Comparative aspects of Comparative aspects of Growth HormoneGrowth Hormone

The routine bioassay for growth hormone The routine bioassay for growth hormone is the rat tibia assay.is the rat tibia assay. Juvenile rats or mice are hypophesectomized.Juvenile rats or mice are hypophesectomized. Animals recover and then are injected with Animals recover and then are injected with

presumed growth hormone.presumed growth hormone. After treatment, the thickness of the After treatment, the thickness of the

epiphyseal plate in the head end of the tibia epiphyseal plate in the head end of the tibia is measured.is measured.

A thickening of the plate indicates a positive A thickening of the plate indicates a positive result.result.

The rat tibia test responds to growth The rat tibia test responds to growth hormones from a wide range of species, hormones from a wide range of species, right from fish up through mammals.right from fish up through mammals. This broad range of activities suggests that This broad range of activities suggests that

the bioactive portion of the GH molecule is the bioactive portion of the GH molecule is conserved through virtually all the conserved through virtually all the vertebrate groups.vertebrate groups.

Sequence data shows that the non-Sequence data shows that the non-bioactive segments of the molecule are bioactive segments of the molecule are variable.variable. This can lead to false readings when using This can lead to false readings when using

antibody-based heterologous assays.antibody-based heterologous assays.

Another antibody-based assay Another antibody-based assay problem is that there is often problem is that there is often considerable cross-reactivity considerable cross-reactivity between GH and prolactin.between GH and prolactin.

This suggests two related things.This suggests two related things. Prolactin and GH evolved from a Prolactin and GH evolved from a

common earlier hormonecommon earlier hormone Structurally, prolactin and GH are close Structurally, prolactin and GH are close

in lower vertebrates.in lower vertebrates.

Function of GH in lower Function of GH in lower vertebrates:vertebrates:

FishFish GH has not been conclusively GH has not been conclusively

demonstrated in agnathans.demonstrated in agnathans. These fish also don’t have a well developed These fish also don’t have a well developed

pars distalis.pars distalis.

Lamprey (cyclostomata)Lamprey (cyclostomata)

Hagfish (cyclostomata)Hagfish (cyclostomata)

Salachians (sharks and rays)Salachians (sharks and rays) Positive response in the rat tibia assay.Positive response in the rat tibia assay. Pituitaries have a distinct pars distalisPituitaries have a distinct pars distalis Insulin-like growth factors have been found Insulin-like growth factors have been found

in at least two species; however, no in at least two species; however, no definitive link has been established between definitive link has been established between these IGFs and salachian GH.these IGFs and salachian GH.

Leopard Shark Leopard Shark (chondrichthys)(chondrichthys)

No GH activity has been identified in No GH activity has been identified in holostean fishes, despite distinct pars holostean fishes, despite distinct pars distalis. *distalis. *

Distinct GH activity has been detected Distinct GH activity has been detected in all other fish groups.in all other fish groups.

Compared to other vertebrate groups, Compared to other vertebrate groups, GH from fish (particularly teleosts) GH from fish (particularly teleosts) show much greater variability between show much greater variability between species.species. For example, there is almost as much For example, there is almost as much

variability between chum and chinook GH variability between chum and chinook GH as between chum and human GH.as between chum and human GH.

Spotted Ratfish Spotted Ratfish (Holocephali)(Holocephali)

In all these groups, GH stimulates the In all these groups, GH stimulates the secretion of somatomedins from the liver.secretion of somatomedins from the liver.

GH is also involved in osmoregulation.GH is also involved in osmoregulation.

GH has been implicated in the adaptation of GH has been implicated in the adaptation of anadromous fish to salt water. anadromous fish to salt water.

GH is elevated during salt water exposure.GH is elevated during salt water exposure. GH may stimulates the expression of CFTR GH may stimulates the expression of CFTR

in the gills of fish.in the gills of fish.

Prolactin and cortisol are also Prolactin and cortisol are also involved in osmoregulation of fish.involved in osmoregulation of fish.

Prolactin and GH may act as Prolactin and GH may act as switches, while cortisol may function switches, while cortisol may function as a “volume” control in regulating as a “volume” control in regulating ion transport.ion transport.

SomatolactinSomatolactin

Somatolactin is a hormone so far Somatolactin is a hormone so far found only in teleosts.found only in teleosts.

Secreted by pars intermediaSecreted by pars intermedia Structurally very similar to both GH Structurally very similar to both GH

and prolatin. and prolatin. Structure appears to be midway Structure appears to be midway

between the two hormones. Antibodies between the two hormones. Antibodies to both hormones will cross-react with to both hormones will cross-react with somatolactin.somatolactin.

It is specifically secreted by fish in a very It is specifically secreted by fish in a very low Calow Ca2+2+ environment. environment. Appears to act as an adaptation to ultrasoft Appears to act as an adaptation to ultrasoft

water.water. May act to specifically stimulate uptake of May act to specifically stimulate uptake of

calcium from low calcium environment.calcium from low calcium environment.

Water softness is based on the amount of Water softness is based on the amount of dissolved calcium. Hard waterdissolved calcium. Hard water CaCa2+2+..

Somatolactin also may be involved in Somatolactin also may be involved in adaptation to seawater during adaptation to seawater during smoltification in salmonids.smoltification in salmonids.

GH in AmphibiansGH in Amphibians

Antibodies to mammalian GH will bind Antibodies to mammalian GH will bind amphibian GH and prolactin equally.amphibian GH and prolactin equally. First taken as evidence that the two First taken as evidence that the two

hormones were just different isoforms of hormones were just different isoforms of the same hormone.the same hormone.

This view has now been discounted.This view has now been discounted. It appears that in larval amphibians It appears that in larval amphibians

prolactin performs the functions of prolactin performs the functions of GH, but once metamorphosis occurs, GH, but once metamorphosis occurs, GH takes over the functions.GH takes over the functions.

Recently, antibodies have been Recently, antibodies have been generated that can distinguish between generated that can distinguish between bullfrog PRL and GH.bullfrog PRL and GH.

Supports the idea that there are two Supports the idea that there are two distinct hormones. distinct hormones.

However, the structures of both are However, the structures of both are significantly different from the mammalian significantly different from the mammalian hormones.hormones.

Amphibian GH is structurally very Amphibian GH is structurally very similar to mammalian GH.similar to mammalian GH.

Amphibian GHs are active in the rat Amphibian GHs are active in the rat tibia assay, but show much lower tibia assay, but show much lower activities than bovine GH.activities than bovine GH.

IGF activity has been show in IGF activity has been show in amphibians. amphibians. However, release of IGFs has not been However, release of IGFs has not been

directly linked to GH or PRL secretion. directly linked to GH or PRL secretion. This maybe a problem associated with the This maybe a problem associated with the

lack of specific amphibian GH and PRL lack of specific amphibian GH and PRL immunoassays.immunoassays.

Area where more research is needed.Area where more research is needed.

GH in ReptilesGH in Reptiles

Little work has been done in this Little work has been done in this area.area.

Mammalian GH stimulates growth in Mammalian GH stimulates growth in juvenile snapping turtles and in at juvenile snapping turtles and in at least one lizard genus (least one lizard genus (LacertaLacerta).).

GH has been purified from adult GH has been purified from adult snapping turtles and sea turtles. snapping turtles and sea turtles. Both forms have been show to be very Both forms have been show to be very

effective in the rat tibia bioassay.effective in the rat tibia bioassay.

GH in BirdsGH in Birds

As might be expected, GH activity has As might be expected, GH activity has been demonstrated in chickens, turkeys been demonstrated in chickens, turkeys and ducks.and ducks. This is not surprising given the economic This is not surprising given the economic

influences.influences. GH stimulates growth and quicker maturation.GH stimulates growth and quicker maturation. This results in a shorter time to market.This results in a shorter time to market.

Avian GH is active in the rat tibia assay, Avian GH is active in the rat tibia assay, but the avian forms are generally but the avian forms are generally NOTNOT recognized by ABs to bovine GH.recognized by ABs to bovine GH.

Duck (Peking duck) GH does not Duck (Peking duck) GH does not react with rat GH Abs.react with rat GH Abs.

GH in birds shows a significant GH in birds shows a significant divergence from mammalian GH.divergence from mammalian GH.

Bird GH appears to be more closely Bird GH appears to be more closely related to reptilian GH than to related to reptilian GH than to mammalian GH.mammalian GH.

ProlactinProlactin

Prolactin is also referred to as a Prolactin is also referred to as a trophic hormone, since it seems to trophic hormone, since it seems to regulate growth in lower regulate growth in lower vertebrates.vertebrates.

The classic bioassay for PRL is the The classic bioassay for PRL is the pigeon crop sac assay. pigeon crop sac assay. Performed on immature pigeonsPerformed on immature pigeons The crop sac is a bilateral extension of The crop sac is a bilateral extension of

the esophagus.the esophagus.

First, animals are “primed” by giving First, animals are “primed” by giving them an injection of PRL (usually them an injection of PRL (usually murine).murine).

After priming (several days is required). After priming (several days is required). One side is injected subcutaneously with One side is injected subcutaneously with a test solution containing putative PRL. a test solution containing putative PRL.

Other side is injected with saline.Other side is injected with saline. After several days, crop sac is removed After several days, crop sac is removed

and the development of the epithelial and the development of the epithelial surface is assayed, either by general surface is assayed, either by general histology, or by scraping and measuring histology, or by scraping and measuring dry weight of removed epithelium.dry weight of removed epithelium.

Prolactin appears to have 4 major Prolactin appears to have 4 major functions in the lower vertebrates.functions in the lower vertebrates. Reproductive.Reproductive. Osmoregulation.Osmoregulation. Growth.Growth. Integument modification.Integument modification.

Fish PRL is structurally different Fish PRL is structurally different from amphibians and amniotes from amphibians and amniotes (mammals, reptiles [turtles, lizards, (mammals, reptiles [turtles, lizards, sphenodon, crocodiles], birds and sphenodon, crocodiles], birds and their extinct relatives)their extinct relatives)

Because of this difference, it has been Because of this difference, it has been suggested that fish PRL be called suggested that fish PRL be called paralactin.paralactin.

However, this difference does not However, this difference does not always hold.always hold. Lungfish PRL is similar to tetrapod PRL.Lungfish PRL is similar to tetrapod PRL.

CoelacanthCoelacanth lungfishlungfish tetrapodstetrapods

Early bony fishEarly bony fish

This probably reflects the evolutionary This probably reflects the evolutionary origin of the tetrapodsorigin of the tetrapods

Functions in fishFunctions in fish As already mentioned: Osmoregulation.As already mentioned: Osmoregulation. CyclostomesCyclostomes

Shown to affect electrolyte balance.Shown to affect electrolyte balance. Appears to act in tandem with Appears to act in tandem with

corticosteroids.corticosteroids. This is based on functional data. The This is based on functional data. The

hormone has not been purified yet.hormone has not been purified yet. Hagfish show PRL-like activity in the rostral Hagfish show PRL-like activity in the rostral

portion of the pituitary gland.portion of the pituitary gland. This activity appears to be involved in electrolyte This activity appears to be involved in electrolyte

regulation.regulation.

There are two classic bioassays for There are two classic bioassays for fish PRL.fish PRL. Colour assayColour assay

Uses the goby Uses the goby Gillichthyes mirabilis.Gillichthyes mirabilis. Administer a subcutaneous injection of Administer a subcutaneous injection of

presumed PRL under the pre-opercular presumed PRL under the pre-opercular skin. skin.

A change in colour (to yellow) is a A change in colour (to yellow) is a positive response.positive response.

Plasma sodiumPlasma sodium Uses hypophysectomized Uses hypophysectomized Fundulus heteroclitusFundulus heteroclitus.. After hypophysectomy, the fish are kept in 50% seawater After hypophysectomy, the fish are kept in 50% seawater

until challenged.until challenged. Inject the fish with presumed PRL.Inject the fish with presumed PRL. Transfer to fresh water and measure plasma NaTransfer to fresh water and measure plasma Na++ 24 hours 24 hours

later. later. Test is considered positive if the fish retain elevated plasma Test is considered positive if the fish retain elevated plasma

NaNa++..

Mammalian PRL is active in both fish Mammalian PRL is active in both fish bioassays.bioassays. SelachiansSelachians

Bioassayable PRL has been purified from Bioassayable PRL has been purified from this group.this group.

This has been determined using an This has been determined using an alternative bioassay- the skin colouration alternative bioassay- the skin colouration bioassay.bioassay.

The bioassayable PRL activity appears to The bioassayable PRL activity appears to be located in the anterior pituitary.be located in the anterior pituitary.

Using the skin colouration assay.Using the skin colouration assay.

TeleostsTeleosts As mentioned,As mentioned, osmoregulation.osmoregulation.

This is certainly true for euryhaline fish.This is certainly true for euryhaline fish. Probably true for freshwater fish as well.Probably true for freshwater fish as well.

Sexual reproduction (in males). *Sexual reproduction (in males). * PRL stimulates the growth of the seminal PRL stimulates the growth of the seminal

vesicle.vesicle. This is very important in seasonal This is very important in seasonal

breaders.breaders.

PRL also is important in PRL also is important in maintaining secondary sexual maintaining secondary sexual characteristics, like the brood characteristics, like the brood pouch in male seahorses. pouch in male seahorses.

In particular, PRL will trigger and In particular, PRL will trigger and maintain breeding-colouration.maintain breeding-colouration.

In both sexes, PRL will stimulate In both sexes, PRL will stimulate overall growth in the gonads.overall growth in the gonads.

PRL also stimulates parental behaviour.PRL also stimulates parental behaviour.

This has been demonstrated in mouth-This has been demonstrated in mouth-brooders and in Sticklebacks.brooders and in Sticklebacks.

Mouth-brooders (usually the male) will pick Mouth-brooders (usually the male) will pick up shed eggs and pump water over them.up shed eggs and pump water over them.

In nest-builders, such as sticklebacks and a In nest-builders, such as sticklebacks and a larger number of marine species, the male larger number of marine species, the male will defend the nest and care for the eggs.will defend the nest and care for the eggs.

Effects on Effects on colouration.colouration. Stimulate Stimulate

proliferation proliferation of of melanocytes.melanocytes.

Not to be Not to be confused confused with the with the actions of actions of MSH (shown MSH (shown here).here).

PRL in AmphibiansPRL in Amphibians

Structurally, PRL is significanly Structurally, PRL is significanly different from teleost PRL (as different from teleost PRL (as reflected by diminished AB reflected by diminished AB recognition).recognition).

However, amphibian PRL is active in However, amphibian PRL is active in both fish bioassays.both fish bioassays.

There is a specific amphibian PRL There is a specific amphibian PRL bioassay, called the “water drive”bioassay, called the “water drive”

Terrestrial newts start out as tadpoles Terrestrial newts start out as tadpoles (fully aquatic).(fully aquatic).

The tadpoles undergo a two-stage The tadpoles undergo a two-stage metamorphosis.metamorphosis. Fist stage: They develop into and “eft”. This Fist stage: They develop into and “eft”. This

stage is an immature adult, with legs, lungs, stage is an immature adult, with legs, lungs, etc.etc.

They are sexually immature and live on land.They are sexually immature and live on land. Second stage: Involves a migration back to Second stage: Involves a migration back to

water.water. Individuals will migrate back into water full-time Individuals will migrate back into water full-time

and undergo a sexual maturation.and undergo a sexual maturation. PRL induces this water drive, as well as PRL induces this water drive, as well as

stimulating sexual maturation (in both stimulating sexual maturation (in both sexes).sexes).

All fish PRLs tested have shown All fish PRLs tested have shown induction of the water drive.induction of the water drive.

All tetrapod PRLs tested have shown All tetrapod PRLs tested have shown induction of the water drive.induction of the water drive.

Conclusion is that although there are Conclusion is that although there are significant structural differences significant structural differences between both amphibian and fish, and between both amphibian and fish, and amphibian and amniote PRLs, both are amphibian and amniote PRLs, both are effective. Amphibian PRL seems to be effective. Amphibian PRL seems to be intermediate in structure.intermediate in structure.

Functions involved with ion regulation:Functions involved with ion regulation:

PRL appears to be required for adult forms PRL appears to be required for adult forms to re-enter the aquatic environment.to re-enter the aquatic environment. In this situation, they tend to take up water and In this situation, they tend to take up water and

lose electrolytes (same problems as fish in lose electrolytes (same problems as fish in freshwater).freshwater).

PRL stimulates ion uptake across skin.PRL stimulates ion uptake across skin. This is also associated with the water drive.This is also associated with the water drive.

In amphibians there is significant ion uptake In amphibians there is significant ion uptake across the urinary bladder and this is across the urinary bladder and this is stimulated by PRL.stimulated by PRL. PRL stimulates NaPRL stimulates Na++ transport across bladder transport across bladder

epithelium.epithelium.

Effects on colouration:Effects on colouration: PRL stimulates the proliferation of PRL stimulates the proliferation of

melanocytes.melanocytes. This has been used as a bioassay, like the goby This has been used as a bioassay, like the goby

colouration assay, but it has proved unreliable.colouration assay, but it has proved unreliable. Interestingly, in the frogs where this occurs, Interestingly, in the frogs where this occurs,

the colour induced is yellow.the colour induced is yellow. Other effects:Other effects:

PRL stimulates growth of gills, tails, and PRL stimulates growth of gills, tails, and brain in tadpoles.brain in tadpoles.

PRL is also involved in regulating limb PRL is also involved in regulating limb regeneration.regeneration.

PRL regulates secretion by the oviducts PRL regulates secretion by the oviducts andregulates ovulation (in part).andregulates ovulation (in part).

PRL in reptilesPRL in reptiles

Very little is known.Very little is known. PRL is still involved in electrolyte PRL is still involved in electrolyte

regulation.regulation. Plasma NaPlasma Na++ levels are regulated by PRL in levels are regulated by PRL in

synergy with cortisol.synergy with cortisol. PRL is involved in tail regeneration in PRL is involved in tail regeneration in

lizards.lizards. PRL appears to be involved in the PRL appears to be involved in the

initiation of moulting.initiation of moulting. PRL has an weak antigonadotropic effect.PRL has an weak antigonadotropic effect.

PRL effects in birds:PRL effects in birds:

More is known about functions in birds.More is known about functions in birds. As already mentioned, PRL stimulates As already mentioned, PRL stimulates

the production of crop milk in pigeons.the production of crop milk in pigeons. In particular, PRL stimulates an In particular, PRL stimulates an

hyperplasia and sloughing of the crop sac hyperplasia and sloughing of the crop sac mucosa.mucosa.

This forms a milky-white liquid with the This forms a milky-white liquid with the epithelial cells in suspension (a process epithelial cells in suspension (a process similar to production of mammalian milk).similar to production of mammalian milk).

This liquid is fed to young.This liquid is fed to young.

As mentioned, crop milk formation As mentioned, crop milk formation can be stimulated with all tetrapod can be stimulated with all tetrapod PRLs, as well as with lungfish PRL.PRLs, as well as with lungfish PRL.

Formation of the brood patch in both Formation of the brood patch in both sexes.sexes. Brood patch is an are of the chest that Brood patch is an are of the chest that

becomes highly vascularized and loses its becomes highly vascularized and loses its feather covering.feather covering.

Used to transmit body from adult to the Used to transmit body from adult to the eggs, to promote egg development.eggs, to promote egg development.

After breeding is over, the After breeding is over, the hyperangiogenesis regresses.hyperangiogenesis regresses.

PRL has a strong antigonadotropic PRL has a strong antigonadotropic effect in birds.effect in birds. Reproductive timing in birds is very Reproductive timing in birds is very

tightly regulated (will be discussed later).tightly regulated (will be discussed later).

PRL strongly suppresses courtship PRL strongly suppresses courtship behaviour.behaviour.

Strongly promotes nesting, brooding Strongly promotes nesting, brooding and food gathering behaviours and food gathering behaviours associated with chick rearing.associated with chick rearing.

PRL stimulates moulting.PRL stimulates moulting. Also appears to be involved in feather Also appears to be involved in feather

pigmentation.pigmentation. Osmoregulation functions:Osmoregulation functions:

This is primarily seen in sea birds with This is primarily seen in sea birds with nasal glands.nasal glands.

Nasal gland secretes a very concentrated Nasal gland secretes a very concentrated NaCl solution.NaCl solution.

This allows them to drink seawater This allows them to drink seawater directly.directly.

PRL stimulates NaPRL stimulates Na++ directly, which directly, which results in increased fluid secretion.results in increased fluid secretion.

PRL in mammalsPRL in mammals

Almost all effects of PRL in mammals Almost all effects of PRL in mammals are reproductive.are reproductive. Stimulation of mammary gland.Stimulation of mammary gland. Stimulation of androgen binding in Stimulation of androgen binding in

prostate gland.prostate gland. Stimulation of estrogen binding in uterus.Stimulation of estrogen binding in uterus. Stimulation of early puberty in rats.Stimulation of early puberty in rats. Stimulates increased cholesterol uptake Stimulates increased cholesterol uptake

in testes.in testes.

Stimulation of parental behaviour in rats. Stimulation of parental behaviour in rats.

See table 4-8 in Norris for other effects on See table 4-8 in Norris for other effects on reproduction.reproduction.

PRL still has some effects on electrolyte PRL still has some effects on electrolyte balance.balance.

Stimulates increased NaStimulates increased Na++ retention in the retention in the kidney.kidney.

CorticotropicCorticotropic