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 Yen & Jaffe’s
Repro­ductive
Endocri­nology
Physiology,
Pathophysiology,
and Clinical
Management
Jerome F. Strauss III, MD, PhD SEVENTH EDITION
Executive Vice President for Medical Affairs
VCU Health System;
Dean, School of Medicine
Virginia Commonwealth University
Richmond, Virginia

Robert L. Barbieri, MD
Kate Macy Ladd Professor
Department of Obstetrics, Gynecology,
and Reproductive Biology
Harvard Medical School;
Chair, Department of Obstetrics and Gynecology
Brigham and Women’s Hospital
Boston, Massachusetts

iii
 CONTRIBUTORS xi

DANNY J. SCHUST, MD ISABELLE STREULI, MD, MSC

Associate Professor Faculty
William T. Griffin, MD, Distinguished Faculty Scholar; Department of Gynecology, Obstetrics, and Reproductive
Director, Division of Reproductive Medicine and Fertility Medicine
Department of Obstetrics, Gynecology, and Women’s Université Paris Descartes, Paris Sorbonne Cité
Health Paris, France;
University of Missouri School of Medicine Department of Gynecology and Obstetrics
Columbia, Missouri Unit for Reproductive Medicine and Gynecological
Chapter 14: Immunology and Reproduction Endocrinology
Hôpitaux Universitaires de Genève
PETER J. SNYDER, MD Geneva, Switzerland
Professor of Medicine Chapter 35: Pelvic Imaging in Reproductive
University of Pennsylvania ­Endocrinology
Philadelphia, Pennsylvania
Chapter 16: Male Reproductive Aging PATRICE SUTTON, MPH
Research Scientist
WEN-CHAO SONG, PhD Program on Reproductive Health and the Environment
Professor of Pharmacology Department of Obstetrics, Gynecology, and Reproductive
Perelman School of Medicine Sciences
University of Pennsylvania University of California, San Francisco
Philadelphia, Pennsylvania San Francisco, California
Chapter 6: Prostaglandins and Other Lipid Mediators Chapter 20: Environmental Factors and Reproduction
in Reproductive Medicine
ROBERT TAYLOR, MD, PhD
FRANK Z. STANCZYK, PhD Professor and Vice Chair for Research
Professor of Research, Obstetrics, and Gynecology Department of Obstetrics and Gynecology
and Preventive Medicine Wake Forest School of Medicine;
University of Southern California Attending Reproductive Endocrinologist
Keck School of Medicine Department of Obstetrics and Gynecology
Los Angeles, California Wake Forest Baptist Health;
Chapter 34: Laboratory Assessment Member
Molecular Medicine and Translational Sciences Graduate
ELIZABETH A. STEWART, MD Program
Professor of Obstetrics and Gynecology Wake Forest School of Medicine
Chair, Division of Reproductive Endocrinology Winston-Salem, North Carolina
Mayo Clinic Chapter 26: Endometriosis
Mayo School of Medicine
Rochester, Minnesota JESSICA TROWBRIDGE, MPH
Chapter 27: Benign Uterine Disorders Research Scientist
Program on Reproductive Health and the Environment
DALE W. STOVALL, MD University of California, San Francisco
Professor San Francisco, California
Department of Internal Medicine Chapter 20: Environmental Factors and Reproduction
University of Virginia
Charlottesville, Virginia; PAUL J. TUREK, MD
Chair and Residency Director Director
Department of Obstetrics and Gynecology The Turek Clinic
Riverside Regional Medical Center San Francisco, California
Newport News, Virginia Chapter 24: Male Infertility
Chapter 14: Immunology and Reproduction
JOHANNES D. VELDHUIS, MD
JEROME F. STRAUSS III, MD, PhD Professor
Executive Vice President for Medical Affairs Mayo Medical School;
VCU Health System; Consultant in Medicine
Dean, School of Medicine Clinical Investigator
Virginia Commonwealth University Department of Medicine and Physiology
Richmond, Virginia Endocrine Research Unit and Biophysics Section
Chapter 4: The Synthesis and Metabolism Mayo School of Graduate Medical Education
of Steroid Hormones Mayo Clinic
Chapter 9: The Ovarian Life Cycle Rochester, Minnesota
Chapter 13: The Hypothalamo-Pituitary Unit, Testis,
and Male Accessory Organs
xii CONTRIBUTORS

ERIC VILAIN, MD, PhD TERESA K. WOODRUFF, PhD

Professor Thomas J. Watkins Professor of Obstetrics and Gynecology
Department of Human Genetics, Pediatrics, and Urology; Northwestern University
Chief, Division of Medical Genetics Feinberg School of Medicine
Department of Pediatrics Chicago, Illinois
University of California, Los Angeles School of Medicine; Chapter 33: Fertility Preservation
Director, Institute for Society and Genetics
University of California, Los Angeles TRACEY J. WOODRUFF, PhD, MPH
Los Angeles, California Professor
Chapter 17: Disorders of Sex Development Director, Program on Reproductive Health and the
Environment
CARMEN J. WILLIAMS, MD, PhD Department of Obstetrics, Gynecology, and Reproductive
Clinical Investigator, Laboratory of Reproductive & Sciences
Developmental Toxicology University of California, San Francisco
National Institute of Environmental Health Sciences San Francisco, California
Research Triangle Park, North Carolina Chapter 20: Environmental Factors and Reproduction
Chapter 9: The Ovarian Life Cycle
STEVEN L. YOUNG, MD, PhD
SELMA FELDMAN WITCHEL, MD Associate Professor
Associate Professor of Pediatrics Department of Obstetrics and Gynecology
Director, Pediatric Endocrinology Fellowship Program Division of Reproductive Endocrinology
Division of Pediatric Endocrinology University of North Carolina School of Medicine
Children’s Hospital of Pittsburgh of UPMC Chapel Hill, North Carolina
University of Pittsburgh Chapter 10: The Structure, Function, and Evaluation
Pittsburgh, Pennsylvania of the Female Reproductive Tract
Chapter 18: Puberty: Gonadarche and Adrenarche
 Preface

A centerpiece of reproductive endocrinology is the utiliza- age of menarche and the age of menopause; genes that pre-
tion of assisted reproductive technologies, including in vitro dispose to endometriosis, uterine fibroids, and polycystic
fertilization (IVF), to build healthy families. In 1978, the ovary syndrome; and genes that influence ovarian reserve
first baby born from IVF, Louise Brown, was proof of the and spermatogenesis. These important discoveries, which
concept that successful human pregnancy was possible fol- will shed light on pathophysiology and new avenues for diag-
lowing in vitro fertilization. In the same year, the first edi- nosis and treatment, are highlighted in the present edition.
tion of Yen and Jaffe was published and represented the birth Since the publication of the last edition of this text, we
of a field with a strong research foundation, whose findings have achieved a better understanding of environmental fac-
had direct applicability to health. Three decades later, the tors influencing fertility, including obesity. These contempo-
2010 Nobel Prize in Medicine or Physiology was awarded to rary issues have been addressed in this edition. Epigenetic
Sir Robert G. Edwards, Ph.D. (b. 1925, d. 2013), for his factors are thought to mediate the impact of environmen-
seminal contributions to the field of reproductive endocri- tal exposures on gametes and embryos and the developing
nology and infertility. The publication of the 7th edition of fetus. They are also believed to be responsible for intergen-
Yen and Jaffe is dedicated to Dr. Edwards and all the pioneers erational effects. Although still in its infancy, the science of
who have devoted their energies to advancing the field. epigenetics holds promise for explaining reproductive phe-
In the first years of the development of the field, a major notypes and reproductive outcomes.
focus was on the endocrine mechanisms that supported the We appreciate the collaboration of past and new authors
optimal development of oocyte and sperm, their interaction for their critical evaluation of the state of their respective
and the implantation of an embryo in the developmentally fields. Their contributions have enriched this text and we
prepared endometrium. Today, interest in germ cell devel- are grateful for their efforts. They have helped carry for-
opment and germ cell biology has gained great prominence, ward the tradition of excellence that Drs. Jaffe and Yen
raising the possibility of application of stem cell-based created when they brought forward the first edition of this
therapeutics to treat infertility and gene disorders. Fertil- text.
ity preservation through the cryopreservation of sperm and
oocytes has emerged as a key element of care for the cancer
patient and others who face concerns regarding retention
Acknowledgments
of reproductive potential. The importance of these transla- The Editors thank William Drone, Kel McGowan, ­Stefanie
tional advances is recognized by the addition of new chap- Jewell-Thomas, and Steven Stave of Elsevier; and Karen
ters to this edition. Olinger and Deborah Weir of Virginia Commonwealth Uni-
The completion of the sequencing of the human genome versity, for their assistance in the preparation of this volume.
and the reduction in cost in whole genome analysis have
opened new opportunities for finding genes that affect dif- Jerome F. Strauss III, MD, PhD
ferent aspects of reproduction, such as genes that affect the Robert L. Barbieri, MD

xiii
 CHAPTER 1

Neuroendocrinology
of Reproduction
Christopher R. McCartney
John C. Marshall

neurobiological principles are similar among all mammals,

The Central Control of Reproduction these animal studies have been (and continue to be) indis-
Successful reproduction is essential to the survival of a spe- pensable. Nonetheless, certain aspects of reproductive neu-
cies. The reproductive system represents a highly-complex roendocrinology may differ markedly among species. Thus,
functional organization of diverse tissues and signaling path- when available, human data will be prioritized throughout
ways that, when properly functioning, ensures a number of this chapter, but animal studies will also be discussed when
key endpoints, the most important of which are the ade- appropriate, recognizing that specific findings may or may
quate production of gametes (ova and sperm); successful not be generalizable to humans.
delivery of gametes for fertilization; and, in women, physi-
ological preparation for possible pregnancy. Neuroendocrine Neuroendocrinology: The Interface
systems are the principal drivers of reproductive function in
both men and women. In particular, hypothalamic gonad-
Between Neurobiology and Endocrinology
otropin-releasing hormone (GnRH) is the primary, if not Endocrinology is the study of cell-to-cell signaling that
exclusive, feedforward signal to gonadotrope cells of the occurs via specific chemicals (hormones) that travel through
anterior pituitary, stimulating the synthesis and secretion of the bloodstream to influence remote targets. The term
both luteinizing hormone (LH) and follicle-stimulating hor- “neuroendocrinology” refers to the involvement of the cen-
mone (FSH). Together, these two gonadotropins direct the tral nervous system—the hypothalamus in particular—in
primary functions of the reproductive axis: gametogenesis this process. This field of study has traditionally focused
and gonadal sex steroid synthesis. on hypothalamic neuron-derived factors that influence vari-
Given its critical importance to a species, the reproduc- ous target tissues, either directly, as with the hormones of
tive system must be robust, continuing to function prop- the neurohypophysis, or indirectly, as with hypothalamic
erly in the face of various internal and external influences. releasing factors that control anterior pituitary hormone
In contrast, in settings of marked physiological stress (e.g., secretion. Neuroendocrine systems direct a wide variety
significantly reduced energy availability), mechanisms that of critical biological processes such as growth and develop-
temporarily limit fertility—the usual outcome of which is ment, energy and fluid homeostasis, responses to stress, and
metabolically expensive in women—are biologically advan- reproduction.
tageous for the individual and, ultimately, the species. Neurons are highly specialized and morphologically
Appropriate function (or quiescence) of the reproductive diverse cells that transmit information via electrical impulses
system is governed by a number of intricate relationships. called action potentials. Neurons have a cell body contain-
For example, feedback signals from the gonads (e.g., sex ing the cell nucleus, mitochondria, and synthetic organ-
steroid concentrations) communicate the status of gonadal elles. Neurons also have cell processes that participate in
function to the hypothalamic-pituitary axis; these signals in the reception and delivery of electrical impulses (Fig. 1.1).
turn influence GnRH and gonadotropin output, rendering Dendrites are short processes—often extensively branched
a coordinated and tightly regulated feedback system that to increase surface area—that typically receive information
maintains gonadal function within narrow limits. The repro- (afferent electrical impulses). The axon is a single cell pro-
ductive system also has extensive interactions with other cess that generally transmits efferent electrical impulses
neuroendocrine systems, such as those involved with energy away from the cell body in a process called “neuronal firing.”
balance and adaptations to stress. The reproductive neuro- In unstimulated neurons, the inner portion of the neuron­al
endocrine network integrates these myriad feedback signals, membrane is negatively charged compared to the outer
and the GnRH-secreting neuronal network represents the membrane surface (e.g., this “resting membrane potential”
final common pathway for the central control of reproduc- is typically between -50 to -75 mV in GnRH neurons). Such
tion. Thus, the regulation of GnRH secretion represents a electrical polarization reflects transmembrane ionic differ-
major focus of reproductive neuroendocrinology. ences, which are maintained by protein channels that govern
Much of our understanding of reproductive neuroendo- transmembrane passage of specific ions (e.g., sodium, potas-
crinology has emerged from the study of rodents, sheep, sium, chloride). Regulated changes of transmembrane ion
and non-human primates, which largely reflects the ethi- differences may cause the membrane potential to become
cal boundaries inherent to human research. Since many more or less negative (hyperpolarization and depolarization,
3
4 PART 1 Endocrinology of Reproduction

Dendrites facilitate nutrient delivery to neurons; and contribute to the

blood-brain barrier. In addition, astrocytes have been impli-
cated in the control of GnRH secretion and the mechanisms
Cell body
underlying pubertal onset.1 For example, astrocytes may
(perikaryon)
impact neuronal activity via secretion of numerous growth
factors, and astrocytes abundantly appose GnRH neurons;
Axon
these contacts can influence synaptic input, and they may
be influenced by estrogen in both rodents and nonhuman
primates. Similarly, specialized ependymal cells (tanycytes)
Axon in the median eminence appear to modify access of GnRH
terminals neuron terminals to the hypophyseal portal blood.
FIGURE 1.1 Morphological components of a neuron.
Anatomy of the Reproductive
Hypothalamic-Pituitary Axis
respectively). Depolarization to a certain threshold results
in a rapid and temporary reversal of membrane potential (an Portions of the hypothalamus and anterior pituitary gland
action potential), which is propagated along the neuronal constitute the primary effector arm of the central reproduc-
membrane. Notably, the amplitude of the action potential tive axis. In particular, hypothalamic neural systems regu-
does not vary with the strength of stimulation; instead, once late GnRH release into the hypophyseal portal veins, with
a threshold is reached, a full action potential occurs—the GnRH being the signal to gonadotropes (anterior pituitary)
so-called “all-or-none” phenomenon. However, the degree to secrete LH and FSH. In turn, these gonadotropins direct
of neuronal stimulation can alter the frequency of action gonadal (ovarian and testicular) function.
potentials generated. In this way, neurons transmit informa-
tion to other neurons and effector tissue cells. Hypothalamus
Neuronal signals are transferred across neuron-to-neuron The hypothalamus is located at the base of the brain (Fig.
connections (synapses) via chemical neurotransmitters. This 1.2). Although small (approximately 10 grams, less than
process begins with bursts of neuronal firing, which results 1% of total brain weight), it performs critical functions for
in the opening of voltage-gated calcium channels at the axo- maintenance of whole-organism homeostasis. In particular,
nal terminal. The influx of calcium promotes exocytosis primary functions include regulation of hunger and weight
of neurotransmitter-containing synaptic vesicles, releasing maintenance, various aspects of metabolism, growth, thirst
neurotransmitters into the synaptic cleft. Neurotransmit- and renal water handling, body temperature, autonomic
ters then bind to specific ligand-­dependent ion channels in function, sleep, circadian rhythms, and emotion. Impor-
the postsynaptic membrane, which can stimulate an action tantly, the hypothalamus is also a primary control center for
potential in the postsynaptic cell membrane. A wide vari- reproduction and influences sexual behavior.
ety of factors serve as neurotransmitters, including amino As an anatomical structure, the hypothalamus does
acids (e.g., acetylcholine, glutamate, γ-aminobutyric acid not have discrete borders; but in general, it forms the
[GABA]), biogenic amines (e.g., norepinephrine, epineph- floor and inferior lateral walls of the third ventricle (Fig.
rine, dopamine, serotonin), and neuropeptides (e.g., kiss- 1.3). The medial portions of the hypothalamus are pri-
peptin, neurokinin B, dynorphin, β-endorphin, somatostatin, marily made up of cell bodies, while the lateral portions
proopiomelanocortin [POMC], neuropeptide Y [NPY]). are mostly composed of neuron fibers (axons), such as
Bursts of neuronal firing can also elicit release of ­neuronal those connecting the medial hypothalamus to other areas
products into the bloodstream to influence remote targets of the brain. (Note that the hypothalamus is extensively
(i.e., “neurosecretion” of “neurohormones”). Hypophysio- interconnected with other brain areas.) By convention,
tropic neurons are specialized hypothalamic neurons that closely associated collections of neuron cell bodies are
secrete peptide releasing factors—GnRH, corticotropin- called nuclei; and the paraventricular, dorsomedial, ven-
releasing hormone (CRH), thyrotropin-releasing hor- tromedial, and arcuate nuclei (the latter can be called the
mone (TRH), and growth hormone-releasing hormone infundibular nucleus in humans) contain a majority of the
(GHRH)—into the hypophyseal portal circulation. These neurons that secrete hypophysiotropic hormones into the
releasing factors in turn stimulate specific anterior pituitary portal circulation. GnRH cell bodies do not form discrete
cell populations. In contrast, hypothalamic release of dopa- nuclei, but are instead diffusely located throughout the
mine into the portal circulation provides tonic inhibition of preoptic area and the mediobasal hypothalamus (Fig. 1.4);
prolactin secretion. Hypothalamic neurosecretion of vaso- the latter is situated caudal to the preoptic area, extend-
pressin and oxytocin, which are released directly into the ing from the retrochiasmatic area (i.e., the area situated
systemic circulation, alter the function of distant tissues behind the optic chiasm) to the mamillary bodies, and
such as the renal tubules and uterus, respectively. including both the arcuate (infundibular) nucleus and the
Neuroglial cells (e.g., astrocytes, ependymal cells, oligo- median eminence.
dendrocytes, and microglia) represent approximately 90%
of cells in the central nervous system (CNS). Neuroglia Median Eminence
do not conduct action potentials, but they perform critical Positioned at the base of the third ventricle, the median emi-
supportive functions. For example, astrocytes form the sup- nence is part of the anatomical link between the hypothala-
portive framework of the CNS; help isolate synaptic junc- mus and anterior pituitary. The internal zone of the median
tions (to prevent nonspecific spread of neuronal impulses); eminence is located along the ventral floor of third ventricle
 CHAPTER 1 Neuroendocrinology of Reproduction 5

Corpus callosum Thalamus

Pineal gland
Fornix attached to
epithalamus

Hypothalamus

Anterior
Midbrain
commissure
colliculi

Midbrain

Lamina
terminalis
Pons
FIGURE 1.2 Cross-sectional rep-
Optic chiasm resentation of the human brain
Pituitary (sagittal plane), including hypo-
in fossa of Medulla thalamus, median eminence, and
sphenoid pituitary gland. (Adapted from
bone Johnson MH, Everitt BJ. Essential
Median Mammillary reproduction, ed 5. Blackwell Sci-
eminence body ence, 2000, Fig. 6.1.)

and is largely composed of axonal fibers from both mag- signals—including hormonal, metabolic, and toxic—via
nocellular neurons (larger neurons that secrete vasopressin macromolecules of peripheral origin that would otherwise
and oxytocin) and hypophysiotropic neurons as they travel be excluded by the blood-brain barrier; accordingly, capil-
from hypothalamic nuclei/areas to their final destinations— laries of the circumventricular organs are fenestrated and
the neurohypophysis (posterior pituitary) and the external permit transcapillary exchange of larger charged molecules
zone of the median eminence, respectively (Fig. 1.5). The (e.g., proteins, peptide hormones). Thus, the median emi-
external zone contains hypophysiotropic neuron terminals, nence represents a key access point for central sensing of
which release hypophysiotropic hormones into an extensive peripheral cues. Similarly, fenestrated vessels readily allow
capillary plexus—the proximal end of the hypophyseal por- entry of hypothalamic releasing factors into portal blood.
tal system. Some nerve terminals in this zone act on other
nerve terminals to influence hormone release (e.g., kiss- Hypophyseal Portal Circulation
peptin neurosecretion at GnRH neuron terminals appears No direct neuronal connections exist between the hypothal-
to influence GnRH release). amus and the anterior pituitary. However, the hypophyseal
The ependymal layer lining the third ventricle includes a portal circulation (hypothalamic-hypophyseal portal system,
population of specialized ependymal cells called tanycytes, pituitary portal system) represents the functional connec-
which have a short process extending toward the ventricu- tion between the median eminence and anterior pituitary
lar surface and a long process extending into the median (Fig. 1.4). The superior hypophyseal artery (a branch of the
eminence toward areas around portal capillaries. The latter internal carotid artery) subdivides to form an extensive cap-
tanycyte projections envelope or retract from GnRH nerve illary network in the external zone of the median eminence,
terminals during high and low GnRH neuronal activity, with loops that reach into the inner zone. Capillary blood
respectively. Thus, tanycytes may influence GnRH secre- then drains into sinusoids that converge into the hypophy-
tion by physically isolating GnRH neuron terminals from seal portal veins. Traversing the pituitary stalk to reach the
portal capillaries, a regulated process.2 Tanycytes have also anterior pituitary, the hypophyseal portal system forms the
been proposed to be a link between cerebrospinal fluid and primary blood supply of the anterior pituitary. The direc-
events at the external zone (e.g., by transporting substances tion of blood flow is primarily, but not exclusively, from the
from the third ventricle to portal blood). hypothalamus to the anterior pituitary; some retrograde
The median eminence is among the so-called circumven- flow allows for short-loop hypothalamic feedback.
tricular organs, which lie adjacent to the ventricular system
and represent openings in the blood-brain barrier. While Pituitary Gland (Hypophysis)
lipid-soluble molecules can diffuse in and out of the CNS The pituitary gland appears as an extension at the base of
relatively easily, and cellular transport mechanisms allow the hypothalamus and resides cradled within the sella tur-
selective entry of ions, the blood-brain barrier functions to cica, a saddle-like structure of the sphenoid bone (Fig. 1.2).
protect certain regions of the brain and hypothalamus from The adenohypophysis (anterior pituitary) is of ectodermal
larger charged molecules, with physical protection pro- origin, derived from an upward invagination of pharyngeal
vided by (a) tight junctions between endothelial cells and epithelium (Rathke pouch) during embryological devel-
(b) neuron-capillary separation by both astrocyte foot pro- opment. The adenohypophysis is comprised primarily by
cesses and microglia. However, the CNS requires feedback the anterior lobe (pars distalis), which contains specialized
6 PART 1 Endocrinology of Reproduction

Paraventricular Dorsal Dorsomedial

nucleus hypothalamic nucleus
area Posterior
Anterior hypothalamic
hypothalamic nucleus
area
Premamillary
Preoptic nucleus
area
Supraoptic
nucleus

Suprachiasmatic Ventromedial
nucleus nucleus
Optic chiasm
Arcuate
Median eminence nucleus

Pituitary
gland
A

Lateral Lateral
hypothalamus III Ventricle hypothalamus III Ventricle

Fornix
Paraventricular
nucleus

Anterior
hypothalamic
area Optic tract Ventromedial
nucleus
Median
Arcuate
eminence
nucleus
Supraoptic region
nucleus
2 Infundibulum
Preoptic
area Suprachiasatic
1 Optic chiasm nuclei
III Ventricle

Mammillothalamic
tract

Posterior
Cerebral hypothalamic
peduncle area
Lateral
hypothalamus

Mammillary
nuclear
B 3 complex
FIGURE 1.3 Nuclei and areas of hypothalamus. A, By custom, the nuclei and areas of the hypothalamus are often divided into three groups
according to their location along the anterior-posterior plane: the anterior group, the tuberal group, and the posterior (or mamillary) group.
The anterior group is formed by the paraventricular, supraoptic, and suprachiasmatic nuclei along with the anterior hypothalamic and pre-
optic areas. The tuberal group—so-called because of its position above the tuber cinereum (from which the infundibulum or pituitary stalk
extends)—contains the dorsomedial, ventromedial, and arcuate nuclei along with the median eminence. Along with the paraventricular
nucleus, the nuclei of the tuberal group contain a majority of the neurons that secrete hypophysiotropic hormones (i.e., hypothalamic hor-
mones regulating hormone synthesis and release from cells in the anterior pituitary). Finally, the posterior group includes the posterior hypo-
thalamic nucleus and mamillary nuclei. B, Cross-sectional representations (coronal planes) of the rostral (1), mid (2), and caudal (3) portions
of the human hypothalamus. (Section B is adapted from Johnson MH, Everitt BJ. Essential reproduction, ed 5. Blackwell Science, 2000, Fig. 6.3.)
 CHAPTER 1 Neuroendocrinology of Reproduction 7

Tanycytes Portal capillary loop

Third ventricle floor

Supraopticohypophysial
Preoptic fibers
area
INTERNAL ZONE
Adrenergic/peptidergic
axon
GnRH axon
Optic chiasm EXTERNAL ZONE
Mamillary body
Arcuate nucleus Portal capillary plexus
Superior hypophyseal
artery
Hypophysial FIGURE 1.5 Diagram of the median eminence.
portal system
Adenohypophysis
and released from a relatively small population of special-
ized hypothalamic neurons. GnRH was initially isolated
Vein from porcine hypothalami and shown to stimulate pitu-
itary gonadotropin release.3 Although the primary function
FIGURE 1.4 Anatomical relationship between hypothalamic GnRH
neurons and their target cell populations in the adenohypophy-
of GnRH is to regulate pituitary gonadotropin secretion,
sis (anterior pituitary). GnRH neuron cell bodies are located in the GnRH also appears to have autocrine and paracrine func-
preoptic area and the mediobasal hypothalmus. GnRH axonal pro- tions in diverse tissues (e.g., ovary, placenta).4
jections terminate at the median eminence, where GnRH is secreted The regulation of GnRH secretion is complex and involves
into the hypophyseal portal system. (Adapted from Johnson MH, overlapping pathways, which likely increases the robustness
Everitt BJ. Essential reproduction, ed 5. Blackwell Science, 2000, Fig. 6.4.) of central reproductive function. However, there are no
known parallel or backup pathways for the stimulation of
gonadotropin secretion. Thus, natural fertility is absolutely
cell populations that produce specific hormones: gonado- dependent on appropriate GnRH secretion. For example,
tropes (the gonadotropins LH and FSH), mammotropes mice with mutations of the GnRH-1 gene are hypogonadal,
(prolactin), corticotropes (adrenocorticotropic hormone), but reproduction can be restored via GnRH-1 gene ther-
thyrotropes (thyroid stimulating hormone [TSH]), and apy5 or transplantation of fetal GnRH neurons.6 Similarly,
somatotropes (growth hormone). The intermediate lobe is a variety of human conditions associated with absent (or
vestigial in adult humans, but includes a small population of near-absent) GnRH secretion lead to pubertal failure, hypo-
cells (e.g., POMC cells) in contact with the posterior lobe; gonadotropic hypogonadism, and infertility, all of which can
and the pars tuberalis is a slender layer of tissue (e.g., LH- be fully reversed with exogenous GnRH therapy.7
producing cells and TSH-producing cells) surrounding the GnRH secretion is influenced by numerous factors
infundibulum and pituitary stalk. including sex steroids, energy availability, and stress. In
In contrast to the adenohypopysis, the neurohypophysis some mammalian species, GnRH secretion is also affected
(posterior pituitary) is comprised of neural tissue and forms by circadian rhythms, photoperiod (e.g., seasonal breeders
as a downward extension of neuroectodermal tissue from the such as sheep), social cues, and pheromones.
infundibulum during embryological development. It is thus a
direct extension of the hypothalamus. The neurohypophysis GnRH Structure
includes the infundibular stalk and the pars nervosa (poste- Gonadotropin-releasing hormone (GnRH-1 in particular)
rior lobe of the pituitary). The supraoptic and paraventricular is a decapaptide, with the amino acid structure (pyro)Glu-
nuclei include magnocellular neurons that produce oxytocin His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2. The amino acid
and arginine vasopressin ([AVP]; also known as antidiuretic structure of GnRH is identical in essentially all mammalian
hormone [ADH]) respectively; these axons project to the species; and with the exception of the central Tyr-Gly-Leu-
posterior lobe of the pituitary, where oxytocin and AVP are Arg segment, the amino acids of GnRH are highly conserved
secreted into a capillary network that drains into the hypoph- among vertebrate species.8 The GnRH-1 gene (GNRH1) is
yseal veins (i.e., directly into the general circulation). The located on human chromosome 8 (8p11.2-p21) and produces
posterior lobe also includes specialized glial cells called pitui- a 92 amino acid precursor peptide called prepro-GnRH,
cytes, which envelope or retract from magnocellular nerve which includes a signal sequence (23 amino acids), GnRH
terminals during high and low neuronal activity, respectively. (10 amino acids), a proteolytic processing site (3 amino
acids), and GnRH-associated peptide (56 amino acids) (Fig.
1.6). The latter peptide can stimulate gonadotropin secretion
Gonadotropin-Releasing Hormone: The Final
and inhibit prolactin secretion, although its precise physio-
Common Pathway for the Central Control
logical role, if any, remains unclear. The actions of GnRH are
of Reproduction
mediated through the GnRH type I receptor.
Gonadotropin-releasing hormone, previously called lutein- Another form of GnRH (GnRH-2) and its receptor
izing hormone-releasing hormone (LHRH), is synthesized have been identified in a variety of animal species including
8 PART 1 Endocrinology of Reproduction

Pro GnRH gene

Cytoplasm
Transcription
Nucleus
GnRH precursor molecule Primary
(prepro-GnRH) Pro GnRH transcript
peptide
–23
Processing Translation Processing
GnRH decapeptide
GAP
mRNA
1 2 3 4 5 6 7 8 9 10 Gly
Lys
Glu His Trp Ser Tyr Gly Leu Arg Pro Gly

Arg
Transport
to cytoplasm

GAP (56aa)
+92
Portal vessel
A B
FIGURE 1.6 Schematic of GnRH synthesis. A, Representation of prepro-GnRH, including a 23 amino acid signal sequence, GnRH, a proteo-
lytic processing site (Gly-Lys-Arg), and GnRH-associated peptide (GAP). The arrow indicates the site of proteolytic cleavage and C-amidation.
B, Schematic of neuronal GnRH synthesis and secretion.

humans.9 GnRH-2 is a decapeptide with similar struc- respectively, remains unclear, although some of these cir-
ture to GnRH-1: (pyro)Glu-His-Trp-Ser-His-Gly-Trp-Tyr- cuits may possibly be involved with various behavioral
Pro-Gly-NH2 (underlined amino acids denote differences responses.
compared to GnRH-1). However, the gene for GnRH-2
is located on human chromosome 20 (20p13). GnRH-2 Embryological Development of the GnRH
is widely expressed in the CNS and extra-CNS tissues, Neuronal Network
and it may contribute to reproductive behavior regulation The ontogeny of GnRH neurons in vertebrate species is
in some species. In lower animals, GnRH-2 can act via its unique among neuronal systems of the CNS: nascent
own receptor, which is structurally and functionally distinct GnRH neurons are initially identified outside of the CNS
from the GnRH type I receptor. Although a homologue of in the nasal placode (sometimes called the olfactory plac-
the GnRH-2 receptor gene has been detected in the human, ode). However, GnRH cells migrate during embryologi-
it includes a frameshift and premature stop codon. Thus, cal development, as directly observed in embryonic nasal
GnRH-2 signaling in humans may act through the GnRH explant cultures and in embryonic head slices (mouse
type I receptor, although the physiological role of GnRH-2 model).11,12 The specific migratory pathway of GnRH
in humans remains unclear. neurons was first demonstrated in mice by documenting
the presence of GnRH-immunoreactive cells in different
Anatomy of GnRH-Secreting Neurons areas at different stages of embryonic development (Fig.
GnRH neurons are a heterogeneous population of hypo- 1.7).13-15 Specifically, GnRH expression is first observed
thalamic neurons. They are relatively few, numbering within the nasal placode circa embryonic day 10 or 11.
approximately 1500 to 2000, and the majority of GnRH By embryonic day 13, GnRH cells are primarily located
neuronal cell bodies are located in the arcuate (infundibu- around the cribiform plate, and GnRH cells begin to reach
lar) nucleus (part of the mediobasal hypothalamus) and the hypothalamus by embryonic day 14, approaching their
the medial preoptic area.10 Although GnRH neurons are final positions around embryonic day 16. This migratory
rather loosely affiliated anatomically, they are function- pathway has been confirmed in both nonhuman primates16
ally integrated and form a complex network with numer- and humans.17
ous interconnections, in addition to connections to other Successful migration of GnRH neurons is inextricably
neuronal populations. The GnRH neurons in the medio- intertwined with olfactory system development, likely
basal hypothalamus appear to be requisite for gonado- reflecting the close functional relationship between repro-
tropin secretion, and GnRH neuronal axons project to duction and the olfactory system (e.g., pheromones) in
the median eminence via the GnRH tuberoinfundibular mammalian phylogeny. The nasal placode gives rise to
tract. The physiological function of other GnRH neurons, nasal epithelium and olfactory sensory neurons, the latter
which arise from the anterior and posterior hypothalamus, of which extend axonal projections to the olfactory bulb.
and project to the limbic system and posterior pituitary, Vomeronasal neurons are a subset of olfactory neurons
 CHAPTER 1 Neuroendocrinology of Reproduction 9

poa
ob
ob poa
vno gt
gt
gt ob
11E
vno
13E vno

14E vno

16E
A

OB

CP

BF

OP/VNO

B
FIGURE 1.7 GnRH neuron migration during embryogenesis. A, Location of GnRH-immunoreactive cells (red circles) as a function of embryo-
logical age (mouse). On embryological day 11 (11E), GnRH cells are located in the nasal (olfactory) placode and presumptive vomeronasal
organ (vno). GnRH cells migrate across the cribiform plate toward the olfactory bulb (ob). GnRH neurons then follow the caudal branch of the
vomeronasal nerve toward the forebrain and hypothalamus. By day 16 (16E), GnRH neurons largely reside in the preoptic area (poa) of the
hypothalamus. Abbreviations: gt, ganglion terminale. (Adapted from Schwanzel-Fukuda M, Pfaff DW. Origin of Luteinizing Hormone-Releasing
Hormone Neurons. Nature 338:161-164, 1989.) B, Sagittal brain slice (mouse, embryonic day 15) demonstrating the migratory route of GnRH-
immunoreactive cells. Staining is for GnRH and peripherin (a neuronal intermediate filament). OP/VNO, olfactory placode-vomeronasal organ;
CP, cribriform plate (CP); OB, olfactory bulb; BF, basal forebrain. (Adapted from Wierman ME, Pawlowski JE, Allen MP, et al. Molecular mechanisms
of gonadotropin-releasing hormone neuronal migration. Trends Endocrinol Metab 15:96-102, 2004.)

believed to be involved with pheromone detection; these of Kallmann syndrome was deletion of the Kallmann syn-
axons originate in the vomeronasal organ and largely extend drome 1 sequence (KAL1 gene), which is located on the X
to the accessory olfactory bulb. At the level of the cribiform chromosome (Xp22.3) and encodes anosmin-1, a secreted
plate, some olfactory (vomeronasal) axons separate and matrix glycoprotein expressed in the presumptive olfactory
form a branch that extends caudally into the forebrain. Of bulb. Although precise mechanisms are unclear, anosmin-1
great importance, migrating GnRH neurons maintain adhe- is believed to be important for the formation of olfactory
sion to these axons; thus, these olfactory neurons form a elements that provide migratory guidance to GnRH neu-
critical guidance track for GnRH neuronal migration across rons as they move out of the nasal placode. Evaluation of
the nasal epithelium and through the forebrain toward the a 19-week-old human fetus with X-linked Kallmann syn-
hypothalamus.18,19 drome demonstrated GnRH-immunoreactive cells within
The dependence of GnRH neuronal migration on nor- a tangle of olfactory and vomeronasal nerves at the dor-
mal olfactory system development is exemplified by Kall- sal surface of the cribiform plate, along with the absence
mann syndrome, a form of congenital hypogonadotropic of olfactory tracts and bulbs.20 In a second human fetus
hypogonadism accompanied by absent sense of smell (anos- (16 weeks) with X-linked Kallmann syndrome, GnRH was
mia). In this syndrome, faulty development of the olfactory detected along terminal nerve fascicles in the nasal mucosa
system renders an inadequate guidance infrastructure for only.21 This syndrome illustrates that, without the guid-
migrating GnRH neurons, leading to failure of GnRH neu- ance framework provided by the olfactory neuronal sys-
rons to reach the hypothalamus. The first identified cause tem, GnRH neurons fail to migrate into the hypothalamus
10 PART 1 Endocrinology of Reproduction

and thus cannot release GnRH into the hypophyseal portal suggest that sex steroid actions on GnRH neuronal activ-
system. ity are mediated primarily by afferent neurons (e.g., those
A number of additional single-gene defects have been secreting glutamate, GABA, kisspeptin, etc.).
associated with Kallmann syndrome, including mutations GnRH neuron cell bodies are relatively scattered across
of prokineticin 2 (PROK2) and its receptor (PROKR2),22 the mediobasal hypothalamus and preoptic area, yet GnRH
fibroblast growth factor-8 (FGF8) and its receptor (fibro- is secreted into the hypophyseal portal system in a coor-
blast growth factor receptor 1 [FGFR1]),23 nasal embryonic dinated, pulsatile fashion. Specifically, GnRH secretion is
LH-releasing hormone factor (NELF),24 and chromodomain marked by episodic bursts of hormone release into the portal
helicase DNA binding protein 7 (CHD7).25 The importance system, as demonstrated in rats,30 sheep,31 and monkeys.32
of these genes in GnRH neuronal development is corrobo- Once released into the portal vascular compartment, GnRH
rated by mouse studies. For example, in fetal mice lacking is rapidly degraded via enzymatic proteolysis, and the half-
either PROK2 or PROKR2, GnRH neurons are trapped in life of GnRH in the blood is very short—approximately 2 to
a tangled web of olfactory/vomeronasal axons, with few, if 4 minutes. Thus, GnRH presentation to gonadotrope cells
any, reaching the forebrain.26 Although these gene products is intermittent.
are clearly important for GnRH neuron ontogeny, their pre- Pulsatile GnRH secretion is absolutely required for
cise roles remain uncertain. long-term stimulation of gonadotropin synthesis and secre-
Mouse studies suggest other important factors underly- tion. Yet there is a relatively narrow window of GnRH
ing GnRH neuron migration during prenatal development. pulse frequency and amplitude that will optimally stimu-
For example, the chemokine (C-X-C motif) receptor 4 late gonadotropin secretion. Intermittent GnRH stimu-
(CXCR4) is expressed on murine GnRH neurons and lation of gonadotrope cells can increase (or maintain)
interacts with a secreted chemokine stromal cell-derived GnRH receptors on gonadotropes—the “self-priming” or
factor-1 (SDF-1), which is present as a gradient in the “autopriming” effect. Thus, intermittent GnRH stimula-
nasal mesenchyme. This gradient, with highest concentra- tion facilitates or maintains gonadotrope responsiveness
tions at the cribriform plate, provides directional informa- to GnRH. However, more frequent exposure to GnRH
tion as GnRH neurons migrate toward the cribiform plate; pulses can reduce gonadotropin responses to GnRH33; at
and GnRH cell migration across the nasal compartment is one extreme, continuous GnRH receptor stimulation leads
markedly impaired in CXCR4 knockout mice.27 As another to marked desensitization of gonadotropin synthesis and
example, extension of the caudal branch of the vomerona- secretion. In a classic experiment involving rhesus monkeys
sal nerve toward the ventral forebrain involves chemoat- with hypothalamic lesions that abolished GnRH secretion,
traction via interactions between netrin-1—a chemokine intermittent (once an hour) exogenous GnRH administra-
expressed as a gradient in the forebrain—and its receptor, tion restored pituitary gonadotropin secretion. However,
deleted in colorectal cancer (DCC). In mice without either changing from intermittent to continuous GnRH adminis-
DCC or netrin-1, the caudal branch of the vomeronasal tration resulted in marked desensitization of gonadotropin
nerve extends toward the cerebral cortex rather than the release (Fig. 1.8).34 This desensitization is largely related
ventral forebrain; GnRH neurons follow this path, ulti- to reduced GnRH receptor expression on gonadotropes
mately residing in the cerebral cortex.28,29 A number of (i.e., receptor downregulation).
such interactions have been implicated in (a) guidance of The foregoing phenomenon can be exploited therapeuti-
olfactory neurons toward the forebrain and (b) the associa- cally with the use of long-acting GnRH receptor agonists.
tion between migrating GnRH neurons and axons of olfac- Such agonists are peptides with structures very similar to that
tory/vomeronasal nerves, but their specific roles in humans of GnRH, but with amino acid substitutions that enhance
remain unclear. For example, no human mutations affecting receptor binding affinity, increase resistance to proteolytic
DCC or netrin-1 have been described to date. degradation, or both (Fig. 1.9), thus providing continuous
After reaching the hypothalamus, GnRH neurons detach GnRH receptor stimulation. Although initial GnRH receptor
from olfactory nerve axons and may disperse further before agonism temporarily increases gonadotropin release (gonado-
resting. A critical next step is extension of GnRH neuro- tropin “flare”), continued agonism leads to desensitization
nal axons to the median eminence, where GnRH may gain of gonadotropin secretion with accompanying reductions of
access to the hypophyseal portal system. gonadal sex steroid concentrations to castrate levels (“medical
oophorectomy,” “medical castration,” “pseudomenopause”),
GnRH Neuronal Firing and GnRH Secretion usually over 4 to 8 weeks. These agents are useful in the
GnRH neuronal activity is marked by bursts of action poten- therapy of gonadotropin-dependent disorders such as central
tials (burst firing), the patterns and rates of which change precocious puberty, endometriosis, and prostate cancer.
across time. Changes of GnRH secretion are presumably Peptide GnRH receptor antagonists are also available
related to changes of GnRH neuron firing rates, although for clinical use. These antagonists reversibly bind to, but do
the precise relationship between these events is unclear. not stimulate, the GnRH receptor (i.e., competitive antag-
Variable firing rate patterns (e.g., times of high and low onism). Thus, these agents do not cause an initial “flare”
firing rates) appear to be intrinsic to GnRH neurons, but of gonadotropin secretion, and they reduce gonadotropins
they can also be altered by neurotransmitters and neuro- more rapidly than GnRH agonists—usually within 24 to 72
modulators (e.g., glutamate, GABA, kisspeptin). Although hours.
sex steroids can markedly influence GnRH neuronal firing
rates, GnRH neurons lack the primary receptors mediating GnRH Stimulation of Gonadotrope Cells
sex steroid feedback (i.e., estrogen receptor alpha, proges- The specialized cells that synthesize and secrete gonado-
terone receptor, androgen receptor); however, many studies tropins (i.e., gonadotropes) are located mainly in the lateral
 CHAPTER 1 Neuroendocrinology of Reproduction 11

20 Pulsatile Continuous Pulsatile 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
Days
FIGURE 1.8 The influence of pulsatile vs. continuous GnRH administration to GnRH-deficient monkeys. Intermittent exogenous GnRH admin-
istration reconstitutes normal gonadotropin secretion. However, continuous GnRH infusion leads to a marked reduction (downregulation) of
luteinizing hormone (green) and follicle-stimulating hormone (purple) concentrations. Resumption of pulsatile GnRH administration restores LH
and FSH secretion. (Adapted from Belchetz PE, Plant TM, Nakai Y, et al. Hypophysial responses to continuous and intermittent delivery of hypoptha-
lamic gonadotropin-releasing hormone. Science 202:631–633, 1978.)

GnRH pGlu His Trp Ser Tyr Gly Leu Arg Pro Gly NH2

Agonists
Leuprolide D Leu NEt

D-amino acid substitution D Ser

Goserelin (tBu) NH2
enhances activity
D His
Histrelin (ImBzl) NH2

Gly Nafarelin D Nal NH2

Tyr Leu
Triptorelin D Trp NH2
Ser Arg
Buserelin D Ser NEt
(tBu)

Receptor binding Trp Pro Receptor

binding only Antagonists
and activation
His Gly Cetorelix D Nal D Cpa D Pal D Cit D Ala NH2
D-amino acid
NH2
substitution
Ganirelix D hArg D hArg NH2
in antagonists pGlu D Nal D Cpa D Pal
(Et)2 (Et)2
D Ala

A B
FIGURE 1.9 Structure of GnRH and GnRH receptor agonists and antagonists. A, Schematic of GnRH-1 in its folded conformation. Folding
around the glycine in position 6 enhances GnRH receptor binding. Substitution of the glycine in position 6 with D-amino acids stabilizes the
molecule in the folded conformation, which increases affinity for the GnRH receptor and reduces metabolic clearance. The amino-terminal
(red) is involved with receptor binding and activation, and GnRH antagonists involve modifications of these residues that prevent receptor
activation. The carboxyl-terminal (green) participates in receptor binding, but not activation. Substitution at position 10 (e.g., replacement
of glycinamide by ethylamide) can increase binding affinity. B, Amino acid structure of GnRH along with selected GnRH receptor agonists
and antagonists. Solid black circles represent amino acids that are unchanged compared to native GnRH. (From Millar RP, et al. Gonadotropin-
releasing hormone receptors. Endocr Rev. 25:235–275, 2004.)

portions of the anterior pituitary gland and constitute 7% to surges35). GnRH receptor density appears to be modulated
10% of the adenohypophysis cell population. GnRH action at primarily by GnRH, with intermittent GnRH stimulation
the pituitary gonadotrope begins with GnRH binding to the leading to increased GnRH receptor expression; this is a
GnRH type I receptor on the plasma membrane.8 The GnRH central facet of the self-priming effect of GnRH, and an
type I receptor is a member of the seven-­transmembrane important mechanism by which GnRH action is modulated
receptor family, a G protein-coupled receptor, and encoded in different physiological states.
on chromosome 4. GnRH receptor density varies in differ- A majority of gonadotropes synthesize and secrete both LH
ent physiological conditions and exhibits a positive correla- and FSH. A detailed description of intracellular mechanisms
tion with gonadotrope responsiveness to GnRH (e.g., with of GnRH action on the gonadotrope is provided in Chapter
both being high in rodents during preovulatory gonadotropin 2. Briefly, GnRH receptor binding activates the guanosine
12 PART 1 Endocrinology of Reproduction

994
1 Pulse/hour 1 Pulse/3 hours 1 Pulse/hour
50 500

45 450
FIGURE 1.10 LH and FSH concen-
trations in gonadectomized (but 40 400
sex steroid-replaced) monkeys after
35 350
arcuate nucleus ablation—a model
of isolated GnRH deficiency. Exoge-

FSH (ng/mL)
30 300

LH (ng/mL)
nous GnRH administered in a pulsa-
tile fashion every hour reconstituted 25 250
LH and FSH secretion. Changing
GnRH pulse administration from a 20 200
relatively high frequency (hourly) to 15 150
a relatively low frequency (every 3
hours) resulted in decreased LH but 10 100
increased FSH secretion. (Adapted
from Wildt L., et al., Frequency and 5 50
amplitude of gonadotropin-releasing
hormone stimulation and gonadotro- 0 0
pin secretion in the rhesus monkey. 20 15 10 5 0 5 10 15 20 25 30 35 40
Endocrinol 109:376–385, 1981.) Days

triphosphate (GTP)-binding protein Gq/11 leading to an

increase of second messengers inositol 1,4,5-triphosphate
(IP3) and 1,2-diacylglycerol (DAG). Further intracellular
signaling involves increased intracellular calcium and activa- LH
tion of various protein kinase C (PKC) isoforms, mitogen-
activated protein kinases (e.g., extracellular signal-regulated
kinase [ERK], c-Jun NH2-terminal kinase [JNK], and p38),
calcium/calmodulin-dependent kinase II (Ca/CaMK II), and
adenylate cyclase.
Each gonadotropin consists of two protein subunits, α and
β. The 92 amino acid α-subunit is common to both LH and
GnRH
FSH—in addition to human chorionic gonadotropin (hCG)
and TSH. β-subunits for LH (LHβ) and FSH (FSHβ) are
121 and 117 amino acids in length, respectively, and account
for the biological specificity of these two hormones. GnRH
stimulates gene expression of LHβ, FSHβ, and α-subunit, 0 1 2 3 4 5
and the latter noncovalently dimerizes with either LHβ or Hours
FSHβ to form LH or FSH, respectively. Gonadotropins also FIGURE 1.11 Close temporal relationship between pulses of LH
undergo variable post-translational modification, primarily (­jugular vein) and GnRH (pituitary portal system) in the sheep model.
glycosylation (addition of oligosaccharide moieties to spe- (Adapted from Moenter SM et al. Dynamics of gonadotropin-releasing
hormone release during a pulse. Endocrinology. 130:503–510, 1992.)
cific amino acids), which influences bioactivity and elimina-
tion half-life.36 The gonadotropins are then packaged into
secretory granules for eventual secretion. reveal that a decrease in the frequency of exogenously-
Although GnRH is the primary stimulus for LH and FSH administered GnRH pulses from one pulse per hour to one
synthesis and release from a common cell type, concentra- pulse every 3 hours results in a 65% increase in plasma FSH,
tions of these two gonadotropins vary differentially through- despite a 50% decrease in LH (Fig. 1.10).33 Similar findings
out ovulatory cycles, with FSH predominance in the early have been described in sheep39 and humans.40,41 Detailed
follicular phase and LH predominance in the late follicular studies in rats demonstrate that rapid GnRH pulse stimu-
phase. This sequential pattern of FSH and LH predominance lation favors α-subunit and LHβ mRNA expression, while
is important for normal follicular maturation, ovarian steroid slow GnRH pulses favor FSHβ mRNA expression.42 The
production, and subsequent ovulation. At least two mecha- mechanisms effecting differential LH and FSH expression
nisms govern differential gonadotropin secretion throughout in response to changes of GnRH pulse frequency include
ovulatory cycles. First, both estradiol and inhibins selectively variations of GnRH receptor number on the gonadotrope
inhibit FSH release from gonadotropes during the midfol- cell surface43 and alterations of gonadotrope activin βB and
licular and luteal phases.37,38 Second, different patterns of follistatin expression (discussed later in the chapter).44
pulsatile GnRH release differentially affect gonadotropin A pulse of GnRH release stimulates a pulse of LH release
synthesis and secretion. Specifically, rapid (high frequency) on a one-to-one basis, and LH (or α-subunit) pulse patterns,
GnRH pulses favor LH, while slower (low frequency) as assessed by frequent sampling of peripheral blood, accu-
GnRH pulses favor FSH synthesis and secretion. For exam- rately mirror GnRH pulse patterns in animal studies (Fig.
ple, studies in ovariectomized, GnRH-deficient monkeys 1.11).31,45 Similarly, exogenous GnRH pulses elicit LH
 CHAPTER 1 Neuroendocrinology of Reproduction 13

1 26 68 121 145
Kp-145, precursor SP 15.4 kDa
FIGURE 1.12 Schematic of the pre-
NH2 COOH
68 121 cursor kisspeptin-145 and the func-
Kp-54, Metastin 5.9 kDa tional kisspeptin (Kp) fragments,
including size and cleavage sites.
108 121
Note that all functional kisspeptin
Kp-14 1.7 kDa
fragments maintain amino acids
109 121 112-121. SP, signal peptide. (From
Kp-13 1.6 kDa Roseweir AK, Millar RP. The role of
kisspeptin in the control of gonadotro-
112 121 phin secretion. Human Reproduction
Kp-10 1.3 kDa Update 15:203–212, 2009.)

pulses in GnRH deficient patients. Since measurable GnRH 1.12). Importantly, all functional kisspeptins maintain
is effectively confined to the hypophyseal portal system, the 10 amino acids of the carboxy-terminal (kisspeptin
which is inaccessible in humans, GnRH pulse frequency is amino acids 112 to 121), which are important for recep-
inferred from LH pulse frequency (or α-subunit pulse fre- tor binding and function. Kisspeptin is the natural ligand
quency46,47) in human studies. While pulses of GnRH stim- of KISS1R—also known as the G-protein coupled receptor
ulate pulsatile release of FSH, the longer serum half-life of 54 (GPR54)—a seven transmembrane domain, G protein-
FSH renders FSH pulses more difficult to identify via fre- coupled receptor.
quent sampling of peripheral blood. Also, while short-term The importance of the kisspeptin system in reproduction
LH secretion is very closely tied to continued GnRH stimu- was initially revealed by members of two consanguineous
lation, FSH secretion is less acutely dependent on GnRH families with KISS1R mutations leading to pubertal failure
stimulation.48,49 For example, with GnRH antagonism, the and normosmic hypogonadotropic hypogonadism.52,53 Inac-
percentage reduction of LH exceeds that of FSH.50 tivating KISS1 mutations leading to pubertal failure and
normosmic hypogonadotropic hypogonadism have also been
described in four sisters.54 Mouse models with targeted
Neuronal Inputs into GnRH Neurons
knockouts of Kiss1 and Kiss1R exhibit hypogonadotropic
The governance of GnRH neurons is highly complex and hypogonadism with impaired sexual maturation, reduced
involves numerous interacting neural systems utilizing vari- gonadal size, failure of estrous cyclicity (females), impaired
ous neurotransmitters and neuromodulators. The neuronal spermatogenesis (males), and infertility.53,55,56 However,
populations upstream of the GnRH neuron play key roles the notion that kisspeptin is an absolute requirement for
in puberty and are important mediators of sex steroid feed- puberty and reproductive function in mice is somewhat
back and the influence of nutritional cues and stress on controversial.57,58 KISS1R and KISS1 mutations neither
the GnRH pulse generator. Numerous neurotransmitters interrupt GnRH neuron migration to the hypothalamus nor
appear to be involved in the regulation of GnRH secretion, impair GnRH synthesis.
including dopamine, norepinephrine, glutamate, GABA, Single boluses of kisspeptin markedly stimulate LH
and nitric oxide. The control of GnRH secretion has been release in rodents, sheep, monkeys, and humans. This effect
the subject of intense investigation, and the recent discov- of kisspeptin is mediated by stimulation of GnRH neurons,
ery of several neuronal populations upstream of the GnRH as supported by the following: kisspeptin fibers appear
neuron (e.g., kisspeptin neurons) has markedly enhanced to project to and form synaptic contacts with GnRH neu-
our understanding of reproductive neuroendocrinology. rons59,60; the kisspeptin receptor is expressed by a majority of
GnRH neurons61; kisspeptin can directly depolarize GnRH
Kisspeptin neurons62,63; and kisspeptin stimulation of gonadotropin
The kisspeptin system is believed to be requisite for normal secretion is completely blocked by GnRH antagonists.64,65
GnRH secretion, serving as a “gatekeeper” of puberty and However, kisspeptin may also work indirectly, as kisspeptin
helping to mediate the effects of sex steroids and metabolic increases GABAergic and glutamatergic postsynaptic cur-
cues on GnRH secretion. Kisspeptin was originally called rents onto GnRH neurons in the presence (but not absence)
metastin because of its ability to suppress metastatic spread of estrogen in the mouse model.66 Kisspeptin does not stimu-
of human melanomas and breast carcinomas. However, in late LH secretion in Kiss1R knockout mice,56 suggesting that
recognition of its discovery at Pennsylvania State Univer- kisspeptin acts exclusively through its cognate receptor.
sity in Hershey, Pennsylvania, it was later named kisspeptin It remains unclear to what degree kisspeptin acts at
after Hershey’s chocolate KISSES®. Herein we will use the GnRH cell bodies versus nerve terminals, but some stud-
following abbreviations51: KISS1 and Kiss1, the human and ies suggest that kisspeptin neurons can form synapses with
nonhuman kisspeptin genes, respectively; KISS1R (Kiss1R) GnRH neuron terminals in the external zone of the median
and KISS1R (Kiss1R), the human (nonhuman) kisspeptin eminence, and that kisspeptin can stimulate GnRH release
receptor genes and gene products, respectively. (exocytosis) from GnRH neuron terminals.67,68 Although
The KISS1 gene product is a 154 amino acid precursor kisspeptin may possibly have direct effects on gonadotropes,
protein (kisspeptin 1-145). Variable proteolytic modifica- available data suggest that this does not play a major role in
tion yields kisspeptins of different lengths: kisspeptin-54, kisspeptin’s ability to stimulate gonadotropin secretion. For
-14, -13, and -10, with the numbers referring to the example, pulsatile GnRH can restore normal reproductive
amino acid length of bioactive kisspeptin fragments (Fig. function in patients with KISS1R mutations.69
14 PART 1 Endocrinology of Reproduction

The numbers of kisspeptin neurons are high in the together, these studies support the contention that NKB
human arcuate (infundibular) nucleus, similar to findings primarily influences pulsatile GnRH secretion indirectly by
in monkeys.65,70,71 Extensive study in the rodent model stimulating kisspeptin release.86,87
discloses two primary populations of kisspeptin-expressing
neurons in the hypothalamus: one in the arcuate nucleus Endogenous Opioid Peptides
(mediobasal hypothalamus) and the other in the anteroven- Endogenous opioid peptides (EOP), which include endor-
tral periventricular nucleus (AVPV) of the preoptic area.72 phins, enkephalins, and dynorphins, participate in myriad
Of interest, kisspeptin expression in the AVPV is much processes such as motor activity, cognitive functions, water
higher in female compared to male rodents, which appears and food intake, and regulation of neuroendocrine func-
to reflect organizational effects of sex steroids during early tion.88 Most active EOPs share a common sequence (Tyr-
development73,74; and the kisspeptin neurons in the AVPV Gly-Gly-Phe-[Met or Leu]) at the amino-terminal, although
appear to be specifically important for LH surge generation endorphins, enkephalins, and dynorphins are derived from
in rodents. Sexual dimorphism of kisspeptin expression has different precursor proteins that undergo regulated post-
also been described in sheep75 and humans.71 However, in translational processing (Fig. 1.13).89 Endorphins such as
primates, including humans, the majority of the kisspeptin β-endorphin are products of the precursor protein proopi-
cell bodies reside in the arcuate nucleus; and although a omelanocortin (POMC). POMC can be preferentially pro-
study of adult women revealed rare kisspeptin neurons in cessed to produce adrenocorticotropin hormone (ACTH)
the medial preoptic area, a population homologous to the and β-lipotropin, as occurs in corticotropes (adenohypophy-
rodent AVPV has not been identified.70 sis) under the control of corticotropin-releasing hormone
(CRH). However, in the hypothalamus, POMC processing
Neurokinin B primarily yields β-endorphin and α-melanocyte-stimulating
Neurokinin B (NKB), a peptide encoded by the tachykinin hormone. Hypothalamic β-endorphin participates in the
3 gene (TAC3), is a member of the tachykinin family— regulation of reproduction, temperature, and cardiovas-
a family that also includes substance P and neurokinin A cular and respiratory functions, and it acts primarily via μ
(products of the TAC1 gene). There are several neuroki- (micro)-opioid receptors. Enkephalins are derived from
nin receptors (NK1R, NK2R, NK3R), and although NKB proenkephalin, and their primary functions appear to relate
can produce some agonism at NK1R and NK2R, NKB binds to autonomic nervous system modulation, mainly via δ
preferentially to and acts primarily via its cognate receptor (delta)-receptor activation. Dynorphins are products of the
NK3R (TACR3 gene).76 Studies of patients with idiopathic precursor prodynorphin and act chiefly at κ (kappa)-opioid
hypogonadotropic hypogonadism from consanguineous receptors. Importantly, while β-endorphin, enkephalins, and
families revealed that homozygous loss-of-function muta- dynorphins acts primarily via μ-, δ-, and κ-opioid receptors,
tions of either TAC3 or TACR3 can cause pubertal failure each can act as agonists at more than one receptor subtype.
and severe hypogonadotropic hypogonadism—highlighting Numerous studies provide evidence that hypothalamic
the importance of NKB in human reproduction.77,78 In con- opiates partly mediate sex steroid negative feedback on
trast to Kiss1 and Kiss1R knockout mice, Tacr3 knockout GnRH release. For example, GnRH neurons express few if
mice remain fertile, although they can demonstrate repro- any progesterone receptors, but β-endorphin concentrations
ductive defects.79,80 increase in hypophyseal blood during the luteal phase—when
Animal studies demonstrate that a selective NK3R ago- sex steroids suppress GnRH secretion—in monkeys.90,91
nist (senktide) stimulates LH secretion in the rat,81 sheep,82 Moreover, naloxone and naltrexone (opiate receptor antago-
and monkey,83 albeit not as potently as kisspeptin. This nists acting primarily at μ- and κ-opioid receptors) increase
effect appears to be influenced by the sex steroid milieu. LH pulse frequency when administered to luteal phase
For example, senktide administration to ewes increases LH women92 or progestin-treated postmenopausal women.93
secretion during the follicular phase, but not during the Similarly, morphine suppresses GnRH secretion from medio-
luteal phase.82 In addition, senktide increases LH release basal hypothalami isolated from fetal and adult humans—an
in the presence of physiological estradiol in rodents, but it effect that is reversed by naloxone94; and chronic high-dose
reduces LH release in estradiol-deficient animals.81 Mecha- opiate administration can cause hypogonadotropic hypogo-
nisms underlying these apparently paradoxical observations nadism by suppressing GnRH and LH secretion.88
are unclear. Several animal studies implicate dynorphin as a principal
Stimulation of LH secretion by NKB is mediated by mediator of progesterone negative feedback on GnRH pulse
GnRH secretion, as GnRH receptor antagonism abolishes frequency in females.95 For example, dynorphin neurons in
LH responses to senktide in the monkey.83 Yet, there are the arcuate nucleus co-localize with progesterone receptors
few or no NKB receptors on GnRH neurons, and LH is not in ewes,96 and dynorphin-containing varicosities are closely
rapidly stimulated with central senktide administration. associated with GnRH neuron cell bodies in the mediobasal
However, kisspeptin neurons express NK3R; and in ovariec- hypothalamus.97 Progesterone treatment in ewes increases
tomized and estradiol-replaced rats, senktide increases c-fos dynorphin A concentrations in third ventricle cerebrospinal
expression in kisspeptin neurons.81 Additionally, desensiti- fluid,98 and central infusion of dynorphin in goats reduces
zation of Kiss1R markedly reduces GnRH responsiveness to volleys of multiple-unit activity in the mediobasal hypothala-
senktide in monkeys, while similar desensitization of NK3R mus and reduces LH pulses.87 In luteal phase ewes, specific
does not impair GnRH responsiveness to kisspeptin.84 κ-opioid receptor antagonists—but not antagonists to δ- or
Moreover, a recent study suggests that continuous kisspeptin μ-opioid receptors—reversed progesterone inhibition of LH
infusion can restore pulsatile LH secretion in patients with secretion and LH frequency when locally administered into
loss-of-function mutations of TAC3 or TACR3.85 Taken the mediobasal hypothalamus.97 However, other EOPs (e.g.,
 CHAPTER 1 Neuroendocrinology of Reproduction 15

α-Neoendorphin
Prodynorphin Dynorphin A

Dynorphin B

Proenkephalin
Peptide F OctaPeptide HeptaPeptide

POMC

γ-MSH α-MSH β-MSH

CLIP

Leu Enkephalin γ-LPH β-Endorphin

ACTH
β-LPH
Met Enkephalin

FIGURE 1.13 Schematic of endogenous opiate precursors. POMC, proopiomelanocortin; MSH, melanocyte-stimulating hormone; CLIP,
­corticotropin-like intermediate lobe peptide; ACTH, adrenocorticotropic hormone; LPH, lipotropin. (From Akil H, et al. Endogenous opioids:
overview and current issues. Drug Alcohol Depend. 51:127–140, 1998.)

β-endorphin) in other hypothalamic areas may be involved Gonadotropin-Inhibitory Hormone

as well; for example, in the aforementioned study,97 κ- and The role of gonadotropin-inhibitory hormone (GnIH)
μ-receptor antagonists locally administered into the preoptic and its orthologues, also called RF-amide related peptides
area increased LH and LH pulse frequency. (RFRP), in the central control of reproduction has been
recently reviewed.101 Briefly, GnIH immunoreactive cells
Kisspeptin, Neurokinin B, Dynorphin (KNDy) have been identified in hypothalami of a number of species
Neurons including monkeys, and GnIH-immunoreactive fibers can
In the arcuate nucleus, kisspeptin, NKB, and dynorphin are be found in close proximity to GnRH neurons and in the
frequently coexpressed in the same neuron. For example, median eminence. GnIH can reduce GnRH neuronal activ-
kisspeptin neurons in the arcuate nucleus have been found ity, and GnIH also appears to have hypophysiotropic actions
to coexpress NKB and dynorphin in mice,86 goats,87 and to directly inhibit pituitary gonadotropin release. A recent
sheep.99 For convenience, and as a playful nod to kisspeptin study in sheep revealed reduced GnIH expression in the
(namesake of Hershey’s chocolate KISSES®), such neurons preovulatory period in ewes, suggesting a reciprocal rela-
are increasingly being called KNDy neurons (Kisspeptin, tionship with GnRH release, and infusion of GnIH blocked
Neurokinin B, Dynorphin). Available data from human the estrogen-induced LH surge.102 GnIH has also been
autopsy studies are consistent: 77% of kisspeptin cell bodies implicated in the regulation of food intake (increase), sexual
(and 56% of kisspeptin axon fibers) in the arcuate (infun- motivation (decrease), and the influence of stress on repro-
dibular) nucleus coexpress NKB.71 duction. In sum, a growing body of data suggests that GnIH
Kisspeptin, Neurokinin B, Dynorphin (KNDy) neurons is an important factor controlling GnRH and gonadotropin
in the arcuate nucleus form an extensively interconnected secretion in a number of animal species, although an under-
network surrounding the third ventricle. KNDy axons also standing of its role in humans awaits further investigation.
appear to project to the median eminence: in rats, Kiss1/
NKB fibers from the arcuate nucleus project to the inter-
The GnRH Pulse Generator
nal zone of the median eminence where they are in close
proximity to GnRH fibers.100 As with kisspeptin neurons, As described above, intermittent GnRH receptor stimu-
KNDy neuron neuroanatomy exhibits sexual dimorphism in lation is an absolute requirement for physiological main-
sheep, possibly related to perinatal sex steroid exposure.75 tenance of gonadotropin secretion. Although the precise
As discussed further below, KNDy neurons appear to basis of pulsatile GnRH release remains unclear, a number
be intimately involved with sex steroid feedback on GnRH of observations support the concept that neuronal systems
secretion, and some investigators have suggested that the within the mediobasal hypothalamus effect pulsatile release
KNDy neuronal network represents a fundamental compo- of GnRH into the hypophyseal portal system. In animal
nent of the GnRH pulse generator, with NKB stimulating models, volleys of multiple unit electrical activity (i.e.,
and dynorphin inhibiting kisspeptin release. detection of activity in multiple neurons near an electrode)
16 PART 1 Endocrinology of Reproduction

250 (KNDy) neurons. Also, kisspeptin release into the hypophy-

seal portal circulation appears to be pulsatile in sheep and
monkeys. Although these kisspeptin pulses were not clearly
coincident with peripheral LH pulses in ovariectomized
150
ewes,114 kisspeptin pulses occurred approximately once per

MUA (spikes/min)
hour and corresponded to GnRH pulses 75% of the time in
LH (ng/mL)

midpubertal rhesus monkeys.115

50
3000 Although kisspeptin administration did not influence the
frequency of LH pulses or volleys of multiple unit electrical
2000 activity in the arcuate nucleus in one rat study,116 admin-
istration of a selective kisspeptin antagonist into the arcu-
1000 ate nucleus suppressed LH pulse frequency in another.117
Also, central administration of dynorphin in goats inhibits
0
both MUA volleys in the mediobasal hypothalamus and pul-
0 120 240 360 480 satile LH release, whereas NKB provokes MUA volleys.87
Time (min) Human studies also imply that kisspeptin may play a role
FIGURE 1.14 Temporal association between volleys of multiple unit in the GnRH pulse generator. For example, continuous
activity (MUA) in the hypothalamus and luteinizing hormone (LH) intravenous infusion of a relatively low-dose of kisspeptin
pulses (green) detected in peripheral blood in an ovariectomized can increase LH pulse frequency in adult men.118 Another
monkey. (Adapted from Knobil E. The electrophysiology of the GnRH pulse recent study in adult men suggested that a single injection
generator in the rhesus monkey. J Steroid Biochem 33:669–671, 1989.) of kisspeptin may reset the GnRH pacemaker.119 In the lat-
ter study, the interval between the kisspeptin-induced LH
pulse and the immediately preceding endogenous LH pulse
was variable, but on average shorter than the normal LH
in the mediobasal hypothalamus coincide with the initia- interpulse interval (as expected). In contrast, the interval
tion of LH pulse secretion (Fig. 1.14).103 Similarly, elec- between the kisspeptin-induced LH pulse and the subse-
trical stimulation via electrodes placed in the mediobasal quent endogenous LH pulse was similar to normal inter-
hypothalamus stimulates GnRH release into the hypophy- pulse intervals (circa 2 hours), suggesting that kisspeptin
seal portal system in monkeys.104 Mediobasal hypothalami administration reset the hypothalamic GnRH clock.
isolated from fetal (20 to 23 weeks gestation) and adult Hypothetical models regarding how KNDy neurons may
humans release GnRH in discrete pulses, with a frequency be involved with GnRH pulse generation are shown in Figure
of approximately one pulse per 60 to 100 minutes94; and 1.15. However, some data suggest that kisspeptin may not be
mediobasal hypothalami separated from the remainder requisite for GnRH pulse generation. In particular, frequent
of the brain can maintain pulsatile LH secretion in mon- sampling studies reveal that humans with KISSR mutations
keys.105 Lastly, selective radiofrequency ablation of the demonstrate pulsatile LH release, albeit at low amplitude.53,120
arcuate nucleus (part of the mediobasal hypothalamus) in Similarly, a recent study suggested that puberty occurs and
adult female monkeys obliterates gonadotropin secretion.106 fertility is preserved in female mice with either (a) congeni-
These data suggest that the mediobasal hypothalamus, the tal absence of kisspeptin neurons or (b) congenital absence of
arcuate nucleus in particular, houses all requisite compo- neurons expressing Kiss1R.57 When taken as a whole, avail-
nents for GnRH pulse generation (i.e., the GnRH pulse able data imply that kisspeptin action may not be an absolute
generator) and that pulsatile GnRH release does not require requirement for pulsatile GnRH secretion, but that kisspeptin
innervation from outside of the mediobasal hypothalamus. is required for normal GnRH pulse secretion and normally
Nonetheless, mechanisms underlying episodic GnRH pulse exerts an important influence on GnRH pulse generation.
generation, and what neuroanatomical components consti-
tute the GnRH pulse generator, are uncertain.
Physiologic Development of Reproductive
Several studies suggest that pulsatility is an intrinsic
Neuroendocrine Function
property of GnRH neurons. For example, pulsatile GnRH
release is exhibited by immortalized GnRH-secreting neu- Patterns of GnRH secretion change markedly across
rons107,108 and by cultured GnRH neurons obtained from human development. Reproductive neuroendocrine events
fetal rats, sheep, and monkeys.109-111 If GnRH pulse genera- throughout early maturation, including both before and
tion is an intrinsic property of GnRH neurons, then coordi- during the establishment of reproductive competence, are
nation of GnRH release could be facilitated by cell-to-cell discussed in detail in Chapter 18. Briefly, GnRH and gonad-
interconnections among GnRH neurons.112,113 otropin secretion is robust in utero, peaking in midgesta-
Recent data suggest that afferent inputs (e.g., kisspeptin tion. In males, gonadotropin secretion markedly stimulates
neurons) are important for normal GnRH secretion, and testicular androgen secretion, which is important for nor-
some investigators have suggested that kisspeptin (KNDy) mal genital differentiation. The gestational increase of sex
neurons represent a key component of the GnRH pulse steroid (e.g., estradiol) production from the fetoplacental
generator, with NKB and dynorphin influencing kisspeptin unit provides negative feedback to limit fetal GnRH and
stimulation of GnRH neurons. As described above, volleys gonadotropin secretion. Birth is followed by a marked but
of multiunit activity in the arcuate nucleus are temporally transient (3 to 9 month) increase of GnRH and gonado-
associated with LH pulses; however, in addition to GnRH tropin secretion (the “mini-puberty of infancy”), perhaps
neurons, the arcuate nucleus contains numerous kisspeptin related to the withdrawal of fetoplacental sex steroids.
 CHAPTER 1 Neuroendocrinology of Reproduction 17

Kisspeptin/NKB/Dyn
Neuron

Pulse onset: ↑NKB→↑NKB Kisspeptin 

Pulse termination: NKB
Dyn 
KNDy ↑NKB→↑Dyn
 GnRH
GnRH
KNDy Kiss1r (kisspeptin receptor) Arcuate
GnRH 
 NK3R (NKB receptor)
 KOR (Dyn receptor)
GnRH (pg/min)

30

20 GnRH
Neuron ?
10  
0 Median
0 0.5 1.0 1.5 2.0
Eminence
A TIME (hr) B
FIGURE 1.15 Working model regarding how KNDy neurons may participate in the generation of GnRH pulses proposed by Lehman and col-
leagues (A) and Wakabayashi and colleagues (B). A, By this model, neurokinin B (NKB, magenta) stimulates and dynorphin (DYN, red) sup-
presses kisspeptin release, with kisspeptin (green) stimulating GnRH neuronal firing. The onset of a GnRH pulse is triggered by an initial increase
of NKB, which stimulates further NKB release (positive feedback loop) and increases kisspeptin output. NKB stimulation of KNDy neurons also
stimulates DYN release; and after a short period of time, the increase of DYN suppresses kisspeptin (and NKB) release. This withdrawal of kis-
speptin stimulation terminates the GnRH pulse. (From Lehman MN, Coolen LM, Goodman RL. Minireview: kisspeptin/neurokinin B/dynorphin (KNDy)
cells of the arcuate nucleus: a central node in the control of gonadotropin-releasing hormone secretion. Endocrinology. 151:3479–3489, 2010.) B, By
this model, KNDy neurons in the arcuate nucleus form a neural circuit, within which neurokinin B (NKB, magenta) accelerates and dynorphin
(Dyn, red) reduces KNDy neuron activation. These reciprocal effects of NKB and Dyn produce episodic activation of KNDy neurons, with KNDy
neuronal activation increasing kisspeptin release at the median eminence. Kisspeptin in turn stimulates GnRH release into the hypophyseal portal
system. KOR, kappa opiate receptor. (Adapted from Lehman MN et al. Minireview: kisspeptin/neurokinin B/dynorphin (KNDy) cells of the arcuate
nucleus: a central node in the control of gonadotropin-releasing hormone secretion. Endocrinology, 151:3479-3489, 2010; and Wakabayashi Y et al.
Neurokinin B and dynorphin A in kisspeptin neurons of the arcuate nucleus participate in generation of periodic oscillation of neural activity driving
p­ulsatile gonadotropin-releasing hormone secretion in the goat. J Neurosci. 30:3124-3132, 2010.)

A marked sex difference of gonadotropin release is evident understood, but likely reflect developmental remodeling
at this time, with LH concentrations being higher in males of inhibitory and stimulatory neural circuits in the hypo-
and FSH levels higher in females. The possibility that kiss­ thalamus. For example, puberty has been associated with
peptin is important for the minipuberty of infancy is sug- reductions of GABAergic inhibitory neurotransmission and
gested by a patient with a compound heterozygote mutation an increase of excitatory neurotransmitters such as gluta-
of KISSR, who had micropenis, undescended testes, and mate. Kisspeptin and NKB also appear to play important
undetectable serum gonadotropins at 2 months of age—a roles in human puberty, as inactivating mutations of KISS1,
time usually marked by robust gonadotropin secretion.121 KISS1R, TAC3, or TACR3 result in pubertal failure; and a
By late infancy or early childhood (earlier in boys than gain of function KISS1R mutation has been reported as a
in girls), GnRH and gonadotropin secretion markedly cause of central precocious puberty.126 In addition to these
decreases, leading to a hypogonadotropic phase of child- transsynaptic mechanisms, neuroglial cells may contribute
hood marked by low sex steroid concentrations—the “juve- to the pubertal reactivation of GnRH secretion (e.g., by
nile pause.” Studies of gonadotropin secretion in children secretion of growth factors).1
reveal low LH and FSH concentrations, a high FSH-to-LH
ratio, and low LH pulse amplitude and frequency.122 Mech-
Patterns of Pulsatile GnRH Secretion in Adults
anisms accounting for low GnRH secretion during this time
appear to include inhibition of the GnRH pulse generator Human studies employing frequent blood sampling and
(“neurobiological brake”) by higher-order neuronal systems pulse detection analysis have documented significant
(e.g., involving γ-aminobutyric acid [GABA]- and neuro- changes of LH (and by inference GnRH) pulse frequency
peptide Y-secreting neurons) and a developmental removal throughout ovulatory cycles. Briefly, average LH (GnRH)
of stimulation (e.g., involving neurons secreting glutamate pulse frequency is around one pulse every 90 minutes in the
and norepinephrine). early follicular phase, and this gradually increases to approx-
Near the close of the first decade, a marked nocturnal imately one pulse per hour by the late follicular phase.
amplification of pulsatile LH secretion indicates the neu- Although monkey studies suggest that GnRH pulse fre-
roendocrine beginnings of puberty. A majority of studies quency slows during the mid-cycle surge,127 human studies
suggest that early pubertal subjects demonstrate sleep- suggest no change in either LH or α-subunit pulse frequency
entrained increases of LH (GnRH) pulse frequency and at midcycle.128,129 LH pulse frequency slows markedly dur-
amplitude.123 Gonadotropin concentrations rise across ing the luteal phase, approximating one pulse every 3 to
puberty,124,125 stimulating gametogenesis, gonadal sex ste- 8 hours. These day-to-day changes of GnRH pulse frequency
roid secretion, and the development of secondary sexual appear to be important for normal hormonal changes across
characteristics. Mechanisms underlying puberty are poorly ovulatory cycles.130,131
Another Random Scribd Document
with Unrelated Content
"These will make us a fine mess of marmalade when I get back to
the ship," panted the perspiring monarch, settling down with his
back cozily to Nikobo's. "How's young Saucebox?"
"All right." The hippopotamus nodded in Tandy's direction. "He is so
small and tired," she murmured worriedly, "and you must know he
has been exposed in an open cage in the jungle for five long months
with only a miserable hippopotamus for company."
"Miserable hippopotamus," snorted Ato indignantly. "You're a very
superior animal, my girl. I'd consider it an honor to converse with
you any day. Did you crack these cocoanuts for me?" As Nikobo,
trying bashfully to conceal her pleasure at Ato's praise, admitted she
had, the King took several long, satisfying draughts from the shells.
"Now, don't you worry about that young sprout," he advised kindly
as Nikobo continued to gaze mournfully at the sleeping boy. "We'll
make allowances for his High and Mighty Littleness and set him
down in his own country. That is, if we ever manage to find it,
though I must say he'll not be much use nor company for us. Ahoy!
Here comes Sammy. Wonder what he's found?" As a matter of fact,
the Royal Explorer of Oz looked more like a walking window box
than a seaman. Long vines hung from his neck and trailed from his
pockets. His arms were crammed with spiked and prickly plants and
on his head he balanced a package of sea shells tied up in his shore-
going coat.
"What you going to do, start a conservatory?" roared Ato as Roger
helped the Captain set his treasures on the ground.
"Rare and unusual, all of 'em," said Samuel, dropping down beside
Ato and looking with complete satisfaction at his curious collection.
"Mind those yellow creepers," warned Nikobo, wiggling her vast
snout warningly. "Those purple flowered plants in the middle are
treacherous, too. They are tumbleweeds, Master Long Legs, and 'tis
from them Patrippany Island gets its name. When the Leopard Men
fought, they would fling these weeds at one another, and I've seen
them falling about for hours, neither side being able to advance a
step or even stand up."
"Tumbleweeds!" breathed Samuel ecstatically. "You don't SAY! Why,
these might come in real handy if we ever get in a tight place. I'll
give a few to the Wizard of Oz and to the Red Jinn when we get
back from this voyage. And what about the yellow creepers, Mate?
Are they fighting plants, too?"
"The creepers, if uprooted and thrown at an animal or man, will
creep rapidly after him, catching him no matter how fast he runs and
tying him up so tight he will not be able to move until the vine
withers," explained Nikobo solemnly. "I happen to know from an
experience I had with one of these vines in my early youth."
"Creeping vines," shivered Ato, moving as far away from Samuel's
collection as possible. "Just keep them away from me, Sammy. What
right have such things on a ship?"
"Oh, they'll be harmless enough when they're potted," answered
Samuel easily. "And a splendid weapon they'll make for some up and
coming country."
"Better keep them for ourselves," advised Roger, fluttering down to
Samuel's shoulder. "Exploring's a dangerous business, if you ask me,
Master Salt."
"Well, you'll have to admit that it's been pretty safe and successful
so far," said Samuel, clasping his hands behind his head and gazing
contentedly up at the waving fronds of the palm trees.
"SAFE!" The ship's cook began to shake and quiver all over. "Ho, ho!
Safe? Especially sailing round that volcano and going swimming with
the crocodiles! Safe! You'll be the death of me yet, Sam-u-el. Have
you planted your Oz flags and told the wild creatures in the jungle
about their new sovereign?"

Roger nodded his head importantly. "We've raised Oz flags on the

tallest trees on the East, South, West and North sides of the Island. I
flew across and got a bird's eye view while the Captain walked clear
'round. We've discovered it's bean shaped, King dear, the exact
shape of a kidney bean, and a fine fertile place for settlers and
prospectors from Oz."
"Yes, all they have to do is cut down a million trees, drain the
swamps and train the wild beasts in the jungle to be as polite and
considerate as Nikobo here."
"Well, what of it? That's their problem." Samuel stretched himself,
luxuriously snapping each finger to see that it was still working. "And
now, since our part is done, what do you say to waking this son of a
King's son and getting aboard the ship? The tide'll run out in a
couple of hours and carry us along." Tazander had been awake for
some time listening to the conversation with closed eyes. Now sitting
up, he calmly spoke his mind.
"I'm not going with you," he stated grandly. "I'm going to stay here
with Kobo till my own people come for me."
"Hah! Mutiny!" Leaping to his feet, Samuel glared down at the puny
youngster with real anger and exasperation. "If you think I'm going
to leave you on this island to be devoured by wild animals when
Nikobo's back is turned, you don't know your pirates. CLIMB up on
that animal. Lively, now!" Samuel looked so fierce and threatening,
Ato felt rather sorry for the stubborn little King, but he was wasting
his sympathy.
"I'm not going," said Tandy, settling more determinedly down into
the sand. "And no one can make me."
"Don't say that! Don't say that!" Blubbering with grief at the thought
of losing her small charge and shivering with anxiety lest he arouse
to further anger this tall sea captain, Nikobo lumbered to her feet
and began to whisper eagerly in Tandy's ear. During this short
conference Samuel gathered up his specimens and Ato his oranges,
and when both had finished the hippopotamus edged nervously
forward.
"I've decided to go with you," she announced in a slightly shaken
voice. "If I go, Tandy'll go, so I'll just GO!"
"WHAT?" roared Samuel Salt, dropping his shells and clapping his
hand to his forehead. "Well, that practically solves everything!"
Looking wildly from the hippopotamus to the Crescent Moon, Samuel
had a dreadful vision of Nikobo rolling dangerously from side to side
of his cherished vessel.
"What'll you eat?" demanded Roger, who was ever more practical
than polite. "How'll we ever feed this enormous lady, Cook dear?
Besides, she'll sink the ship."
"I'll be very quiet and stay wherever you put me," murmured Nikobo
in a meek voice. "I'll go on a diet and eat whatever is left."

"Well, why couldn't she go?" proposed Ato, who already had formed
a great liking for Tandy's devoted guardian. "Why couldn't she? Nice
kind motherly creature that she is!"
"But a hippopotamus needs fresh water and tons of food and—"
Then suddenly Samuel brought his hands together with a resounding
smack.
"Have you thought of something?" asked Ato hopefully, shifting his
oranges from one shoulder to the other.
"Yes," stated the former Pirate solemnly, "I have." Samuel was
secretly delighted to have found a way to carry this superb
herbivorous specimen back to Oz. "I'll build her a raft and tow her
along after the ship. We'll stop at all the islands we come to for fresh
water and grass, and meanwhile she'll have to do with salt baths
and such food as we have in the hold."
"Oh, KOBO! Did you hear that?" Springing up with the first signs of
life or feeling he had yet shown, Tandy flung himself on his huge
champion and friend. "So you're really going. Then I'll go too."
"Can't be all bad, if he's as fond of her as all that," whispered Ato in
Samuel's ear.
"Not bad, just a pest," wheezed Samuel, reaching for his ax. "Needs
a taste of the rope, if you ask me." Then, while Nikobo went for a
last swim in the Biggenlittle River and bade goodbye to her
numerous and wondering relatives, Samuel felled trees, split wood,
and with nails Roger fetched from the ship fashioned a splendid
strong raft for their new pet. Round the edge he built a sturdy railing
to keep Nikobo from sliding off in a rough sea. Ato and Roger, taking
thought for the evening meal, heaped one end of the raft with grass
and twigs and all the jungle roots they could gather. Without moving
or offering to help, Tandy sat watching, and just as the sun sank
down behind the palms, a strange procession started out for the
Crescent Moon. Ahead with the keg of nails soared Roger. Then
came the hippopotamus moving like a small dreadnought through
the water. On her back sat Ato, the haughty young King of
Ozamaland, and Samuel Salt. Samuel rode last, holding in his hand
the long cable he had attached to the raft and with which he meant
to fasten it to the Crescent Moon.
Following his orders, Nikobo swam close to the side of the ship so
Tandy and Ato could climb the rope ladder, then she paddled round
to the stern where Samuel drew his cable through an iron ring in the
ship's hull and made the raft fast. There was a runway at the back of
the raft and the rails on that side let down so that Nikobo had no
trouble clambering aboard. By pulling a rope with her teeth, she
could raise or lower the back of her pen and take a swim whenever
she felt the need of one. After giving her a bit of advice about
voyaging, and seeing her comfortably settled, Samuel climbed the
cable and nimbly pulled himself aboard his ship. Roger had already
stowed their precious specimens in the hold and rubbing his hands
with brisk satisfaction, the Captain of the Crescent Moon weighed
anchor and dropped with the tide down the Biggenlittle River to the
sea. Then touching the automatic controls, he set his sails to catch
the evening breeze, adjusted his steering gear for a course east by
sou'east and strode happily into his cabin. The Salamander chirped
cheerfully as he passed her hot box and after tapping a cheerful
greeting on the lid, the weary explorer stripped off his ruined and
muddy shore-going outfit, took a shower and climbed thankfully
back into his old sea clothes.
"Where's the pest?" he called out as Roger flew past the open port.
"Well, since he was so small and important," sniffed the Read Bird,
waving a claw, "I gave him a large cabin to himself. I didn't think
you and Ato would want him in here."

"Shiver my timbers, NO." Samuel looked ruefully across at the small

berth the Philadelphia boy occupied on their last voyage. "He'll never
be the seaman Peter was, nor the company either. He'd better keep
out of my way, HAH! or I'll give him a taste of my belt." Snatching
up his spyglass and looking as stern as a kind-hearted pirate well
can, Samuel hurried out on deck.

Meanwhile, in the cabin next to the Captain's, Tandy stood regarding

himself mournfully in the small glass over his sea chest. He too had
taken a shower and at Roger's suggestion had donned one of Peter's
old pirate suits.
"I am a King and the son of a King's son," muttered Tandy, staring
sadly at the sallow reflection in the mirror. To tell the truth, the suit
was not in the least becoming to the skinny and sullen young
monarch.
"I am a King and son of a King's son and can bear anything," he
repeated dismally.
"Then bear a hand with the dinner," yelled Roger, who had been
peeking at him through the port hole. "All who eat must work, and
under the hatches with lubbers!"
Pretending not to hear, Tandy sat resignedly on the side of his bunk,
though he really was curious to look around the ship and see what
Kobo was doing. From the galley came the cheerful rattle of pots
and pans and the huge voice of Ato singing as he prepared the
dinner. Gulls flew in excited circles all round the Crescent Moon,
calling out their hoarse challenge and farewell, and Samuel Salt,
leaning on the taffrail, gazed dreamily back at Patrippany Island. The
Oz flags fluttering from the tall palms gave it quite a gay and festive
appearance and in spite of not seeing the Leopard Men, Samuel felt
he had done a good day's discovering.
"Ahoy, below! How you coming?" called Samuel, leaning down to
look at Nikobo. The hippopotamus wagged her huge head.
"Fine! Just fine, Mate," she wheezed pleasantly.
"Hah! Good for you!" Samuel's face broke into a broad grin as Kobo
remembered to call him "Mate." "We'll make an able-bodied
seawoman of you yet, my lass!"
 CHAPTER 8
Maxims for Monarchs

When Ato, banging boisterously on an iron frying pan with a wooden

spoon, summoned all hands to dinner, Samuel and Roger responded
with a rush. But Tandy remained sitting gloomily on his bunk.
"Now what's the matter?" demanded Samuel Salt as Roger, sent to
call the young voyager, came flying back to the table.
"He says I may serve his dinner in the cabin," snickered Roger,
popping a biscuit into his mouth and swallowing it whole.
"Well, don't you do it!" roared the Captain, bringing his fist down
with an angry thump. "No use to start such nonsense!"
"But he's so thin and feeble. The poor child's just full of raw roots
and jungle grass," murmured Ato, beginning to heap a platter with
meat and vegetables. "Wait till he folds himself round some of these
seafarin' rations. He'll be a different person."
"And he'd better be!" rumbled the Captain of the Crescent Moon,
pulling in his chair. "And if you and Roger want to spoil the little
pest, go ahead, but he'd better keep out of MY way. HAH!"
"I could drop the dinner on his head," suggested Roger helpfully as
Ato handed him an appetizing tray for Tandy. "How would that be?"
"Utterly reprehensible, and conduct unbecoming in a Royal Read Bird
and able-bodied seaman," chuckled the ship's cook, shaking his
finger at Roger. "Why don't you try to help the little beggar and set
him a good example?"
Now Roger, in spite of his sharp tongue, was really a sociable and
kind-hearted bird and the sight of Tandy sitting so forlornly on his
bunk made him regret his teasing speeches. After all, the little fellow
was far from home and had had a hard time in the jungle.
"Here!" he puffed, setting down the tray and lighting the lantern.
"This'll put feathers on your chest, young one, and mind you eat
every scrap."

"Thank you," answered Tandy, so drearily that Roger with a shudder

of distaste fled back to the cheerful company of Samuel and Ato. But
later, when Samuel had gone below to pot the precious plants from
Patrippany Island and the ship's cook was leaning over the rail
conversing cozily with the hippopotamus, Roger flew back to Tandy's
cabin resolved to help him if he could. With calm satisfaction he
noted that Tandy had eaten everything on the tray. Lying on his
back, the young King of Ozamaland was staring solemnly up at the
beams over his bunk.
"Ahoy! And what goes on here?" cried Roger, setting down on the
old sea chest. "How about a turn on deck, my lad, and a bit of
chatter with the crew?"
"It is not seemly for a King and son of a King's son to talk with his
inferiors," observed Tandy coldly.
"In-feer-iors!" screamed Roger, forgetting all his good intentions and
mad enough to nip the youngster's nose right off. "Are you by any
chance referring to me?"
"Ozamaland is a great and powerful country and I am its King,"
stated Tandy, turning his back on the Read Bird. At this Roger let out
another screech, and then suddenly remembering the purpose of his
visit, took a long breath to steady himself. When he spoke again his
voice was both calm and reasonable.
"Ozamaland may be a great and powerful country and you may also
be its King, but remember you are no longer in Ozamaland,"
explained Roger firmly. "You are on this ship by the express wish and
kindness of the Captain and in the company of Kings and BETTER.
WAIT!" Shaking a claw at Tandy's back, Roger flew off to fetch one
of Ato's books from the shelf above the stove. Tandy was in the
same position when he returned, but paying him no further
attention, Roger pulled the lamp nearer and opened his volume.
"When a King is in the company of Kings," began the Read Bird
impressively, "he is no longer a special or royal being, but merely a
man among men, and as such must maintain his honor and standing
by sheer worth and ability alone."
"Who says that? What are you reading?" Tandy sat up with sudden
interest, for his whole life had been spent in study and reflection and
the voice of the Read Bird was not unlike the voice of
Woodjabegoodja, his royal instructor at home.
"I am reading Maxims for Monarchs," answered Roger calmly, "a
book of great authority and antiquity that has been used by the
Rulers of Oz and Ev and the Nonestic Islands these many thousand
years. No great and important country would think of being without
a copy of this book," he continued severely.
"Strange, then, that I should not have heard of it," mused Tandy,
looking not quite so sure of himself. "We have no Maxims for
Monarchs in Ozamaland."

"Pooh, Ozamaland!" Roger dismissed the whole country with a shrug

of his wing. "A country as young and unimportant as that would
probably know nothing about such matters."
"You mean my country is not so old nor important as Oz and this
two-penny island of your fat Master?" shouted Tandy angrily.
"Of course not. Why, it's not even been discovered, and whoever has
been there?" demanded Roger disdainfully. "Take you, as its King,
acting in this small up-country fashion—what CAN a fellow think?
Here—" Shoving the book toward the disagreeable young monarch,
the Read Bird urged him to look for himself. With a puzzled frown
Tandy reread the passage Roger had just quoted.
"Well, even though your Master is a King, you're not a King and
neither is Samuel Salt," said Tandy, looking at Roger with some of
his former arrogance.
"Oh, isn't he? Well, just lay to this, young fellow," Roger shook his
claw under Tandy's upturned nose. "Samuel Salt is Captain of this
ship, a Knight and the Royal Discoverer of Oz, which makes him
seventy times as important as you, King Pins. He not only is boss of
the Crescent Moon, but he rules the sea, discovering countries for
other Kings to govern, and if it were not for Samuel Salt and people
like him, there wouldn't be any Kingdoms nor people like you to run
them. See? As for me, I'm a Royal Read Bird and wouldn't be a King
for a minute. I can live my own life and go and come as I please."
"Then while I'm on this ship I'm not a King at all," said Tandy
wonderingly. "Then what am I? What am I supposed to do?" The
little boy looked puzzled and positively frightened.
"Why, you're supposed to act like a person, that is, if possible,"
sniffed Roger, reaching over for his book and looking at Tandy
sideways down his bill. "What are you besides a King? What can you
do that is useful or interesting?"
"Do, DO?" Tandy's voice rose shrilly. "Why—er—why, I can draw
pictures and ride an elephant."
"Good!" Roger put up his claw to hide the grin that, in spite of his
best efforts, began to spread round his bill. "Well, there isn't much
call for drawing or elephant riding on a ship, but you can draw water
to swab the decks and I'll teach you to ride the yards and follow the
crosstrees to the main topgallant mast in the blowingest blow that
ever blowed. And depend upon it, young one, you'll have more fun
as a person than you ever had as a King. There's no place for having
fun like a ship!"
"Fun!" said Tandy flatly and inquiringly. "What's that?"
"Tar and tobaccy jack! What are you tellin' me?" Roger almost
toppled off the sea chest. "Do you mean to sit there like a dumb
image and tell me you've never had any fun? Never felt so bursting
full of ginger and happiness you could sing or do a sailor's horn
pipe?"
"It is not seemly—" began the boy in a staid voice. "It is—"
"Seemly! Great goosefeathers, are you alive or aren't you?" gasped
Roger. "What in paint did you do in that cussed country of yours
before you got carried off and penned up like a pig in the jungle?"

Considering Roger's question, Tandy clasped and unclasped his

hands nervously. "Well, you must know," he began in a very grown-
up voice, "the King of Ozamaland is not allowed to mingle with the
common people. In all things he is alone and set apart. So it was
with my father and mother before they disappeared. So it is with
me. Furthermore, it being prophesied that I would be carried off by
an aunt in the middle years of my youth, it was deemed expedient
and necessary to keep me locked away from danger in the White
Tower of the Wise Men."
"Hurumph!" grunted the Read Bird, who had not heard so many long
words since the voyage began. "And what did you do in this precious
tower?"
"I studied," sighed Tandy, reclining wearily back on his pillows, "for
there are many things a King must learn. But one hour of every
evening I was permitted to walk about the garden on top of the
tower and look down upon my Kingdom. On very great occasions I
was allowed to come out and ride the white elephant in the grand
processions of state."
"Humph!" grunted Roger again, looking at Tandy with round
dismayed eyes. "And with whom did you play?" he asked after a
little silence.
"Play?" Again Tandy's voice was politely inquiring.

"The word was play," insisted the Read Bird doggedly. "With whom
did you run about, play tag, checkers, pirates or go fishing?"
Tandy looked confused and Roger shook his head sorrowfully. "Never
heard of such things!" he exclaimed indignantly. "Well, all I can say
is, whoever carried you off and shut you up in that jungle cage did
you a real service. If you had not been there we never would have
found you and I'm here to tell you that from now on things are
going to be different. You're discovered now and aboard the
grandest ship afloat. You can forget all about being a King and start
right in being a person and an able-bodied seaman. I for my part
mean to see you have some fun or break a wing in the attempt."
"But would a King—"
"King! Never let me hear that terrible word again," shuddered Roger,
sticking his head under his wing and then popping it comically out
again. "From now on, you're plain Tandy and can do as you plain
please so long as it does no harm to yourself or the ship.
Understand? And tomorrow we'll start having fun, so be ready."
Roger's promise sounded almost like a threat, but there was such a
merry twinkle in his eye, Tandy began to feel interested. "You might
even begin tonight," sniffed Roger, taking up the tray. "Just begin by
thinking of something you want to do. Think about it hard and then
DO it." Winking cheerfully over the empty plates, the Read Bird
spread his wings and sailed through the port.
For several minutes Tandy lay where he was, turning Roger's last
injunction over and over in his stiff, precise little mind. What DID he
really want to do? At first he could think of nothing. Then suddenly
he knew. Why, of course—he wanted to talk to Kobo and he just
plain WOULD. There was a frosted cake left from his supper, and
slipping it into his blouse, Tandy stepped quietly out on deck. The
ship, with only a slight roll, was moving briskly through the water,
white foam falling in lacy spray from her sides, the moon-white sails
spread like giant wings above his head. There was no one in sight,
and almost holding his breath, Tandy tiptoed aft and leaned
adventurously over the taffrail.
"Kobo—Yo KOBO!" he called huskily.
"Hello! I thought you'd be out soon." Swinging round and turning
her vast smile upward, the hippopotamus gazed fondly at her young
charge. "Are you comfortable? Did you have a good dinner?" she
asked anxiously.
"Yes, and look what I saved for you!" As he spoke, Tandy glanced
over his shoulder as if he were almost afraid to have anyone see him
enjoying himself. "Open your mouth, Kobo!" he whispered eagerly.
Without hesitation or question the hippopotamus stretched her jaws
wide and Tandy with the first real thrill of his life flung the frosted
cake into that immense pink cavern. As Kobo neatly caught and
snapped her lips on the tempting morsel Tandy let out a faint cheer
and began to think there might be something in Roger's suggestions
after all. "I'll throw you lots of things tomorrow," he promised gaily.
"Good night, Kobo. Good night, Kobo dear."

Humming a tuneless little song, the young King hurried almost

cheerfully back to his cabin. Pausing in the doorway of his tidy
quarters, he looked about complacently. What did he want to do
next? There was no one to tell him to go to bed, so he just plain
wouldn't. He'd sit up as late as he plain pleased. Rummaging
through Peter's sea chest, which Ato had placed near his bunk,
Tandy found a large tablet of stiff paper, a box of paints and some
crayons. Settling himself cross-legged on his bunk, he began
drawing, not pictures of the castles and courtiers of Ozamaland, but
pictures of the queer jungle beasts and Leopard Men he had seen on
Patrippany Island.
When Roger, on first watch, called out eight bells, he saw Tandy's
light still burning, and flying down to investigate, found his new pupil
fast asleep in the middle of his masterpieces. The whole bunk was
covered with bright drawings and pictures and even to Roger's
inexperienced eye they seemed excellently done. So, carefully the
Read Bird stowed them in the sea chest, then, without bothering to
waken or undress the little King, he covered him with a light blanket
and went quietly from the cabin.
 CHAPTER 9
Sea Legs for Tandy

"If what Roger tells us is so, little Sauce Box yonder has had a pretty
dull life," said Ato as he and the Captain sat finishing their breakfast
next morning. "Lucky for him we happened along and anyway, the
hippopotamus will be good company, eh, Samuel? She seems
downright sensible and jolly. Reminds me of Pigasus and I suppose
she does belong to the pig family when you come to think of it."
"Well, she's a pretty big pig if she does," laughed Samuel Salt,
swallowing his coffee with gusty relish. "Pretty big any way you take
her. Personally, I like the animal, but the King and son of a King's
son! PAH! Reminds me of Peter, he's so different, and the sooner we
reach Ozamaland and set him ashore, the better. Meals in his own
cabin. Hoh!"
"Oh, give him time," drawled Ato, helping himself a second time to
fried potatoes. "If there's any good in the lad, a sea voyage will
bring it out, and what chance has he had shut up in a tower for ten
years and in a cage for five months? Though how an aunt managed
to have him carried so far and why she left him with those savages
in the jungle I can't get through my head at all."
"Maybe it was a gi-ant," whistled Roger, swooping down on Ato's
plump shoulder and flapping his wings cheerfully. "How far do you
figure it is to Ozamaland, Master Salt?"
"Well, that I couldn't just say," answered Samuel in a milder voice.
Pushing back his chair, he stepped over to the map on the west wall.
"Maybe a thousand leagues or so from Patrippany Island, maybe
more, in a line east by sou'east from Ev. If that is so, we're bound to
bump into it sometime, as I've set my course east by sou'east, and
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