1. Comparative aspects of Growth Hormone The routine bioassay for growth hormone is the rat tibia assay. The routine bioassay for growth hormone is the rat tibia assay. Juvenile rats or mice are hypophesectomized. Juvenile rats or mice are hypophesectomized. Animals recover and then are injected with presumed growth hormone. 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. 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. A thickening of the plate indicates a positive result.

2. The rat tibia test responds to growth hormones from a wide range of species, right from fish up through mammals. The rat tibia test responds to growth hormones from a wide range of species, right from fish up through mammals. This broad range of activities suggests that the bioactive portion of the GH molecule is conserved through virtually all the vertebrate groups. 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. Sequence data shows that the non-bioactive segments of the molecule are variable. This can lead to false readings when using antibody- based heterologous assays. This can lead to false readings when using antibody- based heterologous assays.

3. Another antibody-based assay problem is that there is often considerable cross-reactivity between GH and prolactin. Another antibody-based assay problem is that there is often considerable cross-reactivity between GH and prolactin. This suggests two related things. This suggests two related things. Prolactin and GH evolved from a common earlier hormone Prolactin and GH evolved from a common earlier hormone Structurally, prolactin and GH are close in lower vertebrates. Structurally, prolactin and GH are close in lower vertebrates.

4. Function of GH in lower vertebrates: Fish Fish GH has not been conclusively demonstrated in agnathans. GH has not been conclusively demonstrated in agnathans. These fish also don’t have a well developed pars distalis. These fish also don’t have a well developed pars distalis.

5. Lamprey (cyclostomata)

6. Hagfish (cyclostomata)

7. 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 distalis 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. 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.

8. Leopard Shark (chondrichthys)

9. No GH activity has been identified in holostean fishes, despite distinct pars distalis. * No GH activity has been identified in holostean fishes, despite distinct pars distalis. * Distinct GH activity has been detected in all other fish groups. 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. 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. For example, there is almost as much variability between chum and chinook GH as between chum and human GH.

10. Spotted Ratfish (Holocephali)

11. In all these groups, GH stimulates the secretion of somatomedins from the liver. In all these groups, GH stimulates the 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 anadromous fish to salt water. GH has been implicated in the adaptation of 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 in the gills of fish. GH may stimulates the expression of CFTR in the gills of fish.

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

24. Somatolactin Somatolactin is a hormone so far found only in teleosts. Somatolactin is a hormone so far found only in teleosts. Secreted by pars intermedia Secreted by pars intermedia Structurally very similar to both GH and prolatin. Structurally very similar to both GH and prolatin. Structure appears to be midway between the two hormones. Antibodies to both hormones will cross- react with somatolactin. Structure appears to be midway between the two hormones. Antibodies to both hormones will cross- react with somatolactin.

25. It is specifically secreted by fish in a very low Ca 2+ environment. It is specifically secreted by fish in a very low Ca 2+ environment. Appears to act as an adaptation to ultrasoft water. Appears to act as an adaptation to ultrasoft water. May act to specifically stimulate uptake of calcium from low calcium environment. May act to specifically stimulate uptake of calcium from low calcium environment. Water softness is based on the amount of dissolved calcium. Hard waterCa 2+. Water softness is based on the amount of dissolved calcium. Hard waterCa 2+. Somatolactin also may be involved in adaptation to seawater during smoltification in salmonids. Somatolactin also may be involved in adaptation to seawater during smoltification in salmonids.

26. GH in Amphibians Antibodies to mammalian GH will bind amphibian GH and prolactin equally. 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. First taken as evidence that the two hormones were just different isoforms of the same hormone. This view has now been discounted. This view has now been discounted. It appears that in larval amphibians prolactin performs the functions of GH, but once metamorphosis occurs, GH takes over the functions. It appears that in larval amphibians prolactin performs the functions of GH, but once metamorphosis occurs, GH takes over the functions.

27. Recently, antibodies have been generated that can distinguish between bullfrog PRL and GH. Recently, antibodies have been generated that can distinguish between bullfrog PRL and GH. Supports the idea that there are two distinct hormones. Supports the idea that there are two distinct hormones. However, the structures of both are significantly different from the mammalian hormones. However, the structures of both are significantly different from the mammalian hormones. Amphibian GH is structurally very similar to mammalian GH. Amphibian GH is structurally very similar to mammalian GH.

28. Amphibian GHs are active in the rat tibia assay, but show much lower activities than bovine 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. IGF activity has been show in amphibians. However, release of IGFs has not been directly linked to GH or PRL secretion. 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. This maybe a problem associated with the lack of specific amphibian GH and PRL immunoassays. Area where more research is needed. Area where more research is needed.

29. GH in Reptiles Little work has been done in this area. Little work has been done in this area. Mammalian GH stimulates growth in juvenile snapping turtles and in at least one lizard genus (Lacerta). 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. 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. Both forms have been show to be very effective in the rat tibia bioassay.

30. GH in Birds As might be expected, GH activity has been demonstrated in chickens, turkeys and ducks. As might be expected, GH activity has been demonstrated in chickens, turkeys and ducks. This is not surprising given the economic influences. This is not surprising given the economic 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, but the avian forms are generally NOT recognized by ABs to bovine GH. Avian GH is active in the rat tibia assay, but the avian forms are generally NOT recognized by ABs to bovine GH.

31. Duck (Peking duck) GH does not react with rat GH Abs. Duck (Peking duck) GH does not react with rat GH Abs. GH in birds shows a significant divergence from mammalian GH. 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. Bird GH appears to be more closely related to reptilian GH than to mammalian GH.

33. Prolactin Prolactin is also referred to as a trophic hormone, since it seems to regulate growth in lower vertebrates. 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. The classic bioassay for PRL is the pigeon crop sac assay. Performed on immature pigeons Performed on immature pigeons The crop sac is a bilateral extension of the esophagus. The crop sac is a bilateral extension of the esophagus.

34. First, animals are “primed” by giving them an injection of PRL (usually murine). 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. After priming (several days is required). One side is injected subcutaneously with 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 and the development of the epithelial surface is assayed, either by general histology, or by scraping and measuring dry weight of removed epithelium. 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.

35. Prolactin appears to have 4 major functions in the lower vertebrates. Prolactin appears to have 4 major functions in the lower vertebrates. Reproductive. Reproductive. Osmoregulation. Osmoregulation. Growth. Growth. Integument modification. Integument modification. Fish PRL is structurally different from amphibians and amniotes (mammals, reptiles [turtles, lizards, sphenodon, crocodiles], birds and their extinct relatives) Fish PRL is structurally different from amphibians and amniotes (mammals, reptiles [turtles, lizards, sphenodon, crocodiles], birds and their extinct relatives)

37. Because of this difference, it has been suggested that fish PRL be called paralactin. Because of this difference, it has been suggested that fish PRL be called paralactin. However, this difference does not always hold. However, this difference does not always hold. Lungfish PRL is similar to tetrapod PRL. Lungfish PRL is similar to tetrapod PRL. Coelacanthlungfishtetrapods Coelacanthlungfishtetrapods Early bony fish This probably reflects the evolutionary origin of the tetrapods This probably reflects the evolutionary origin of the tetrapods

38. Functions in fish Functions in fish As already mentioned: Osmoregulation. As already mentioned: Osmoregulation. Cyclostomes Cyclostomes Shown to affect electrolyte balance. Shown to affect electrolyte balance. Appears to act in tandem with corticosteroids. Appears to act in tandem with corticosteroids. This is based on functional data. The hormone has not been purified yet. 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. Hagfish show PRL-like activity in the rostral portion of the pituitary gland. This activity appears to be involved in electrolyte regulation. This activity appears to be involved in electrolyte regulation.

39. There are two classic bioassays for fish PRL. There are two classic bioassays for fish PRL. Colour assay Colour assay Uses the goby Gillichthyes mirabilis. Uses the goby Gillichthyes mirabilis. Administer a subcutaneous injection of presumed PRL under the pre-opercular skin. Administer a subcutaneous injection of presumed PRL under the pre-opercular skin. A change in colour (to yellow) is a positive response. A change in colour (to yellow) is a positive response.

40. Plasma sodium Plasma sodium Uses hypophysectomized Fundulus heteroclitus. Uses hypophysectomized Fundulus heteroclitus. After hypophysectomy, the fish are kept in 50% seawater until challenged. After hypophysectomy, the fish are kept in 50% seawater until challenged. Inject the fish with presumed PRL. Inject the fish with presumed PRL. Transfer to fresh water and measure plasma Na + 24 hours later. Transfer to fresh water and measure plasma Na + 24 hours later. Test is considered positive if the fish retain elevated plasma Na +. Test is considered positive if the fish retain elevated plasma Na +.

41. Mammalian PRL is active in both fish bioassays. Mammalian PRL is active in both fish bioassays. Selachians Selachians Bioassayable PRL has been purified from this group. Bioassayable PRL has been purified from this group. This has been determined using an alternative bioassay- the skin colouration bioassay. This has been determined using an alternative bioassay- the skin colouration bioassay. The bioassayable PRL activity appears to be located in the anterior pituitary. The bioassayable PRL activity appears to be located in the anterior pituitary. Using the skin colouration assay. Using the skin colouration assay.

42. Teleosts Teleosts As mentioned, osmoregulation. As mentioned, 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 vesicle. PRL stimulates the growth of the seminal vesicle. This is very important in seasonal breaders. This is very important in seasonal breaders.

43. PRL also is important in maintaining secondary sexual characteristics, like the brood pouch in male seahorses. 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 particular, PRL will trigger and maintain breeding-colouration. In both sexes, PRL will stimulate overall growth in the gonads. In both sexes, PRL will stimulate overall growth in the gonads.

44. PRL also stimulates parental behaviour. PRL also stimulates parental behaviour. This has been demonstrated in mouth-brooders and in Sticklebacks. 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. 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. In nest-builders, such as sticklebacks and a larger number of marine species, the male will defend the nest and care for the eggs.

45. Effects on colouration. Effects on colouration. Stimulate proliferation of melanocytes. Stimulate proliferation of melanocytes. Not to be confused with the actions of MSH (shown here). Not to be confused with the actions of MSH (shown here).

46. PRL in Amphibians Structurally, PRL is significanly different from teleost PRL (as reflected by diminished AB recognition). Structurally, PRL is significanly different from teleost PRL (as reflected by diminished AB recognition). However, amphibian PRL is active in both fish bioassays. However, amphibian PRL is active in both fish bioassays. There is a specific amphibian PRL bioassay, called the “water drive” There is a specific amphibian PRL bioassay, called the “water drive”

48. Terrestrial newts start out as tadpoles (fully aquatic). Terrestrial newts start out as tadpoles (fully aquatic). The tadpoles undergo a two-stage metamorphosis. The tadpoles undergo a two-stage metamorphosis. Fist stage: They develop into and “eft”. This stage is an immature adult, with legs, lungs, etc. Fist stage: They develop into and “eft”. This stage is an immature adult, with legs, lungs, etc. They are sexually immature and live on land. They are sexually immature and live on land. Second stage: Involves a migration back to water. Second stage: Involves a migration back to water. Individuals will migrate back into water full-time and undergo a sexual maturation. 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). PRL induces this water drive, as well as stimulating sexual maturation (in both sexes).

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

50. Functions involved with ion regulation: Functions involved with ion regulation: PRL appears to be required for adult forms to re- enter the aquatic environment. PRL appears to be required for adult forms to re- enter the aquatic environment. In this situation, they tend to take up water and lose electrolytes (same problems as fish in freshwater). In this situation, they tend to take up water and lose electrolytes (same problems as fish in 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 across the urinary bladder and this is stimulated by PRL. In amphibians there is significant ion uptake across the urinary bladder and this is stimulated by PRL. PRL stimulates Na + transport across bladder epithelium. PRL stimulates Na + transport across bladder epithelium.

51. Effects on colouration: Effects on colouration: PRL stimulates the proliferation of melanocytes. PRL stimulates the proliferation of melanocytes. This has been used as a bioassay, like the goby colouration assay, but it has proved unreliable. 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. Interestingly, in the frogs where this occurs, the colour induced is yellow. Other effects: Other effects: PRL stimulates growth of gills, tails, and brain in tadpoles. PRL stimulates growth of gills, tails, and brain in tadpoles. PRL is also involved in regulating limb regeneration. PRL is also involved in regulating limb regeneration. PRL regulates secretion by the oviducts andregulates ovulation (in part). PRL regulates secretion by the oviducts andregulates ovulation (in part).

52. PRL in reptiles Very little is known. Very little is known. PRL is still involved in electrolyte regulation. PRL is still involved in electrolyte regulation. Plasma Na + levels are regulated by PRL in synergy with cortisol. Plasma Na + levels are regulated by PRL in synergy with cortisol. PRL is involved in tail regeneration in lizards. PRL is involved in tail regeneration in lizards. PRL appears to be involved in the initiation of moulting. PRL appears to be involved in the initiation of moulting. PRL has an weak antigonadotropic effect. PRL has an weak antigonadotropic effect.

53. PRL effects in birds: More is known about functions in birds. More is known about functions in birds. As already mentioned, PRL stimulates the production of crop milk in pigeons. 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. 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 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. This liquid is fed to young.

54. As mentioned, crop milk formation can be stimulated with all tetrapod PRLs, as well as with lungfish PRL. 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. 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. 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. Used to transmit body from adult to the eggs, to promote egg development. After breeding is over, the hyperangiogenesis regresses. After breeding is over, the hyperangiogenesis regresses.

55. PRL has a strong antigonadotropic effect in birds. PRL has a strong antigonadotropic effect in birds. Reproductive timing in birds is very tightly regulated (will be discussed later). Reproductive timing in birds is very tightly regulated (will be discussed later). PRL strongly suppresses courtship behaviour. PRL strongly suppresses courtship behaviour. Strongly promotes nesting, brooding and food gathering behaviours associated with chick rearing. Strongly promotes nesting, brooding and food gathering behaviours associated with chick rearing.

56. PRL stimulates moulting. PRL stimulates moulting. Also appears to be involved in feather pigmentation. Also appears to be involved in feather pigmentation. Osmoregulation functions: Osmoregulation functions: This is primarily seen in sea birds with nasal glands. This is primarily seen in sea birds with nasal glands. Nasal gland secretes a very concentrated NaCl solution. Nasal gland secretes a very concentrated NaCl solution. This allows them to drink seawater directly. This allows them to drink seawater directly. PRL stimulates Na + directly, which results in increased fluid secretion. PRL stimulates Na + directly, which results in increased fluid secretion.

58. PRL in mammals Almost all effects of PRL in mammals are reproductive. Almost all effects of PRL in mammals are reproductive. Stimulation of mammary gland. Stimulation of mammary gland. Stimulation of androgen binding in prostate gland. Stimulation of androgen binding in 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 in testes. Stimulates increased cholesterol uptake in testes.

59. Stimulation of parental behaviour in rats. Stimulation of parental behaviour in rats. See table 4-8 in Norris for other effects on reproduction. See table 4-8 in Norris for other effects on reproduction. PRL still has some effects on electrolyte balance. PRL still has some effects on electrolyte balance. Stimulates increased Na + retention in the kidney. Stimulates increased Na + retention in the kidney. Corticotropic Corticotropic