phase of the cycle, also exerts an inhibitory ef- E6AP BRG1 fect on gonadotropin secretion. In contrast to estradiol, proges- terone affects mainly the GnRH pulse generator by slowing the Fig. 4.15 A connections map for the human estrogen receptor (ER). frequency of pulses. This effect is responsible for the significant The ER interacts with a large number of proteins that can either posi- decrease in LH pulse frequency observed during the luteal phase tively or negatively regulate target gene transcription. ER! cofactors of the cycle, when high levels of progesterone are present, and interact with different target proteins linking the receptor to other signal which becomes more pronounced as the luteal phase progresses transduction pathways. Some of the key connections that positively (discussed later). (#) or negatively (–) regulate ER! transcriptional activity are shown. There is good evidence that the slowing action of progester- (From McDonnell DP, Norris JD. Connections and regulation of the one on GnRH-LH pulse frequency is mediated by central human estrogen receptor. Science. 2002;296:1642.) !-endorphin. Indeed, brain levels of this opioid peptide, as mea- sured in hypophyseal portal blood in the nonhuman primate, are elevated during the luteal phase (Fig. 4.17, A). Furthermore, administration of naloxone (an opiate antagonist) in women dur- ing the luteal phase results in a significant acceleration in pulse frequency (Fig. 4.17, B). In view of these estradiol and progesterone inhibitory