ENDOCRINE MANUAL FOR REPRODUCTIVE ASSESSMENT OF DOMESTIC ...

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3. CREATININE ASSAY RECIPE

NaOH (0.75 N) 3.0 g
100 ml
NaOH Pellets
H2O
This is a strong base so keep in a glass bottle

Picric Acid (0.4 M)

Saturated Picric acid 100 ml S925-40
H2O 900 ml

OR….

Crystallized Picric acid in H2O 5g S4255
H2O 500 ml

Safety Precautions

Picric Acid - Saturated Solution

• Store tightly capped - explosive when dried
• Store in a flame proof cabinet
• Poisonous - avoid contact with eyes and mouth
• Irritant - avoid contact with skin - wear gloves when handling
• Dilute 1:100 in dH2O for working solution

• Flush with water for 5 minutes if it contacts skin or eyes
• Flush with water for 5 minutes when disposing diluted solution down the drain
• Incompatible with metals, oxidizing and reducing agents, strong bases, ammonia, concrete

and plaster

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Assay Reagents for Steroid and LH enzymeimmunoassays:

Na2CO3 Sigma, S-2127 FW=106.0 Anhydrous 1kg
NaHCO3 Sigma, S-8875 FW=84.01 1kg
NaH2PO4 Sigma, S-9638 MW=138.00 Monobasic 1kg
Na2HPO4 Sigma, S-0876 MW=142.00 Dibasic 1kg
NaCl Enzyme Grade
BSA Sigma, A-7906 FW=192.1 Fraction V 100g
Tween 20 Sigma, P-1379 500ml
Citric acid Sigma, C-0759 FW=34.01 Anhydrous 500g
ABTS Sigma, A-1888 FW=121.1 5g
H2O2 Sigma, H-1009 30% w/w solution 500ml
Tris (Trizma base) Sigma, T-1503 Reagent Grade 1kg
Sodium azide Sigma, S-8032
Tween 80 Sigma, P-1754 Good for 100-500 500ml
Phosphate citrate Sigma, P-4922 plates. 100 capsules
buffer (with sodium
perborate capsules) Sigma T-3405 Good for 50 plates 100 tablets
TMB Reagent grade Make
H2SO4 FW=98.08 a 0.6M solution 1mg
Reagent grade
HCL Sigma, S-5881 Needed for pH 1) 500U
NaOH pellets 1) pH 2) need 0.75M (3.0g
2) Crt assay in 100ml H2O) CAUTION:
Anti-mouse IgG Good for 20 plates Explosive
Sigma M8645 whole molecule when dried/
Streptavidin purified Will last ~ 6 months poisonous
Roche Diagnostics Boehringer /irritant
Picric acid 1089153 1) Dilute
1) Saturated End up with 0.4M saturated
2) Crystalized 1) Sigma, solution stock 1:100
in H20 925-40 in H2O

Creatinine Standards 2) Sigma, C- 2) Weigh out
Ethyl Alcohol 4255 5g (using
plastic) into
Methanol Sigma, 925-11 500ml H20

Dehydrated 200 For fecal extraction
proof – use ~
20ml/extraction
Reagent grade For fecal extraction
– use 1ml/extraction

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Supplies Nunc mircotitre 96 flat well Supplier 1 HX 24870A Daigger
plates maxisorp Supplier 2 12-565-135 Fisher Scientific
Mircotitre Mircotitre plate sealer For running EIA’s 3501 Dynex Laboratories
plates and 12 x 75 mm glass culture 14 961 26 Fisher Scientific
Sealers tubes For boiling fecal extracts 60917-500 VWR
Small Glass Rack - 72 holes, holds 10- 14-961-30 Fisher Scientific
Tubes and 13mm To store neat urine and
Racks 16 x 125 mm glass culture fecal extracts 62-526-003 Sarstedt Inc.
tubes To store fecal samples 65-809-003 Sarstedt Inc.
Large Glass Rack - 72 holes, holds 2650 Perfector Scientific
Tubes and 16mm tubes To store antibody
Racks Clear 12 x 75 mm plastic Store for urine and LX2860BX Sarstedt Inc.
tubes extracted fecal samples Daigger
Small Purple 12 mm stopper caps LX2860EX
Plastic Conical tubes (50 ml) blue For washing plates and Daigger
Tubes caps rinsing glassware Need 3
Large ziplock bags with white
Plastic write-on labels
tubes
and/or Daigger cardboard storage
baggies boxes and lids, 3"
O-ring vials
Freezer Daigger cardboard storage
Boxes and box grids (100 cell)
grids 500 ml

Plastic
wash
bottles

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EQUIPMENT

Pipettes Pipette (Brinkman 05-402-87 Fisher Scientific
05-402-88 Fisher Scientific
and Tips Eppendorf 20) 05-402-89 Fisher Scientific
05-402-90 Fisher Scientific
Pipette (Brinkman 1670-Y Perfector Scientific
21-380-8 Fisher Scientific
Eppendorf 100) 2.5ml, 5ml, 12.5ml, 25ml,
50ml Need 3-4
Pipette (Brinkman
To hold buffers Need 10
Eppendorf 200) Should hold up to 4 plates
at a time Need 3-4
Pipette (Brinkman For fecal extraction
To dry down fecal Extra Filters and
Eppendorf 1000) extracts lamps
Manuel
200ul tips Disks for revelation
software
1000ul tips

Repeater Reapter (Brinkman

and Tips Eppendorf)

Repeater Fisherbrand

Dispenser Tips

Vortex

PH meter Calibrators (pH 4, 7, 10),

rinse and holding rack

Sonicator For fecal extractions

Magnetic And magnetics

mixer

Glass 1 litre

Bottles

Plate

Shaker

Boiling H20
bath

Drying Multitube manifold

apparatus

Centrifuge To spin 16 x 125 mm tubes

for fecal extraction

Rubber One to weigh out fecals and To pulverize dried fecal
mallot One to weigh out chemicals samples
Scale Must have 450nm and Both Sensitivity of 0.000g
405nm filters; reference
Plate filter 540nm or above
Reader and
Printer

Printer

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OTHER…… Milli-Q, distilled, Smithsonian's National Zoological Park
Timer deionized or reverse Conservation & Research Center
Sharpie Markers osmosis Endocrine Workbook
Kimwipes Plate Reader
Parafilm Printer Hard and disk copies
Calculator Covers
Glass Critical element
Scintillation
vials
Clipboards
Protocols and
Assay sheets
Colored Tape
Good H20
supply

Adaptors and
voltage converts
for equipment

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XVIII. REVIEW OF STEROID METABOLISM

Adapted from: Th. Steimer, Division of Clinical Psychopharmacology, University Institute of
Psychiatry,2, ch. du Petit Bel-Air, 1225 Chêne-Bourg, Geneva, Switzerland

Introduction

Steroids are lipophilic, low-molecular weight compounds derived from cholesterol that play a
number of important physiological roles. The steroid hormones are synthesized mainly by
endocrine glands such as the gonads (testis and ovary), the adrenals and, during gestation, by the
fetoplacental unit, and are then released into the blood circulation. They act both on peripheral
target tissues and the central nervous system (CNS). An important function of steroid hormones is
to coordinate physiological and behavioral responses for specific biological purposes, like
reproduction. Thus, gonadal steroids influence the sexual differentiation of the genitalia and of the
brain, determine secondary sexual characteristics during development and sexual maturation,
contribute to the maintenance of their functional state in adulthood and control or modulate sexual
behavior.

Despite their relatively simple chemical structure, steroids occur in a variety of biologically active
forms. This is due in part to the fact that circulating steroids are extensively metabolized by the
liver and in target tissues where conversion to an active form is sometimes required. Steroid
metabolism is therefore important not only for the production of these hormones, but also for the
regulation of their cellular and physiological actions.

Steroid hormones: Structure, nomenclature and classification

The parent compound from which all steroids are derived is cholesterol. As shown in Fig. 1a,
cholesterol is made up of three hexagonal carbon rings (A,B,C) and a pentagonal carbon ring (D)
to which a side-chain (carbons 20-27) is attached (at position 17 of the polycyclic hydrocarbon).
Two angular methyl groups are also found at position 18 and 19. Removal of part of the side-
chain gives rise to C21-compounds termed pregnanes (progestins and corticosteroids). Total
removal produces C19-steroids, androstanes (including the androgens), whereas loss of the 19-
methyl group (usually after conversion of the A-ring to a phenolic structure, hence the term
"aromatization") yields the estranes, to which estrogens belong. Individual compounds are
characterized by the presence or absence of specific functional groups (mainly hydroxy, keto(oxo)
and aldehydes groups) on the carbon skeleton.

Given that at most positions, the functional groups can be oriented either in equatorial or axial
position (see Fig. 1b), this type of structure gives rise to a great number of possible stereoisomers
(i.e., molecules having the same chemical formula, but a different three-dimensional
conformation). Stereoisomerism is very important for biological activity (i.e., for steroid-protein
interactions). Substituent groups above the plane of the molecule are said to be in the "ß" position,
whereas those situated under the plane of the molecule are said to be in the "α" position. Double-
bonds are indicated by the suffix -ene. A complete description of a steroid molecule must
therefore include the name of the parent compound (pregnane, androstane or estrane series), and
the name, number, position and orientation (α or ß) of all functional groups. Commonly occurring
steroids are usually identified by a "trivial" name (e.g., cortisol, testosterone, etc.). Thus,
testosterone (trivial name) becomes "17ß-hydroxy-androst-4-ene-3-one".

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Steroid hormones can be grouped in various classes according to a number of criteria. Based on
their chemical structure they can belong to one of the classes (series) mentioned above (e.g., "a
pregnane derivative"). If their site of production is considered to be more important, one can
distinguish for example between "ovarian" or "adrenal" steroids. If their biological function is
essential, terms like "a glucocorticoid" or "sex steroids" can be used. Finally, classification can
also be based on their molecular actions ("an estrogen-receptor agonist") or biochemical effects.

Steroid hormone biosynthesis

A general outline of the major biosynthetic pathways

The adrenals produce both androgens and corticosteroids (mineralo- and glucocorticoids), the
ovaries (depending on the stage of the ovarian cycle) can secrete estrogens and progestins, and the
testis mainly androgens. However, the biochemical pathways involved are strikingly similar in all
tissues, the difference in secretory capacity being mostly due to the presence or absence of specific
enzymes. It is therefore possible to give a general outline of the major biosynthetic pathways
which is applicable to all steroid-secreting glands, as shown in Fig. 2.

From acetate to cholesterol.

Cholesterol can be synthesized in all steroid-producing tissues from acetate, but the main
production sites are the liver, the skin and the intestinal mucosa. Steroid hormone formation in
endocrine glands probably relies mostly on exogenous cholesterol (plasma cholesterol). The 27-
carbon skeleton of cholesterol is derived from acetyl-CoA through a series of reactions which
involve the following intermediate products: (1) Mevalonate (by condensation of 3 molecules of
acetyl-CoA), which requires the enzyme HMG-CoA-reductase, an important enzyme in the
control of cholesterol biosynthesis; (2) Squalene, a 30-carbon linear structure which undergoes
cyclization to yield (3) Lanosterol; and (4) after removal of 3 carbons, cholesterol.

From cholesterol to progestins, androgens and estrogens.

The first committed step in steroid biosynthesis is the conversion of the 27-carbon skeleton of
cholesterol to a C21-compound, pregnenolone (Fig. 2). This critical step, which is subject to
hormonal control by adrenocorticotropic hormone (ACTH) in the adrenals and by luteinizing
hormone (LH) in the gonads, is catalyzed by a P-450 enzyme, the cholesterol side-chain cleavage
enzyme P-450scc (also called 20,22-desmolase, or 20,22-lyase). Pregnenolone can be converted
either to progesterone, which branches to the glucocorticoid and androgen/estrogen pathways, or
to 17α-hydroxypregnenolone, which is another route for the formation of androgens and estrogens
(Fig. 2, top-left part). Androgen formation in the adrenals is limited to dehydroepiandrosterone
and androstenedione, whereas in the testes 17ß-hydroxysteroid dehydrogenase (17HSD) in Leydig
cells (under the control of LH) stimulates production of testosterone, the principal "male"
hormone. Estrogen formation requires another P-450 enzyme, the aromatase complex (P-
450Arom). The substrate is either androstenedione (for estrone) or testosterone (for estradiol).
Estrone and estradiol are interconvertible through a reversible reaction involving another 17ß-
hydroxysteroid dehydrogenase, as in the androstenedione-testosterone conversion. Aromatase
activity is present in the ovary and the placenta. In the ovary, aromatase activity and estrogen
formation occur in granulosa cells and are controlled by follicle-stimulating hormone (FSH),
whereas production of the androgenic substrates (testosterone, 4-androstenedione) requires LH
stimulation of the theca cells.

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From progesterone and 17α-hydroxyprogesterone to gluco- and mineralocorticoids.

Hydroxylation of progesterone at carbon 21 yields 11-deoxycorticosterone (DOC), and
corticosterone after another hydroxylation step at carbon 11. Corticosterone is a major
glucocorticoid in rats and other species (e.g., birds) which do not produce cortisol. Two further
steps (hydroxylation and oxydoreduction at carbon 18) result in the formation of aldosterone.
Cortisol is formed from 17α-hydroxyprogesterone, with 11-deoxycortisol as an intermediate.
Cortisol is the main glucocorticoid secreted by the adrenal glands in most mammals.

Steroid hormones in the blood

It is generally assumed that steroids are released into the blood circulation as soon as they are
formed, i.e. there are no active transport and/or release mechanisms. Secretion rates are therefore
directly related to the biosynthetic activity of the gland and to the blood flow rate.

Steroid binding proteins

Because of their lipophilic properties, free steroid molecules are not highly soluble in water. In
biological fluids they are usually found either in a conjugated form, i.e. linked to a hydrophilic
moiety (e.g. as sulfate or glucuronide derivatives) or bound to carrier proteins (non-covalent,
reversible binding). Binding to plasma albumin (which accounts for 20-50% of the bound
fraction) is rather unspecific, whereas binding to either corticosteroid-binding globulin (CBG) or
sex hormone-binding globulin (SHBG) [sometimes called "sex steroid-binding protein", or SBP]
is based on more stringent stereospecific criteria. The "free fraction" (1-10% of total plasma
concentration) is usually considered to represent the biologically active fraction (i.e., hormone that
is directly available for action), although this idea has been challenged by recent evidence that, in
some cases at least, the specific binding proteins may facilitate steroid entry into target tissues.
Apart from the two functions mentioned above, the major roles of plasma binding proteins seem to
be (a) to act as a "buffer" or reservoir for active hormones (because of the non-covalent nature of
the binding, protein-bound steroids are released into the plasma in free form as soon as the free
concentration drops according to the law of mass action) and (b) to protect the hormone from
peripheral metabolism (notably by liver enzymes) and increase the half-life of biologically active
forms.

Peripheral metabolism of circulating steroids

Because steroids are lipophilic, they diffuse easily through the cell membranes, and therefore have
a very large distribution volume. In their target tissues, steroids are concentrated by an uptake
mechanism which relies on their binding to intracellular proteins (or "receptors", see below). High
concentration of steroids are also found in adipose tissue, although this is not a target for hormone
action. In the human male, adipose tissue contains aromatase activity, and seems to be the main
source of androgen-derived estrogens found in the circulation. But most of the peripheral
metabolism occurs in the liver and to some extent in the kidneys, which are the major sites of
hormone inactivation and elimination, or catabolism (see below).

Steroid interaction with target tissues

Formation of active metabolites in target tissues

For certain classes of hormones and particular target tissues, steroids must be converted in situ to
an active form before they can interact with their specific receptor(s). For example, conversion of
testosterone to 5α-DHT (Fig. 3, top) is required for its action on prostate growth and function,

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whereas aromatization to estradiol-17ß in the brain is mandatory for some of its developmental,
neuroendocrine and behavioral effects. Unlike its parent compound, the progesterone metabolite
5α-DHP (Fig. 3, bottom) has no effect on the uterus, but is more effective than progesterone itself
regarding the facilitation and/or inhibition of GnRH-induced LH release in vitro. The two main
classes of hormones for which metabolic activation has been shown to play a role are the
progestins and the androgens, but catecholestrogens (2- or 4-OH derivatives of estrogens) may
also constitute another class of biologically active compounds resulting from target organ
metabolism.

Correlation between structure and function: the role of metabolism

The biological activity of a steroid molecule depends on its ability to interact with a specific
binding site on the corresponding receptor. In most cases, biological activity can be directly
correlated with binding affinity. The affinity (usually characterized by the binding constant KD,
which is the molar concentration required to saturate half of the available binding sites) of a
steroid for its specific receptor is dependent upon the presence or absence of particular functional
groups and the overall three-dimensional structure of the molecule. Stereoisomerism may play an
important role in this respect: molecules with the same chemical composition but a different
spatial orientation of their substituents may have totally different binding properties and biological
effects. Thus, 5α-reduced dihydrotestosterone (DHT) is a potent androgen, with a strong affinity
for intracellular androgen receptors, whereas its 5ß-epimers do not bind to these receptors and are
totally devoid of androgenic properties.

Steroid inactivation and catabolism

General principles

Inactivation refers to the metabolic conversion of a biologically active compound into an inactive
one. Inactivation can occur at various stages of hormone action. Peripheral inactivation (e.g., by
liver enzymes) is required to ensure steady-state levels of plasma hormones as steroids are more or
less continuously secreted into the bloodstream. Moreover, if a hormone is to act as a "chemical
signal", its half-life in the circulation must be limited so that any change in secretion rate is
immediately reflected by a change in its plasma concentration (particularly when secretion rates are
decreased). But hormone inactivation can also occur in target tissues, notably after the hormone has
triggered the relevant biological effects in order to ensure termination of hormone action. The main
site of peripheral steroid inactivation and catabolism is the liver, but some catabolic activity also
occurs in the kidneys. Inactive hormones are mainly eliminated as urinary (mostly conjugated)
metabolites. Usually, steroids are eliminated once they have been inactivated (i.e., they are not
"recycled"). This elimination requires conversion to hydrophilic compounds in order to ensure their
solubility in biological fluids at rather high concentrations. A few examples of steroid excretion
products are shown in Table 1.

Formation of steroid conjugates

Conjugation (formation of hydrophilic molecules) is an important step in steroid catabolism. Most
excretory products in urine are in conjugated form. Two major pathways are used:

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1. Formation of glucuronides. This reaction requires uridine diphosphoglucuronic acid
(UDPGA) and a glucuronyl transferase. Glucuronic acid is attached to a HO-group on
the steroid molecule.

2. Formation of sulphates. This conversion is catalyzed by sulphokinases, which occur in
the cytosol of liver, testicular, adrenal and fetal tissues. The substrates are steroids with
an HO-group and phosphoadenosine-5’-phosphosulphate (PAPS).

Two examples of conjugated derivatives are shown in Fig. 4. In addition to being excretory
products, sulphates are also found in endocrine tissues and/or the plasma as precursors for
hormone synthesis. This is the case of dehydroepiandrosterone sulphate (DHEAS), which is used
notably for estrogen biosynthesis in the fetoplacental unit. Sulphatases occurring in the
microsomal fraction of liver, testis, ovary, adrenal and placenta catalyze the hydrolysis of
sulphated steroids to free steroids. The digestive juice of the snail Helix pomatia contains both
sulphatase and glucuronidase activity, and extracts from this source are used to hydrolyse urinary
conjugates in vitro for clinical assessment of total and conjugated excretion products.

In most species, fecal steroid metabolites are not excreted in a conjugated form, although
exceptions exist (e.g., felids).

Summary

Metabolism plays many important roles in steroid hormone action. Various biosynthetic pathways
occurring in endocrine glands such as the gonads, the adrenals and the fetoplacental unit are
required to produce and secrete circulating hormones. These hormones are partly metabolized in
the periphery, either before reaching their target tissues (to control plasma levels of active
compounds), or after termination of their action (inactivation and elimination). But many of them
are also metabolized within their target tissues, where a complex interplay between activation and
inactivation mechanisms serves to regulate the specificity and the amplitude of the hormonal
response. The proportion of steroid excreted in urine or feces usually is species or taxon specific.
For example, most felids excrete >90% of gonadal steroids into feces, whereas baboons excrete
>80% of gonadal steroids into urine. Steroids vary in the extent to which they are metabolized
before excretion, both within and among individuals and species. The time course of steroid
excretion and the degree to which steroids are excreted in urine or feces are determined by
infusing unlabeled or radiolabeled steroid and quantifying hormonal metabolites in excreta. The
lag-time from steroid production/secretion to appearance in excreted urine is generally <12 hours,
but can range from 12-24 hours in ruminants and 24-48 hours in primates and hindgut fermenters.

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1a 1b

FIGURE 1. Structure and classification of steroid hormones.
(a) Top: structure of cholesterol, with numbering of carbons (1 to 27) and identification of cycles
(A to D). Bottom: The three main classes of steroids ("parents compounds") derived from
cholesterol, with number of carbons in brackets (C21 to C18). Pregnanes (C21) form the basic
structure of progestins and corticosteroids, Androstanes (C19) of androgens and Estranes (C18) of
estrogens (with of phenolic A- ring).
(b) Three-dimensional conformation of naturally occurring steroid hormones. Functional groups
(or hydrogen atoms) are either in axial (a) or equatorial (e) position. Functional groups (or
hydrogens) above the plane of the molecule are said to be in the "ß " position (e.g. the HO-group
at C), those below the plane in "α" position (e.g. the hydrogen at position C5, bottom figure).
Orientation of substituents at certain positions can be critical for the overall conformation of the
molecule. Thus, orientation of the hydrogen atom at the A-B ring junction (marked by open
circles in Fig. 1b) will determine whether the A and B rings are fused in a trans- (as in the
molecule on top) or cis- (bottom drawing) conformation. This type of conformation (trans- or cis-
A-B ring fusion) is often critical for biological activity. For example, 5α-DHT (see Fig. 3) has
androgenic properties not shared by its 5ß-reduced analog, 5ß-DHT.

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FIGURE 2. Major Pathways of Steroid Biosynthesis.
The pathways outlined here are common to the adrenals, the gonads and, to some extent, to the
fetoplacental unit. The first committed step is the conversion of cholesterol to pregnenolone,
catalyzed by the P-450scc enzyme, which is under pituitary hormone control (ACTH or LH
depending on the tissue). Cholesterol side-chain removal is blocked specifically by
aminoglutethimide, a steroid biosynthesis inhibitor. From pregnenolone, steroid biosynthesis can
proceed either through the so-called "delta-5" pathway (17α-hydroxypregnenolone,
dehydroepiandrosterone, testosterone), or through the "delta-4" pathway (progesterone onwards).
Progesterone is the starting point for mineralocorticoid synthesis, whereas glucocorticoids are
derived from its metabolite, 17α-hydroxyprogesterone. Estrogens are formed from androgens
(androstenedione and/or testosterone). Most reactions are irreversible (as denoted by a single
arrow). Reversible reactions (double arrows) depend on cofactor availability (e.g. the
NADP/NADPH ratio). [Abbreviations used here for the various enzymes are listed in the figure].

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FIGURE 3. Steroid metabolism in target tissues.
Two examples showing pathways of steroid metabolism in target tissues which results in the
formation of biologically active metabolites. Top: 5α-reduction (left) or aromatization (right) of
testosterone. Bottom: 5α-reduction (left) or 5ß-reduction (right) of progesterone.

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TABLE 1. Steroid excretion products (examples).

Steroid class Starting steroid Excretion product Type of conjugate

Progesterone Pregnanediol Glucuronide
Glucuronide
Progestins 17α-
hydroxyprogesterone
Pregnanetriol

Androgens Testosterone Androsterone Glucuronide and/or
Etiocholanolone Sulphate

Glucocorticoids Cortisol 11ß- Glucuronide
hydroxyandrosterone

Allotetrahydrocortisone

FIGURE 4. Glucuronide and sulphate derivatives.
Testosterone glucuronide (left) and estrone sulphate (right), two steroid conjugates found in
human urine as excretory product for testosterone and estrone, respectively.

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XIX. REVIEW OF REPRODUCTIVE PHYSIOLOGY

GENERAL REPRODUCTIVE PHYSIOLOGY

Figure 1. Example of hormonal interactions within the HHG axis.

GnRH Reproduction is controlled by the complex
interaction of hormones within the
HYPOTHALAMUS hypothalamo-hypophyseal-gonadal (HHG)
axis as shown in Figure 1. In brief,
ANTERIOR gonadotropin-releasing hormone (GnRH)
PITUITARY from the hypothalamus stimulates the release
of gonadotropins, LH and FSH, from the
Estradiol FSH Testosterone anterior pituitary gland. In the female,
Progesterone gonadotropins are responsible for follicular
(eCG) LH Inhibin development, ovulation and corpus luteum
function. In the male, these same hormones
(hCG) stimulate testicular development,
spermatogenesis and testosterone production.

In both males and females, products from the

OVARY TESTIS gonads (e.g., estradiol, progesterone, inhibin,
testosterone) feedback to the pituitary gland
Follicle development Tubule formation and hypothalamus to regulate secretion of the

Ovulation Sperm production gonadotropins. Thus, gonadal activity is

CL development Leydig cell function tightly regulated by the interaction of

intrinsic stimulatory factors and resulting

products. Reproduction also is affected by external factors, many of which are seasonally

mediated (e.g., nutrition, photoperiod, climate). When studying a new species it is important to

collect data for periods exceeding one year to determine the influence of season or other extrinsic

factors on reproductive activity.

Female reproduction

Ovarian cycle dynamics are particularly complex and mechanisms of control not completely
understood. In general, during mammalian reproductive cycles a species-specific number of
follicles is selected to complete differentiation and ovulate. This occurs after regression of the
corpus luteum and withdrawal of progesterone. In most species, ovulation will not occur until this
‘progesterone block’ is removed. During the early follicular phase in mammals such as cattle,
horses and primates, after the recruitment of a cohort of follicles, one follicle is selected to become
dominant and continues to grow, while growth of the subordinate follicles is curtailed. Shortly
after selection, concentrations of gonadotropin receptors and steroidogenic enzymes increase in
the dominant follicle. The granulosa cells of the selected follicle acquire LH receptors to allow
them to increase aromatization in response to LH as well as FSH. The increased P450 aromatase
activity within the follicle causes an increase in concentrations of estradiol that eventually elicits

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the preovulatory LH/FSH surge. Recent evidence also suggests that intrafollicular insulin-like
growth factor (IGF) synergizes with FSH to promote follicular growth and aromatization and
helps complete dominant follicular selection. After ovulation, LH works to reorganize the
collapsed follicle into a corpus luteum and initiates and maintains progesterone production. If the
female becomes pregnant, the corpus luteum is maintained and progesterone of luteal and/or
placental sources sustains the pregnancy. If conception does not occur, prostaglandin-F2_ and/or
estrogens of uterine and/or ovarian sources cause luteal regression and the ovarian cycle begins
again.

Figure 2. Longitudinal secretory profile of the major reproductive hormones during a spontaneous
ovarian cycle.

The female ovarian cycle - Spontaneous ovulation

Follicular Development Ovulation CL Development

LH Progesterone

FSH

Estradiol

Day 0

Females of some species are known as "induced ovulators" in that a mating or similar stimulus is
required to induce the ovulatory LH surge and cause ovulation of the follicle. In these species,
follicles grow and then regress with no luteal phase unless an ovulatory stimulus occurs. In many
of these species, a non-pregnant luteal phase or pseudopregnancy results after non-conceptive
matings. These luteal phases can be of similar (e.g., ferrets) or shorter (e.g., felids) duration than a
pregnant luteal phase.

In developing assisted reproductive techniques, like artificial insemination (AI) and in vitro
fertilization/embryo transfer (IVF/ET), hormonal therapies are used to control reproductive
processes. Thus, equine chorionic gonadotropin (eCG or PMSG) which has FSH-like activity can
stimulate follicular development, whereas human chorionic gonadotropin (hCG) which has LH-

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like activity is used to induce ovulation (see Figure 1). Development of these technologies
requires an understanding of the mechanisms controlling ovarian function and relies on the
availability of adequate endocrine information to determine how well exogenous chorionic
gonadotropin therapy mimics natural responses.
Male reproduction
In the male, LH stimulates the production of testosterone from the interstitial cells, also known as
Leydig cells. FSH is important for seminiferous tubule formation and works in conjunction with
testosterone to stimulate spermatogenesis. Testosterone further acts to maintain secondary sex
characteristics (e.g., lion’s mane, facial hair, increased muscle mass, colored plummage, etc.) and
accessory sex gland function in part through local conversion to dihydrotestosterone (DHT), and
sexual behavior.
Non-invasive hormone monitoring
Obviously, the ability to track gonadal activity is essential for understanding the fundamentals of
reproduction. Fecal and urinary steroid metabolite monitoring are now well-established tools for
evaluating reproductive processes in diverse mammalian species. Steroids are easily extracted
from feces by boiling in 90% aqueous ethanol, whereas urinary steroids often are directly
measurable by immunoassay using group-specific antibodies that crossreact with excreted
metabolites or metabolite conjugates. With assisted reproductive techniques, like AI and IVF/ET,
becoming increasingly important for managing species ex situ, steroid metabolite monitoring has
provided an especially useful tool for examining the efficacy of associated hormonal therapies on
reproductive responses. Because of its enormous utility and noninvasive nature, excreted hormone
metabolite monitoring has become one of the most powerful tools available in wildlife research
today.

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Smithsonian's National Zoological Park
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Endocrine Workbook

XX. REVIEW OF ADRENAL PHYSIOLOGY

Although not a reproductive hormone per se, the potential impact of cortisol (i.e., stress) on
reproduction cannot be overlooked. Since the early 70’s “stress” has become an increasingly
popular and widely applied term and usually conjures up negative images; however,
disagreements continue over a clear definition of the term. It has been defined as a general
syndrome occurring in response to any stimulus that threatens or appears to threaten the
homeostasis (or the physiological and physical integrity) of an individual. The stimuli are called
“stressors” and the syndrome “stress”. It also has been described as the “fight or flight” response.
This physiological stress response appears to have evolved as an adaptive mechanism that allows
organisms to adjust to and cope with less predictable circumstances in their environment and to
respond rapidly to a wide variety of stimuli. Thus, “stress” represents an important part of life and
should not be considered inherently bad. Some researchers have recently argued that the term
“stress” should only be used for events that are detrimental for an individual. However, it often is
difficult to distinguish a “normal” and an adaptive response from ones that lead to negative effects.
Although we can clearly identify some of the negative effects, we are not yet able to distinguish
positive and negative stressors and associated responses reliably.

Figure 3. Hormonal interactions within the HPA axis.

The perception of a stressor by an organism activates

CRF the hypothalamic-pituitary-adrenal (HPA) axis (see

HYPOTHALAMUS Figure 3). The hypothalamus releases CRF

(corticotropin releasing factor) which stimulates the

ANTERIOR pituitary gland to secrete ACTH (adrenocorticotropin),
PITUITARY thus causing the release of corticoids (often termed
“stress” hormones) from the adrenal gland. A number

ACTH of acute events such as mating, fighting, chasing,
temperature shock, pain, etc. can evoke the stress

response and activate the adrenal cortex. The resulting

ADRENAL rapid changes in heart rate, blood pressure, and gastro-
GLAND intestinal activity are all designed to allow the organism
to quickly respond to the situation. However, it is when

acute stress occurs repeatedly without allowing for

coping responses or recovery, and/or when the stress

Cortisol response is chronically activated that stress becomes a
problem, called “distress”. An accumulation of

“Fight or flight” response biological costs through a series of acute stressors

Immune suppression and/or a consistent chronic stressor have been shown to

Reproductive inhibition lead to various pathological conditions, such as immune

deficiency, reproductive suppression, growth reduction,

muscle wasting, gastrointestinal dysfunction and impaired brain function. Thus, although acute

stress can have a stimulatory or facilitatory effect on certain aspects of reproduction, chronic stress

can lead to an overall inhibition of reproduction. Identifying early symptoms of distress and

pinpointing chronic as well as repetitive acute stressors is essential for evaluating animal well-being.

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Smithsonian's National Zoological Park
Conservation & Research Center
Endocrine Workbook

However, it also must be recognized that responses to stress can be very individual-specific.
Animals born and raised in captivity may react very differently compared to their wild counterparts
even when faced with the same stressors. In captivity many stressors have been removed, but the
lack of stimulation in and of itself may present a source of distress due to boredom.

Measuring stress and distress is difficult. No single biochemical or behavioral measure can be
used to assess animal well-being or stress. Whereas behavioral observations may frequently
provide a first indicator of distress, they can also be misleading. Similarly, measures of
reproductive success, growth rate, and general health, although important for examining certain
aspects of animal well-being, often are not reliable as early indicators of distress when examined
by themselves. Only through a combination of these measure, including physiological analyses of
circulating and excreted hormones like corticoids, can we begin to examine the influence of stress
and distress on animal well-being.

The recent development of non-invasive monitoring of adrenal steroids has provided a new tool
for these investigations. Measuring glucocorticoid concentrations in blood samples has long been
used as an indicator of stress in mammals. However, the invasive nature and inherent stress of
collecting blood samples has limited its usefulness for studies on many captive and wild animals.
Alternatively, fecal or urinary corticoid monitoring can be used in combination with behavioral
observations and other measures of overall health in longitudinal studies without additional stress
to the animal. Careful biochemical and physiological validation is necessary for the application of
this technique and resulting measures cannot provide a “litmus” test for distress. Increases in
excreted glucocorticoids may be due to “negative” (i.e., nonadaptive) or “positive” (i.e., adaptive)
stress responses. Furthermore, glucocorticoid levels may decline due to intrinsic hormone control
and negative feed back mechanisms rather than elimination or decrease of the external stressor. In
addition, although a wide variety of stressors stimulate the HPA axis, not all types of stressors will
affect an increase in glucocorticoids. Nevertheless, the combined use of all available measures
can help us better understand the impact of various stressors on animal well-being and make
significant advances in our assessment of distress.

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