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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 document with
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 "Yes!" he said, "what about the expert? If there had been two they would
have disagreed. And mind you at a distance of twelve years a signature
would be difficult of absolute identification. Every one's handwriting
undergoes certain modifications in the course of years. Experts," he
reiterated. "Bah!"

"But," I went on, impatiently, "I don't see the object of the whole
scheme."

"The object was blackmail," the whimsical creature retorted, "and it has
succeeded admirably. Already we read that Messrs. Shap and Lloyd are
staying at expensive hotels in London, that they have granted interviews to
pressmen and written articles for half-penny newspapers. We shall hear of
them as cinema stars presently. They have had the most gorgeous, the most
paying publicity, and presently Sir David Carysfort will have had enough of
them and will put a few more hundreds in their pockets just to be rid of
them. That was the object of the whole scheme, my dear young lady! And
see how well it was carried out.

"Of course the fuddle-headed Dutchman never thought of it. I imagine

that the whole scheme originated in the fertile brain of Mr. Julian Lloyd.
And it was thoroughly well thought out from the manufacture of the
documents and letters down to the assault on the silly old country attorney.
And, mind you, the rascals originally went to a silly country attorney; they
would have been afraid to go to a London lawyer, lest he be too sharp for
them.

"The only mistake they made were the letters purported to be written to
Berta Shap by the husband who is supposed to have disappeared, and the
copy of Berta's marriage certificate. It is those letters that gave me the clue
to the whole thing; old Stonebridge was too dull to have seen through those
letters. If they were genuine why should Felix Shap have brought them over
to England? They had nothing whatever to do with any contract about the
Shap Fuelettes. If they were genuine, how could he guess that he would
have to disprove a story of a secret marriage and of young Alfred being the
son of Sir Alfred Carysfort? By wanting to prove too much, he, to my mind,
gave himself away, and one can but marvel that neither lawyers nor police
saw through the roguery.
 "Of course the moment one understands that one set of papers was
spurious, it is easily concluded that all the others were forgeries. And the
late Sir Alfred Carysfort, anxious only to obliterate every vestige of that
early marriage of his, unwittingly played into the hands of those two
scoundrels by destroying all the correspondence that he had ever had with
Shap.

"Think it all over, you will see that I am right. Look at this paragraph
again in the Evening Post, does it not bear out what I say?"

The paragraph in the evening paper to which the Old Man in the Corner
was pointing read as follows:

"Among the passengers on the Dutch liner Stadt Rotterdam is Mr. Felix
Shap, the hero of a recent celebrated case. He is returning to Batavia,
having, through a misadventure which has remained an impenetrable
mystery to this day, been deprived of all the proofs that would have
established his claim to a substantial share of the profits in the Shap
Fuelettes Company. Fortunately Mr. Shap had enlisted so many sympathies
in England that his friends had no difficulty in collecting a considerable
sum of money which was presented to him on his departure in the form of a
purse and as a compensation for the ill-luck which has attended him since
he set foot in this country. Mr. Shap will now be able to take abroad with
him the assurance that British public opinion is always on the side of the
victims of an adverse and unmerited fate."

"Yes!" the funny creature concluded with a cackle, "until the victims are
found out to be rogues. Mr. Felix Shap and his friend, Mr. Julian Lloyd, will
be found out some day."

The next moment he had gone with that rapidity which was so
characteristic of him, and I might have thought that he was just a spook who
had come to visit me whilst I dozed over my cup of tea, only that on the
table by the side of an empty glass was a piece of string adorned with a
series of complicated knots.
 VIII

THE MYSTERY OF BRUDENELL COURT

§1

"Did you ever make up your mind about that Brudenell Court affair?"
the Old Man in the Corner said to me that day.

"No," I replied. "As far as I am concerned the death of Colonel Forburg

has remained a complete mystery."

"You don't think," he insisted, "that Morley Thrall was guilty?"

"Well," I said, "I don't know what to think."

"Then don't do it," he rejoined, with a chuckle, "if you don't know what
to think, then it's best not to think at all. At any rate wait until I have told
you exactly what did happen—not as it was reported in the newspapers, but
in the sequence in which the various incidents occurred.

"On Christmas Eve, last year, while the family were at dinner, there was
a sudden commotion and cries of 'Stop, thief!' issuing from the back
premises of Brudenell Court, the country seat of a certain Colonel Forburg.
The butler ran in excitedly to say that Julia Mason, one of the maids, was
drawing down the blinds in one of the first-floor rooms, when she saw a
man fiddling with the shutters of the French window in the smoking-room
downstairs. She at once gave the alarm, whereupon the man bolted across
the garden in the direction of the five-acre field. The Colonel and his
stepson, as well as two male guests who were dining with them,
immediately jumped up and hurried out to help in the chase. It was a very
dark night, people were running to and fro, and for a few moments there
was a great deal of noise and confusion, through which two pistol-shots in
close succession were distinctly heard.

"The ladies—amongst whom was Miss Monica Glenluce, the Colonel's

stepdaughter—had remained in the dining-room, and the dinner was kept
waiting, pending the return of the gentlemen. They straggled in one by one,
all except the Colonel. The ladies eagerly asked for news; the gentlemen
could not say much—the night was very dark and they had just waited
about outside until some of the indoor men who had given chase came back
with the news that the thief had been caught.

"This news was confirmed by young Glenluce, Miss Monica's brother,

who was the last to return. He had actually witnessed the capture. The thief
had bolted straight across the five-acre meadow, but doubled back before he
reached the stables, turned sharply to the right through the kitchen garden,
and then jumped over the boundary wall of the grounds into the lane
beyond, where he fell straight into the arms of the local constable who
happened to be passing by.

"Young Glenluce had great fun out of the chase; he had guessed the
man's purpose, and instead of running after him across the meadow, he had
gone round it, and had reached the boundary wall only a few seconds after
the thief had scaled it. There was some talk about the gunshots that had
been heard, and every one supposed that Colonel Forburg, who was a
violent-tempered man, had snatched up a revolver before giving chase to
the burglar, and had taken a potshot at him; it was fortunate that he had
missed him.

"The incident would then have been closed and the interrupted dinner
proceeded with, but for the fact that the host had not yet returned. Nothing
was thought of this at first, for it was generally supposed that the Colonel
had been kept talking by one of his men, or perhaps by the constable who
had effected the capture; it was only when close on half an hour had gone
by that Miss Monica became impatient. She got the butler to telephone both
to the stables and the lodge, but the Colonel had not been seen at either
place, either during or after the incident with the burglar; communication
with the police station brought the same result; nothing had been seen or
heard of the Colonel.
 "Genuinely alarmed now, Miss Monica gave orders for the grounds to be
searched; it was just possible that the Colonel had fallen whilst running, and
was lying somewhere, helpless in the dark, perhaps unconscious.... Every
one began recalling those pistol-shots and a vague sense of tragedy spread
over the entire house. Monica blamed herself for not having thought of all
this before.

"A search party went out at once; for a while stable-lanterns and electric-
torches gleamed through the darkness and past the shrubberies. Then
suddenly there were calls for help, the wandering lights centred in one spot,
somewhere in the middle of the five-acre meadow near the big elm tree.
Obviously there had been an accident. Monica ran to the front door,
followed by all the guests. Through the darkness a group of men were seen
slowly wending their way towards the house; one man was running ahead,
it was the chauffeur. Young Glenluce, half guessing that something sinister
had occurred, went forward to meet him.

"What had happened was indeed as tragic as it was mysterious; the

search party had found the Colonel lying full-length in the meadow. His
clothes were saturated with blood; he had been shot in the breast and was
apparently dead. Close by a revolver had been picked up. It was impossible
to keep the terrible news from Miss Monica. Her brother broke the news to
her. She bore up with marvellous calm, and it was she who at once gave the
necessary orders to have her stepfather's body taken upstairs and to fetch
both the doctor and the police.

"In the meanwhile the guests had gone back into the house. They stood
about in groups, awestruck and whispering. They did not care to finish their
dinner, or to go up to their rooms, as in all probability they would be
required when the police came to make enquiries. Monica and Gerald
Glenluce had gone to sit in the smoking-room.

"It was the most horrible Christmas Eve any one in that house had ever
experienced."

§2
 "Murder committed from any other motive than that of robbery," the Old
Man in the Corner went on after a moment's pause, "always excites the
interest of the public. There is nearly always an element of mystery about it,
and it invariably suggests possibilities of romance. In this case, of course,
there was no question of robbery. After Colonel Forburg fell, shot, as it
transpired, at close range and full in the breast, his clothes were left
untouched; there was loose silver in his trousers pocket, a few treasury
notes in his letter-case, and he was wearing a gold watch and chain and a
fine pearl stud.

"The motive of the crime was therefore enmity or revenge, and here the
police were at once confronted with a great difficulty. Not, mind you, the
difficulty of finding a man who hated the Colonel sufficiently to kill him,
but that of choosing among his many enemies one who was most likely to
have committed such a terrible crime. He was the best-hated man in the
county. Known as 'Remount Forburg,' he was generally supposed to have
made his fortune in some shady transactions connected with the Remount
Department of the War Office during the Boer War, more than twenty years
ago.

"His first wife was said to have died of a broken heart, and he had no
children of his own; some ten years ago he had married a widow with two
young children. She had a considerable fortune of her own, and when she
died she left it in trust for her children, but she directed that her husband
should be the sole guardian of Monica and Gerald until they came of age;
moreover, she left him the interest of the whole of the capital amount for so
long as they were in his house and unmarried. After his death the money
would revert unconditionally to them.

"Of course it was a foolish, one might say a criminal will, and one
obviously made under the influence of her husband. One can only suppose
that the poor woman had died without knowing anything of 'Remount
Forburg's' character. Since her death his violent temper and insufferable
arrogance had alienated from the children every friend they ever had. Only
some chance acquaintances ever came anywhere near Brudenell Court now.
Naturally every one said that the Colonel's behaviour was part of a scheme
for keeping suitors away from his stepdaughter Monica, who was a very
beautiful girl; as for Gerald Glenluce, Monica's younger brother, he had
been sadly disfigured when he was a schoolboy through a fall against a
sharp object that had broken his nose and somewhat mysteriously deprived
him of the sight of one eye.

"Those who had suffered most from Colonel Forburg's violent tempers
declared that the boy's face had been smashed in by a blow from a stick,
and that the stick had been wielded by his stepfather. Be that as it may,
Gerald Glenluce had remained, in consequence of this disfigurement, a shy,
retiring, silent boy, who neither played games nor rode to hounds and had
no idea how to handle a gun; but he was essentially the Colonel's favourite.
Where Forburg was harsh and dictatorial with every one else, he would
always unbend to Gerald, and was almost gentle and affectionate toward
him. Perhaps an occasional twinge of remorse had something to do with this
soft side of his disagreeable character.

"Certainly that softness did not extend to Monica. He made the girl's life
almost unbearable with his violence which amounted almost to brutality.
The girl hated him and openly said so. Her one desire was to get away from
Brudenell Court by any possible means. But owing to her mother's foolish
will she had no money of her own, and the few friends she had were not
sufficiently rich, or sufficiently disinterested, to give her a home away from
her stepfather, nor would the Colonel, for a matter of that, have given his
consent to her living away from him.

"As for marriage, it was a difficult question. Young men fought shy of
any family connection with 'Remount Forburg.' The latter's nickname was
bad enough, but there were rumours of secrets more unavowable still in the
past history of the Colonel. Certain it is that though Monica excited
admiration wherever she went, and though one or two of her admirers did
go to the length of openly courting her, the courtship never matured into an
actual engagement. Something or other always occurred to cool off the
ardour of the wooers. Suddenly they would either go on a big-game
shooting expedition, or on a tour round the world, or merely find that
country air did not suit them. There would perhaps be a scene of fond
farewell, but Monica would always understand that the farewell was a
definite one, and, as she was an intelligent as well as a fascinating girl, she
put two and two together, and observed that these farewell scenes were
invariably preceded by a long interview behind closed doors between her
stepfather and her admirer of the moment.

"Small wonder then that she hated the Colonel. She hated him as much
as she loved her brother. A great affection had, especially of late, developed
between these two; it was a love born of an affinity of trouble and sense of
injustice. On Gerald's part there was also an element of protection towards
his beautiful sister; the fact that he was so avowedly the spoilt son of his
irascible stepfather enabled him many a time to stand between Monica and
the Colonel's unbridled temper.

"Latterly, however, some brightness and romance had been introduced

into the drab existence of Monica Glenluce by the discreet courtship of her
latest admirer, Mr. Morley Thrall. Mr. Thrall was a wealthy man, not too
young and of independent position, who presumably did not care whether
county society would cut him or no in consequence of his marriage with the
stepdaughter of 'Remount Forburg.'

"Subsequent events showed that he had observed the greatest discretion

while he was courting Monica. No one knew that there was an
understanding between him and the girl, least of all the Colonel. Mr. Morley
Thrall came, not too frequently, to Brudenell Court; while there he appeared
to devote most of his attention to his host and to Gerald, and to take little if
any notice of Monica. She had probably given him a hint of rocks ahead,
and he had succeeded in avoiding the momentous interview with the
Colonel which Monica had learned to look on with dread.

"Mr. Morley Thrall had been asked to stay at Brudenell Court for
Christmas, the other guests being a Major Rawstone, with his wife and
daughter, Rachel. They were all at dinner on that memorable Christmas Eve
when the tragedy occurred, and all the men hurried out of the dining-room
in the wake of their host when first the burglary alarm was given.

§3
 "Thus did matters stand at Brudenell Court when, directly after the
holidays, Jim Peyton, a groom recently in the employ of Colonel Forburg,
was brought before the magistrates charged with the murder of his former
master. There was a pretty stiff case against him too. It seems that he had
lately been dismissed by Colonel Forburg for drunkenness, and that before
dismissing him the Colonel had given him a thrashing which apparently
was well deserved, because while he was drunk he very nearly set fire to the
stables, and an awful disaster was only averted by the timely arrival of the
Colonel himself upon the scene.

"Be that as it may, the man went away swearing vengeance.

Subsequently he took out a summons for assault against Colonel Forburg
and only got one shilling damages. This had occurred a week before
Christmas. There were several witnesses there who could swear to the
threatening language used by Peyton on more than one occasion since then,
and of course he had been caught in the very act of trying to break into the
house through the French window of the smoking-room.

"On the other hand, the revolver with which 'Remount Forburg' had been
shot, and which was found close to the body with two empty chambers, was
identified as the Colonel's own property, one which he always kept, loaded,
in a drawer of his desk in the smoking-room. And—this is the interesting
point—the shutters of the smoking-room were found by the police
inspector, who examined them subsequently, to be bolted on the inside, just
as they had been left earlier in the evening by the footman whose business it
was to see to the fastening of windows and shutters on the ground floor.

"This fact—the shutters being bolted on the inside—was confirmed by

Miss Monica Glenluce, who had been the first to go into the smoking-room
after the tragic event. Her brother joined her subsequently. Both of these
witnesses said that the room looked absolutely undisturbed, the shutters
were bolted, the drawer of the desk was closed: they had remained in the
room until after the visit of the police inspector.

"After the positive evidence of these two witnesses, the police

prosecution had of necessity to fall back on the far-fetched theory that
Colonel Forburg himself, before he hurried out in order to join in the chase
against the burglar, had run into the smoking-room and picked up his
revolver, and that, having overtaken Peyton, he had threatened him; that
Peyton had then jumped on him, wrenched the weapon out of his hand and
shot him. It was a far-fetched theory certainly, and one which the defence
quickly upset. Gerald Glenluce for one was distinctly under the impression
that the Colonel ran from the dining-room straight out into the garden, and
the young footman who was watching the fun from the front door, and saw
the Colonel run out, was equally sure that he had not a revolver in his hand.

"Peyton got six months hard for attempted house-breaking, there really
was no evidence against him to justify the more serious charge; but when
the charge of murder was withdrawn, it left the mystery of 'Remount
Forburg's' tragic end seemingly more impenetrable than before.
Nevertheless the coroner and jury laboured conscientiously at the inquest.
No stone was to be left unturned to bring the murder of 'Remount Forburg'
to justice, and in this laudable effort the coroner had the able and
unqualified assistance of Miss Glenluce. However bitter her feelings may
have been in the past towards her stepfather while he lived, she seemed
determined that his murderer should not go unpunished. Nay more, there
appeared to be in all her actions during this terrible time a strange note of
vindictiveness and animosity, as if the unknown man who had rid her of an
arrogant and brutal tyrant had really done her a lasting injury.

"It was entirely through her energy and exertions that certain witnesses
were induced to come forward and give what turned out to be highly
sensational evidence. The police who were convinced that James Peyton
was guilty had turned all their investigations in the direction of proving
their theories; Miss Monica, on the other hand, had seemingly made up her
mind that the murderer was to be sought for inside the house; it even
appeared as if she had certain suspicions which she only desired to confirm.
To this end she had questioned and cross-questioned every one who was in
the house on that fatal night, well knowing how reluctant some people are
to be mixed up in any way with police proceedings. But at last she had
forced two persons to speak, and it was on the first day of the inquest that at
last a glimmer of light was thrown upon the mysterious tragedy.

"After the medical evidence which went to establish beyond a doubt that
Colonel Forburg died from a gunshot wound inflicted at close range, both
balls having penetrated the heart, Miss Glenluce was called. Replying to the
coroner, who had put certain questions to her with regard to the Colonel's
state of mind just before the tragedy, she said that he appeared to have a
premonition that something untoward was about to happen. When the butler
ran into the dining-room saying that a burglar had been seen trying to break
into the house, the Colonel had jumped up from the table at once.

"'I did the same,' Miss Monica went on, 'as I was genuinely alarmed; but
my stepfather, in his peremptory way, ordered me to sit still. "I believe," he
said to me, with a funny laugh, "that it's a put-up job. It's some friend of
Thrall's giving him a hand." I could not, of course, understand what he
meant by that, and I looked at Mr. Thrall for an explanation. I must add that
Mr. Thrall had been extraordinarily moody all through dinner; he appeared
flushed, and I noticed particularly that he never spoke either to my step-
father, to my brother, or to me. However at the moment I failed to catch his
eye, and the very next second he was out of the room, on the heels of
Colonel Forburg.'

"This was remarkable evidence to say the least of it, but nevertheless it
was confirmed by two witnesses who heard the Colonel make that strange
remark: one was Rachel Rawstone, the young friend who was dining at
Brudenell Court that Christmas Eve, and the other was Gerald Glenluce. Of
course, by this time the public was getting very excited: they were like so
many hounds heading for a scent, and the jury was beginning to show signs
of that obstinate prejudice which culminated in a ridiculous verdict. But
there was more to come. Thanks again to Miss Monica's insistence, the
footman at Brudenell Court, a lad named Cambalt, had been induced to
come forward with a story which he had evidently intended to keep hidden
within his bosom, if possible. He gave his evidence with obvious reluctance
and in a scarcely audible voice. It was generally noticed, however, that Miss
Monica urged him frequently to speak up.

"Cambalt deposed that just before dinner on Christmas Eve, he had gone
in to tidy the smoking-room before the gentlemen came down from
dressing. As he opened the door he saw Mr. Morley Thrall standing in the
middle of the room facing Colonel Forburg who was seated at his desk.
Young Mr. Glenluce was standing near the mantelpiece with one foot on the
fender, staring into the fire. Mr. Thrall, according to witness, was livid with
rage.

"''E took a step forward like,' Cambalt went on, amidst breathless silence
on the part of the public and jury alike, 'and 'e raised 'is fist. But the Colonel
'e just laughed, then 'e opened the drawer of the desk and took out a
revolver and showed it to Mr. Thrall and says: "'Ere y'are, there's a revolver
'andy, any way." Then Mr. Thrall 'e swore like anything, and says: "You
blackguard! You d—— scoundrel! You ought to be shot like the cur you
are." I thought he would strike the Colonel, but young Mr. Glenluce 'e just
stepped quickly in between the two gentlemen and 'e says: "Look 'ere,
Thrall, I won't put up with this! You jess get out!" Then one of the
gentlemen seed me, and Mr. Thrall 'e walked out of the room.'

"'And what happened after he had gone?' the coroner asked.

"'Oh!' the witness replied, 'the Colonel 'e threw the revolver back into the
drawer and laughed sarcastic like. Then 'e 'eld out 'is 'and to Mr. Gerald, and
says: "Thanks, my boy. You did 'elp me to get rid of that ruffian." After
that,' Cambalt concluded, 'I got on with my work, and the gentlemen took
no notice of me.'

"This witness was very much pressed with questions as to what

happened later on when the burglary alarm was given and the gentlemen all
hurried out of the house. Cambalt was in the hall at the time and he made
straight for the front door to see some of the fun. He said that the Colonel
was out first, and the other three gentlemen, Mr. Gerald, Mr. Rawstone and
Mr. Morley Thrall went out after him; Mr. Thrall was the last to go outside;
he ran across the garden in the direction of the five-acre field. Major
Rawstone remained somewhere near the house, but it was a very dark night,
and he, Cambalt, soon lost sight of the gentlemen. Presently, however, Mr.
Thrall came back toward the house. It was a few minutes after the shots had
been fired and witness heard Mr. Thrall say to Major Rawstone: 'I suppose
it's that fool Forburg potting away at the burglar; hell get himself into
trouble, if he doesn't look out.' Soon after that Mr. Gerald came running
back with the news that the burglar had fallen into the arms of a passing
constable and Cambalt then returned to his duties in the dining-room.
 "As you see," the Old Man in the Corner went on glibly, "this witness's
evidence was certainly sensational. The jury, which was composed of farm
labourers, with the local butcher as foreman, had by now fully made up its
silly mind that Mr. Morley Thrall had taken the opportunity of sneaking into
the smoking-room, snatching up the revolver, and shooting 'Remount
Forburg,' whom he hated because the Colonel was opposing his marriage
with Miss Monica. It was all as clear as daylight to those dunderheads, and
from that moment they simply would not listen to any more evidence. They
had made up their minds; they were ready with their verdict and it was:
Manslaughter against Morley Thrall. Not murder, you see! The dolts who
had all of them suffered from 'Remount Forburg's' arrogance and violent
temper would not admit that killing such vermin was a capital crime.

"What I am telling you would be unbelievable if it were not a positive

fact. It is no use quoting British justice and dilating on the absolute fairness
of trial by jury. A coroner's inquest fortunately is not a trial. The verdict of a
coroner's jury, such as the one which sat on the Brudenell Court affair,
though it may have very unpleasant consequences for an innocent person,
cannot have fatal results. In this case it cast a stigma on a gentleman of high
position and repute, and the following day Mr. Morley Thrall, himself J.P.,
was brought up before his brother magistrates on an ignominious charge.

§4

"It is not often," the Old Man in the Corner resumed after a while, "that
so serious a charge is preferred against a gentleman of Mr. Morley Thrall's
social position, and I am afraid that the best of us are snobbish enough to be
more interested in a gentleman criminal than in an ordinary Bill Sykes.

"I happened to be present at that magisterial enquiry when Mr. Morley

Thrall, J.P., was brought in between two warders, looking quite calm and
self-possessed. Every one of us there noticed that when he first came in, and
in fact throughout that trying enquiry, his eyes sought to meet those of Miss
Glenluce who sat at the solicitor's table; but whenever she chanced to look
his way, she quickly averted her gaze again, and turned her head away with
a contemptuous shrug. Gerald Glenluce, on the other hand, made pathetic
efforts at showing sympathy with the accused, but he was of such
unprepossessing appearance and was so shy and awkward that it was small
wonder Morley Thrall took little if any notice of him.

"Very soon we got going. I must tell you, first of all, that the whole point
of the evidence rested upon a question of time. If the accused took the
revolver out of the desk in the smoking-room, when did he do it? The
footman, Cambalt, reiterated the statement which he had made at the
inquest. He was, of course, pressed to say definitely whether after the
quarrel between Mr. Morley Thrall and the Colonel which he had
witnessed, and before every one went in to dinner, Mr. Thrall might have
gone back to the smoking-room and extracted the revolver from the drawer
of the desk; but Cambalt said positively that he did not think this was
possible. He himself, after he had tidied the smoking-room, had been in and
out of the hall preparing to serve dinner. The door of the smoking-room
gave on the hall, between the dining-room and the passage leading to the
kitchens. If any one had gone in or out of the smoking-room at that time,
Cambalt must have seen them.

"At this point Miss Glenluce was seen to lean forward and to say
something in a whisper to the Clerk of the Justices, who in his turn
whispered to the chairman on the Bench, and a moment or two later that
gentleman asked the witness:

"'Are you absolutely prepared to swear that no one went in or out of the
smoking-room while you were making ready to serve dinner?7

"Then, as the young man seemed to hesitate, the magistrate added more
emphatically:

"Think now! You were busy with your usual avocations; there would
have been nothing extraordinary in one of the gentlemen going in or out of
the smoking-room at that hour. Do you really believe and are you prepared
to swear that such a very ordinary incident would have impressed itself
indelibly upon your mind?'
 "Thus pressed and admonished, Cambalt retrenched himself behind a
vague: 'No, sir! I shouldn't like to swear one way or the other.'

"Whereat Miss Monica threw a defiant look at the accused, who,

however, did not as much as wink an eyelid in response.

"Presently when that lady herself was called, no one could fail to notice
that she, like the coroner's jury the previous day, had absolutely made up
her mind that Morley Thrall was guilty, otherwise her attitude of open
hostility toward him would have been quite inexplicable. She dwelt at full
length on the fact that Mr. Thrall had paid her marked attention for months,
and that he had asked her to marry him. She had given him her consent, and
between them they had decided to keep their engagement a secret until after
she, Monica, had attained her twenty-first birthday, when she would be free
to marry whom she chose.

"'Unfortunately,' the witness went on, suddenly assuming a dry, pursed-

up manner, 'Colonel Forburg got wind of this. He was always very much set
against my marrying at all, and between tea and dinner on Christmas Eve he
and I had some very sharp words together on the subject, at the end of
which my stepfather said very determinedly: "Christmas or no Christmas,
the fellow shall leave my house by the first available train to-morrow, and
to-night I am going to give him a piece of my mind."'

"Just for a moment after Miss Glenluce had finished speaking, the
accused seemed to depart from his attitude of dignity and reserve, and an
indignant 'Oh!' quickly repressed, escaped his lips. The public by this time
was dead against him. They are just like sheep, as you know, and the verdict
of the coroner's jury had prejudiced them from the start, and the police,
aided by Miss Glenluce, had certainly built up a formidable case against the
unfortunate man. Every one felt that the motive for the crime was fully
established already. 'Remount Forburg' had had a violent quarrel with
Morley Thrall, then had turned him out of the house, and the latter, furious
at being separated from the girl he loved, had killed the man who stood in
his way.

"I should be talking until to-morrow morning were I to give you in detail
all the evidence that was adduced in support of the prosecution. The
accused listened to it all with perfect calm. He stood with arms folded, his
eyes fixed on nothing. The 'Oh!' of indignation did not again cross his lips,
nor did he look once at Miss Monica Glenluce. I can assure you that at one
moment that day things were looking very black against him.

"Fortunately for him, however, he had a very clever lawyer to defend

him in the person of his distinguished cousin, Sir Evelyn Thrall. The latter,
by amazingly clever cross-examination of the servants and guests at
Brudenell Court, had succeeded in establishing the fact that at no time, from
the moment that the burglary alarm was given until after the two revolver
shots had been heard, was the accused completely out of sight of some one
or other of the witnesses. He was the last to leave the dining-room. Mrs.
Rawstone and her daughter testified to that. He had stayed behind one
moment after the other three gentlemen had gone out in order to say a few
words to Monica Glenluce. Miss Rawstone was standing inside the dining-
room door and she was quite positive that Mr. Thrall went straight out into
the garden.

"On the other hand Major Rawstone saw him in the forecourt coming
away from the five-acre meadow only a very few moments after the shots
were fired, and gave it absolutely as his opinion that it would have been
impossible for the accused to have fired those shots. This is where the
question of time came in.

"'When a man who bears a spotless reputation,' Major Rawstone argued,

'finds that he has killed a fellow creature, he would necessarily pause a
moment, horror-struck with what he has done; whether the deed was
premeditated or involuntary he would at least try and ascertain if life was
really extinct. It is inconceivable that any man save an habitual and
therefore callous criminal, would just throw down his weapon and with
absolute calm, hands in pocket and without a tremor in his voice, make a
casual remark to a friend. Now I saw Mr. Morley Thrall perhaps two
minutes after the shots were fired; in that time he could not have walked
from the centre of the field to the forecourt where I was standing; and he
had not been running as his voice was absolutely clear and he came walking
towards me with his hands in his pockets.'
 "As was only to be expected, Sir Evelyn Thrall made the most of Major
Rawstone's evidence, and I may say that it was chiefly on the strength of it
that the charge of murder against the accused was withdrawn, even though
the Clerk to the Magistrates, perpetually egged on by Miss Glenluce, did his
best to upset Major Rawstone. When the lady found that this could not be
done, she tried to switch back to the idea that accused had abstracted the
revolver out of the smoking-room before dinner and immediately after his
quarrel with Colonel Forburg. The footman Cambalt's evidence on this
point had been somewhat discounted by his refusing to state positively that
no one could have gone into the smoking-room at that time without his
seeing them. But against this theory there was always the argument—of
which Sir Evelyn Thrall made the most as you know—that before dinner
the accused could not have known that there would be an alarm of burglary
which would give him the opportunity of waylaying the Colonel in the open
field. With equal skill, too, Sir Evelyn brought forward evidence to bear out
the statement made by the accused on the matter of his quarrel with Colonel
Forburg.

"'Just before dinner,' Mr. Thrall stated, 'Colonel Forburg told me he had
something to say to me in private. I followed him into the smoking-room,
and there he gave me certain information with regard to his past life, and
also with regard to Miss Glenluce's parentage, which made it absolutely
impossible for me, in spite of the deep regard which I have for that lady, to
offer her marriage. Miss Glenluce is the innocent victim of tragic
circumstances in the past, and Forburg was just an unmitigated blackguard,
and I told him so, but I had my family to consider and very reluctantly I
came to the conclusion that I could not introduce any relation of Colonel
Forburg into its circle. Colonel Forburg did not stand in the way of my
marrying his stepdaughter; it was I who most reluctantly withdrew.'

"Whilst the accused was cross-examined upon this statement, and he

gave his answers in firm, dignified tones, Miss Monica never took her eyes
off him, and surely if looks could kill, Mr. Morley Thrall would not at that
moment have escaped with his life, so full of deadly hatred and contempt
was her gaze. The accused had signed a much fuller statement than the one
which he made in open court; it contained a detailed account of his
interview with Colonel Forburg, and of the circumstances which finally
induced him to give up all thoughts of asking Miss Glenluce to be his wife.

"These facts were not made public at the time for the sake of Miss
Monica and of the unfortunate, Gerald, but it seems that the transactions
which had earned for the Colonel the sobriquet of 'Remount Forburg' were
so disreputable and so dishonest that not only was he cashiered from the
army, but he served a term of imprisonment for treason, fraud, and
embezzlement. He had no right to be styled Colonel any longer, and quite
recently had been threatened with prosecution if he persisted in making
further use of his army rank.

"But this was not all the trouble. It seems that in his career of improbity
he had been associated with a man named Nosdel, a man of Dutch
extraction whom he had known in South Africa. This man was
subsequently hanged for a particularly brutal murder, and it was his widow
who was 'Remount Forburg's' second wife, and the mother of Monica and
of Gerald, who had been given the fancy name of Glenluce.

"Obviously a man in Mr. Morley Thrall's position could not marry into
such a family, and it appears that whenever there was a question of a suitor
for Monica, 'Remount Forburg' would tell the aspirant the whole story of his
own shady past and, above all, that of Monica's father. Sir Evelyn Thrall
had been clever enough to discover one or two gentlemen who had had the
same experience as his cousin Morley; they, too, just before their courtship
came to a head had had a momentous interview with 'Remount Forburg,'
who found this means of choking off any further desire for matrimony on
the part of a man who had family connections to consider. But it was very
obvious that Mr. Morley Thrall had no motive for killing 'Remount
Forburg'; he would have left Brudenell Court that very evening, he said,
only that young Glenluce had begged him, for Monica's sake, not to make a
scene; anyway, he was leaving the house the next day and had no intention
of ever darkening its doors again.

"Poor Monica Glenluce or Nosdel, ignorant of the hideous cloud that

hung over her entire life, ignorant, too, of what had passed between her
stepfather and Mr. Morley Thrall, felt nothing but hatred and contempt for
the man whose love, she believed, had proved as unstable as that of any of
her other admirers. For charity's sake one must suppose that she really
thought him guilty at first, and hoped that when the clouds had rolled by he
would return to her more ardent than before. Presumably he found means to
make her understand that all was irrevocably at an end between them as far
as he was concerned, whereupon her regard for him turned to bitterness and
desire for revenge.

"And, indeed, but for the cleverness of a distinguished lawyer, poor

Morley Thrall might have found himself the victim of a judicial error
brought about by the deliberate enmity of a woman. Had he been committed
for trial, she would have had more time at her disposal to manufacture
evidence against him, which I am convinced she had a mind to do."

"As it is," I now put in tentatively, for the Old Man in the Corner had
been silent for some little while, "the withdrawal of the charge of murder
against Morley Thrall did not help to clear up the mystery of 'Remount
Forburg's' tragic death."

"Not so far as the public is concerned," he retorted dryly.

"You have a theory?" I asked.

"Not a theory," he replied. "I know who killed 'Remount Forburg.'"

"How do you know?" I riposted.

"By logic and inference," he said. "As it was proved that Morley Thrall
did not kill him, and that Miss Monica could not have done it, as the ladies
did not join in the chase after the burglar, I looked about me for the only
other person in whose interest it was to put that blackguard out of the way."

"You mean——?"

"I mean the boy Gerald, of course. Openly and before the other witness,
Cambalt, he stood up for his stepfather against Thrall who was not
measuring his words, but just think how the knowledge which he had
gained about his own parentage and that of his sister must have rankled in
his mind. He must have come to the conclusion that while this man—his
stepfather—lived, there would be no chance for him to make friends, no
chance for the sister whom he loved ever to have a home, a life of her own.
Whether that interview on Christmas Eve was the first inkling which he had
of the real past history of his own and Forburg's family, it is impossible to
say. Probably he had suspicions of it before, when, one by one, Monica's
suitors fell away after certain private interviews with the Colonel. Morley
Thrall must have been a last hope, and that, too, was dashed to the ground
by the same infamous means.

"I am not prepared to say that the boy got hold of the revolver that night
with the deliberate intention of killing his stepfather at the earliest
opportunity; he may have run into the smoking-room to snatch up the
weapon, only with a view to using it against the burglar; certain it is that he
overtook 'Remount Forburg' in the five-acre field and that he shot him then
and there. Remember that the night was very dark, and that there was a
great deal of running about and of confusion. The boy was young enough
and nimble enough after he had thrown down the revolver to run across the
field and then to go back to the house by a roundabout way. It is easy
enough in a case like that to cover one's tracks, and, of course, no one
suspected anything at the time. Even the sound of firing created but little
astonishment; it was so very much on the cards that the Colonel would use
a revolver without the slightest hesitation against a man who had been
trying to break into his house. It was just the sort of revenge that a man of
Gerald's temperament—disfigured, shy, silent and self-absorbed—would
seek against one whom he considered the fount of all his wrongs."

"But," I objected, "how could young Glenluce run into the smoking-
room, pick up the revolver out of a drawer, and run back through the hall
with servants and guests standing about? Some one would be sure to see
him."

"No one saw him," the funny creature retorted, "for he did it at the
moment of the greatest confusion. The butler had run in with the news of
the burglary, the Colonel jumped up and ran out through the hall, the guests
had not yet made up their minds what to do. In moments like this there are
always just a few seconds of pandemonium, quite sufficient for a boy like
Gerald to make a dash for the smoking-room."