Transcript Biol 155 Human Physiology - University of British Columbia

Comparative aspects of Growth
Hormone

The routine bioassay for growth hormone is the
rat tibia assay.
Juvenile rats or mice are hypophesectomized.
 Animals recover and then are injected with
presumed growth hormone.
 After treatment, the thickness of the epiphyseal plate
in the head end of the tibia is measured.
 A thickening of the plate indicates a positive result.
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The rat tibia test responds to growth hormones
from a wide range of species, right from fish up
through mammals.

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This broad range of activities suggests that the
bioactive portion of the GH molecule is conserved
through virtually all the vertebrate groups.
Sequence data shows that the non-bioactive
segments of the molecule are variable.

This can lead to false readings when using antibodybased heterologous assays.

Another antibody-based assay problem is that
there is often considerable cross-reactivity
between GH and prolactin.

This suggests two related things.
Prolactin and GH evolved from a common earlier
hormone
 Structurally, prolactin and GH are close in lower
vertebrates.

Function of GH in lower vertebrates:

Fish
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GH has not been conclusively demonstrated in
agnathans.

These fish also don’t have a well developed pars distalis.
Lamprey (cyclostomata)
Hagfish (cyclostomata)
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Salachians (sharks and rays)
Positive response in the rat tibia assay.
 Pituitaries have a distinct pars distalis
 Insulin-like growth factors have been found in at least two
species; however, no definitive link has been established
between these IGFs and salachian GH.

Leopard Shark (chondrichthys)
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No GH activity has been identified in holostean
fishes, despite distinct pars distalis. *

Distinct GH activity has been detected in all
other fish groups.

Compared to other vertebrate groups, GH from
fish (particularly teleosts) show much greater
variability between species.

For example, there is almost as much variability
between chum and chinook GH as between chum
and human GH.
Spotted Ratfish (Holocephali)

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In all these groups, GH stimulates the secretion of
somatomedins from the liver.
GH is also involved in osmoregulation.
GH has been implicated in the adaptation of
anadromous fish to salt water.
 GH is elevated during salt water exposure.
 GH may stimulates the expression of CFTR in the
gills of fish.


Prolactin and cortisol are also involved in
osmoregulation of fish.

Prolactin and GH may act as switches, while
cortisol may function as a “volume” control in
regulating ion transport.
Somatolactin

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Somatolactin is a hormone so far found only in
teleosts.
Secreted by pars intermedia
Structurally very similar to both GH and
prolatin.

Structure appears to be midway between the two
hormones. Antibodies to both hormones will crossreact with somatolactin.

It is specifically secreted by fish in a very low
Ca2+ environment.
Appears to act as an adaptation to ultrasoft water.
 May act to specifically stimulate uptake of calcium
from low calcium environment.


Water softness is based on the amount of
dissolved calcium. Hard water
Ca2+.

Somatolactin also may be involved in adaptation
to seawater during smoltification in salmonids.
GH in Amphibians

Antibodies to mammalian GH will bind
amphibian GH and prolactin equally.
First taken as evidence that the two hormones were
just different isoforms of the same hormone.
 This view has now been discounted.

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It appears that in larval amphibians prolactin
performs the functions of GH, but once
metamorphosis occurs, GH takes over the
functions.

Recently, antibodies have been generated that
can distinguish between bullfrog PRL and GH.

Supports the idea that there are two distinct
hormones.

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However, the structures of both are significantly
different from the mammalian hormones.
Amphibian GH is structurally very similar to
mammalian GH.

Amphibian GHs are active in the rat tibia assay,
but show much lower activities than bovine GH.

IGF activity has been show in amphibians.
However, release of IGFs has not been directly
linked to GH or PRL secretion.
 This maybe a problem associated with the lack of
specific amphibian GH and PRL immunoassays.
 Area where more research is needed.
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GH in Reptiles
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Little work has been done in this area.
Mammalian GH stimulates growth in juvenile
snapping turtles and in at least one lizard genus
(Lacerta).
GH has been purified from adult snapping
turtles and sea turtles.

Both forms have been show to be very effective in
the rat tibia bioassay.
GH in Birds

As might be expected, GH activity has been
demonstrated in chickens, turkeys and ducks.
This is not surprising given the economic influences.
 GH stimulates growth and quicker maturation.
 This results in a shorter time to market.

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Avian GH is active in the rat tibia assay, but the
avian forms are generally NOT recognized by
ABs to bovine GH.

Duck (Peking duck) GH does not react with rat
GH Abs.

GH in birds shows a significant divergence from
mammalian GH.

Bird GH appears to be more closely related to
reptilian GH than to mammalian GH.
Prolactin


Prolactin is also referred to as a trophic
hormone, since it seems to regulate growth in
lower vertebrates.
The classic bioassay for PRL is the pigeon crop
sac assay.
Performed on immature pigeons
 The crop sac is a bilateral extension of the
esophagus.

First, animals are “primed” by giving them an
injection of PRL (usually murine).
 After priming (several days is required). One side is
injected subcutaneously with a test solution
containing putative PRL.
 Other side is injected with saline.
 After several days, crop sac is removed and the
development of the epithelial surface is assayed,
either by general histology, or by scraping and
measuring dry weight of removed epithelium.

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Prolactin appears to have 4 major functions in
the lower vertebrates.
Reproductive.
 Osmoregulation.
 Growth.
 Integument modification.

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Fish PRL is structurally different from
amphibians and amniotes (mammals, reptiles
[turtles, lizards, sphenodon, crocodiles], birds
and their extinct relatives)
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Because of this difference, it has been suggested
that fish PRL be called paralactin.
However, this difference does not always hold.

Lungfish PRL is similar to tetrapod PRL.
Coelacanth
lungfish
tetrapods
Early bony fish

This probably reflects the evolutionary origin of
the tetrapods
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Functions in fish
As already mentioned: Osmoregulation.
 Cyclostomes

Shown to affect electrolyte balance.
 Appears to act in tandem with corticosteroids.
 This is based on functional data. The hormone has not
been purified yet.
 Hagfish show PRL-like activity in the rostral portion of
the pituitary gland.

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This activity appears to be involved in electrolyte regulation.

There are two classic bioassays for fish PRL.
 Colour
 Uses
assay
the goby Gillichthyes mirabilis.
 Administer a subcutaneous injection of presumed
PRL under the pre-opercular skin.
 A change in colour (to yellow) is a positive response.

Plasma sodium
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Uses hypophysectomized Fundulus heteroclitus.
After hypophysectomy, the fish are kept in 50% seawater until
challenged.
Inject the fish with presumed PRL.
Transfer to fresh water and measure plasma Na+ 24 hours later.
Test is considered positive if the fish retain elevated plasma Na+.

Mammalian PRL is active in both fish bioassays.
 Selachians
 Bioassayable
PRL has been purified from this
group.
 This has been determined using an alternative
bioassay- the skin colouration bioassay.
 The bioassayable PRL activity appears to be
located in the anterior pituitary.
 Using the skin colouration assay.

Teleosts
 As
mentioned, osmoregulation.
 This
is certainly true for euryhaline fish.
 Probably true for freshwater fish as well.
 Sexual
 PRL
reproduction (in males). *
stimulates the growth of the seminal vesicle.
 This is very important in seasonal breaders.
 PRL
also is important in maintaining
secondary sexual characteristics, like the
brood pouch in male seahorses.
 In
particular, PRL will trigger and maintain
breeding-colouration.
 In
both sexes, PRL will stimulate overall
growth in the gonads.
 PRL
also stimulates parental behaviour.
 This
has been demonstrated in mouth-brooders
and in Sticklebacks.
 Mouth-brooders
(usually the male) will pick up
shed eggs and pump water over them.
 In
nest-builders, such as sticklebacks and a larger
number of marine species, the male will defend
the nest and care for the eggs.
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Effects on
colouration.
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Stimulate
proliferation of
melanocytes.
Not to be
confused with
the actions of
MSH (shown
here).
PRL in Amphibians
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Structurally, PRL is significanly different from
teleost PRL (as reflected by diminished AB
recognition).
However, amphibian PRL is active in both fish
bioassays.
There is a specific amphibian PRL bioassay,
called the “water drive”
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Terrestrial newts start out as tadpoles (fully
aquatic).
The tadpoles undergo a two-stage
metamorphosis.

Fist stage: They develop into and “eft”. This stage is
an immature adult, with legs, lungs, etc.
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Second stage: Involves a migration back to water.
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They are sexually immature and live on land.
Individuals will migrate back into water full-time and
undergo a sexual maturation.
PRL induces this water drive, as well as
stimulating sexual maturation (in both sexes).
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All fish PRLs tested have shown induction of
the water drive.
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All tetrapod PRLs tested have shown induction
of the water drive.
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Conclusion is that although there are significant
structural differences between both amphibian and
fish, and amphibian and amniote PRLs, both are
effective. Amphibian PRL seems to be intermediate
in structure.

Functions involved with ion regulation:
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PRL appears to be required for adult forms to reenter the aquatic environment.
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PRL stimulates ion uptake across skin.
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In this situation, they tend to take up water and lose
electrolytes (same problems as fish in freshwater).
This is also associated with the water drive.
In amphibians there is significant ion uptake across
the urinary bladder and this is stimulated by PRL.

PRL stimulates Na+ transport across bladder epithelium.
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Effects on colouration:

PRL stimulates the proliferation of melanocytes.
This has been used as a bioassay, like the goby colouration
assay, but it has proved unreliable.
 Interestingly, in the frogs where this occurs, the colour
induced is yellow.

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Other effects:
PRL stimulates growth of gills, tails, and brain in
tadpoles.
 PRL is also involved in regulating limb regeneration.
 PRL regulates secretion by the oviducts andregulates
ovulation (in part).
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PRL in reptiles
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Very little is known.
PRL is still involved in electrolyte regulation.
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Plasma Na+ levels are regulated by PRL in synergy
with cortisol.
PRL is involved in tail regeneration in lizards.
PRL appears to be involved in the initiation of
moulting.
PRL has an weak antigonadotropic effect.
PRL effects in birds:

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More is known about functions in birds.
As already mentioned, PRL stimulates the
production of crop milk in pigeons.
In particular, PRL stimulates an hyperplasia and
sloughing of the crop sac mucosa.
 This forms a milky-white liquid with the epithelial
cells in suspension (a process similar to production
of mammalian milk).
 This liquid is fed to young.
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As mentioned, crop milk formation can be
stimulated with all tetrapod PRLs, as well as with
lungfish PRL.
Formation of the brood patch in both sexes.
Brood patch is an are of the chest that becomes
highly vascularized and loses its feather covering.
 Used to transmit body from adult to the eggs, to
promote egg development.
 After breeding is over, the hyperangiogenesis
regresses.

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PRL has a strong antigonadotropic effect in
birds.

Reproductive timing in birds is very tightly regulated
(will be discussed later).
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PRL strongly suppresses courtship behaviour.

Strongly promotes nesting, brooding and food
gathering behaviours associated with chick
rearing.

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PRL stimulates moulting.
Also appears to be involved in feather
pigmentation.
Osmoregulation functions:
This is primarily seen in sea birds with nasal glands.
 Nasal gland secretes a very concentrated NaCl
solution.
 This allows them to drink seawater directly.
 PRL stimulates Na+ directly, which results in
increased fluid secretion.

PRL in mammals

Almost all effects of PRL in mammals are
reproductive.
Stimulation of mammary gland.
 Stimulation of androgen binding in prostate gland.
 Stimulation of estrogen binding in uterus.
 Stimulation of early puberty in rats.
 Stimulates increased cholesterol uptake in testes.
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Stimulation of parental behaviour in rats.
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See table 4-8 in Norris for other effects on
reproduction.
PRL still has some effects on electrolyte balance.
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Stimulates increased Na+ retention in the kidney.
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Corticotropic