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Chapter 7: Principles of reproductive endocrinology.

CHAPTER OBJECTIVES

* Define endocrinology.

* Describe the nature of hormones.

* Describe the synthesis, secretion, transport, and actions of hormones.

* Describe how endocrine systems are regulated.

* Describe how hormones are measured.

HOMEOSTASIS

All living things have the ability to metabolize various nutrients to support vital life functions. The numerous biochemical reactions that regulate an organism's metabolism occur in an internal environment that is maintained in a constant state relative to the external environment in which an organism lives. The processes through which living bodies maintain such conditions are collectively referred to as homeostasis. In animals, homeostasis requires complex interactions among various organs. Communication among various organ systems involves the nervous and the endocrine systems. Various neuronal reflexes between sense and effector organs provide a means of communication within the nervous system. In contrast, communication within the endocrine system involves hormones, which can be thought of as extracellular chemical messengers. These systems do not operate independently; rather, they interact with each other to maintain homeostasis. Like other vital functions, the mechanisms controlling the reproductive activity of mammals are subject to homeostatic regulation involving complex interactions between nervous and endocrine tissues. We have already examined the anatomic basis for neuronal regulation of reproduction. In this chapter we will consider how hormones regulate reproductive activity. The field of study that deals with hormones is known as endocrinology.

WHAT IS ENDOCRINOLOGY?

A traditional approach to studying the regulation of reproductive tissues is to distinguish between the nervous and endocrine systems. This distinction is largely based on the way biologists originally defined a hormone. Based on the work of Starling in the early 1900s, a hormone is a chemical messenger that is released into the blood and exerts actions on cells that are distant from its site of origin (Figure 7-1). Neurotransmitters-chemical messengers that allow communication between adjacent nerve cells-are not considered to be hormones in the traditional sense of the word because these chemicals are released into synapses and do not enter the blood.

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The distinction between the endocrine and nervous systems has blurred during the past 30 years primarily because of two major discoveries. First, some nerve cells were found to release neurohormones, chemicals that enter the circulation and affect distant cells (e.g., neurosecretory cells that release oxytocin). Second, scientists learned that substances traditionally classified as hormones do not have to enter the bloodstream to affect other cells. A hormone can also diffuse within the extracellular fluid to effect changes in nearby cells and, in some cases, the very cell that produces the hormone. Based on these insights, the definition of hormone has been broadened to include any chemical that acts as an extracellular messenger.

Based on the current understanding, it may seem that almost any biochemical (e.g., vitamins, energy substrates, and minerals) can be classified as a hormone. However, this is not the case. One important characteristic that distinguishes hormones from other chemical messengers is that hormones circulate in extracellular fluids in very low concentrations; that is, at concentrations ranging from picograms ([10.sup.-12] g) to nanograms ([10.sup.-9] g) per mL of blood. This is much lower than concentrations of vitamins, minerals, and metabolites, which are present in concentrations of micrograms ([10.sup.-6] g) and milligrams ([10.sup.-3] g) per mL of blood.

[FIGURE 7-2 OMITTED]

Hormones coordinate the activities of cells in several ways (Figure 7-2). When hormones act via the so-called classic or endocrine mode of action, they are released into the circulation and exert effects on distant target cells. If the hormone is released into the blood by a neurosecretory cell, the mode of action is called neuroendocrine. A hormone that is released into the extracellular fluid and effects changes in nearby cells is said to act via a paracrine mechanism. A specialized case of this type of action is neurocrine; that is, involving chemical messengers that are released by nerves and exert local effects on nearby cells. Finally, a cell can release a chemical that regulates its own activity; that is, autocrine regulation.

Contemporary endocrinology is concerned with all types of hormones and all modes of action, as well as the tissues that produce and respond to hormones. Reproductive endocrinology is a branch of endocrinology that is concerned specifically with the hormones produced by reproductive tissues.

Hormones and Reproduction

Before the mid-nineteenth century, studies of the reproductive system were confined to anatomic descriptions of reproductive organs. This changed in 1849 when Berthold completed a series of experiments that attributed development of secondary sex traits in roosters to a humoral factor that is produced by the testes and carried to target tissues via the blood (see Box 7-1).
BOX 7-1 Focus on Fertility: The First Reported Endocrine Experiment

In 1849, the French scientist A.A. Berthold
reported what appears to be the first endocrine
experiment. The focus of his work was the
development of secondary sex traits in cockerels
(Figure 7-3). Male chicks that were bilaterally
castrated did not display male sexual and
aggressive behaviors, failed to develop a comb and
wattles, and had a weak crow (masculine vocalizations).
When Berthold implanted one or two testes
from the same or a different animal into the
abdominal cavities (the location of testes in birds)
of castrated cockerels, the birds developed as
normal males. The loss of masculine traits
following removal of the testes together with the
fact that implanted testes prevented these effects
demonstrated that these organs were the source of
some factor that regulates development of male
secondary sex traits in chickens. The fact that the
transplanted testes were effective suggests that
the relationships between the testes and target
tissues are humoral rather than neuronal. Years
later, researchers demonstrated that testicular
extracts alone replaced the activity of the testes.
In the 1930s, testosterone, the chemical
responsible for these actions, was purified from
testicular extracts.

[FIGURE 7-3 OMITTED]

The methods employed by Berthold represent
a standard experimental approach to demonstrating
that a particular physiologic event is controlled
by a particular hormone, and for studying the
physiologic effects of hormones. This approach is
commonly called the ablation-replacement
paradigm. For example, experiments designed to
characterize the role of estradiol on LH secretion
in females typically involve measuring LH concentrations
before and after injection of estradiol in
ovariectomized (surgical removal of the ovaries)
animals. In this case the source of estradiol is
ablated by surgery, and the hormone is then
replaced by injecting it. If ablation of a particular
hormone produces some physiologic effect and the
effect is reversed by treatment with the hormone,
then the effect can be attributed specifically to
the action of the hormone. Neither ablation nor
hormone injection alone can link a particular
response with a particular hormone. Today,
methods other than surgery are used to ablate
endocrine tissues. Some of the more common
approaches include chemical ablation of the
endocrine tissue, chemical disruption of hormone
synthesis and secretion, immunization against
specific hormones, use of receptor antagonists,
and use of transgenic animals that do not express
genes for certain hormones ("genetic knockouts").


Such studies shifted focus away from anatomy and toward the analysis of mechanisms controlling the activity of reproductive tissues. The importance of hormones in the control of reproduction cannot be overemphasized. Reproductive hormones control virtually all aspects of reproductive activity including: gametogenesis, sexual differentiation, sexual maturation, sexual behavior, and function of internal and external genitalia. Thus it is virtually impossible to develop a comprehensive understanding of reproductive physiology without understanding basic principles of endocrine physiology.

CLASSIFICATION OF REPRODUCTIVE HORMONES

An understanding of how a particular hormone regulates reproductive activity requires knowledge of where the hormone is produced, where it acts, and what it does as well as insight into how the hormone is synthesized, secreted, transported, metabolized, and acts, on target tissues. As we explore the reproductive physiology of mammals, we will encounter 15 hormones that play significant roles in regulating reproductive activity (Table 7-1). The task of learning the aforementioned information for each of these hormones may seem daunting. However, this process can be facilitated by first learning some general characteristics of reproductive hormones and then learning specific details about them as we study the physiologic processes in which they play particularly important roles.

Making general inferences about the reproductive hormones is facilitated by various schemes for classifying hormones. Table 7-1 illustrates a few of these methods. Classifying hormones based on their chemical characteristics is the most useful for our purposes. The hormones listed in Table 7-1 fall into one of several chemical classes: polypeptides (including proteins and peptides), steroids, and fatty acids. These classes can be further reduced to two major categories; that is, hydrophilic (water-soluble) and hydrophobic (water-insoluble or fat-soluble). In general, the polypeptide and prostaglandin hormones are hydrophilic, whereas the steroid hormones are hydrophobic. We will use this classification scheme to explore some general features of hormone synthesis, secretion, transport, and action. However, before we can do this we must establish some basic principles of endocrine physiology.

OVERVIEW OF ENDOCRINE PHYSIOLOGY

Whether a hormone is acting in an endocrine, paracrine, or autocrine manner several concepts of fundamental importance apply to its regulatory activity (Figure 7-4):

* Cells that produce hormones can also serve as targets for hormones.

* A particular hormone can induce the same or different biological effects in several different target cells.

* Different hormones can exert the same or different actions on the same target cell.

* Whether or not a particular target cell responds to a particular hormone depends on whether or not the target cell has the ability to respond to the hormone.

[FIGURE 7-4 OMITTED]

The fourth concept may seem obvious, but it is of central importance for understanding endocrinology. Specifically, this idea raises the following question: What determines whether or not a cell can respond to a particular hormone?

Hormone Receptors

The biological activity (i.e., the ability to evoke a biological response) of a particular hormone depends on it interacting with its receptor. Thus, a particular hormone can affect only those target cells that express the receptor for the hormone. A hormone receptor is a protein that interacts chemically with a hormone. They are located on the cell membrane or within the cytosolic or nuclear compartments, depending on the receptor type. Receptors mediate the effects of hormones by triggering intracellular processes that regulate activities of enzymes and/or expression of genes. All receptors have the following characteristics:

* A receptor has high affinity for the hormone.

* A receptor is specific for the hormone.

* Hormone-receptor binding is a saturable phenomenon.

[FIGURE 7-5 OMITTED]

One approach to conceptualizing these characteristics is to consider the following experiment. Imagine that different amounts of a hormone (HA) are allowed to react with a constant amount of receptor (e.g., a fixed volume of cellular extract from target tissue). After a period of time the reaction is stopped and the amount of hormone bound to the receptor is measured for each concentration of hormone. Figure 7-5 shows data derived from this type of experiment. Note that the relationship between hormone concentration and amount of binding is not linear. Initially the amount of hormone bound to the receptor is directly proportional to the amount of hormone added. Eventually, the curve flattens; that is, the amount of bound hormone is about the same no matter how much hormone is added. This demonstrates the characteristic of saturability. In other words, at a particular concentration, the receptors cannot bind additional hormone. This is also referred to as the binding capacity of the sample, which is an indirect measure of the number of receptors present.

Affinity is the chemical attraction between the hormone and its receptor. In our example, affinity is defined as the concentration of hormone that results in 50 percent of the binding capacity. To further illustrate this concept, imagine that we run a second experiment using a second hormone (HB), one that isn't as biologically potent as the first. The binding curve for HB is similar to the one for H with one important exception; that is, the curve for HB has shifted to the right. Thus the concentration of HB required to produce 50 percent of the binding capacity is greater than that for HA. In other words, the receptor has less affinity for HB than for HA. A particular receptor might not have any affinity for a hormone (e.g., HC in Figure 7-5). In this case, the hormone will never saturate the receptor. When endocrinologists speak of the potency of a hormone, they are referring its affinity with the receptor relative to other hormones. You have already encountered an example of this phenomenon. Recall that a potent metabolite of testosterone (DHT) induces development of the male external genitalia. DHT is approximately 100 times more potent than testosterone, meaning that 100 times more testosterone is required to produce the same effects as DHT; that is, the affinity of the receptor for DHT is 100 times that for testosterone.

Specificity is measured by determining what hormones will bind to a particular receptor. High specificity means that a particular receptor binds only one type of hormone with high affinity. For example, receptors for androgens will bind hormones such as testosterone and DHT with high affinity, but not other hormones such as estradiol or progesterone. In our example [H.sub.A] and [H.sub.B] could be DHT and testosterone, whereas [H.sub.C] could be progesterone, a hormone for which the receptor has no affinity.

Kinetics of Hormone-Receptor Interactions

The binding of a hormone to its receptor can be understood as a chemical reaction that is governed by the law of mass action, where the hormone (H) and receptor (R) are the reactants and the hormone-receptor complex (HR) is the product:

H + R [??] HR

At equilibrium,

[k.sub.f][H][R] = [k.sub.r] [HR]

or

[H][R]/[HR] = [k.sub.r]/[k.sub.f] = [K.sub.D]

where [k.sub.f] is the rate constant for HR formation, [k.sub.r] is the rate constant for HR dissociation, [K.sub.D] is the equilibrium dissociation constant (a measure of affinity) and [H], [R], and [HR] are concentrations of hormone, receptor, and the hormone-receptor complex, respectively. It is important that you take a minute and think about these equations because they provide the basis of all endocrine experiments. At equilibrium, the rate of HR formation is equal to the rate of HR dissociation, and the ratio of [H] [R] to [HR] is [K.sub.D], a measure of the affinity of between the hormone and its receptor. In the case of a hormone with high affinity for a receptor, the ratio of [H][R] to [HR] at equilibrium is low. In the case of a hormone with lower affinity for a receptor, the ratio of [H][R] to [HR] at equilibrium is higher.

ASSESSING A HORMONE'S EFFECTS

As noted earlier, receptors mediate the effects of hormones on target cells. The degree of the response induced by a hormone is directly related to the amount of HR complex generated by the interaction between H and R. The law of mass action implies that the amount of HR is a function of the concentration of hormone, concentration of receptor, and the affinity of the receptor. This provides a theoretical basis for evaluating endocrine systems. Assessing the biological effects of a hormone on its target cell requires the following information (Figure 7-6):

* Concentration of the hormone in extracellular fluids (e.g., blood).

* Concentration of receptors in target tissues.

* Affinity of the receptor.

Any change in activity of an endocrine system is the result of changes in one or more of these parameters.

One of the most common approaches to assessing hormonal control of reproduction is to measure concentrations of hormones in the blood. Much of what we know about the physiologic mechanisms regulating the reproductive activity in mammals is based on experiments that have characterized patterns of reproductive hormones in blood during various physiologic states as well as ones that have assessed the concentrations and affinities of receptors in target tissues. Our study of the reproductive physiology of mammals relies heavily on understanding circulating patterns of reproductive hormones. Therefore, some cautions regarding interpreting such data are warranted. First, it is important to emphasize that a change in circulating concentrations of a hormone can reflect changes in synthesis and secretion of the hormone by endocrine cells, transport of the hormone in blood and clearance of the hormone from the circulation. Second, a change in concentration of a hormone will not induce changes in target tissues unless the target tissue is responsive to the hormone. Changes in tissue response to hormones can be attributed to changes in number of receptors (which is a function of synthesis and degradation) and affinity of receptors (which is largely attributed to its physical-chemical properties) as well as the ability of receptors to induce changes within the target cell (so-called post-receptor mechanisms). The major implication of these caveats is that assessment of the activity of a particular hormone may require more than simply measuring its concentration in the blood. A more comprehensive analysis of a hormone's activity may require assessing its synthesis, secretion, clearance, the number and affinity of its receptor, and finally the mechanisms linking hormone-receptor binding and induction of a biological response.

[FIGURE 7-6 OMITTED]

ACTIVITY OF HYDROPHILIC HORMONES

Having established a theoretical framework for assessing the activity of endocrine systems, we can now turn our attention to analyzing how the two major classes of hormones (hydrophilic and hydrophobic) are synthesized, secreted, transported, cleared, and exert their effects on target cells. We will first deal with hydrophilic hormones (Figure 7-7).

Synthesis

Polypeptide hormones are synthesized and packaged in membrane vesicles which act as storage depots until the hormones are released. We will not concern ourselves with the details of polypeptide synthesis. Only the highlights of this process will be addressed. Briefly, a particular gene that codes for precursor of a hormone is transcribed to yield messenger RNA (mRNA) that is translated along the rough endoplasmic reticulum (RER) to produce a hormone precursor known as a pre-pro-hormone. As the emerging peptide enters the RER, a small (pre-) fragment is cleaved off, leaving the pro-hormone. As the pro-hormone travels along the RER and is packaged into vesicles by the Golgi apparatus, a second (pro-) fragment is removed, leaving the actual hormone. The pro-peptide and the hormone are packaged and stored in secretory vesicles, which accumulate in the cytosol in peripheral regions of the cell.

[FIGURE 7-7 OMITTED]

The synthesis of prostaglandins (not shown) is quite different from that of polypeptide hormones. Prostaglandins are derived from fatty acid derivatives found in the cell membrane. These hormones are not packaged in vesicles and leave the cell by facilitated diffusion.

Secretion

Secretion of polypeptide hormones involves exocytosis. Stimulation of endocrine cells causes secretory vesicles to move towards and fuse with the cell membrane thereby releasing their contents into the surrounding extracellular space. The packaging and storage of hormones in vesicles allows the processes of synthesis and secretion to be uncoupled. In other words, the synthesis and secretion of a hormone can occur independently. On the one hand, a cell may be secreting, but not producing a hormone. On the other hand, synthesis can occur while secretion has ceased. The former situation results in a decrease in intracellular concentrations of the hormone, whereas the latter situation results in an increase in cellular stores of the hormone.

Due to a lack of storage, prostaglandin synthesis is tightly coupled to secretion. In other words, a change in synthesis results in a corresponding change in secretion of the hormone.

Transport and Clearance

Hydrophilic hormones are readily soluble in the aqueous blood. Therefore, most of these types of hormones circulate in the free form; that is, not associated with hydrophilic serum proteins. Polypeptide hormones do not last long in the circulation. The half-life (time required for the concentration to decrease by half) of polypeptide hormones is typically no longer than several hours and in some cases as short as several minutes. Clearance of polypeptide hormones from the blood involves two processes. Small amounts of these hormones are degraded by blood proteases. The major route for removal of polypeptide hormones is via uptake and degradation by target cells.

The half-lives of prostaglandins are also very short. Unlike the polypeptide hormones, these hormones are rapidly degraded by various enzymes that are widely distributed in tissues such as the lungs.

Action

Water-soluble hormones do not diffuse freely across the lipid bilayer of cell membranes. These hormones exert their effects on target cells by interacting with receptors that are located on the membranes of target cells (Figure 7-8). Membrane receptors are very complex proteins consisting of several major domains. The binding domain extends into the extracellular region and consists of hydrophilic amino acids that bind to the hormone. An intracellular domain is also hydrophilic, but is linked to membrane-associated enzymes. These receptors also contain a transmembrane domain. The transmembrane domain of the receptor consists of alternating sequences of hydrophobic amino acids (within the membrane) linked by hydrophilic sections that protrude outward and inward into the extracellular and intracellular compartments, respectively.

[FIGURE 7-8 OMITTED]

Binding of the hormone to the binding domain of the receptor induces conformational changes in the receptor, allowing it to activate intracellular enzymes that are associated with the inner surface of the cell membrane. This leads to a chain reaction that ultimately results in generation of intracellular (second) messengers. These compounds induce changes in the target cell by activating and/or inhibiting various regulatory proteins. For example, the interaction between LH and its receptor in Leydig cells of the testes generates a second messenger that enhances activity of the rate-limiting enzyme in the synthesis of testosterone.

As noted in the previous section, uptake of polypeptide hormones by target cells accounts for most of the clearance of hormone from the circulation. Target cells internalize hormone-receptor complexes via endocytosis; that is, sections of cell membrane containing hormone-receptor complexes invaginate to form vesicles. The hormone is typically metabolized within the endocytotic vesicle and the unoccupied receptors are recycled to the cell membrane.

ACTIVITY OF HYDROPHOBIC HORMONES

The synthesis, secretion, transport, and mode of action of hydrophobic hormones (Figure 7-9) is markedly different from that of hydrophilic hormones. Many of these differences reflect the different solubilities of these two classes of hormones.

[FIGURE 7-9 OMITTED]

Synthesis

All of the hydrophobic hormones regulating reproductive activity are steroids. Steroids are members of a chemical class of compounds that are characterized by a four-ring (tetracyclic cyclopental[a]phenanthrene) skeleton. Reproductive tissues produce three major classes of steroid hormones: progestins, estrogens, and androgens. Corticosteroid hormones, another general type of steroid, are produced by the adrenal gland and play a role in parturition. We will be concerned with only one to three specific steroids within each class. Figure 7-10 shows the biosynthetic pathway for these steroid hormones. Each of these hormones is derived from cholesterol. It is important to note that the complement of steroid hormones produced varies among steroidogenic tissues. For example, the corpus luteum produces only progesterone whereas the ovarian follicle produces large amounts of estradiol and some androgens. The type of steroid hormones produced by a particular cell type is a function of which steroidogenic enzymes are expressed by the cell. For example, the corpus luteum produces only progesterone because it lacks the enzymes necessary to convert progesterone to estradiol.

[FIGURE 7-10 OMITTED]

The major source of cholesterol is low-density lipoproteins (LDL), which consist of a lipid shell containing phospholipids, cholesteryl esters, and a protein. The protein binds to membrane receptors on many cell types including cells that produce steroid hormones. The LDL-receptor complex is internalized by the cell via endocytosis. Vesicles containing this complex fuse with lysosomes which contain enzymes that liberate free cholesterol. The cholesterol is then re-esterified and stored in lipid droplets. Although cells are capable of de novo cholesterol synthesis, most of the cholesterol used for steroid hormone synthesis is derived from pools stored in lipid droplets.

Synthesis of steroid hormones from cholesterol involves enzymes located in the mitochondria and smooth endoplasmic reticulum. The rate-limiting step in steroid hormone production is the transfer of cholesterol to the mitochondria, the site of the first step in steroid hormone synthesis. Because steroids are not very soluble in water, only a minute amount exists in the free form in cytosol. Most of the cholesterol and steroid hormones are bound to low-affinity carrier proteins.

Secretion

Unbound steroid hormones readily diffuse across the cell membrane into extracellular fluids. Unlike cells that produce polypeptide hormones, steroid-producing cells do not have a large capacity for storing their hormones. Aside from a small storage pool provided by carrier proteins, most steroids leave the cell soon after they are synthesized. Thus, secretion of steroid hormones closely reflects the synthetic activity of these cells.

Transport and Clearance

Once steroid hormones enter the blood they interact with several serum-binding proteins. The affinity of these proteins for steroid hormones is much lower than that of receptors. However, because binding proteins are present in large concentrations most (>90 percent) of the circulating steroid hormones exist in the bound form. Serum-binding proteins protect steroid hormones from metabolic degradation, thereby providing a substantial storage pool for these chemical messengers. Unbound steroid hormones are readily metabolized to water-soluble forms by peripheral tissues. These metabolites are then eliminated in the urine and feces.

[FIGURE 7-11 OMITTED]

Action

Unbound molecules of steroid hormones that are not metabolized diffuse across the hydrophobic cell membrane of target cells, where they interact with their receptors. There has been considerable controversy regarding the precise intracellular location of steroid hormone receptors. Some steroid hormones interact with a receptor located in the cytosol and then the hormone-receptor complex is translocated to the nuclear compartment where it binds to DNA. The receptors for other steroid hormones reside in the nuclear compartment associated with specific regions of DNA. In either case, interaction of the hormone-receptor complex with the DNA enhances transcription of certain genes that give rise to proteins that regulate activity of the target cell. The intracellular receptors for hydrophobic hormones are complex proteins that express conformational changes upon binding with a hormone. One notable change is that the receptors form dimers and then activate specific genes. These receptors consist of three major domains (Figure 7-11): 1) an amino terminal sequence, 2) a DNA binding domain, and 3) a ligand-binding domain. The ligand-binding domain interacts with the hormone, whereas the DNA-binding domain interacts with the DNA to regulate gene transcription.

REGULATION OF ENDOCRINE SYSTEMS

Our analysis of interactions between endocrine and target cells raises an important question: How are the relationships between these two cell types regulated? We will consider two conditions that require communication between endocrine and target cells. The first case deals with homeostasis: that is, what prevents a hormone from overstimulating its target cell? The second case deals with situations where a rapid response is required; that is, how can a hormone induce a rapid and robust stimulation of its target cell?

Negative Feedback

Homeostasis in endocrine systems is typically maintained by a negative feedback relationship between target and endocrine cells (Figure 7-12a). In its simplest form, a negative feedback system consists of a hormone inducing a response in a target cell, and the response in turn inhibiting further release of the hormone. The response that inhibits hormone secretion can be another hormone or some other physiologic change. The negative feedback signal can inhibit hormone release by suppressing synthesis and/or secretion of the hormone.

A negative feedback system with which you are undoubtedly familiar involves home heating systems (Figure 7-12b). In this example, a thermostat, sending an electronic signal to a furnace, is analogous to an endocrine cell producing a hormone that evokes some physiologic effect in a target cell. In the case of the heating system, a drop in temperature is detected by the thermostat. This opens an electronic circuit that causes the furnace to generate heat. Once a rise in temperature is detected by the thermostat the signal to the furnace is shut off and the furnace turns off.

[FIGURE 7-12 OMITTED]

Positive Feedback

Imagine that instead of shutting off in response to a temperature increase, a furnace responds by accelerating its production of heat (Figure 7-13b). This would be an example of a positive feedback system. Such a system is potentially dangerous; that is, the furnace could overheat and self-destruct. Even though such systems seem to defy homeostasis, they exist in some biological systems and play important regulatory roles. A simple example of such a system is shown in Figure 7-13a. In this case an endocrine cell releases a hormone that evokes a response in a target cell which in turn further stimulates the endocrine cell. We will encounter two positive feedback systems in reproductive physiology. In each case these systems serve the purpose of causing rapid increases in hormone levels. Positive feedback systems shut down because the sharp increase in hormone concentrations eliminates the source of the response. For example, during the birthing process, stimulation of the cervix by the emerging fetus stimulates release of oxytocin by the posterior pituitary gland, which induces uterine contractions, which stimulate a greater release of oxytocin. The system shuts down once the fetus leaves the birth canal (i.e., no stimulation of the cervix).

[FIGURE 7-13 OMITTED]

OVERVIEW OF THE ENDOCRINE SYSTEM CONTROLLING REPRODUCTION

Having described the major reproductive hormones as well as general principles of endocrine physiology it is now possible to provide an overview of the endocrine system that regulates reproductive activity in mammals. Fig ure 7-14 shows major endocrine tissues, reproductive hormones, target tissues upon which these hormones act, and the feedback systems that sustain homeostasis within this systems. For the time being, it is only necessary for you to become familiar with the general flow of information between the various organs controlling reproduction. Information from higher brain centers converge upon the hypothalamus which integrates numerous neuronal inputs and transduces this type of information into two major hormone signals: oxytocin and gonadotropin-releasing hormone (GnRH). Oxytocin affects the reproductive tract and the mammary gland (not shown). GnRH stimulates the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which in turn regulate gonadal activity. In some species (rodents) another pituitary hormone (prolactin) also influences gonadal activity. Release of prolactin by the anterior pituitary gland is controlled by dopamine, a biogenic amine that suppresses release of prolactin. One of the major effects of these gonadotrophic hormones is to stimulate release of various hormones from the ovaries and testes. These gonadal hormones regulate the reproductive ducts and exert positive and negative feedback effects on the brain and pituitary gland to regulate gonadotropin secretion.

[FIGURE 7-14 OMITTED]

ASSESSING ENDOCRINE PHYSIOLOGY

As indicated earlier, most of our understanding of reproductive endocrinology is based on interpretation of hormone patterns in blood. Characterizing patterns of hormones is one of the most commonly-used methods for characterizing activity of endocrine cells. The experimental approach for doing so involves measuring concentrations of a hormone in blood samples taken sequentially over a period of time. Because so much of our knowledge of reproductive physiology is based on analysis of hormone patterns it is necessary to understand how hormone concentrations are determined as well as how they should be interpreted.

Measuring Hormones

A standard approach to evaluating the reproductive status of an animal is to collect blood samples and measure concentrations of particular reproductive hormones in serum or plasma. Before the 1960s it was impossible to characterize patterns of hormones in blood. None of the traditional chemical methods for measuring various compounds were sensitive enough to detect the low concentrations of hormones found in biological fluids. This all changed in 1969 when Berson and Yalow discovered that it was possible to generate highly-specific antibodies to hormones. This breakthrough led to development of the radioimmunoassay (RIA), a highly sensitive and specific method for measuring physiologic levels of hormones in biological fluids.

Unlike chemical assays, the RIA indirectly measures the amount of hormone present in a sample. Reagents for the assay include a highly purified form of hormone that is labeled with a radioactive isotope ([sup.125]I or [sup.3]H), purified (nonradioactive) hormone in known concentrations (standard), and an antibody that specifically binds radioactive and nonradioactive forms of the hormone with the same affinity. The interaction among these reagents can be expressed as a chemical reaction, as follows:

H + H* + Ab [??] HAb + H*Ab

Where H is the nonradioactive hormone, H* is the radioactively labeled hormone (label) and Ab is the antibody. The reaction is allowed to proceed to equilibrium. Due to the law of mass action, the amount of product formed depends on the affinity of the antibody and the initial concentration of reactants. Suppose we conduct this reaction in a number of different test tubes where the amount of H is varied among tubes, but the amount of antibody and the amount of H* are held constant. Under these circumstances, the amount of H is the only variable that determines the amount of H*Ab formed. Thus the amount of H*Ab formed is inversely proportional to the initial amount of H (Figure 7-15). Now suppose we measure the amount of H*Ab in each of the test tubes and create a graph of the concentration of H added verses the concentration of H*Ab formed (Figure 7-16). From this graph it is clear that there is a mathematical relationship between the amount of H present in the assay tube and the amount of H*Ab formed by the reaction. Thus we can represent the concentration of a hormone in a blood sample as a function of the amount of radioactive label bound to the antibody. This means we can subject a sample containing an unknown amount of H to the assay, and measure the resulting H*Ab. This corresponds to a particular concentration of standard H, which provides an accurate estimate of hormone concentration in our sample.

[FIGURE 7-15 OMITTED]

Radioimmunoassay is still the most common way to assess hormone concentrations. However, a derivative of this technique called the enzyme-linked immunosorbent assay (ELISA) has become more popular in recent years. The only difference between the RIA and ELISA is the nature of the labeled hormone. Instead of a radioactive label, the hormone is labeled with an enzyme that catalyzes a chemical reaction that produces a color change. After the labeled hormone, unlabeled hormone, and antibody are allowed to react, and antibody-bound hormone is separated from free hormone, the enzyme substrate is added, and the amount of color generated is directly proportional to the amount of labeled hormone bound to the antibody. This of course is inversely proportional to the amount of unlabeled hormone in the sample.

[FIGURE 7-16 OMITTED]

Interpretation of Hormone Profiles

The ability to monitor circulating concentrations of reproductive hormones made it possible for researchers to describe patterns of hormones associated with a variety of physiologic states including sexual development, puberty, reproductive cycles, and pregnancy. As noted earlier, changes in blood concentrations of a hormone can be affected by various mechanisms. Therefore, it is important that you understand how to interpret circulating hormone profiles.

Hormones usually fluctuate at basal levels within a particular homeostatic range (Figure 7-17). These tonic or basal concentrations reflect negative feedback control of hormone secretion as well as the rate of hormone clearance from the circulation. In some cases hormone concentrations rise rapidly to amounts that greatly exceed basal levels. If these elevations are prolonged, lasting several hours, they are referred to as surges. Some surges can be induced by positive feedback (Figure 7-17). The ascending portion of the response is attributed to enhanced release of the hormone. Concentrations begin to decrease once release of the hormone stops. In a true positive feedback system a stimulus enhances hormone release and the resultant increase in hormone concentration further enhances generation of the stimulus. Not all hormone surges are generated by positive feedback. In some cases a stimulus induces massive release of the hormone, but the hormone does not influence the stimulus.

[FIGURE 7-17 OMITTED]

It is important to understand that the pattern of hormone observed depends on how often and how long blood samples are collected. Measuring hormone concentrations in samples taken infrequently (e.g., once per day or once per week) permit characterization of long-term changes (over days, weeks, or months), but does not reveal short-term (over hours or minutes) fluctuations. Assessing hormone concentrations in blood samples taken at frequent intervals for several hours can reveal a great deal about regulation of hormone secretion. Figure 7-18 depicts a typical pattern of LH in blood. The most striking feature of this type of profile is that concentrations of the hormone fluctuate in a pulsatile or episodic manner. This type of pattern is characterized by a rapid increase (pulse) in hormone concentration, followed by a more gradual decrease. The highest concentration achieved within a particular pulse is called the peak, whereas the lowest concentration between pulses is called the nadir. Such patterns are usually described in terms of the frequency and average amplitude of the pulses. Frequency refers to the number of pulses per unit of time. Amplitude is the difference between the peak and nadir. In some cases, the average time between pulses is quantified. This is known as the period, and it is the inverse of the pulse frequency.

[FIGURE 7-18 OMITTED]

Other terms refer to other types of hormone patterns. In some cases, hormone concentrations fluctuate in accordance with time of day or time of year. When a hormone rhythm has a period of about 24 hours, it is called a circadian rhythm. A rhythm with a period of approximately 1 year is called a circannual rhythm.

Circulating patterns of hormones are of little use unless we understand their physiologic bases; that is, understanding how they are generated and what effects they perpetuate. It is generally assumed that hormone patterns in blood reflect secretion of the hormone. This is not a reasonable assumption because mechanisms such as metabolism and clearance also play insignificant roles in determining hormone patterns in blood. With respect to the pulsatile pattern of LH, it appears that each pulse is the result of a short-term (several minutes) release of LH by the pituitary gland, followed by clearance from the blood. Another concern associated with interpreting hormone patterns deals with the specificity of the assay used to quantify hormone concentrations. Some assays detect only the free form of the hormone, whereas others measure bound and free forms. Other considerations include the extent to which assays detect metabolites or isoforms of hormones. These considerations are important because only some forms of the hormone have biological activity. Thus if one is unaware of what the assay is measuring, it is possible to generate hormone profiles that have little to do with the biological status of the animals studied.

SUMMARY OF MAJOR CONCEPTS

* Homeostatic relationships among various tissues are maintained by the endocrine and nervous systems.

* Hormones are intercellular messengers present in low concentrations in extracellular fluids.

* Mechanisms of synthesis, secretion, transport, metabolism, and action differ between hydrophilic and hydrophobic hormones.

* The ability of a hormone to induce a biological effect depends on its concentration, the concentration of its receptors in target cells, and the affinity of its receptor for the hormone.

* Endocrine systems are typically regulated by negative feedback loops; positive feedback loops exist, but are rare.

* Circulating patterns of hormones can be easily monitored, but their physiologic significances may be unclear in the absence of other information.

DISCUSSION

1. Glucose can be released from the liver and transported to many other tissues where it is taken up and induces a variety of biological effects. Glucose concentrations in animals range between 40 and 150 mg/100 mL. Based on this information, would you consider glucose to be a hormone? Why or why not?

2. GnRH, a peptide hormone produced by hypothalamic neurosecretory cells, and estradiol, a steroid hormone produced by the ovary, each exert effects on LH-producing cells of the anterior pituitary gland. In general terms, compare and contrast how these hormones act to affect LH release by pituitary cells.

3. Plasma concentrations of LH increase in males following removal of both testes. If the castrated male is then injected with testosterone, concentrations of LH fall to levels observed before removal of the testes. Explain these results based on your understanding of how endocrine systems are regulated.

4. Suppose you want to assess the potency of a synthetic estrogen (relative to the naturally occurring estradiol) with respect to effects on epithelial cells of uterine endometrium. Describe how you would do this. Show a graph of your expected results.

5. Suppose you measure plasma concentrations of testosterone in bull elk throughout the year and discover that concentrations increase during the rutting season. You conclude that this is due to increased secretion by the testes. Is this a valid conclusion? Why or why not?

REFERENCES

Berthold, A.A. 1849. Transplantation der Hoden. Archives of Anatomy and Physiology. Wissenschaftliche Medicin. 16:42-46.

Cannon, W.B. 1960. The Wisdom of the Body. New York: W.W. Norton.

Hadley, M.E. and J.E. Levine. 2007. Endocrinology, sixth edition. Upper Saddle River, NJ: Pearson Education.

Starling, E.H. 1905. The chemical correlation of the functions of the body. Lancet 1:340-341.

Wilson, J.D., D.W. Foster, H.M. Kronenberg and P.R. Larsen. 1998. Principles of endocrinology. In Williams Textbook of Endocrinology, ninth edition. Philadelphia: W.B. Saunders, pp. 1-10.

Keith K. Schillo, PhD

Department of Animal and Food Sciences

University of Kentucky

Lexington, Kentucky
TABLE 7-1 Classification of Major Reproductive Hormones

                                                 Class of
Hormone               Major Source(s)            Molecule

Gonadotropin-         Hypothalamus               Decapeptide
Releasing Hormone
(GnRH)

Luteinzing            Anterior lobe of           Glycoprotein
Hormone (LH)          pituitary gland

Follicle-             Anterior lobe of           Glycoprotein
Stimulating           pituitary gland
Hormone (FSH)

Prolactin (PRL)       Anterior lobe of           Protein
                      pituitary gland

Oxytocin (OT)         Hypothalamus               Octapeptide

Estradiol             Ovaries (granulosa         Steroid
([E.sub.2])           cells), placenta, and
                      testes (Sertoli cells)

Progesterone          Ovaries (corpus luteum)    Steroid
([P.sub.4])           and placenta

Testosterone (T)      Testes (Leydig cells)      Steroid
                      and ovaries (theca
                      interna cells)

Inhibin               Ovary (granulosa           Glycoprotein
                      cells) and testes
                      (Sertoli cells)

Activin               Ovary (granulosa           Glycoprotein
                      cells) and testes
                      (Sertoli cells)

Prostaglandin         Uterus                     Fatty acid
[F.sub.[2[alpha]]     (endometrium) and
([PGF.sub.2[alpha]]   vesicular glands

Prostaglandin         Ovaries, uterus and        Fatty acid
[E.sub.2]             extra-embryonic
([PGE.sub.2])         membranes

Human chorionic       Trophectoderm of           Glycoprotein
gonadotropin (hGC)    blastocyst

Equine chorionic      Chorion (girdle cells)     Glycoprotein
gonadotropin (eCG)

Placental lactogen    Placenta                   Protein

                      Major Female Target        Major Male
Hormone               Tissue(s)                  Target Tissue(s)

Gonadotropin-         Anterior lobe of           Anterior lobe of
Releasing Hormone     pituitary gland            pituitary gland
(GnRH)

Luteinzing            Ovary (theca cells of      Testes (Leydig
Hormone (LH)          follicle and cells of      cells)
                      corpus luteum)

Follicle-             Ovary (granulosa cells     Testes (Sertoli
Stimulating           of follicle)               cells)
Hormone (FSH)

Prolactin (PRL)       Mammary gland              Testes
                      (glandular epithelium);
                      ovary (corpus luteum)
                      in some species

Oxytocin (OT)         Uterus (myometrium         Cauda epididymis,
                      and endometrium)           ductus deferens, and
                      and mammary gland          ampulla (smooth
                      (myoepithelial cells)      muscle cells)

Estradiol             Internal and external      Brain and
([E.sub.2])           genitalia, brain,          pituitary gland
                      pituitary gland, and
                      mammay gland

Progesterone          Brain, pituitary gland,    No major target
([P.sub.4])           uterus (myometrium         tissue
                      and endometrium), and
                      mammary gland

Testosterone (T)      Brain and ovaries          Accessory sex
                      (granulose cells)          glands, external
                                                 genitalia, and
                                                 testes (seminiferous
                                                 epithelium)

Inhibin               Anterior lobe of           Anterior lobe of
                      pituitary gland            pituitary gland

Activin               Anterior lobe of           Anterior lobe of
                      pituitary gland            pituitary gland

Prostaglandin         Ovaries (corpus luteum     Epididymis
[F.sub.[2[alpha]]     and follicles) and
([PGF.sub.2[alpha]]   uterus (myometrium)

Prostaglandin         Ovaries (corpus luteum)    No known targets
[E.sub.2]             and oviduct
([PGE.sub.2])

Human chorionic       Ovaries (corpus luteum)
gonadotropin (hGC)

Equine chorionic      Ovary
gonadotropin (eCG)

Placental lactogen    Mammary gland
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Author:Schillo, Keith K.
Publication:Reproductive Physiology of Mammals, From Farm to Field and Beyond
Geographic Code:1USA
Date:Jan 1, 2009
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