Regulation of Hormone Action

Mechanism of action of Steroidal hormone:

The steroid hormones belong to a heterogeneous class of small
polycyclic organic compounds. These compounds play a vital role in
many developmental and homeostatic processes. Steroid hormones are
produced in the adrenal glands, ovaries, and testes, and regulate a wide
range of physiologic functions. Sex steroids, which include estrogens,
progesterone and testosterone, are involved in both normal and
pathologic embryonic sex. In order to obtain a better understanding of
the role that steroids play in both health and disease, it has been
necessary to investigate the molecular biology of hormone responses.
The past few years have brought many advances in the field of steroid
hormone action and make it possible to now formulate a general model
of steroid hormone action.
Steroid hormones are transported through the circulatory system to the
target organs where they exert their specific hormonal actions. Steroids
bind to a variety of macromolecules in the circulation. The affinity of
these macromolecules for steroids can vary from weak to strong, and can
be present in significant concentrations. The measurement of serum or
plasma steroid concentration generally is a measure of the total steroid
present and does not reflect the equilibrium that exists between steroid
hormone bound to these macromolecules and those free in the blood.
The bound and free forms of the steroid are in an equilibrium that is
controlled by many physiologic factors. It is important to remember that
generally only the free hormone can leave the circulation and enter the
target cells, where it can bind to specific intracellular receptors to initiate
the biochemical expression of specific sex steroids. Examples of these
specific steroid-binding macromolecules include a testosterone-estrogen-
binding globulin, also known as sex hormone-binding globulin, which
binds both testosterone and estradiol with high affinity. Its production is
stimulated by increasing estrogen concentration and decreased by
increasing testosterone concentration. A second specific binding
globulin is the corticoid-binding globulin, also known as transcortin,
whose main function is to bind glucocorticoids, such as cortisol, but
which also binds progesterone with a high affinity. The plasma
concentration of this binding globulin is increased by estrogen;
consequently, it is increased dramatically during pregnancy.
In addition to the binding of steroids by these high-affinity, low-capacity
macromolecules, albumin is a sex steroid binder with low affinity but
tremendous capacity in the circulation. The concentration of albumin in
the blood can be as high as 4%. At such a high concentration, it can alter
the equilibrium distribution of steroids in blood, and therefore is an
important consideration in the circulating economy of steroid hormones.
Because of the greater concentration of albumin and the rapid
dissociation of steroids from this low-affinity binder, albumin may serve
a more important regulatory role than do the high-affinity, low-capacity
binding proteins.
INTRACELLULAR STEROID BINDING AND MECHANISM OF
STEROID ACTION –

Steroids hormones circulate throughout the body predominantly

complexed with binding proteins. However, perhaps as much as 5% of
the circulating steroid hormones are uncomplexed. Only these free
steroid molecules may enter cells and they do so probably by passive
diffusion. Autoradiographic experiments have demonstrated that
following in vivo hormone administration only certain cells retain
steroids for significant time periods. These cells are termed target cells.
A class of protein molecules called receptors resides within the
cytoplasm of target cells in the absence of hormone. Classical
pharmacology defines a receptor as a sort of biologic transducer which
converts input information, i.e., circulatory steroids, into biologic
response, i.e., specific RNA and protein synthesis. These
macromolecules are present in limited numbers but only in target cells.
They bind specific steroids with very affinity. Receptors for steroid
hormones are located inside target cells, in the cytoplasm or nucleus, and
function as ligand-dependent transcription factors. That is to say, the
hormone-receptor complex binds to promoter regions of responsive
genes and stimulate or sometimes inhibit transcription from those genes.
Thus, the mechanism of action of steroid hormones is to modulate gene
expression in target cells. By selectively affecting transcription from a
battery of genes, the concentration of those respective proteins are
altered, which clearly can change the phenotype of the cell.

It is important to realize that the reversible reaction between the steroid

moiety and the receptor is highly selective, progesterone binds to
progesterone receptor but not to testosterone or estrogen receptors. In
vitro studies using a cell-free system have revealed that the receptors are
not retained by purified nuclei in the absence of hormone. Thus one
postulates that interaction of steroid with receptor induces some subtle
alteration in the structure of the receptor molecules which facilitates
translocation of the receptor- hormone complex to the nuclear
compartment.
Structure of Intracellular Receptors -

Steroid hormone receptors are members of a large group ("superfamily")

of transcription factors. In some cases, multiple forms of a given
receptor are expressed in cells, adding to the complexity of the response.
All of these receptors are composed of a single polypeptide chain that
has, in the simplest analysis, three distinct domains:

• The amino-terminus: In most cases, this region is involved in

activating or stimulating transcription by interacting with other
components of the transcriptional machinery. The sequence is
highly variable among different receptors.
• DNA binding domain: Amino acids in this region are responsible
for binding of the receptor to specific sequences of DNA.
• The carboxyl-terminus or ligand-binding domain: This is the
region that binds hormone.
In addition to these three core domains, two other important regions of
the receptor protein are a nuclear localization sequence, which targets
the protein to nucleus, and a dimerization domain, which is responsible
for latching two receptors together in a form capable of binding DNA.
Hormone-Receptor Binding and Interactions with DNA -

Being lipids, steroid hormones enter the cell by simple diffusion across
the plasma membrane. Steroid hormones enter the cell by facilitated
diffusion. The receptors exist either in the cytoplasm or nucleus, which
is where they meet the hormone. When hormone binds to receptor, a
characteristic series of events occurs:

• Receptor activation is the term used to describe conformational

changes in the receptor induced by binding hormone. The major
consequence of activation is that the receptor becomes competent
to bind DNA.
• Activated receptors bind to "hormone response elements", which
are short specific sequences of DNA which are located in
promoters of hormone-responsive genes. In most cases, hormone-
receptor complexes bind DNA in pairs, as shown in the figure
below.
• Transcription from those genes to which the receptor is bound is
affected. Most commonly, receptor binding stimulates
transcription. The hormone-receptor complex thus functions as a
transcription factor.
Translocation of the Hormone-Receptor complex –

Nuclear uptake of receptor- hormone complexes has been studied in

many laboratories. In vitro studies using partially purified receptors and
highly purified nuclei have shown that translocation is a time- and
temperature-dependent process. Rate studies are consistent with a
diffusion-limited translocation process. The time course for binding of
receptors to chromatin or to nuclei is very similar and thus it is unlikely
that the nuclear membrane is rate limiting in the nuclear accumulation of
steroid receptor complexes.

Steroid hormone action on the genome-

Steroid hormones modulate the metabolism, differentiation, and growth

of a large number of cell types in eukaryotic organisms. It is generally
assumed that steroid hormones control some of these processes by
regulating the transcription of specific genes. The processes that are
controlled by a steroid hormone in a particular cell type constitute the
domain of response which is generally directed toward a limited number
of cellular functions. Steroid hormones produce many of their
physiological responses by altering the amounts of specific gene
products in target cells. In general, steroid hormones regulate the levels
of specific proteins by altering their rates of synthesis. Since steroid
hormones alter a small fraction of the physiological properties of target
cells, and since these properties are dependent, in part, on the nature of
the hormone-dependent proteins synthesized, it follows that the synthetic
rates of only a small subset of total cell proteins should be selectively
altered by hormonal treatment.

The transcription of DNA to messenger RNA (mRNA) is the most

important process regulated by steroid hormones. All genes share a
common basic design, composed of a structural region in which the
DNA encodes the specific amino acids of the protein and regulatory
region that interacts with various proteins to control the rate of
transcription. Several key elements in the regulatory region of the target
gene must be activated before mRNA synthesis can occur. These
elements, called cis-acting elements because they are located on the
same DNA as the gene itself, are usually located near the 5' end
(beginning) of the gene and consist of four main groups: promoters,
hormone-responsive enhancers, silencers, and hormone-independent
enhancers. The promoter, essential for gene activation, sets the basal rate
of transcription and controls the accuracy of transcription initiation. The
promoter is located closest to the transcription start site and consists of
two sub-elements: the TATA box and the upstream promoter. Located
further upstream are one or more Hormone Response Elements (HREs),
the specific DNA-binding sites to which steroid receptors bind, and
conferring hormone sensitivity to the gene. Silencers are elements that
inhibit transcription of adjacent genes in the absence of hormone
activation. Hormone-independent enhancers are DNA sequences bound
by other transcription factors that can further increase the rate of gene
expression.The synergistic interaction between regulatory cis-acting
elements permits fine tuning of the rates of transcription of target genes
in response to the local cellular and hormonal milieu.
Mechanism of action of Non-Steroidal hormone:

Protein and peptide hormones, catecholamines like epinephrine, and

eicosanoids such as prostaglandins find their receptors decorating the
plasma membrane of target cells.

Nonsteroid hormones (water soluble) are made of amino acids. They are
not fat soluble, so they cannot diffuse across the plasma membrane of
target cells so they do not enter the cell but bind to plasma membrane
receptors, generating a chemical signal (second messenger) inside the
target cell. Five different second messenger chemicals, including cyclic
AMP have been identified. Second messengers activate other
intracellular chemicals to produce the target cell response.

Binding of hormone to receptor initiates a series of events which leads to

generation of so-called second messengers within the cell (the hormone
is the first messenger). The second messengers then trigger a series of
molecular interactions that alter the physiologic state of the cell. Another
term used to describe this entire process is signal transduction.
Structure of Cell Surface Receptors -

Cell surface receptors are integral membrane proteins and, as such, have
regions that contribute to three basic domains:

• Extracellular domains: Some of the residues exposed to the

outside of the cell interact with and bind the hormone - another
term for these regions is the ligand-binding domain.
• Transmembrane domains: Hydrophobic stretches of amino acids
are "comfortable" in the lipid bilayer and serve to anchor the
receptor in the membrane.
• Cytoplasmic or intracellular domains: Tails or loops of the
receptor that are within the cytoplasm react to hormone binding by
interacting in some way with other molecules, leading to
generation of second messengers. Cytoplasmic residues of the
receptor are thus the effector region of the molecule.

Several distinctive variations in receptor structure have been identified.

As depicted below, some receptors are simple, single-pass proteins;
many growth factor receptors take this form. Others, such as the receptor
for insulin, have more than one subunit. Another class, which includes
the beta-adrenergic receptor, is threaded through the membrane seven
times.
Receptor molecules are neither isolated by themselves nor fixed in one
location of the plasma membrane. In some cases, other integral
membrane proteins interact with the receptor to modulate its activity.
Some types of receptors cluster together in the membrane after binding
hormone. Finally, as elaborated below, interaction of the hormone-
bound receptor with other membrane or cytoplasmic proteins is the key
to generation of second messengers and transduction of the hormonal
signal.

Second Messenger Systems –

Currently, four second messenger systems are recognized in cells, as
summarized in the table below. Note that not only do multiple hormones
utilize the same second messenger system, but a single hormone can
utilize more than one system. Understanding how cells integrate signals
from several hormones into a coherent biological response remains a
challenge.

Examples of Hormones Which Utilize This

Second Messenger
System
Epinephrine and
norepinephrine, glucagon, luteinizing hormone,
Cyclic AMP follicle stimulating hormone, thyroid-stimulating
hormone, calcitonin, parathyroid
hormone, antidiuretic hormone
Insulin, growth
Protein kinase
hormone, prolactin, oxytocin, erythropoietin,
activity
several growth factors
Epinephrine and norepinephrine, angiotensin
Calcium and/or
II, antidiuretic hormone, gonadotropin-releasing
phosphoinositides
hormone, thyroid-releasing hormone.
Cyclic GMP Atrial naturetic hormone, nitric oxide
In all cases, the seemingly small signal generated by hormone binding
its receptor is amplified within the cell into a cascade of actions that
changes the cell's physiologic state.

Cyclic AMP Second Messenger Systems

Cyclic adenosine monophosphate (cAMP) is a nucleotide generated

from ATP through the action of the enzyme adenylate cyclase. The
intracellular concentration of cAMP is increased or decreased by a
variety of hormones and such fluctuations affect a variety of cellular
processes. One prominent and important effect of elevated
concentrations of cAMP is activation of a cAMP-dependent protein
kinase called protein kinase A.

Protein kinase A is nominally in a catalytically-inactive state, but

becomes active when it binds cAMP. Upon activation, protein kinase A
phosphorylates a number of other proteins, many of which are
themselves enzymes that are either activated or suppressed by being
phosphorylated. Such changes in enzymatic activity within the cell
clearly alter its state.

Now, let's put this information together to understand the mechanism of

action of a hormone like glucagon:

• Glucagon binds its receptor in the plasma membrane of target cells

(e.g. hepatocytes).
• Bound receptor interacts with and, through a set of G proteins,
turns on adenylate cyclase, which is also an integral membrane
protein.
• Activated adenylate cyclase begins to convert ATP to cyclic AMP,
resulting in an elevated intracellular concentration of cAMP.
• High levels of cAMP in the cytosol make it probable that protein
kinase A will be bound by cAMP and therefore catalytically active.
 • Active protein kinase A "runs around the cell" adding phosphates
to other enzymes, thereby changing their conformation and
modulating their catalytic activity.
• Levels of cAMP decrease due to destruction by cAMP-
phosphodiesterase and the inactivation of adenylate cyclase.

In the above example, the hormone's action was to modify the activity of
pre-existing components in the cell. Elevations in cAMP also have
important effects on transcription of certain genes.

Tyrosine Kinase Second Messenger Systems

The receptors for several protein hormones are themselves protein

kinases which are switched on by binding of hormone. The kinase
activity associated with such receptors results in phosphorylation of
tyrosine residues on other proteins. Insulin is an example of a hormone
whose receptor is a tyrosine kinase.
The hormone binds to domains exposed on the cell's surface, resulting in
a conformational change that activates kinase domains located in the
cytoplasmic regions of the receptor. In many cases, the receptor
phosphorylates itself as part of the kinase activation process. The
activated receptor phosphorylates a variety of intracellular targets, many
of which are enzymes that become activated or are inactivated upon
phosphorylation.

As was seen with cAMP second messenger systems, activation of

receptor tyrosine kinases leads to rapid modulation in a number of target
proteins within the cell. Interestingly, some of the targets of receptor
kinases are protein phosphatases which, upon activation by receptor
tyrosine kinase, become competent to remove phosphates from other
proteins and alter their activity. Again, a seemingly small change due to
hormone binding is amplified into a multitude of effects within the cell.

In some cases, binding of hormone to a surface receptor induces a

tyrosine kinase cascade even through the receptor is not itself a tyrosine
kinase. The growth hormone receptor is one example of such a system -
the interaction of growth hormone with its receptor leads to activation of
cytoplasmic tyrosine kinases, with results conceptually similar to that
seen with receptor kinases.
Calcium and/or phosphoinositides pathway –

Inositol trisphosphate or inositol 1,4,5 trisphosphate abbreviated as IP3 is

an inositol phosphate signaling molecule. It is made by hydrolysis of
phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid that is
located in the plasma membrane, by phospholipase C (PLC).Together
with diacylglycerol (DAG), IP3 is a second messenger molecule used
in signal transduction in biological cells. While DAG stays inside the
membrane, IP3 is soluble and diffuses through the cell, where it binds
to its receptor, which is a calcium channel located in the endoplasmic
reticulum. When IP3 binds its receptor, calcium is released into the
cytosol, thereby activating various calcium regulated intracellular
signals.

When a ligand (hormone) binds to a G protein-coupled receptor (GPCR)

that is coupled to a Gq heterotrimeric G protein, the α-subunit of Gq can
bind to and induce activity in the PLC isozyme PLC-β, which results in
the cleavage of PIP2 into IP3 and DAG. If a receptor tyrosine
kinase (RTK) is involved in activating the pathway, the isozyme PLC-γ
has tyrosine residues that can become phosphorylated upon activation of
an RTK, and this will activate PLC-γ and allow it to cleave PIP2 into
DAG and IP3. This occurs in cells that are capable of responding
to growth factors such as insulin, because the growth factors are the
ligands responsible for activating the RTK.
IP3 is a soluble molecule and is capable of diffusing through the
cytoplasm to the ER, or the sarcoplasmic reticulum (SR) in the case
of muscle cells, once it has been produced by the action of PLC. Once at
the ER, IP3 is able to bind to the IP3 receptor on a ligand-gated
Ca2+ channel that is found on the surface of the ER. The binding of
IP3 (the ligand in this case) to IP3 receptor triggers the opening of the
Ca2+ channel, and thus release of Ca2+ into the cytoplasm. In heart
muscle cells this increase in Ca2+ activates the ryanodine receptor-
operated channel on the SR, results in further increases in Ca 2+ through a
process known as calcium-induced calcium release. IP3 may also
activate Ca2+ channels on the cell membrane indirectly, by increasing the
intracellular Ca2+ concentration.

Cyclic GMP pathway –

Cyclic guanosine monophosphate (cGMP) is a cyclic
nucleotide derived from guanosine triphosphate (GTP). cGMP acts as
a second messenger much like cyclic AMP. Its most likely mechanism
of action is activation of intracellular protein kinases in response to the
binding of membrane-impermeable peptide hormones to the external cell
surface.Guanylate cyclase (GC) catalyzes cGMP synthesis. This enzyme
converts GTP to cGMP. Peptide hormones such as the atrial natriuretic
factor activate membrane-bound GC.
Once produced cGMP can have a number of effects in cells, but many of
those effects are mediated through the activation of protein kinase G
(PKG). cGMP binds to sites on the regulatory units of PKG and
activates the catalytic units, enabling them to phosphorylate their
substrates. Unlike with the activation of some other protein kinases,
notably PKA, the PKG is activated but the catalytic and regulatory units
do not disassociate.
Activation of PKG by cGMP leads to activation of myosin phosphatase
which in turn leads to release of calcium from intracellular stores in
smooth muscle cells. This in turn leads to relaxation of the smooth
muscle cells. In the case of vasodilation the Nitric Oxide is originally
produced in the neighbouring endothelial cells before diffusing into the
smooth muscle cells where it actiavtes sGC and cGMP production.
PKG can also have other effects in cells, for example by activating a
number of transcription factors which can lead to changes in gene
expression which in turn can alter the response of the cell to a variety
of stimuli. cGMP can also be converted back to GTP by proteins
known as phosphodiesterases. cGMP is involved in the regulation of
some protein-dependent kinases. For example, PKG (protein kinase G)
is a dimer consisting of one catalytic and one regulatory unit, with the
regulatory units blocking the active sites of the catalytic units.

Homeostatic Process

The goal of homeostasis is the maintenance of equilibrium around a

point or value called a set point. While there are normal fluctuations
from the set point, the body’s systems will usually attempt to go back to
this point. A change in the internal or external environment is called a
stimulus and is detected by a receptor; the response of the system is to
adjust the deviation parameter toward the set point. For instance, if the
body becomes too warm, adjustments are made to cool the animal. If the
blood’s glucose rises after a meal, adjustments are made to lower the
blood glucose level by getting the nutrient into tissues that need it or to
store it for later use.

Calcium homeostasis in mammals:

The concentration of calcium in the blood of mammals is ~10 mg/dL,
with some variation due to species, age, dietary intake, and analytic
method. Calcium in plasma or serum exists in three forms or fractions:
1) Protein-bound calcium accounts for approximately one-third of the
total serum calcium concentration. Protein-bound calcium cannot
diffuse through membranes and thus is not usable by tissues. 2) Ionized
or free calcium is the physiologically active form that accounts for
50%–60% of total calcium concentration. 3) Complexed or chelated
calcium is bound to phosphate, bicarbonate, sulfate, citrate, and lactate
and accounts for ~10% of the total calcium concentration.
The calcium ion is an essential structural component of the skeleton
and plays a key role in muscle contraction, blood coagulation, enzyme
activity, neural excitability, secondary messengers, hormone release,
and membrane permeability. Precise control of calcium ion in
extracellular fluids is vital to health. Three major hormones (PTH,
vitamin D, and calcitonin) interact to maintain a constant concentration
of calcium, despite variations in intake and excretion. Other hormones,
such as adrenal corticosteroids, estrogens, thyroxine, somatotropin,
and glucagon, may also contribute to the maintenance of calcium
homeostasis. Calcium homeostasis in mammals, as in other tetrapods,
appears to be dominated by the fact that these animals rely on their food
and drink for the calcium they require, and that they eat and drink only
at intervals. As such, they have but intermittent access to calcium and
this poses two problems. Firstly, during absorption of ingested calcium
the animal must limit the postprandial hypercalcemia which could result
from excessively rapid calcium absorption. Secondly, as ingestion of
calcium is intermittent, there must be some premium placed on calcium
storage during those times when calcium is available in order to provide
for those periods when external calcium is not available.
In mammals, the regulatory requirements are met predominantly by the
interaction of three hormones, the vitamin D3 complex, parathyroid
hormone (PTH) and calcitonin, which act primarily on three target
organs: intestinal mucosa, kidney and bone.

The most common perturbation to calcium homeostasis in mammals, as

in all tetrapods, is the absorption of ingested calcium from the intestine.
Although the rate at which this occurs is under the control of vitamin D
dependent uptake mechanisms, the potential exists for the development
of elevated plasma calcium levels. This is, however, attenuated by the
action of two hormones. As the plasma calcium levels rise, the release of
PTH is reduced with a consequent reduction in active bone
calcium mobilization. Also, following rising calcium levels, calcitonin is
released and inhibits the action of PTH on osteoclastic activity and
osteocytic osteolysis, enhancing the rate at which the action of PTH is
inhibited. As a consequence, the movement of calcium from the blood
predominates over the reverse movement and favors the storage of the
absorbed calcium in bone. Between periods of intestinal calcium
absorption, previously stored calcium is mobilized by the stimulatory
action of PTH on bone resorbing cells. Additionally, PTH enhances
renal retention of calcium and at the same time seems to stimulate the
production of 1,25-dihydroxy vitamin D3, in order to prepare the
intestinal mucosa for the next calcium load or to increase the efficiency
of the intestinal mucosa to extract calcium from the intestinal contents.

Parathyroid hormone –

PTH is synthesized and stored in the chief cells of the parathyroid

glands. Synthesis is regulated by a feedback mechanism involving the
level of blood calcium (and, to a lesser degree, that of magnesium). In
addition, biological amines, peptides, steroids, and several classes of
drugs can influence PTH secretion.
The primary function of PTH is to control calcium concentration in the
extracellular fluid, which it does by affecting the rate of transfer of
calcium into and out of bone, resorption in the kidneys, and absorption
from the GI tract. The effect on the kidneys is the most rapid, causing
reabsorption of calcium and excretion of phosphorus. The major initial
effect on bone is to mobilize calcium from the bone to the extracellular
fluid; later, bone formation may be enhanced. PTH does not directly
affect calcium absorption from the gut. Its effect is mediated indirectly
by regulation of synthesis of the active metabolite of vitamin D.
Vitamin D –

The second major hormone involved in the regulation of calcium

metabolism and skeletal remodeling is vitamin D, which
includes cholecalciferol (vitamin D3) of animal origin, as well
as ergocalciferol (vitamin D2) of plant origin. Vitamin D has long been
considered an essential dietary ingredient, but in several species,
including sheep, cattle, horses, pigs, and people, vitamin D can be
formed in the skin from a cholesterol metabolite (7-dehydrocholesterol)
after exposure to ultraviolet light. In contrast, dogs and cats are not able
to synthesize vitamin D3 adequately in the skin and mainly depend on
dietary intake.
Vitamin D must be metabolically activated before it can function
physiologically. The biologic actions of vitamin D depend on
hydroxylation in the liver and kidneys to form the biologically active
1,25-dihydroxyvitamin D (calcitriol). This conversion in the kidneys is
the rate-limiting step in vitamin D metabolism, and it is partly
responsible for the delay between vitamin D administration and
expression of its biologic effects. PTH and conditions that stimulate its
secretion, as well as hypophosphatemia, increase the formation of the
active vitamin D metabolite. High circulating phosphorus
concentrations have the opposite effect. Under certain conditions,
prolactin, estradiol, placental lactogen, and possibly somatotropin have
a similar enhancing effect. Increased secretion of these hormones,
either alone or in combination, appears to be important in the efficient
adaptation to the major calcium demands of pregnancy, lactation, and
growth.

Calcitonin –

Calcitonin is a 32-amino acid polypeptide hormone secreted by the

parafollicular cells (C-cells) of the thyroid gland in mammals and by
ultimobranchial tissue in avian and other nonmammalian species. The
concentration of calcium ion in extracellular fluids is the principal
stimulus for the secretion of calcitonin by C-cells. In hypercalcemia,
the rate of secretion of calcitonin is increased greatly by rapid
discharge of stored hormone from C-cells into interfollicular
capillaries. Hyperplasia of C-cells occurs in response to longterm
hypercalcemia. When blood calcium is lowered, the stimulus
for calcitonin secretion is diminished. The storage of large amounts of
preformed hormone in C-cells and rapid release in response to a
moderate rise in circulating calcium probably reflect the physiologic
role of calcitonin as an “emergency” hormone to protect against
development of hypercalcemia.

Calcitonin exerts its effects by interacting with target cells, primarily in

bone and kidney. The actions of PTH and calcitonin are antagonistic on
bone resorption but synergistic on decreasing the renal tubular
reabsorption of phosphorus. The hypocalcemic effects of calcitonin are
primarily the result of decreased entry of calcium from the skeleton
into plasma, resulting from a temporary inhibition of PTH-stimulated
bone resorption. The hypophosphatemia develops from a direct action
of calcitonin, which increases the rate of movement of phosphorus out
of plasma into soft tissue and bone and inhibits the bone resorption
stimulated by PTH and other factors. Although many effects have been
attributed to calcitonin at pharmacologic doses, their physiologic
relevance is suspect. Physiologically, calcitonin has at best a minor role
in regulating blood concentrations of calcium. Neither chronically high
(eg, as in animals with medullary thyroid cancer) nor chronically low
(eg, as in animals after surgical removal of the thyroid gland)
circulating calcitonin concentrations result in any changes in the serum
calcium concentration.

Glucose homeostasis in mammals:

Glucose is the main source of fuel for the cells in human body and its
normal homeostasis is the pre-requisite for maintaining health. Blood
sugar levels are tightly regulated and maintained within a narrow range
by interplay of hormones - insulin and glucagon. In normal course,
excess glucose gets stored in the liver as glycogen and if the levels are
more it gets converted into fat and gets stored in liver as well as in
adipose tissues. However, as more and more glucose is pumped into the
system through high intake of caloric food, insulin keeps pace with it for
some time, but at some point, the cells become resistant to insulin
resulting in pre-diabetes and ultimately diabetes and excess weight.
There are vegetative controls in the central nervous system, which keep
the food intake and energy expenditure in check, so that energy reserves
remain constant and thus the body weight as well. When the fat cells
increase their fat storage, the adipose tissue releases leptin, the
circulating satiety hormone, which signals the hypothalamus to regulate
the food intake and inhibition of fatty acid synthesis. Abnormality in
glucose and energy homeostasis can exist independent of each other, or
these can co-exist as well.
Blood glucose concentration is the net result of the difference between
rates of glucose entry and removal from the circulation. Glucose
homeostasis is maintained by a feedback mechanism designed to keep
blood glucose levels close to a set point characteristic for each species.
In vertebrates blood glucose levels are positively correlated to metabolic
rate and display an allometric relationship with body weight. Key to the
homeostatic control of glucose is the existence of sensors located in
different parts of the body that continuously monitor blood glucose
variations. The molecular basis of glucodetection is relatively well
understood in mammalian pancreatic β-cells and neurons excited by
glucose (GE) found in areas of the brain such as the septum, amygdale,
striatum, motor cortex, hindbrain and hypothalamus.

All mammalian cells use glucose as a fuel for their basic functions.
Interestingly, despite the fluctuations in food consumption and activity
level throughout the course of a day, most mammals maintain stable
blood glucose levels. Blood glucose levels are determined by relative
rates of glucose entry into the blood and uptake into the tissues. Hence,
stable glucose levels are the result of whole-body glucose production
that matches whole-body glucose use.

Glucosensing –
The body continuously adjusts its metabolism to keep blood
glucose concentrations at a constant value. Glucose homeostasis in
humans and most other studied mammals is maintained by a
feedback mechanism designed to keep the blood glucose close to a
set point characteristic for each species (see above). Key to this
homeostatic control is the existence of sensors located in different
parts of the body that continuously monitor blood glucose variations.
They respond to changes in glycemia by triggering hormonal
secretion or activation of the autonomic nervous system to control
glucose uptake, utilization or production and also to control energy
expenditure and food intake. The molecular basis of glucodetection is
relatively well understood for insulin secretion by pancreatic β-cells.
Apart from the classical glucosensor in pancreatic β-cells, glucosensing
cells have been found in peripheral locations such as the L-cell of
the intestine (Reimann et al., 2008), glucose-inhibited α-cells (Rorsman
et al., 2008), hepatoportal vein (Donovan, 2002), liver (Magnuson and
Matschinsky, 2004) and carotid body (Pardal and López-Barneo, 2002),
as well as in central locations within the brain where glucosensing
neurons have been located to areas such as the septum, amygdala,
striatum, motor cortex, hindbrain and hypothalamus (Levin et al.,
2004c; Moran, 2010).

The Pancreas –
The pancreas has key roles in the regulation of macronutrient digestion
and hence metabolism/energy homeostasis by releasing various digestive
enzymes and pancreatic hormones. It is located behind the stomach
within the left upper abdominal cavity and is partitioned into head, body
and tail. The majority of this secretory organ consists of acinar—or
exocrine—cells that secrete the pancreatic juice containing digestive
enzymes, such as amylase, pancreatic lipase and trypsinogen, into the
ducts, that is, the main pancreatic and the accessory pancreatic duct. In
contrast, pancreatic hormones are released in an endocrine manner, that
is, direct secretion into the blood stream. The endocrine cells are
clustered together, thereby forming the so-called islets of Langerhans,
which are small, island-like structures within the exocrine pancreatic
tissue.
There are five different cell types releasing various hormones from the
endocrine system: glucagon-producing α-cells ; insulin, amylin and C-
peptide -producing β-cells; pancreatic polypeptide (PP)-producing γ-
cells; somatostatin-producing δ-cells ; and ghrelin-producing ɛ-cells.
Each of the hormones has distinct functions. Glucagon increases blood
glucose levels, whereas insulin decreases them. Somatostatin inhibits
both, glucagon and insulin release, whereas PP regulates the exocrine
and endocrine secretion activity of the pancreas. These hormones
regulate glucose homeostasis in vertebrates. Although the islets have a
similar cellular composition among different species, that is, human, rat
and mouse, their cytoarchitecture differs greatly. Although islets in
rodents are primarily composed of β-cells located in the center with
other cell types in the periphery, human islets exhibit interconnected α-
and β-cells.
Through its various hormones, particularly glucagon and insulin, the
pancreas maintains blood glucose levels within a very narrow range of
4–6 mM. This preservation is accomplished by the opposing and
balanced actions of glucagon and insulin, referred to as glucose
homeostasis. During sleep or in between meals, when blood glucose
levels are low, glucagon is released from α-cells to promote hepatic
glycogenolysis. In addition, glucagon drives hepatic and renal
gluconeogenesis to increase endogenous blood glucose levels during
prolonged fasting. In contrast, insulin secretion from β-cells is
stimulated by elevated exogenous glucose levels, such as those
occurring after a meal. After docking to its receptor on muscle and
adipose tissue, insulin enables the insulin-dependent uptake of glucose
into these tissues and hence lowers blood glucose levels by removing the
exogenous glucose from the blood stream. Furthermore, insulin
promotes glycogenesis, lipogenesis and the incorporation of amino acids
into proteins; thus, it is an anabolic hormone, in contrast to the catabolic
activity of glucagon.

Glucagon -
Glucagon is a peptide hormone, produced by alpha cells of the pancreas.
It works to raise the concentration of glucose and fatty acids in the
bloodstream, and is considered to be the main catabolic hormone of the
body.[3] It is also used as a medication to treat a number of health
conditions. Its effect is opposite to that of insulin, which lowers
extracellular glucose. When the blood glucose level falls to dangerously
low levels (as during very heavy exercise or lack of food for extended
periods), the alpha cells of the pancreas release glucagon,
a hormone which travels through the blood to the liver, where it binds
to glucagon receptors on the surface of liver cells and stimulates them to
break down glycogen stored inside the cells into glucose (this process is
called glycogenolysis). The cells release the glucose into the
bloodstream, increasing blood sugar levels. Hypoglycemia, the state of
having low blood sugar, is treated by restoring the blood glucose level to
normal by the ingestion or administration
of dextrose or carbohydrate foods. It is often self-diagnosed and self-
medicated orally by the ingestion of balanced meals. In more severe
circumstances, it is treated by injection or infusion of glucagon.
Glucagon generally elevates the concentration of glucose in the blood by
promoting gluconeogenesis and glycogenolysis. Glucagon also
decreases fatty acid synthesis in adipose tissue and the liver, as well as
promoting lipolysis in these tissues, which causes them to release fatty
acids into circulation where they can be catabolised to generate energy
in tissues such as skeletal muscle when required.
Glucose is stored in the liver in the form of the polysaccharide glycogen,
which is a glucan (a polymer made up of glucose molecules). Liver cells
(hepatocytes) have glucagon receptors. When glucagon binds to the
glucagon receptors, the liver cells convert the glycogen into individual
glucose molecules and release them into the bloodstream, in a process
known as glycogenolysis. As these stores become depleted, glucagon
then encourages the liver and kidney to synthesize additional glucose
by gluconeogenesis. Glucagon turns off glycolysis in the liver, causing
glycolytic intermediates to be shuttled to gluconeogenesis.
Glucagon also regulates the rate of glucose production through lipolysis.
Glucagon induces lipolysis in humans under conditions of insulin
suppression (such as diabetes mellitus type 1).

Insulin -
Insulin is a peptide hormone produced by beta cells of the pancreatic
islets; it is considered to be the main anabolic hormone of the body. It
regulates the metabolism of carbohydrates, fats and protein by
promoting the absorption of glucose from the blood
into liver, fat and skeletal muscle cells. When levels of blood sugar rise,
whether as a result of glycogen conversion, or from digestion of a meal,
a different hormone is released from beta cells found in the islets of
Langerhans in the pancreas. This hormone, insulin, causes the liver to
convert more glucose into glycogen (this process is called glycogenesis),
and to force about 2/3 of body cells (primarily muscle and fat tissue
cells) to take up glucose from the blood through, thus decreasing blood
sugar. When insulin binds to the receptors on the cell surface, enabling a
facilitated diffusion of glucose into the cell. As soon as the glucose
enters the cell, it is phosphorylated into Glucose-6-Phosphate in order to
preserve the concentration gradient so glucose will continue to enter the
cell. Insulin also provides signals to several other body systems, and is
the chief regulator of metabolic control in humans.
There are also several other causes for an increase in blood sugar levels.
Among them are the 'stress' hormones such as epinephrine (also known
as adrenaline), several of the steroids, infections, trauma, and of course,
the ingestion of food.
Diabetes mellitus type 1 is caused by insufficient or non-existent
production of insulin, while type 2 is primarily due to a decreased
response to insulin in the tissues of the body (insulin resistance). Both
types of diabetes, if untreated, result in too much glucose remaining in
the blood (hyperglycemia) and many of the same complications. Also,
too much insulin and/or exercise without enough corresponding food
intake in diabetics can result in low blood sugar (hypoglycemia).
The role of insulin in glucose level regulation include –
• Increase of cellular intake of certain substances, most prominently
glucose in muscle and adipose tissue
• Stimulates the uptake of glucose – Insulin decreases blood glucose
concentration by inducing intake of glucose by the cells. This is
possible because Insulin causes the insertion of the GLUT4
transporter in the cell membranes of muscle and fat tissues which
allows glucose to enter the cell.
• Increased fat synthesis – insulin forces fat cells to take in blood
glucose, which is converted into triglycerides; decrease of insulin
causes the reverse.
• Induce glycogen synthesis – When glucose levels are high, insulin
induces the formation of glycogen by the activation of the
hexokinase enzyme, which adds a phosphate group in glucose, thus
resulting in a molecule that cannot exit the cell. At the same time,
 insulin inhibits the enzyme glucose-6-phosphatase, which removes
the phosphate group. These two enzymes are key for the formation
of glycogen. Also, insulin activates the enzymes
phosphofructokinase and glycogen synthase which are responsible
for glycogen synthesis.
• Decreased gluconeogenesis and glycogenolysis – decreases
production of glucose from noncarbohydrate substrates, primarily
in the liver (the vast majority of endogenous insulin arriving at the
liver never leaves the liver); decrease of insulin causes glucose
production by the liver from assorted substrates.

Cortisol –
Cortisol is a steroid hormone, in the glucocorticoid class of hormones.
When used as a medication, it is known as hydrocortisone.
It is produced in many animals, mainly by the zona fasciculata of
the adrenal cortex in the adrenal gland. It is produced in other tissues in
lower quantities. It is released with a diurnal cycle and its release is
increased in response to stress and low blood-glucose concentration. It
functions to increase blood sugar through gluconeogenesis, to suppress
the immune system, and to aid in the metabolism of fat, protein,
and carbohydrates. It also decreases bone formation.
In general, cortisol stimulates gluconeogenesis (the synthesis of 'new'
glucose from non-carbohydrate sources, which occurs mainly in
the liver, but also in the kidneys and small intestine under certain
circumstances). The net effect is an increase in the concentration of
glucose in the blood, further complemented by a decrease in the
sensitivity of peripheral tissue to insulin, thus preventing this tissue from
taking the glucose from the blood. Cortisol has a permissive effect on
the actions of hormones that increase glucose production, such
as glucagon and adrenaline.
Cortisol also plays an important, but indirect, role in liver and
muscle glycogenolysis (the breaking down of glycogen to glucose-1-
phosphate and glucose) which occurs as a result of the action of
glucagon and adrenalin. Additionally, cortisol facilitates the activation
of glycogen phosphorylase, which is necessary for adrenaline to have an
effect on glycogenolysis. Paradoxically, cortisol promotes not only
gluconeogenesis in the liver, but also glycogenesis. Cortisol is thus
better thought of as stimulating glucose/glycogen turnover in the liver.
This is in contrast to cortisol's effect in the skeletal muscle where
glycogenolysis is promoted indirectly through catecholamines.

Cortisol counteracts insulin, contributes to hyperglycemia by

stimulating gluconeogenesis and inhibits the peripheral use of glucose
(insulin resistance) by decreasing the translocation of glucose
transporters (especially GLUT4) to the cell membrane. Cortisol also
increases glycogen synthesis (glycogenesis) in the liver, storing glucose
in easily accessible form. The permissive effect of cortisol on insulin
action in liver glycogenesis is observed in hepatocyte culture in the
laboratory, although the mechanism for this is unknown.
Estrous cycle in Rat:
The estrous cycle or oestrous cycle is the set of recurring physiological
changes that are induced by reproductive hormones in
most mammalian therian females. Estrous cycles start after sexual
maturity in females and are interrupted by anestrous phases or
by pregnancies. Typically, estrous cycles repeat until death. Some
animals may display bloody vaginal discharge, often mistaken
for menstruation.
The reproductive cycle of female rats is called estrous cycle and is
characterized as proestrus, estrus, metestrus (or diestrus I) and diestrus
(or diestrus II). The ovulation occurs from the beginning of proestrus to
the end of estrus. From the onset of sexual maturity up to the age of 12
months, the mean cycle length in the female rat is 4 days, and this short
cycle length makes the rat an ideal animal for investigation of changes
occurring during the reproductive cycle. During the estrous cycle,
prolactin, LH and FSH remain low and increase in the afternoon of the
proestrus phase. Estradiol levels begin to increase at metestrus, reaching
peak levels during proestrus and returning to baseline at estrus.
Progesterone secretion also increases during metestrus and diestrus with
a decrease afterwards. Then the progesterone value rises to reach its
second peak towards the end of proestrus.

A four-phase terminology is used in reference to animals with estrous

cycles -
Proestrus
One or several follicles of the ovary start to grow. Their number is
species-specific. Typically this phase can last as little as one day or as
long as three weeks, depending on the species. Under the influence of
estrogen, the lining of the uterus (endometrium) starts to develop. Some
animals may experience vaginal secretions that could be bloody. The
female is not yet sexually receptive; the old corpus luteum degenerates;
the uterus and the vagina distend and fill with fluid, become contractile
and secrete a sanguinous fluid; the vaginal epithelium proliferates and
the vaginal cytology shows a large number of non-cornified nucleated
epithelial cells. Variant terms for proestrus include pro-
oestrus, proestrum, and pro-oestrum.
Estrus
Estrus or oestrus refers to the phase when the female is sexually
receptive ("in heat"). Under regulation by gonadotropic
hormones, ovarian follicles mature and estrogen secretions exert their
biggest influence. The female then exhibits sexually receptive
behavior,[4] a situation that may be signaled by visible physiologic
changes. Estrus is commonly seen in the mammalian species, including
primates. This phase is sometimes called estrum or oestrum.
In some species, the labia are reddened. Ovulation may occur
spontaneously in others. Especially among quadrupeds, a signal trait of
estrus is the lordosis reflex, in which the animal spontaneously elevates
her hindquarters.
Metestrus or diestrus
This phase is characterized by the activity of the corpus luteum, which
produces progesterone. The signs of estrogen stimulation subside and
the corpus luteum starts to form. The uterine lining begins to appear. In
the absence of pregnancy the diestrus phase (also termed pseudo-
pregnancy) terminates with the regression of the corpus luteum. The
lining in the uterus is not shed, but is reorganized for the next cycle.
Anestrus
Anestrus refers to the phase when the sexual cycle rests. This is typically
a seasonal event and controlled by light exposure through the pineal
gland that releases melatonin. Melatonin may repress stimulation of
reproduction in long-day breeders and stimulate reproduction in short-
day breeders. Melatonin is thought to act by regulating
the hypothalamic pulse activity of the gonadotropin-releasing hormone.
Anestrus is induced by time of year, pregnancy, lactation,
significant illness, chronic energy deficit, and possibly age. Chronic
exposure to anabolic steroids may also induce a persistent anestrus due
to negative feedback on the hypothalamus/pituitary/gonadal axis. After
completion (or abortion) of a pregnancy, some species have postpartum
estrus, which is ovulation and corpus luteum production that occurs
immediately following the birth of the young. For example,
the mouse has a fertile postpartum estrus that occurs 14 to 24 hours
following parturition.

Menstrual cycle in Human:

Menstruation describes the female period. The menstruation cycle
begins when a woman gets her periods. The menstrual blood which
leaves her body are products shed from the uterus (the uterine lining also
called the endometrium). During the remainder of the menstrual cycle
the uterine lining regrows. It does so in preparation for pregnancy, which
occurs if the egg (oocyte) a woman releases about half way through her
menstrual cycle is fertilised. When fertilisation occurs, the lining stays in
place to nourish the fertilised egg. When fertilisation does not occur the
menstrual cycle continues and the uterine lining is shed marking the start
of the woman’s next menstrual period. Women begin menstruation at an
average age of 13 (called menarche) and on average continue
menstruating till age 51 (called menopause).
Menstruation involves highly complex hormonal interactions. The
key hormones involved in menstruation are oestrogen and progesterone
(produced by the ovaries) and luteinising hormone and follicle
stimulating produced by the pituitary gland, under the influence of
hormones secreted by the hypothalamus. The interactions between these
organs are referred to as the hypothalamic-pituitary-ovarian axis (HPO
axis).

Phases of the menstrual cycle -

The menstruation cycle refers to the cycles in which a woman’s uterus

grows and sheds a lining (the endometrium) which could support the
development of a fertilised egg. It typically occurs in 28 day cycles, so a
woman generally gets her period every 28 days. However, cycle length
may be as short as 21 days or as long as 40 days in some women. The
inner lining of the uterus (the endometrium) goes through three phases
during the typically 28 day menstrual cycle: the menstrual phase (days
1-5), the proliferative phase (days 6-14) and the secretory phase (days
15-28).

The ovarian cycle, refers to the cycle in which a woman’s ovaries

prepare an egg to be released during ovulation. It is divided into two
phases: the follicular phase (days 1-14) and the luteal phase (days 15-
28), during which different levels of hormones are released. These two
cycle occur in a synchronised manner; day 1 of the ovarian cycle is
always also day 1 of the menstrual cycle.

Hormonal regulation of menstrual cycle -

Gonadotrophin releasing hormone- Release of this hormone is

responsible for the stimulation of specific cells called gonadotrophs in
the pituitary gland. This stimulation results in the production of two
important hormones called luteinising hormone (LH) and follicular
stimulating hormone (FSH) from the pituitary. Gonadotropin Releasing
Hormone (GnRH) is of great importance in the menstrual cycle. One of
the most important features of GnRH release is the fact that its release
occurs in a pulsatile fashion. At the start of puberty there is a marked
increase in the frequency and amplitude of GnRH release.
A part of the brain called the surge centre controls the timing of this
increased release of GnRH. The surge centre is present in females very
early in life, however it is only as puberty approaches that this centre
becomes more responsive to hormonal changes. Throughout the
menstrual cycle there is pulsatile release of GnRH. Anything that
interferes with the pulse frequency of GnRH can stop the menstrual
cycle from occurring. Restoration of this pulsatile GnRH by
administering hormones can produce a return to ovulation.
The pituitary gland -The pituitary gland is an out pouching of the base
of the brain which lies under the hypothalamus. The close proximity of
these two parts of the brain is a reflection of their closely linked
function. The pituitary gland is divided into two different parts, each of
which have different functions. The anterior pituitary is responsible for
housing the gonadotrophs, these are the cells that release hormones
important in controlling the menstrual cycle.
The anterior pituitary gland is composed of six different cell types and
produces six different hormones. The cell type that is of importance in
menstruation is the gonadotroph. These cells release follicle stimulating
hormone (FSH) and luteinising hormone (LH) and are also responsible
for production and storage of these hormones.

FSH
The granulosa in the ovaries are the main target for the action of FSH. In
response to FSH stimulation the granulosa cells release oestrogen. The
combined effect of oestrogen and FSH is to cause growth and increased
oestrogen production.

LH
LH stimulates cells in the ovary, called the theca cells, to produce
hormones called androgens which are then transported to the granulosa
cells in the ovary for conversion into oestrogens.

Gonadotrophin secretory patterns

The normal ovulatory cycle is divided into two phases called the
follicular and luteal phases.

• Follicular phase: is initiated from the day bleeding stops and

finishes with a mid-cycle surge of LH.
• Luteal phase: this is initiated with the mid-cycle surge of LH
which coincides with ovulation and ends with the first day of onset
of the period.
Ovaries
The female ovaries are paired, flat, elliptical structures which measure
approximately 5cm in diameter. The ovaries are in the abdomen and are
suspended by various ligaments. The ovary itself consists of two parts,
the outer cortex and the inner medulla. The cortex is where development
of the eggs occurs, and the medulla carries nerves and blood vessels.
Females are born with approximately 2 to 4 million primary follicles.
These fetal follicles contain a developing egg called a primary oocyte
surrounded by a layer of granulosa cells. These primary oocytes are part
way through a cell division. This process of division doesn’t resume
until the time of ovulation. With each ovarian cycle, a handful of ovarian
follicles are recruited and usually only one of these ovulates, the
remaining unrecruited follicles remain in an inactive state. Development
of follicles occurs until menopause.

Hormones in the ovarian cycle

Oestrogen
This is low at the beginning of the menstrual cycle and peaks at the
middle and then once again towards the end.

Progesterone
There is little production of this in the first half of menstruation but a
significant increase in the second half. The progesterone remains high if
pregnancy occurs. Progesterone is responsible for an increased body
temperature in pregnancy as well.

Endometrium
The endometrium is the inner layer of the uterus and is attached to the
muscle layer of the uterus. It is functionally divided into two distinct
zones. The outer part is the part that sheds during the menstrual cycle,
and the inner part contains stem cells that helps to regenerate the lost
cells.
The endometrium goes through three stages during the menstrual
cycle:
• Menstrual phase
• Proliferative phase
• Secretory phase

Menstrual phase
This phase begins with the first day of menstruation. Contraction of the
muscle layer occurs expelling the blood and endometrial cells through
the vagina. Occurs when estrogen and progesterone are at their lowest
levels.

Proliferative phase
There is estrogen mediated renewal of the endometrial tissue due to the
migration of stem cells from the inner layer. There are new blood vessels
and glands that form during this phase.

Secretory phase
Increased secretory activity by the endometrial glands is stimulated by
progesterone. The endometrial glands in this phase become more
developed. The increased secretory activity in this phase of menstruation
creates an ideal environment in the uterus for development of
an embryo.

A woman’s menstrual cycle is divided into four phases:

• Follicular phase
• Ovulation phase
• Luteal phase
• Menstrual phase
Follicular phase

The follicular phase starts on the first day of the period (so there is some
overlap with the menstrual phase) and ends when you ovulate.

It starts when the hypothalamus sends a signal to the pituitary gland to

release follicle-stimulating hormone (FSH). This hormone stimulates the
ovaries to produce around 5 to 20 small sacs called follicles. Each
follicle contains an immature egg. Only the healthiest egg will
eventually mature. (On rare occasions, a woman may have two eggs
mature.) The rest of the follicles will be reabsorbed into the body. The
maturing follicle sets off a surge in estrogen that thickens the lining of
your uterus. This creates a nutrient-rich environment for an embryo to
grow. The average follicular phase lasts for about 16 days. It can range
from 11 to 27 days, depending on your cycle.

Ovulation phase

Rising estrogen levels during the follicular phase trigger the pituitary
gland to release luteinizing hormone (LH). This is what starts the
process of ovulation. Ovulation is when the ovary releases a mature egg.
The egg travels down the fallopian tube toward the uterus to be fertilized
by sperm.

The ovulation phase is the only time during the menstrual cycle when
the person get pregnant. The manifestations of the ovulation are:

• a slight rise in basal body temperature

• thicker discharge that has the texture of egg whites
Ovulation happens at around day 14 if you have a 28-day cycle right in
the middle of the menstrual cycle. It lasts about 24 hours. After a day,
the egg will die or dissolve if it isn’t fertilized.

Luteal phase

After the follicle releases its egg it changes into the corpus luteum. This
structure releases hormones, mainly progesterone and some estrogen.
The rise in hormones keeps the uterine lining thick and ready for a
fertilized egg to implant.

If pregnant occurs, the body will produce human chorionic gonadotropin

(hCG). This is the hormone pregnancy tests detect. It helps maintain the
corpus luteum and keeps the uterine lining thick. If pregnant doesn’t
occur, the corpus luteum will shrink away and be resorbed. This leads to
decreased levels of estrogen and progesterone, which causes the onset of
the period. The uterine lining will shed during period.

Menstrual phase

This phase starts when an egg from the previous cycle isn’t fertilized.
Because pregnancy hasn’t taken place, levels of the hormones estrogen
and progesterone drop. The thickened lining of your uterus, which
would support a pregnancy, is no longer needed, so it sheds through
your vagina. During your period, you release a combination of blood,
mucus, and tissue from your uterus. Menstruation is the elimination of
the thickened lining of the uterus (endometrium) from the body through
the vagina. Menstrual fluid contains blood, cells from the lining of the
uterus (endometrial cells) and mucus. The average length of a period is
between three days and one week.
Bioasssay of hormones using RIA and ELISA:

RIA –

A radioimmunoassay (RIA) is an immunoassay that

uses radiolabeled molecules in a stepwise formation of immune
complexes. A RIA is a very sensitive in vitro assay technique used to
measure concentrations of substances, usually
measuring antigen concentrations (for example, hormone levels
in blood) by use of antibodies. Although the RIA technique is
extremely sensitive and extremely specific, requiring specialized
equipment, it remains among the least expensive methods to perform
such measurements. It requires special precautions and licensing, since
radioactive substances are used.
Radioimmunoassays (RIAs) use antibodies to detect and quantitate the
amount of antigen (analyte) in a sample. These assays are typically very
sensitive and specific. It is possible to detect as low as a few picograms
of analyte in the experimental tube when using antibodies of high
affinity. The basic principle of radioimmunoassay is competitive
binding, where a radioactive antigen ("tracer") competes with a non-
radioactive antigen for a fixed number of antibody or receptor binding
sites. When unlabeled antigen from standards or samples and a fixed
amount of tracer (labeled antigen) are allowed to react with a constant
and limiting amount of antibody, decreasing amounts of tracer are bound
to the antibody as the amount of unlabeled antigen is increased.

Classically, to perform a radioimmunoassay, a known quantity of

an antigen is made radioactive, frequently by labeling it with gamma-
radioactive isotopes of iodine, such as 125-I, attached to tyrosine. This
radiolabeled antigen is then mixed with a known amount of antibody for
that antigen, and as a result, the two specifically bind to one another.
Then, a sample of serum from a patient containing an unknown quantity
of that same antigen is added. This causes the unlabeled (or "cold")
antigen from the serum to compete with the radiolabeled antigen ("hot")
for antibody binding sites. As the concentration of "cold" antigen is
increased, more of it binds to the antibody, displacing the radiolabeled
variant, and reducing the ratio of antibody-bound radiolabeled antigen to
free radiolabeled antigen. The bound antigens are then separated and the
radioactivity of the free (unbound) antigen remaining in
the supernatant is measured using a gamma counter.
This method can be used for any biological molecule in principle and is
not restricted to serum antigens, nor is it required to use the indirect
method of measuring the free antigen instead of directly measuring the
captured antigen. For example, if it is undesirable or not possible to
radiolabel the antigen or target molecule of interest, a RIA can be done
if two different antibodies that recognize the target are available and the
target is large enough (e.g., a protein) to present multiple epitopes to the
antibodies. One antibody would be radiolabeled as above while the other
would remain unmodified. The RIA would begin with the "cold"
unlabeled antibody being allowed to interact and bind to the target
molecule in solution. Preferably, this unlabeled antibody is immobilized
in some way, such as coupled to an agarose bead, coated to a surface,
etc. Next, the "hot" radiolabeled antibody is allowed to interact with the
first antibody-target molecule complex. After extensive washing, the
direct amount of radioactive antibody bound is measured and the amount
of target molecule quantified by comparing it to a reference amount
assayed at the same time. This method is similar in principle to the non-
radioactive sandwich ELISA method.

ELISA –

The enzyme-linked immunosorbent assay (ELISA) is an

immunological assay commonly used to measure antibodies, antigens,
proteins, glycoproteins and hormones in biological samples. ELISA
assays are generally carried out in 96 well plates, allowing multiple
samples to be measured in a single experiment. These plates need to be
special absorbant plates to ensure the antibody or antigen sticks to the
surface. Each ELISA measures a specific antigen, and kits for a variety
of antigens are widely available.
The ELISA is what is known as a sandwich ELISA, here two sets of
antibodies are used to detect secreted products. The method is stepwise
in the order shown. The 1st step is to coat the ELISA plate with capture
antibody, any excess, unbound antibody is then washed from the plate.
The capture antibody is an antibody raised against the antigen of
interest.
Next the sample (e.g. urine, serum,cell supernatant, hormones) is added.
Any antigen found in the sample will bind to the capture antibody
already coating the plate. Samples are usually added in duplicate or
triplicate (to allow for statistical analysis), and in varying concentrations
to guarantee it falls within the levels of detection of the assay. Again any
excess sample is washed from the plate.
In step 3, detection antibody is added. This antibody is labelled with an
enzyme, usually horse radish peroxidase or alkaline phosphatase.
Detection antibody binds to any target antigen already bound to the
plate. Finally, a substrate is added to the plate. ELISA assays are
usually chromogenic using a reaction that converts the substrate into a
coloured product which can be measured using a plate reader.
Determination of antigen concentration in a sample requires production
of a standard curve using antigens of a known concentration. The
concentration of antigen in a sample can then be calculated using the
optical density (OD).

ELISA (enzyme-linked immunosorbent assay) is a plate-based assay

technique designed for detecting and quantifying peptides, proteins,
antibodies, and hormones. In ELISA, an antigen must be immobilized to
a solid surface and then complexed with an antibody that is linked to an
enzyme. Detection is accomplished by assessing the conjugated enzyme
activity via incubation with a substrate to produce a measurable product.
The most crucial element of the detection strategy is a highly specific
antibody-antigen interaction. ELISAs are typically performed in 96-well
(or 384-well) polystyrene plates, which will passively bind antibodies
and proteins. The binding and immobilization of reagents makes
ELISAs simple to design and perform. Having the reactants of the
ELISA immobilized to the microplate surface enables easy separation of
bound from non-bound material during the assay. This ability to wash
away non-specifically bound materials makes the ELISA a powerful tool
for measuring specific analytes within a crude preparation.