TENROlD HORMONES AND GOITROGEN-INDUCED METAMORPHOSIS IN THE SEA LAMPREY (Pett-omyzonmarinus).

Richard Giuseppe Manzon

A thesis submitted in confodty with the requirements for the degree of Doctor of Philosophy Graduate Department of Zoology University of Toronto

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THYROlD HORMONES AND GOLTROGEN-INDUCED METAMORPHOSIS IN

THE SEA LAMPREY (Petromyzon mariBus).

Richard Giuseppe Manzon

Doctor of Philosophy, 2000

Department of Zoology, University of Toronto

The larval sea lamprey undergoes a tme metamorphosis fiom a sedentary, filter- feeding larva to a fiee-swimming, parasitic juvenile that feeds on the blooà and body fluids of teleost fishes, As is the case with arnphibians and teleosts, thyroid hormones (TH) are believed to be involved in lamprey metamorphosis. However, the onset of lamprey metamorphosis is characterized by a sharp decline in semm TH titers rather than the typical increase observed in ail other vertebrates known to undergo this type of postembryonic developmental event. Furthermore, the goitrogen potassium perchlorate (KC104), which lowers serum TH titers and prevents metamorphosis in most vertebrates, can induce precocious metamorphosis in Iarval lampreys. The primary goals of this thesis were to elucidate the role of depressed serum TH titers in the goitrogen-induced metamorphosis of lampreys and to provide some insight into the mechanisms involved in this induction. 1have shown that KCI04-induced Iamprey metamorphosis cmbe bIocked by using exogenous TH to prevent the decline in serum TH titers associateci with induced metamorphosis. The ability to induce metamorphosis was not unique to KC104. The goitrogens sodium perchlorateTpotassium thiocyanate and methimmole aiso induced metamorphosis in Iarval sea lampreys, and the incidence of metamorphosis was positively correlated to the magnitude of the decline in serum TH concentrations, These data are consistent with the proposed ideas that either a decline in serum TH concentrations is essentiai for the onset of metamorphosis in Iampreys, or elevated serum TH concentrations are inhibitory to metamorphosis (Le., high

TH titers have an anti-metamorphic effect in lampreys). In addition, 1have shown that

KC104 adrninistered to lamal Iamprey endostyles in vim reduces iodide uptake and organification, as weli as totai endostylar thyroglobuiin. The fact that the goitrogen KC104 acts directly on the endostyle to inhibit thyroidai activity Mersupports the notion that goitrogens induce metamorphosis via their effects on TH synthesis. In summary, 1have shown that goitrogens act directly on the larval lamprey endostyle to inhibit thyroidal activity, thus resulting in a decline in serum TH titers that is Luiked to and essential for the onset of metamorphosis in larnpreys. As 1approached the completion of my doctoral thesis, 1came to the conclusion that this was the most ciifficuit and rewarding endeavor that I have ever tackled. However, my definition of "ciifficuit'' changed when 1set out to acknowledge the many people that helped me along the way, without these individuals 1would not have succeeded at completing thïs thesis. 1think 1may have procrastinated in writing these acknowledgments as much as 1did with any section of this thesis. In fact, atone point 1thought 1 wouid provide myself with a restriction in the length of these acknowledgments. My acknowledgments with this approach would have been: 1thank aLl those peopIe kaown and unknown to me which have crossed my path during my doctoral studies and did not intentionally act to impede my progress.

However, 1 realized that I was just trying to avoid the inevitable, which is kind of what 1am doing now, and that certain people in my Life really do deserve to be thanked and acknowledged for helping; so here goes (as you will see 1did not impose a length restriction),

1will begin by thanking the many members, past and present, of Dr. J.H. Youson's laboratory who slogged it out in the trenches with me. These postdoctoral fellows, technicians, graduate students and undergraduates provided many hours of stimulating, intellectual discussion as well as nonsensical banter, that helped me get through the day.

Most days in the lab were truly enjoyable, and this was due to the people 1worked with. in particular 1 would to thank IIya Adam, Mehmet Danis, JulieeHeinig, Bojan Lipicnïk, Rod

Roopsingh and Paul Robson, who left their work behind to help me collect the thousands of larval larnpreys used dunng the course of this thesis, and more importantly, were the reason 1 managed to collect the serum fiom all these lampreys before my retirement* 1wodd also

like to thank four @ed undergraduate students: QUOCHuynh, Nancy Manjovski, Bnan

Peck and Preshi Shanmugathasan; a11 were a pleasure to teach and train, 1learned something

from each of you and in exchange for training, you put up with me and helped me get

through the many radioimmunoassays that always seemed to fail on my Iab bench- Thanks

also to Sevana Yaghoubian for introducing me to the art of poiyacrylamide gel electrophoresis and Western blotting-

A special thanks to DE. John Holmes, Mike Wiïeand Luciano Marra, al1 of you taught me dot about being a graduate student and biologist. John, you introduced me to the lamprey and taught me a great deai about their biology, ecology and how to catch and care for them. You tnily were "The Source"- It was a rare occasion when you did not have an answer to my question and you always helped me find my way back to the cabin when we were out in the field. Lu, you tdywere a fkiend and mentor to me; you were aiways there to help whether it was with my lab work, grant applications, schoobng, teaching duties, or life in generai.

Thanks to all the Sea Lamprey Control personnel in both Canada and the USA, and the US Fish and WildLSe Service, Essex Junction, Vermont for your help with larval lamprey collections. Whether you were answering my questions, providing me with survey data, sending me survey maps or electrofistiing dongside me, you were always an excellent resource. John Gersmehl, Jeff Slade and EueKoon frequently went above and beyond the cal1 of duty to ensure that my iamprey hunting was successfiil.

Thanks to Dr. John Leatheriand for my introduction to the radioimmunoassay technique and Dr. Geoffrey Edes and Audrey Waytiuk for performing the deiodinase assays presented in Chapter 2 of this thesis. Thanks to Drs. Rudy Boonstra and Mark Sheridan for being my mentors; even though you were not necessary dÏrectLy involved in this thesis, you always provided me with sound advice-

1would LÏke to thank the members of my advisory committee, Drs. Rick Elinson and

Mike Filosa for providing me with guidance throughout this thesis and keeping me on the right track. Speciai thanks to Dr. Mike Filosa, you were a teacher in the tmest sense of the word befitting of the titLe "SENSEI"that so few deserve- The contribution you made to my thesis in the final year was invaluable, You not only helped me with the technical and academic aspects of my doctoral thesis, but you taught me about culture, the arts, history, and life in general.

Thanks to Dr. John Youson, my doctoral supervisor, for providing me with the atmosphere and guidance to excel in my graduate studies. John, you saw abiiities in me that

1did not know 1 had and you always knew when to push and challenge me to bring my work to the next level. You were teacher, supervisor, and mentor, but over the years you have also become my fiend.

Last, but not least, 1would like to acknowtedge the four people who have provided me with the love, support, £iiendship and encouragement that fueled the completion of this thesis: my parents Giuseppe and Teresa Manzon, my wife Lori, and my second mother,

Sally Wood. To my parents Giuseppe and Teresa, what can 1Say but thanks, you sacrificed so much to help me both financidy and emotionally. Thanks for putting up with my me through al1 the rough spots and for ignoring me when 1was out of fine. You both showed a tremendous interest in my schooLing and my research, and you were always anxious to know how things were going and what I was up to- You were there for me when 1needed you, supported me in all my decisions and provided me with this opportunity. SalIy, th& for

continually pestering me with questions about my work, providing me with encouragement

when 1needed it, and barking at me to get back to work when 1was taking tmmany breaks-

Most of A,thanks forjust putting up with me, giving me space when 1needed it and being my friend-

To my wife, best fiiend, and colleague, Lori, from the bottom of my heart, THANKS!

You are the one person without whom 1reaily could not have finished thïs thesis and words could never describe my appreciation. You provided me with help in ali aspects ofthis work frorn the field, aquatics room and laboratory to the editing of endless manuscn'pts- More importantly, you gave me your unconditional Love, support and encouragement, You helped me to find the confidence in myself that 1needed to succeed; we really do make a good team-

The research presented in this thesis was supported by operating grants fiom the

Naturd Sciences and Engineering Research Council of Canada @TSERC) to J.H. Youson and contracts between The Great Lakes Fisheries Commission and J.H. Youson, 1wouid like to sincerely thank NSERC, the Ontario Graduate Scholarship program and the University of

Toronto for their scholarship support throughout this doctoral thesis.

CHAPTERONIE:

THE EFFECTS OF EXOGENOUS THYROXINE (Ta) OR TEUIODOTHYRONINE

(T3).IN THE PRESENCE AND ABSENCE OF POTASSIUM PERCHLORATE.ON

THE INCIDENCE OF METAMORPHOSIS AND ON SERmT4 AND T3 CONCENTRATIONS IN LARVAL SEA LAMPREYS (Petnmyzon marinus L.) ...... 38

ABSTRACT ...... 39

INTRODUCTION ...... 40

MATERIALS AND METHODS ...... 42

Animal collection and maintenance ...... 42

Treatment regime...... 43

Experirnental sampling. senun collection. and radioimmunoassay (RIA) ...... 44

Data analysis ...... 45

RESULTS ...... 45

Animal size ...... 45

Metamorphosis ...... 46

Serum T4 and T3 concentrations ...... 47

DISCUSSION ...... -49

C-RTWO:

BLOCKING OF KCIO4-INDUCED METAMORPHOSIS IN PREMETAMORPHIC

SEA LAMPREYS BY EXOGENOUS THYROID HORMONES (TH); EFFECTS OF

KC104 AND TH ON SERUM TH CONCENTRATIONS AND INTESTINAL THYROXINE OUTERIRING DEIODINATION...... 66 ABSTRACT ...... 67 0 ...... 68

MATERIALS AND METHODS ...... 70

RESULTS ...... w...... w...... 74

Size and incidence of metamorphosis ...... 74

Comparison of serum TK concentrations between sampling periods within an

experimental group ...... 75

Comparison of serum TH concentrations between experimentd groups within a sampling

period ...... 76

T40RD activity ...... 78

DISCUSSION ...... 78

CHAPTER THREE:

VARLABLE EFFECTS OF GOITROGENS IN INDUCING PRECOCIOUS METAMORPHOSIS IN SEA LAMPREYS (Petromyzon mannus)...... 91 ABSTRACT...... -92

INTRODUCTION ...... ,...... 93

MATERIALS AND METHODS ...... 95

Experimental Protocols and Animals ...... 95

Experiment 1 : PiJpyI thiouracil and triiodothyronine ...... 97 Experiment 2: Propylthiouracil ...... 98

Experiment 3: Potassium perchlorate, potassium thiocyanate and methimazole...... 98

Experiment 4: Potassium perchlorate, sodium perchlorate and potassium chloride ...... 99

Serum collection and TH measurement ...... ,...... 99 . . Statisficd analyses ...... 100 RESULTS ...... 101

Experiment 1: Propy IthiouraciI and triiodothyronine ...... 101

Experiment 2: Propylthiouracil ...... 102

Experiment 3 : Potassium perchlorate, potassium thiocyanate and methimazole...... , 103

Experiment 4: Potassium perchiorate, sodium perchlorate, and potassium chioride .... 105

DISCUSSION ...... 106

CHAPTER FOUR:

TEMPERATURE AND KC104-INDUCED METAMORPHOSIS IN THE SEA

LAMPREY (Petromyzon mm*nus)...... l.27 ABSTRACT ...... 128

INTRODUCTION ...... 129

MATERIALS AND METKODS ...... 130

RESULTS ...... 132

DISCUSSION ...... 134

CHAPTERFIVE:

KC104 INHIBITS THYROIDAL ACTIVITY IN THE: LARVAL LAMPREY

ENDOSTYLE IN VZTRO...... 141 ABSTRACT ...... 142

LNTRODUCTION ...... 143

MATERIALS AND METHODS ...... ,, ...... *.....145

Animais ...... 145

In vitro experimental protocol ...... 146

Light microscopy...... 147 EIectrophoresis ...... 148

ensitoe...... 150

RESULTS ...... 151

DISCUSSION...... ,.-...... 153

GENERAL DISCUSSIONooeeeeeeeeeeeemeeeœeeeememeeeooeoeomeeeeeeomeeeeeeoeemeemmooeeeommmoeemeeeeemeemeeeoemeeeemo~eeeo171

Conjecture 1...... 181 Conjecture 2...... 182

Surnrnary and Conclusions ...... 183

LITERATURE CITEDeeeeee-ee.oeeeoee.eeeoooeeeeeeeeeeeoeeeeeoe~eoeeeeeeoeeeeeeoeeeoeoeooee.eeoeeeoeeeeeeeoeeeeeeeeeeeeeeee 187

APPEmm A eemeeeeeeeeeeoeeeœeemeeoeeeeeeeeeeeeeoeeeoemeoemeeemmeeeeo~eeeeee~eemeeeeeeeeee~eeœeemeaemmeeemeemme~~~~~eoeo~eoee~e222 LIST OF TABLES

Table 1- Experimental groups and nominal ambient aquarium concentrations to which larval sea lampreys (Petromyzorz marinus) were exposed for 4 - 24 weeks ...... 57

Table 2. Treatment groups and nominai ambient aquarium concentrations to which larvai sea lampreys (Pe~omyzonmarinus) were exposed for 4, 8 or 16 weeks ...... 82

Table 3. Baseline and experimentai groups in four separate experiments, the nominal ambient aquarium concentration of the various experimentai treatments, and mean sea lamprey (Petromyzon maninus) size at the time of sampiing, ...... 115

Table 4. Cornparison of mean senun thyroxine and triiodothyronine concentrations in larval (A) and metamorphosing (M) sea lampreys (Petromyzan murinus) following various goitrogen treatments (Experiment 3)- ...... 116

Table 5. Radioiodide (PJalZSJJdoses and potassium perchlorate (KCLo4) treatment concentrations administered to larvai sea lamprey (Petromyzon marfnus)endostyles in vitro and the data coiiected fiom the endostyles in experiments 1 - 5...... 159

Table 6. Summary of the incidence of metamorphosis following treatment with one of several goitrogens at different concentrations. The incidence of metamorphosis is related to the percent decrease in serum thyroxine (T4) and triiodothyronine (33 concentrations fiom control values...... 185

xiii Figure 1. Schematic representation of the major outer-ring (ORD) and inner-ring (IRD) deiodination pathways of the thyroid hormones thyroxine and 3,5,3'-triiodothyronine. ... 8

Figure 2. Diagramatic representation of (A) the gross anatomical morphology and (B) the histology of the lanrai Iamprey endostyle...... 26

Figure 3. Mean (+ 2SE) lengths (A) and weights (B) of sea lamprey (Petromyzon markus) larvae exposed to various treatrnents for 4 - 24 weeks ...... 58

Figure 4. Number of metamorphosing 1arvaI sea Iampreys (Petromyzon mannus) following exposure to potassium perchlorate (KC104; 0.0 1 %) for 4 - 24 weeks,...... 60

Figure 5. Comparison of the mean (k 2SE) serum thyroxine u4)concentrations of sea lamprey (Petromyzon manirus) larvae exposed to either 5 (10 mgfiter) or triiodothyronine CIj; 1 mgniter), in the presence or absence of potassium perchlorate (KC104; 0.0 1%), for 4 to 24 weeks...... 62

Figure 6. Comparison of the mean (k 2SE) serum triiodothyronine (T3) concentrations of sea lamprey (Petromyzon marinus) larvae exposed to either thyroxine (T4; 10 mgniter) or T3 (1 mgfiter), in the presence or absence of potassium perchlorate (KCi04; 0.0 l%),for 4 to 24 weeks., ...... 64 Figure 7. Number of metamorphosing larval sea lampreys foliowing exposure to KC104 (0.05%) for either 4, 8, or 16 weeks...... 83

Figure 8. Mean (2 2SE) semm thyroxine (T4) concentrations in Iarval sea lamprey following exposure to either a low (L)or high 0 T4 (0.56 and 1.12 CLM, respectively) or triiodothyronine (T3; 0.37 and 1.48 pM, respectively) treatment in the presence (+) or absence (-) of the goitrogen potassium perchlorate (KC104; 0.05%) for 48or 16 weeks...... ,...... ,.*...... 85

Figure 9. Mean (k 2SE) serum triiodothyronine (T3)concentrations in larval sea lamprey following exposure to either a low (L)or high OI) thyroxine (T4; 0.56 and 1-12 pM, respectively) or T3 (0.37 and 1.48 pM, respectively) treatment in the presence (+) or absence (-) of the goitrogen potassium perchlorate (KC104; 0.05%) for 4,8 or 16 weeks...... 87

Figure 10. Activity (fmols T4 deiodinated/hour/incubationtube) of intestinal Tq outer-ring (5') deiodination to T3(T40RD), in larval sea lamprey exposed to either a Iow (L) or high CH) T4 (0.56 and 1.12 pM, respectively) or T3(0.37 and 1-48 pM, respectively) treatment in the presence (+) or absence (-) of potassium perchlorate (Kclo4) for 4 or 16 weeks...... 89 Figure 11. Mean (k 2 standard errors) serum thyroxine (T4;A and C) and triiodothyronine m; B and D) concentrations in sea lampreys of two different groups (based on length)...... 117 Figure 12. Stage and totaI number of metamorphosing sea lampreys in untreated (control) and goitrogen-treated individuals foUowing 6 weeks (A) or 16 weeks (B)...... 119

Figure 13. Mean (& 2 standard errors) semm thyroxine (T4) and tniodothyronine (73 concentrations in sea Iampreys. Lampreys were either untreated as in the baseline (start of experiment) and control groups, or treated with potassium perchlorate (KClû4) or potassium thiocyanate (KSCN) for 16 weeks, or methimazole (MMI) for 6 weeks...... 121 Figure 14. Stage and total number of metamorphosing sea lampreys in untreated (control) and treated individuais fiom two different size groups (based on length)...... 123

Figure 15. Mean (k 2 standard errors) senun thyroxine (Tq) and triiodothyronine a) concentrations in untreated (control) and treated sea lampreys fiom two dIfferent size groups (based on length)...... 125 Figure 16. Stage and incidence of metamorphosis in untreated and potassium perchlorate- (KC104; 0.05%) treated Iarval sea Iampreys (Petromyzon marinus) at wann (18 OC) and cold (3 OC) water temperatures after 23 weeks...... 137

Figure 17. Mean (+ 2SE) serum thyroxine fl4; A) and 3,5,3'-triiodothyronine (T3;B) concentrations in untreated and potassium perchlorate- (KCl04; 0.05%) treated Iarval sea lampreys at warm (18 OC) and cold (3 OC) water temperatures after 23 weeks...... 139

Figure 18. Routine light microscopy (A and C) and autoradiography (B and D) of transverse sections through the antenor portion of the larval lamprey (Lampetra appendix) endostyle folIowing a four hour in vitro incubation with 3 pCi N~~~~I...... 160

Figure 19. The percentage of uptake, as determined by gamma radiation emissions, by potassium perchlorate- @CIO4) treated endostyles relative to untreated (control) endostyles following a four hour in vitro incubation with Na12s 1...... 162 Figure 20. (A) Autoradiogram to show radioiodide incorporation and organification into a lam rey thyroglobulin (Tg) by larval lamprey endostyles incubated in vitro with Na L5: 1...... 164 Figure 21. Immunodetection of thyroglobulin (Tg) in larval lamprey endostyles by Western blot using a rabbit anti-human Tg antibody, following an in vitro incubation with Na 1251...... -...... -...... --.-...... -.-...... 166 Figure 22. (A) The percentage of incorporath (organincation) into lamprey thyroglobulin (Tg) by endostyles treated with either low potassium perchlorate (L- KCm; 0.72 mM) or hïgh potassium perchlorate (H-KC104; 3.6 mM) dative to untreated (control) eudostyles. ..o...... 168

Figure 23. (A) Stage and number of metamorphosing sea Iamprreys at 28 weeks foilowing O (controls), 2,4, 8, or 16 weeks of treatment with 3.6 mM potassium perchlorate

xvi ANOVA analysis of variance N sampIe size Bv blood vessels NaC104 sodium perchlorate CF condition factor OD adjusted optical density X mm2 ClOï perchlorate anion ORD outer-ring deiodination CON control PAGE polyacrylamide gel electrophoresis ce* counts per minute Pi pigment DIT P-Tg porcine thyroglobuh ECL enhanced PTU propylthiouracil cherniluminescence Fig. figure RIA Gi rT3 reverse T3 (3 ,3',Sr-triiodothyronine) Gt glandular tract SDS sodium-dodecyl sulfate H high SE standard error Il3 incubation buffer SS-14 somatostatin- 14 IO iodide organification T2 dïiodothyronine

IRD inner-ring deiodination T3

IU iodide uptake T4 thyroxine KCIO~ potassium perchlorate Tg KSCN potassium thiocyanate TH thyroid hormone(s) L low TR thyroid hormone nuclear receptor LM Iight microscopy TRa TR - a subtype

MIT monoiodotyrosine TRB TR - subtype MM1 rnethimazole TRH thyrotropin releasing hormone MS-222 tricaine methanesulfonate TSH thyrotropin (thyroid stimulating hormone)

xvii GENERAL INTRODUCTION Metamorphosis is a life history strategy which has been exploited by several classes of organisms in the phylum Chordata. The term, or perhaps more appropriately the

"concept", of metamorphosis has been used to describe a wide vm-ety of postembryonic changes in animal form and Mestyle (Cohen, 1985). Unfortunately, there is no clear consensus as to what constitutes a "truey'metamorphosis within the various Chordate classes-

Just et al- (198 1) suggested that metamorphosis shodd encompass three criteria: i) A dramatic change in form that is not related to either embryogenesis, sexual maturation, or aging. ii) The Iarvai form differs fiom that of the embryo and adult such that it pennits the larva to exploit a different ecological niche, iii) The morphological changes associated with metamorphosis occur in response to one or more exogenous (environmental) andor endogenous (physiologicai) cues.

Youson (1988) distinguishes between a "true" or first metamorphosis and transforrnations in fish associated with sexual maturation which he terms a second metamorphosis. Fit metamorphoses involve dramatic changes in form and lifestyie that are not associated with sexuai maturation (Youson, 1988). However, inconsistencies and confusion stili exist regarding the application of the term metamorphosis to postembryonic transformations in fish (Hall and Wdce, 1999). One reason for this confusion is that the degree of change which is necessary to denote a metamorphosis has not been specified

(Williamson, 1992; Hall and Wake, 1999). It is within the context described by Just et al.

(198 1) and Youson (198 8) that 1use the term metamorphosis- Regardless of the definition or cnteria which one adopts to study metamorphosis and other postembryonic transformations, these systems make excellent models to examine the control of developmental and

endocnnologicaI processes. Moreover, comparative studies of metamorphosis across classes

within the animal kingdom provide a heworkfor studybg both the evolution of

regulatory signals controlling metamorphosis and the evolution of Life history strategies,

Among the Chordates, a tme metamorphosis has been observed in the protochordates

(Urochordates and Cephalochordates) and severaI vertebrate classes including the

Agnathans, Osteichthyes and Amphr'bians, but not in amniotes (lust et ai-, 198 1). Amphibian

metamorphosis has been the most extensively studied of these systems, and much of this

work has focused on anuran (frog and toad) metamorphosis- Anurans are one of the primary,

non-mammalian, mode1 systerns for the study of vertebrate developmental biology and

endocrinology. More recently, there has been an increased interest in studying the

metamorphosis and deveiopment of other veaebrates, including a wide variety of amphibian and fish species- Among the fishes, the first metamorphoses of the Petromyzoatifonnes

(lampreys) and the Pleuronectiformes (flat fishes) have received considerable attention, less focus has been placed on the metamorphosis of the Anguilliformes (eels).

The foundation of our understanding of the exogenous and endogenous factors controlling vertebrate metamorphosis lies in studies on anuran amphibians. Many exogenous and/or ecological cues are important to the onset and progression of anuran metamorphosis including: temperature, photoperioci, nutrition, pond desiccation, predatory pressure and density (see Hayes, 1997a for references). In addition to this vast array of exogenous factors, there are many endogenous factors, primarily hormonal, controlling anuran metamorphosis.

Thyroid hormones (TH), prolactin, and more recently, the corticoids have been the rnost extensively studied hormones, but numerous other hormones and regulatory factors are lïkely

3 to be involved in the contcol of metamorphosis. AU of these regdatory idluences aside, TH are the prïmary, obligatory signal that initiates the morphologicd and biochemiçal changes associated with anuran metamorphosis- The importance of TH in regulating anuran metamorphosis has been displayed at the systemic, tissue and cellular levels in numerous morphological, expenmentai, physiological, blochemical, and gene expression studies (see

Gilbert et al., 1996; Tata, 1998; Shi, 2000).

The fact that TH pIay such a pivotai role in the regulation of anuran metamorphosis prompted investigations into the role of TH in the metamorphoses of other vertebrates, including the Petromyzontiformes @mpreys). Contrary to the resdts obtained with anurans

(Etkin, 1935), bony fishes such as the PIeurooectifonnes (Miwa and Inui, 1985), and the

Anguilliforrnes (Kitajima et al., 1967 in Yamano et al., 1991a), attempts at inducing the metamorphosis of larval Larnpreys with exogenous TH treatments andlor thyroid stimulants were unsuccessful (see below), However, classicai anti-thyroid agents (goitrogens) which inhibit anuran metamorphosis (Men, 1929) induce precocious metamorphosis in lampreys

(Hoheisel and Sterba, 1963). The fact that this goitrogen-induced metamorphosis is unique to lampreys was a major catalyst for this thesis. The primary aim of my doctoral thesis was to investigate the nature of goitmgen-induced metamorphosis in lampreys and to determine whether a reduction or inhibition of thyroidal activity plays a role in the initiation of precocious and/or spontaneous lamprey metamorphosis*

Thyroidal reguurtion of anuran rneîkzmorphosis

The hormonal infiuences involved in the initiation and regulation of metamorphosis are a complex network of signals fiom various tissues and glands. Many of these chernical signals are generated in response to other cues or secretions and any number of them might üiteract to ensure normal development, Much of the research to date has focused on the function of signds from the hypothalamus, pituitary, thyroid and adrenals. In particular, the

TH thyroxine (T4)and triiodothyronhe (73 are paramount in the initiation and stimulation of anuran metamorphosis. At this time 1wili focus on TH; however, the involvement of other signals in the regdation of metamorphosis should not be overlooked as they may be essential for enhancing or dampeuhg the effects of TH to ensure a normal metamorphosis.

Foiiowing Gudematsch's (19 12) discovery that tadpoles fed a diet of equine thyroid tissue metamorphosed precociously, W. Etkin made a tremendous contribution toward characterizing the involvement of TH in amphibian metamorphosis. It became evident that simply treating tadpoles with either TH or thyroid extracts was not suficient for a normal and complete metamorphosis. The timing and concentration of TH treatments were critical.

In 1935, Etkin showed that in order to approximate a normal metamorphosis in thyroidectomized tadpoles the concentration of exogenous TH treatments must increase throughout the developmental event. He described three phases to metamorphosis: i) premetamorphosis, characterized by larval growth and requiring TH at constant, low concentrations; ii) pmmetamorphosis, characterized by the onset of morphogenesis and gradually increasing TR concentrations; and fi) metamorphic climax, charactenzed by rapidly increasing TH concentrations to complete the final and most dramatic phases of morphogenesis (Etkin, 1935, 1964 1968).

Etkin's studies were supported by a similar study conducted by Koliros (1961) on hypophysectomized tadpoles and ultimately by suesshowing that thyroidal activity increased dunng spontaneous metamorphosis. The earliest of these works demonstrated that the height of the cells in thyroid follicles increased dunng metamorphosis, a feature consistent with increased thyroidal activity (see Etkin, 1968)- Later, Kaye (196 1) reporteci

that radioiodide uptake by tadpole thyroid glands increased throughout prometamorphosis

until the middle of metamorphic ciimax. Radioiodide uptake at the start of metamorphic

climax was 15 fold greater than at the end of premetamorphosis. Lastly, Just (1972) found

that changes in the levels of plasma protein-bound iodine foiiowed a similar trend,

With the advent of radioirnmunoassays (RIA) came the ability to mesure minute

quantities of TH in small volumes of sera or whole-body extracts. Severai laboratories

proceeded to describe the changes in T4 and concentrations throughout the We cycles of a

variety of different amphibian species, including nwnerous anurans (for review see White

and Nicoll, 198 1, Kikuyama et al,, 1993, Kdtenbach, 1996). However, the resuIts of these

studies varied both between species and Iaboratorïes. The timing of the changes in TH concentrations as determined by RIA were not always consistent with either the changes

proposed by Etkin or those described for radioiodide uptake and plasma protein-bound

iodine. Despite these differences, an increase in TH concentrations during metamorphic development was always describeci (Leloup and Buscaglia, 1977; Miyauchi et al-, 1977;

Regard et al., 1978; Mondo and Kaltenbach, 1979; Sudand Suzuki, 1981). In general,

TH concentrations are below the detection limits of most assays during premetamorphosis,

îhey are quanufilable at some point during prometamorphosis at which thethey may gradually increase, and rise rapidly to a peak at some point between the onset and the middle of metamorphic climax, and decline thereafter.

When investigating the regulatory influences of TH on developmental processes, it is essential to remember that the measurement of serum or whole-body T4 and T3 concentrations is just a starting point. Tissue morphogenesis is temporally regulated during anuran metamorphosis even though dS tissues are exposed to similar semTH

concentrations. This differential timing suggests that some regulation of TH action on

metamorphosis must occur periphedy, at the tissue or cellular IeveIs (j3ecker et al., 1997).

Regdation may occur by altering either the cellular concentrations of TE& in particular 4, or

the number of thyroid hormone nuclear receptors (TEQ rt is generally accepted that T3is the

more biologically active TH; TR have a greater affinity for T3 than Tq, T3is a more potent

stimulator of amphibian metamorphosis and physiologicd processes in general, and the

induction of metamorphosis by exogenous T4 is dependent on its conversion to T3 (Buscaglia et al., 1985; Becker et al., 1997).

The conversion of Tq to T3occurs via the enzymatic removal of the 5' iodine on the

T4 molecule. The TH deiodination pathways found in most vertebrates cm be simplified by referring to them as outer-ring and inner-ring deiodinations (Fig. 1). The outer-ring (5') deiodination of T4('ï@RD) produces 4, and thus can be considered an activation pathway.

In contrat, inner-ring deiodinations (Ml)result in the production of biologically inactive molecules. The IRD of Tq by removal of the 5 iodine, produces reverse T3 (3,3',5'- triiodothyronine; rT3) and the IRD of T3 produces 3,3'-diiodothyronine pz). The enzymes catalyzing these deiodination reactions have ken identined in several mammaiian and non- mamrnalian vertebrates as type 1, type 2 and type 3 deiodinases. GeneraUy, the type 1 and type 2 deiodinases catalyzes ORD and the type 3 deiodinase cataiyze iRD (Refetoff and

Nicoioff, 1995).

The importance of deiodinases in the regulation of tissue and cellular T3 concentrations and the coordination of anuran metamorphosis bas been demonstrated by

Becker et al. (1997). They examineci the activity and mRNA levels of type 2 and type 3 Figure 1. Schernatic representation of the major outer-ring (ORD) and inner-ring (IRD) deiodination pathway s of the thyroid hormones thyroxine and 3,5,3'-triiodothyronine. 1 - 1 - THYROXINE (T4) / IRD ,/

1 - REVERSE T3 (1T3) deiodinases in IO different tissues of metamorphosïng Rrma catesbeiana. The most significant finding of thelr study was that the activity and mRNA Levels of the type 2 deiodinase were highest in those tissues that were actively undergoing morphogenesis. Type

2 deiodinase activirj in the hind-iimbs was highest during prometamorphosis when they are developing, and decreased to levels below detection by mid-metamorphic climax (Becker er al., 1997). In contrast, the tail and intestine, which do not begin morphogenesis until metarnorphic climax, had very low LeveIs of type 2 deiodinase activity dunng prometamorphosis (Becker et al., 1997). At the time of metamorphic ciimax, when tail cegression and intestine remodeling begin a dramatic increase in type 2 deiodinase activity is observed in these structures (Becker et al., 1997). The eye and skin develop gradually tbroughout metamorphosis and have consistent, moderate levels of type 2 deiodinase activity

(Becker et al., 1997).

Surprisingly, the activity of the type 3 deiodinase during tissue morphogenesis in R. catesbeianna parailels that of the type 2 deiodinase (Becker et al., 1997). In contrast, the mRNA levels of the type 3 deiodinase are different nom those of the type 2 deiodinase

(Becker et al., 1997). This similarity in activity levels, despite ciifferences in mRNA expression, between the two deiododinases suggests that there may be a post-transcriptionai control mechanism Galton and Hiebert (1988) describe a trend of high IRD activity during premetamorphosis and prometamorphosis foiiowed b y a decrease in IRD activity during metamorphic climax. These results are consistent with the changes observed in serum TH concentrations. More recently, Huang et al. (1999) used transgenic techniques to overexpress the type 3 deiodinase gene in Xenopus laevis. They observed a delayed metamorphosis and reduced response to TH, which is consistent with the inactivation of TH by the type 3 deiodinase. Becker et al. (1997) suggested that ORDs and IRDs act in a "push- pull" fashion to stringentiy regulate celluIarT3 concentrations and ensure normal morphogenesis at the tissue and organ leveIs, They hypothesized that the differential expression of deiodinase transcripts ensures that the appropriate cellular T3concentrations are attained to facilitate stage-dependent morphogenesis. Moreover, post-translationai regulation of these deiodinases and the reguIation of their activity may dso be involved in the maintenance of ceUuIar TH concentrations,

In the 19603, Tata and coworkers presented the fust evidence that the nucleus was the site of action of TH (fiom WiIliams, 1994). They demonstrated that exogenous TH treatments produce rapid increases in mEWA synthesis in anurans (Tata and Widnell, 1966;

Tata and Williams-Ashman, 1967). These experiments stimdated investigations into the function of TR in metamorphosis and ultimately Ied to their isolation and charactenzation_

The earliest studies utilized binding assays to determine the number of receptors, their relative affinities for T4 and Tj, and changes in these two parameters over the anuran Life cycle (for review see Galton, 1983, 1988)- In these studies, the number of receptors increased during metamorphic climax and this increase could be triggered with exogenous

TH. More recentiy, cDNAs for both the a and f3 TR subtypes (TRa and TRP,respectively) of X. laevis were isolated and characterized (Yaoita and Brown, 1990; Yaoita et al., 1990;

Shi et al., 1992). Yaoita and Brown (1990) found that TRa mRNA levels increase prior to premetarnorphosis, peak by the start of prometamorphosis and remain constant until the end of metamorphic climax. In contrast, TRB mlWA Ievels do not increase substantialiy untiI late prometamorphosis. At this time TRP mRNA levels increase rapidly in concert with serurn T3 titers and the onset of metamorphic climax (Leloup and Buscaglia, 1977; Yaoita and Brown, 1990). Moreover, the "auto" up regdation of TR mRNA in response to exogenous TH has been shown in several studies, and the increases in TRP are more pronounced than T'Ra (Tata, 1996)-

Thyroid hormone receptor expression and the expression of its heterodimeric partner, the retinoid X receptor, are important factors in the differentîai timing of tissue morphogenesis (Shi, 2000). Messenger RNA levels of these receptors Vary both temporally and spatially throughout metamorphosis- In generd, the mRNA levels of TR and retinoid X receptors are high in tissues during times of morphogenesis, but are low before and after rnorphogenesis (Shi, 2000). Thyroid hormone responsive genes have ken identified in numerous organs and tissues of metamorphosing anurans including the tail, hbs, intestine, liver, skin, and brain. Shi (2000) has proposed 3 different models for gene regdation by TH based on the regdation of a cascade of early or direct response genes and late or indirect response genes. Those genes which respond to TH within 24 hours of treatment are considered eady response genes, whereas genes which require the presence of TH for longer than 24 hours are considered Iate response genes. The activation of late response genes is dependent upon either the activation of other genes or protein synthesis.

Early TH responsive genes have been identified in the hind-limbs, intestine, tail and brain using various techniques. Treatment of X. laevis tadpoles with T3 for 24 hours resuIted in the upregulation of 14 genes and the downregulation of 5 genes in the hind-hbs

(Buckbinder and Brown, 1992). Shi and Brown (1993) identined 22 upregdated genes and 1 downregulated gene in the intestine of X. laevis following 18 hours of treatment with 4.

Twenty four hours of treatment with T3 resulted in the upregulation of 15 genes and the downregulation of 4 genes in the tail of X. faevis (Wang and Brown, 1991, 1993)- In the brain, a greater proportion of eady response genes are downreguIated relative to other tissues

(Denver et al., 1997). Denver et al. (1997) identïfied L4 downreguIated and 20 upregulated

genes in the brain of X. CaevrS foilowing 24 hours of treatment with T3- Among the

upregulated early response genes that have been idenîiZed, 15, L 1, and 12 in the intestine,

tail, and brain, respectively, were upregulated independent of protein synthesis. Late TH

responsive genes have aIso been identified in the intestine, tail, Iimbs, skin, epidennis, Iiver

and pancreas of X, laevis and R. catesbeiana (see Shi, 2000)-

The obligatory role that TH play in anuran metamorphosis has been clearly

demonstrated at the systemic, tissue, cellular and molecular levels, but numerous other

regulatory systems are important for the initiation and completion of metamorphosis.

Aithough TH are the primary morphogens, their synthesis and secretion is regulated by the

hypothalamic-pituitary axis and their action is regulated at the tissue and cellular level via deiodinases and TR. The hypothalmic-pituitary axis may be the primary avenue through which anurans perceive essentiai environmental stimuli for the initiation and regulation of metamorphosis (Denver, 1996, 1997). Moreover, other hormonal signals (i.e,, corticotropin releasing hormone, corticoids, prolactin, sex steroids) may serve to modulate or fine-tune this developmentai event and ensure a successful metamorphosis (Kikuyama et al., 1993;

Kaltenbach, 1996; Hayes, 1997b; Denver, 1999; Shi, 2000).

Thymidal control of Pieuronectiforme nounder) metamophosis

Al1 species of fish belonging to the order Pleuronectiformes undergo a me metamorphosis (Youson, 1988) fkom a bilateraiiy symmetrical, pelagic lama to an asymmetrical, benthic juvenile (Keefe and Able, 1993). The hormonal control of metamorphosis has been examined in the Japanese flounder (Paralichthys olivaceus) and, more recentiy, in the summer flounder (Parafichthysdentatus). FIounder metamorphosis has

been charactenzed and divided into a senes of stages based on externai morphology. These

morphological changes are sunilar in P. olibaceus (Minami, 1982; Miwa and Iiiui, 1987;

Miwa et ai-, 1988) and P- dentatus (Keefe and Able, f 993 ;Huang et al., 1998; Schreiber and

Specker, 1998)- Hounder metamorphosis can be dassified into the foIiowing general stages:

premetamorphosis, prometamorphosis, metamorphic climax, pst-climax and juveniIe;

however, each of these stages has severai subdivisions (see references above). The following

is a bnef description of the afiorementioned general stages of flounder metamorphosis.

Premetamorphic flounders are pelagic and completely bilaterdy symmetrical, during this

stage severd dorsal fin rays begin to elongate. Prometamorphosis begins with the onset of

eye migration and ends when the nght eye reaches the dorsal midiine and dorsal fin ray

resorption begins. Metamorphic climax is characterized by the translocation of the right eye

across the dorsaI mid-line to the Ieft side of the body and ends when fin ray resorption is

complete. FIounder larvae may exhibit periodic settling ont0 the benthos during climax. The

completion of eye migration and benthic settling occw during postclirnax, giving rise to a juvenile flounder.

Thyroid hormones appear to mediate flounder metamorphosis in a similar manner as in amphibians. The treatment of P. olivaceus larvae with Tq (0.01 - 0.1 rnmter) accelerated fin resorption, right eye translocation and the rate of settling relative to control larvae (Inui and Miwa, 1985). These, T4-treated larvae also reached each stage of development 1 - 2 weeks in advance of the controIs. Consistent with the stimulatory role of T4 was the inhibition of metamorphosis by the goitrogen thiourea (0.03 mgIliter; Inui and Miwa, 1985).

Treatment with thiourea resulted in metamorphic stasis producing giant, pelagic larvae. When exogenous TH were adrninistered to these thiourea-treated Iarvae, the inhibitory

effects of thiourea on metamorphosis were overcome- This aiIeviation of metamorphic stasis

was dose-dependent, and T3was severai tirnes more potent than T4 (Miwa and Inui, 1987).

Schreiber and Specker (1998) also found that exogenous T4 treatments accelerated,

and thiourea inhibited, the metarnorphosis of P, dentatus. Their data indicated that this

acceleration and inhibition was stage-dependent. The treatment of premetamorphic and

prometamorphic flounders with thiourea (0.03 mgfliter) slowed metamorphic development and the Iarvae never developed past mid- and Iate metamophic climax, respectively

(Schreiber and Specker, 1998). The administration of exogenous T4 (0.1 mgniter) to premetamorphic Iarvae resuited in an accelerated metamorphosis, and these larvae reached mid-climax 1 week prior to controls (Schreiber and Specker, 1998). However, these T4- treated larvae reached iate climax and postclimax at the same theas the untreated controls, indicating that T4 treatment at 0.1 mgfiter can accelerate oniy the early stages of metamorphosis (Schreiber and Specker, 1998). Perhaps these T4-treated flounders would have completed their metamorphosis prior to the untreated controls ifT4 treatment concentrations were increased throughout metamorphosis, in a manner similar that which is necessary for amphibians (Etkin, 1935)- This suggestion is supported by the observed changes in whole-body T4 concentrations during flounder metamorphosis.

The whole-body T4 concentrations of ffounders change throughout larval development and metamorphosis (Miwa et al., 1988; Tagawa et al., 1990; de Jesus et al.,

199 1; Schreiber and Specker, 1998). Whole-body Tq concentrations peak at the end of metamorphic climax at approximately 14 ng/g body weight in P. olivaceus (Miwa et al.,

1988; Tagawa et al., 1990; de Jesus et al., 1991), and 13 ng/g body weight (71 ng/g dry body weight) in P. dentam (Schreiber and Specker, 1998). GeneraUy, whole-body Tq

concentrations remain bw throughout premetamorphosis and prometamorphosis, increase

rapidly at the onset of metamorphic ciïmax, peak at the end of metamorphic ciimax and

decline to 50 % of peak concentrations dwing postchax Wwaet ai-, 1988; Tagawa et al.,

1990; de Jesus et al., 1991; Sc-hreiber and Specker, 1998). However, Schreiber and Specker

(1998) measured Tq concentrations in P- dentatus that were 80 % of peak values in early premetamorphosis. Interestingly, the whole-body Tjconcentrations of P. olNaceus did no t fluctuate much during metamorphosis. Triiodothyronine concentrations are very Iow (c 1 ng/g body weight) during premetamorphosis and prometamorphosis, increase slightly during metamorphic clunax and decline to very low Ievels thereafter uagawa et al., 1990; de Jesus et al-, 1991)-

Thyroid hormone-rnediated tissue morphogenesis has been demonstrated in several organs and tissues of the Iapanese and summer flounders. During metamorphosis, the skeletal muscle fibers of fiounders undergo several morphological and biochemical changes.

These include an increase in muscle fiber thickness and a shift fiom 1arva.l to adult myosin light chah and troponin T isoforms manoet al., 1991b). The addition of exogenous T4 accelerated these changes in the skeletai muscle and thiourea prevented the appearance of the adut t muscle fibers (Yamano et al., 1991b, 1994a). During metamorphic climax, the erythrocyte population of P. olivaceus undergoes a shift from large, round, iarval erythrocytes to small, elliptical, adult erythrocytes, and exogenous T4 can precociously induce this shift (Miwa and Inui, 1991). In both P. olivaceus and P. dentatus, gastric glands and pepsinogen production appeared 1 - 2 weeks earlier in &-treated animals than in controls

Mwaet al., 1992; Huang et al., 1998). Moreover, Huang et al. (1998) found that T4 treatment could initiate gastric gland development in as üttie as three days, prior to signs of extemal morphogenesis. Thiourea treatment of premetamorphic larvae delayed gastric gland development and prevented pepsinogen production (Miwa et al., 1992; Huang et al., 1998).

The mitochondria-rich ceiis of the giUs are important for osmoregulation and have been implicated in ion uptake as juvenile Aounders enter estuaries with low salinlties

(Schreiber and Specker, 1999)- The gills of larvai flounders have one type of rnitochondria- rich celI, these are light-staining and Iack a well-defined apical pit (Schreiber and Specker,

1999). Juvenile flounders have two types of mitochondria-rich cells: light-staining cells and dark-staining cells (Schreiber and Specker, L999). Schreiber and Specker (1999) found that both types of juvenile mitochondna-rich cens differed in ultrastructure fiom the larval mitochondria-rich ceus. Juvenile ceUs have weil defined apical pits and smaiier, more electron-dense mitoc hondria, Moreover, these juvenile mitochondria-rich cells show a much etronger immunoreactivity to a Na+=-ATPase antibody than those of the larvai gills

(Schreiber and Specker, 1999). Thiourea inhibited both metamorphosis and the appearance of juvenile mitochondria-rich ceils. Treatment of these animais with exogenous Tq counteracted the effects of thiourea and promoted the development of juvenile mitoc hondria-rich ceils (Sc hreiber and Specker, 2000).

The ability of T4 to accelerate tissue-specific changes that are consistent with metamorphosis suggests that TH may mediate morphogenesis in the flounder in a similar fashion to that observed in amphibians. However, gene expression studies are required to confirm this suggestion. The cloning of four P. olivaceus TR isoforms is a significant step towards an understanding of TH-mediated morphogenesis in the flounder. Yamano et al-

(1994b) have cloned two TR cDNAs with hîgh homology to the TRcc of other vertebrates, and designated them as TRa-A and TRa-B (Yamano et al., 1994b). Two additional TR cDNAs (mp-I and TRB-2) were isolateci whose sequeaces were homologous to the subtype of other vertebrates (Yamano and M,1995)-

The expression of these TR isoforms is regulated both temporaiIy and spatially during

P. olivaceus metamorphosis (Yamano and Miwa, 1998). TRa-A and TRp (ïR&l and TRP-

2 combined) are expressed at approximately 50 and 30 %, respectively, of their peak values throughout prernetamorphosis and decline to their Iowest leveIs in prometamorphosis

(Yamano and Miwa, L998). The expression of TRa-A increases rapidly between prometarnorphosis and the onset of metamorphic climax, peaks at the end of ciïmax and then decreases to 25 % of peak values shoaly after metamorphic climax (Ymano and Miwa,

1998). TRB expression also increases following prometamorphosis; however, this increase is not as rapid, peak values are detected towards the end of postclimax, and levels remain high dunng the juvenile period. Conversely, TRa-B expression remains low throughout the larval and juveoile periods (Yamano and Miwa, 1998).

Spatial variations in TR receptor expression also occur throughout P. olivaceus metamorphosis, and the distribution patterns of TRa and TRB dBer (Yarnano and Miwa,

1998). Both TRa and TRB expression is observed in skeletal muscle, but TRB expression is concentrated on the myosepta whereas TRa expression is unifody distributed. TRP is also strongly expressed in the cartilage and in regions just beneath the epidermis. In the developing stomach, TRa is expressed in the epithelia of developing gastric glands, whereas

TRB is predorninately expressed in the lamina propria and submucosa (Yamano and Miwa,

1998). These TR expression studies, coupled with eariier TES and thiourea studies, support the idea that TH exert their effects on tissues undergoing morphogenesis. Moreover, the temporal and spatial regdation of dinerent TR isofomis may be involved in mediating the progression of metamorphosis at the cellular level.

The Urmprey I$ie cycle

The lamprey has a complex Life cycle with clearly dernarcated larvai, juvenile and spawning periods. During the larval period, the blind, relatively sedentary larvae remain burrowed in sandy river beds, filter-feeding on detritus and organic matter in the water colurnn. The duration of the larval period varies fiom 3 - 7 years and is characterized by a growth phase to attain the minimum size for metamorphosis (Potter, 1980)- When the appropriate endogenous and exogenous conditions are present, larval Lampreys metamorphose into juveniles.

Lamprey metamorphosis has been well-characterized and is divided into 7 stages (1 -

7, earliest to latest) that are universal among most lamprey species (Bird and Potter, 1979;

Youson and Potter, 1979; Potter et al., 1982; Youson, 1994). The metamorphic event Iasts 3

- 4 months and involves dramatic morphological, physiological and biochemical changes in body proportions, integument, brain and spinal cord, sense organs, and the respiratory, digestive, excretory and endocrine systems (see Youson, 1980, 1988). Following the completion of metamorphosis, juveniIe lampreys usually over-winter in their natal streams.

In the spring, juveniles either migrate downstream to a large body of water to commence parasitic feeding on the blood and body fluids of teleost fishes (some species of lamprey feed wi thin their natal stream) or complete sexual maturation, spawn and die (nonparasitic species). Parasitic larnpreys feed for 1 - 2 years and then commence their migration to the river spawning grounds where they undergo sexual maturation, spawn and die- Over the last five decades our knowledge about the morphologid, physiological, and biochemical changes occumng during lamprey metamorphosis has expanded greatiy; however, our understanding of the rnechanisms which uigger and regulate this developmental event is Iimited. Researchers have examined several environmental, physio1ogicaI and hormonal cues which may be relevant to the initiation, progression ador completion of lamprey metamorphosis. The remainder of this introduction will focus on the factors that have been found to have the greatest impact on lamprey metamorphosis: the pineal gland and photoperiod, water temperature, lamprey size and lipid reserves, and TH.

Environmentul factors and Urmprey mefantophosis

The observation that the onset of metamorphosis is remarkably syncironous for larnpreys within a paaicular region provides compelling evidence that metamorphosis is occumng in response to some environmentai signal. This idea is best demonstrated by the sea lamprey (Petrumyzon marinus). in North America, anadromous sea larnpreys in New

Brunswick (Potter et al., 1978; Youson and Potter, 1979) and landlocked sea lampreys in the

Great Lakes drainage basin (Manion and Stauffer, 1970) begin to metamorphose during the second week of July. The most Iikely environmental candidates for ensunng this synchrony in the initiation of metamorphosis are photoperiod and temperature.

Most vertebrates perceive changes in photoperiod via the pineal gland. The pineal gland transmits information about day length and time of year to various systems by changing the secretion rates of its primary product, melatonin. This function of the pineal gland is important in the regdation of biologicai rhythrns such as seasonal reproduction

(Bentley, 1998). The function of the pineal gland and photoperiod in the timing and initiation of lamprey metamorphosis is somewhat puuling. The pineal gland, but not photoperiod, appears to be important for metamorphosis. Pinealectomy experiments on two southern hemisphere Iamprey species (Mordacia mordax and Geotriiz australis) (Eddy and

Strahan, 1968) and two northem hemisphere lamprey species (Larnpetra p1met-i and P. marinus) (Eddy, 1969; Cole and Youson, 1981) indicate that the pined gIand is essential for the initiation of metamorphosis, In these studies, significantly fewer pineaiectomized larnpreys began to metamorphose than unoperated and sham-operated control lampreys-

However, the incidence of metamorphosis was not aBected by continuous Iight, continuous darkness or variations in photoperiod (Cole and Youson, 1981; Youson et al., L993; Holmes et ai., 1994). Eleven months is the Iongest time period that larval lampreys have been exposed to an artificial photoperiod regime- Perhaps Youson (1997) is correct in suggesting that photoperiod should not be omitted as a factor in metamorphosis until the cumulative effects have been examined over the entire Iarvai period- However, due to the length of the larval penod and the difF1culties associated with maintaining larval Iamprey populations in the laboratory for several years, a more miiüi11approach may be to fwus on the pineal gland and its secretion(s).

Unlike photoperiod, the importance of temperature in the initiation and progression of metamorphosis in lampreys has been extensively studied and weU-established. The fïrst report that temperature can influence metamorphosis was made by Potter (1970) following a prelirninary study on lampreys of the genus Morducia. He reported that larvae maintained at

15 OC were delayed in their metamorphosis by 4 - 5 weeks in cornparison to larvae kept at 22

- 25 OC. At approximately the same tirne, Manion and Stauffer a970) presented inconclusive data suggesting that the rate of metamorphosis was slower for sea lampreys maintained at cooler water temperatures (13 or 14 OC) than for those kept at warmer water temperatures (18 or 21 OC). PuMs (L980) conducted an extensive study over a 4 year pend

and reported that the incidence of metamorphosis was greater at increased water

temperatures; 8 %, 59 % and 88 % of sea lampreys metamorphosed when maintained at

water temperatures of 7 - 11 OC, 14 - 16 OC and 20 - 21 OC, respectively, However, in this study, the three temperature regimes were achieved under different environmental conditions

(Le., laboratory, river, and lake). More recently, controlled laboratory studies have confirmed that the incidence of metamorphosis in Iarvae of premetamorphic size (see below) was greater in wann water (21 OC) than in cool water (13 OC) (Youson et al,, 1993)-

Furthemore, it was shown that a rise fiom cold (winter) to warm (siimmer) temperatures produces the highest incidences of metamorphosis (Holmes and Youson, 1994; HoImes et al,, 1994).

Physiological condirion and hmprey meîamophosis

Larval sea lampreys must attain a minimum size (iength and weight) and condition factor (CF; [weight (g)/length X 106) pnor to undergoing metamorphosis (Youson et al., 1993). Severai studies have shown that these parameters are valuable tools for predicting which larvae will begin to metamorphose in the spring months. For example, the minimum length, weight and CF criteria for metamorphosis has been established for several populations of sea lampreys in the Great Lakes drainage basin (see Youson et al., 1993;

Holmes et al,, 1994; Holmes and Youson, 1994; Holmes and Youson, 1998). A reiiable approximation of these criteria for predicting metamorphosis in lampreys captured in the spring is 120 mm and 3.0 g with a CF of 1.5 or greater. This CF value is similar to that obtained by Potter et ai. (1978) for anadromous sea lampreys which metamorphose at a similar size. The ability to accurately identify immediately premetamorphic individuds is a

necessity when investigating the factors which regdate the initiation and progression of

metamorphosis.

The fact that larval lampreys must reach a minimum size and CF prior to the

commencement of metamorphosis underscores the importance of physiologicai condition in

this developmental event. Towards the end of the Iarvai period, lampreys undergo a so-

called "arrested-growth phase" whereby there is a proportiondy greater increase in Iarval

weight versus Iength (Potter, 1980; Youson, 1988). This "arrested-growth phase" results in

an increase in the CF value and is related to the accumulation of sufncient lipid reserves to survive the protracted, non-trophic phase associated with metamorphosis. Shortiy after the onset of metamorphosis, tampreys enter a prolonged, non-trophic phase that may last up to

11 months (Youson, 1994); however, the exact point at which feeding stops is not known- In the case of nonparasitic lampreys, feeding never resumes.

The first experimental evidence showing that lipid accumulation occurs prior to the onset of metamorphosis was presented by Lowe et al, (1973)- Carcass lipid content in the early stages of metamorphosis (July) was 13.5 % of the wet body weight, whereas the largest nonmetamorphosing larval sea lampreys at the same the of year had a lipid content of oniy

6.5 % (Loweet al., 1973). Moreover, lipid accumulation began in the fidi prior to metarnorphosis; premetamorphic Iampreys had a carcass lipid content of 8 % in September, compared to the larval average of only 4 %. Similady, O'BoyIe and Beamish (1977) reported that the Iipid content in the musde of larvai and premetamorphic lampreys was 4.5 and 11 % of the wet weight, respectively, The primary sites of storage for lipids in lampreys are the fat column, nephric fold, beneath the skin, and in regions surrounding the body cavity (Youson et al., 1979). These fipid stores are utiked but not depleted, to fuel morphogenesis as metamorphosis proceeds (Lowe et al,, L973; Youson et al,, 1979). This utiIization of

Lipids is evidenced by a decrease in the total body Lipid content fiom a peak of 135 % of the wet body weight at the onset of metamorphosis to 7.8 96 towards the end of metarnorphosis

(Lowe et aL, 1973). It is likely that these Lipid reserves present at the end of metarnorphosis, which are elevated cornpared to the larval average, provide a source of fuel for those juvenile

Iampreys that remain in their natal streams and do not begin feeding until the foUowing sprïng.

The Zamprey endostyle and thyroid ghnd

The larval lamprey is unique among vertebrates in that it is the ody representative of this subphylum that lacks follicular thyroid tissue. The typical vertebrate thyroid tissue of the juvenile lamprey develops fiom a subpharyngeal gland early in metamorphosis

(Schneider, 1879). This gland is called an endostyle because of its similarity to the protochordate endostyle (Schneider, 1879). Since the tirne that their stimulatory role in arnphibian metamorphosis was established, TH and potential thyroid stimulants have been the pnmary focus when studying the regulation of lamprey metamorphosis, despite the knowledge that folLicular thyroid tissue appears post-metamorphically in larnpreys. In fact,

Horton (1934) tested the effects of thyroid extracts on lamprey metamorphosis pnor to the first experimental evidence indicating that larval lampreys had the potential to synthesize iodinated compounds (Gorbman and Creaser, 1942). In the foilowing sections, 1will briefly describe the larval lamprey endostylehhyroid axis, the transformation of the endostyle dwing metamorphosis, and the current state of our knowledge on the involvement of the thyroid axis in lamprey metamorphosis. Numerous detailed descriptions and reviews of the anatomy, histology and fine cellular structure of the larval lamprey endostyle have ken pubfished (Marine, 1913; Leach,

1939; Barrington and Franchi, 1956; Clements-Merlini, 1960a, 1960b; Egeberg, 1965; Fujita and Honma, 1968,1969; Barrington and Sage, 1972; Wright and Youson, 1980). The following is a brief consensus description of the gland and its celIs to facilitate a discussion regarding thyroidal activity and metamorphosis. The larvai lamprey endostyle is a subpharyngeal gland embedded in the muscle and connective tissue between the first and fifth gill arches and is visible through the skin of the ventral body wall in the intact animal or the pharynx with which it communicates via the hypobranchiai duct (Fig. 2A). Antenor to the hypobranchial duct, the endostyle has a pair of srraight anterior chambers separated by a thin septum, while posteriorally three chambers can be described: a posterior medial charnber, which coils towards the hypobranchial duct, and two posterior Iateral chambers that extend to the caudai limits of the gland (Fig. 2A). The entire endostyle is covered by a connective tissue sheath and the Iumen is lined with several different ceii types.

The rnost prominent histological features of each chamber are the paired ventral and dorsal glandular tracts which consist exclusively of type 1 ceils (Fig. 2B). These tall, wedge- shaped cells have basaily located nuclei and a narrow apex that curls centrally towards the opening of the glandular tract. Each type 1cell has a large cilium and numerous microvilli which converge to form a plug at the openings of the glandular tracts. The consensus is that the glandular tracts of the endostyle do not have a direct role in thyroidal activity nor do they contribute to the formation of the follicular thyroid gland during metamorphosis. The primary function of the type 1 ceus is not clear, but histochemical evidence suggests that they actively produce and secrete mucus (Barrington and Sage, 1972). This mucous Figure 2. Diagramatic representation of (A) the gross matornical morphology and (B) the histology of the larval lamprey endostyle. Figure 2B is a transverse section through the right anterior chamber of the endostyle and cleariy displays the histological organization of the various tissues and epitheiïal ceU types within the lamprey endostyle. (Modified fkom

Barrington and Franchi, 1956; Barrington and Sage, 1972) / ventral glandular type tract secretion may be involved in tiltet-feeding (Newth, 1930). lining the burrow to aid in respiration, or in the hanspoa of hormonal secretions into the pharynx where they will ultimately be shuttled to the intestine for absorption (CIements-Merlini, 196ûa, b).

Located ventrally and dors* to the opening of the ventral gianduiar tract are the type 2a and 2b cells, respectively (Fig. 2B). Type 2a and 2b cells are columnar with basally located nuclei and densely packed microvilli. They may differentiate into the type 1 cells of the glandular tracts (Barrington and Sage, L963a)- The type 2c and type 3 cells are aIso columnar (varying in height) and possess numerous microvilli and/or short cilia (Fig. 2B).

These are the pcïmary iodide-binding ceiis of the endostyle. Bound iodide is localized on both the surface of the celis among the microvilli and ciiia and over the apical cytoplasm

(Barrington and Sage, 1972). Another feature of these type 2c and type 3 cells that supports their involvement in thyroidal activity is their immunoreactivity to an anti-human thyroglobulin (Tg) antibody (Wright et al., 1978). Type 4 ceUs are low columnar and may be ciliated, they are best charactenzed by their extrusions of the apical cytoplasm or nuclei.

Lining the wall and septa of the chambers are the Low cuboidal, type 5 ceus. Type 4 and type

5 cells have a limited capacity to bind iodide (Clements-Merlini, 1960a; Barrington and

Sage, 1972; Wright and Youson, L976).

The transformation of the larval lamprey endostyle into a follicular thyroid gland was first descnbed by Schneider in 1879. Since this initial observation, many others have exarnined this phenornenon in several lamprey species (see Barrington and Sage, 1972;

Wright and Youson, 1980). Despite this extensive research, there has been no definitive resolution as to which endostyle ce11 types contribute to the formation of the foilicular thyroid gland. A series of studies conducted by G.M. Wright and coworkers using numerous metamorphosing anïmals and a welL-defined system for detemiinhg metamorphic stages have contributed greatly to the clarification of this issue. Data fiom autoradiographic, immunohistochemical, and electron microscopy studies indicate that the endostylar ceii types

2c and 3 most certainly take part in the formation of thyroid foiïicles and it is WeIy that the type 5 cells do as weIl (Wright and Youson, 1976, 1980; Wnght et al., 1980). However, based on the current evidence, it is possible that al1 cell types with the exception of type 1 cells make some contribution to the formation of thyroid folLicles (Wright and Youson,

1976). The transformation of the larvai endostyle into a juvenile, foilïcular, thyroid gland begins at stage 1 or 2 of metamorphosis (staging according to Youson and Potter, 1979) with the breakdown of the glandular tracts; the £ktfolJicles appear by stage 3 of metamorphosis and the newly transformed, follicular thyroid is present by stage 5 (Wnght and Youson,

19 80). Low levels of radioiodide binding and immunoreactivïty to an anti-human Tg antibody are seen throughout the transformation process (Wright and Youson, 1976; Wright et al., 1980).

It has now been well-established that the endostyle is the site of TH biosynthesis in the larval Iarnprey. Initiaily, thyroidal activity in the endostyle was implied because of its ability to concentrate and bind iodide; however, other structures such as the notochord were also able to concentrate iodide (Clements-Merlini, 1962a). Definitive thyroidai activity in the larval lamprey endostyle was confirmed by a series of biochemical studies that showed the presence of iodoproteins and TH in both the endostyle and the systemic circulation-

These studies demonstrated that the method of TH synthesis in thé Iarval lamprey endostyle is 1ikeIy to be similar to that observed in a folIicular thyroid giand (Barrington and Sage,

1972). Numerous studies have confkmed that when radiohiide Ïs concentrated by the lamprey endostyle, it is incorporated into the protein bction. Several different iodoproteins have been isolated and charactenzed, including moIecuIes with sedhentation coefficients of approximately 19 S, 12 S, and 3 - 8 S (Roche et al., 1961; Salvatore et al., 1965; AIoj et d,

1967; Roche et al., 1968; Su& and Kondo, 1973; Suzuki er al,, 1975; Monaco et al,, 1977,

1978). These sedimentation coefficients were similar to those found in other vertekate thyroids. The relative proportion of each of these iodoproteins in the larval lamprey endostyle differed in the various studies, but this variation was likely due to dlfferences in experimental methodology, such as variations in the length of incubation with radioiodide or endogenous proteolytic activity.

In addition to identwng iodoproteins simllar in size to those found in other vertebrates, time-lape iodide incorporation studies suggested that the smaiier iodoproteins were precursors for the synthesis of the 19 S iodoprotein- These resuits were similar to those obtained for mammalian Tg, which suggested that the mammalian 19 S Tg was made up of several different iodoproteins < 12 S in size (Ekholm, 1990). However, it was later confirmed that mammalian Tg (the 19 S, 660 kDa molecule) is made up of two identical 12 S

(330 kDa) molecules and that iodoproteins srnalier than 12 S were the products of endogenous proteolytic activity during TH synthesis @unn et al., 1983; Ekholrn, 1990). A definitive size for lamprey Tg has not yet been established, and lamprey Tg should be characterized using modem biochemical and molecular techniques. However, there is Iittle doubt that a Tg-like molecule is synthesizeci by the Iarval lamprey endostyle and that its synthesis is a step in the production of TH. Hydrolysis studies have shown that the iodoproteins isolated fiom the lamprey endostyle contain T4,T3, monoiodotyrosine (MIT), and diiodotyrosine (DIT) (Roche et aL, 196 L; Suzuki and Kondo, 1973; Monaco et al,, 1977,

1978). me hypothalamic-pifrrirapthyroid axis and hpreymefamorphoIms

B iologists have tried to determine the importance of the hypothaImic-pituitary- thyroid ais in lamprey metamorphosis since the time of Gudematsch (1912)- Young and

Bellerby (1935) injected larval L planen with ox pituitary extracts and observed changes in the rnorphology of the cloaca similar to those that occur during sexuai maturation, but a morphogenesis indicative of metamorphosis was not reported. Knowles (1941) suggested that the lack of metamorphosis in this study and other earlier ones was related to the fact that the larvae used were not necessarily near the age or size of metamorphosis. To address this concern, Knowles (1941) detennined the size and age of metamorphosis of larvai L. planeti from his collection stream and injected what he considered to be premetarnorphic larvae with the thyrotropic fraction of an extract fiom the anterior pituitary of an ox. He observed some changes in body proportions (i.e., decreased length), but the larvae did not metamorphose.

More recently, Joss (1985) provided evidence that the lamprey pituitary is essential for the metamorphosis of G. australis. Removal of the rostrai pars distalis fiom premetamorphic lampreys completely inhibits spontaneous metmorphosis and removal of the caudal pars distalis results in a metamorphosis that arrests at stage 3 (Joss, 1985). These results suggested that the rostrai pars distalis is required for the initiation of metamorphosis and the caudal pars distaüs is important once metamorphosis has begun. Wi5ght (1989) observed dramatic ultrastructurai changes indicative of increased synthetic and secretory activity in the caudal pars distalis dunng stages 3,4 and 5 of sea lamprey metamorphosis, but a secretory product has yet to be identified. Whether the lamprey pituitary produces a signai or signals which affect metamorphosis directly or indirectly via either the thyroid mis or

another endocrine axis has not kenconfinned, To date, it is unclear whether the lamprey

hypothalamus and pituitary produce thyrotropin releasing hormone and thyrotropin

(thyroid stimulating hormone; TSH), respectively- Moreover, the regdatory influence of the

hypothalamic-pituitary axis on the lamprey endostyle is still in question-

The first experiments that examined the role of the thyroid and TH in lamprey metamorphosis suggested that they were not significant, Horton (1934) treated larvai sea lampreys by immersion and injection with iodine-containing compounds and thyroid extracts, but did not observe metamorphosis- Leach (1946) was also unable to induce metamorphosis in lampreys by injecting them with T4. TheSe results were in contrast to the clear, stimulatory function ofTH on amphibian metamophosis (Etkin, 1964, 1968).

Eventually, Hoheisel and Sterba (1963) presented results that supported the notion that TH may be involved in Iamprey metamorphosis; however, their resuk were not consistent with the stimulatory role observed in amphibian metamorphosis. They showed that the anti- thyroid agent (goitrogen) potassium perchlorate (KC104) could stimulate precocious metarnorphic development in lmaiL. planeri In the first, second, and third years of Iarval growth.

The effects of various goitrogens on endostyle morphology and iodine metabolism indicated that KC104 inhibited thyroidal activity (Jones, 1947; Klenner and Schipper, 1954;

Clements-MerIini, 1962c; Barrington and Sage, 1963a, b), but how did this relate to the induction of metamorphosis? That goitrogen-induced metamorphosis may be related to the inhibition of thyroidal activity was later supported by the observations that both semm Tq

(Wright and Youson, 1977) and serum T3 concentrations (Lintlop and Youson, 1983) decreased at approximately the same time that the ktexternal signs of spontaneous metarnorphosis became evident, Suzuki (1986, 1987,1989), in a series of preliminary studies, confinned the resuIts of Hoheisel and Sterba (1963)- He reported a complete metamorphosis in Iarge L, reissnen' folIowlng treatment with either KCl04, sodium perchlorate (NaC104), propylthiouracil 0 or thiourea. Moreover, KC104 treatment resulted in very low serum Tq concentrations in Iampreys prior to the observation of extemai morphogenesis.

It now appeared that the role of TH in lamprey metamorphosis was different from its role in the metamorphosis ofother vertebrates. The initiation of lamprey metamorphosis may be related to a decrease rather than an increase in thyroidal activity. However,

Leatherland et al. (1990) pcesented data that could be interpreted to indicate that this observed decrease in thyroidal activity was a consequence of metarnorphosis rather than an initiator. Precocious metamorphosis was not induced in G. australis following a 70 day exposure to the goitrogen PTU, despite a significant decrease in both serum T4 and T3 concentrations. Leatherland et al, (1990) also suggested that since semm T4 and T3 concentrations do not decline until after the tirst visible signs of spontaneous metamorphosis, this decline could not be the stimulus that initiates morphogenesis.

More recently, a comprehensive series of experiments on goitrogen-induced lamprey metamorphosis and semm TH concentrations suppoaed the idea that an inhibition of thyroidal activity is related to lamprey metamorphosis. Holmes and Youson (1993) exposed

180 larval sea larnpreys of three diffecent sizes (65 - 95, 110 - 119, > 130 mm in length) to two concentrations of KC104 (0.0 1% or 0.05%) in a controlled experiment conducted dunng the winter months when spontaneous metamorphosis does not occur. The induction of precocious metamo~hosiswas observed in ail KC104 treatment aupsbut not in the untreated control groups. The incidence of metamorphosis was not affected by the KC104 treatment concentration, but signincantly more larvae metamorphosed in the larger size groups than the smailer size groups; 22,53 and 98 % of KClO+-treated larvai lampreys metamorphosed in the 65 - 95,110 - 119, and > 130 mm size groups, respectively-

Serum TH concentrations increase graduaiIy throughout the 3 - 7 year iarval penod of the Iamprey life cycIe (Youson, 19941, peak, then decline rapidly early in spontaneous metarnorphosis (Wright and Youson, 1977; Lintlop and Youson, 1983; Leatherland et al.,

1990; Youson et al,,1994). In a cornpanion study to that of Holmes and Youson (1993),

Youson et al. (1995) reported that the serum T4 concentrations of KC104-treated lampreys of the two smalIer size groups were 27 - 3 1 % lower than the values for control lampreys, but those of animais in the > 130 mm group did not Merfrom those of the controls. Moreover, the serum T3concentrations of Km4-treated lampreys of all three size groups were 91 - 95

% Iower than those of the control group. These declines in semm T3 concentrations were comparable in magnitude to the declines associated with the eady stages of spontaneous metamorphosis (Lintlop and Youson, 1983; Youson et al., 1994); however, the magnitude of the decline in serum T4concentrations in the induced metarnorphosis was smaller than that observed between premetamorphic larvae and stage 1 or 2 of spontaneous metamorphosis

(Wright and Youson, 1977; Youson et al., 1994).

Rationule and objectives

Over the Iast century, scientists have studied anuran metamorphosis and have confirmed without question that elevations in thyroidal activity are essential for the initiation and progression of metamorphosis. Furthemore, they have shown that TH act via their receptors to alter gene expression. More recently, the stimulatory role of TH on metamorphosis has been shown in uumerous other amphibians as well as several bony fishes.

Larnpreys appear to differ fiom other vertebrates wïth respect to the influence ofTH on their metamorphosis. Severai studies have shown that Iamprey metamorphosis can be initiated by inhibiting thyroidal activity with goitrogens, Furthemore, the onset of spontaneous metamorphosis in lampreys coincides with a decrease, rather than an increase, in serum TH concentrations-

These unique qualities of the lamprey thyroid system and lamprey metamorphosis were paramount in the formulation of the central question upon which my doctoral thesis is based. Are thyroid hormones a primary factor in the initiation and regulation of lamprey metamorphosis, and if so, is their role similar in nature to that observed in other vertebrates or is their role fundamentally different? 1have focused my research on determining whether a decline in semm TH concentrations is an event which initiates metamorphosis, is permissive to metamorphosis, or is simpIy a consequence of metamorphosis. Alternatively, are elevated serurn TH concentrations inhibitory to metamorphosis? To answer these questions, 1have used goitrogens and their ability to induce lamprey metamorphosis as an experimental tool. The primary objectives of this thesis were:

1. To determine the role of depressed serum TH concentrations in the KCL04-

induced metarnorphosis of larval sea lampreys.

2. To confimi that the goitmgen-induced metamorphosis of sea lampreys is related

to an inhibition of thyroidal activïty and is not specinc to KC104.

3. To ensure that KC104 acts directly on the larval endostyle to inhibit thyroid

hormone synthesis and thus induce metarnorphosis. By developing an understanding of the role of Iowered senun TH concentrations in goitrogen-induced metamorphosis and the mechankm through which goitrogens induce metamorphosis, 1feel that our knowledge and understanding about the role of TH in spontaneous lamprey metamorphosis can be greatiy enhanced. RESEARCH CHAPTERS CHAPTER ONE

THE EFFECTS OF EXOGENOUS THYROXINE (T4)OR

TRIIODOTHYRONINE (T3), IN TEIE PRESENCE AND ABSENCE OF

POTASSIUM PERCHLORATE, ON TEE INCIDENCE OF

METAMORPHOSIS AND ON SERUM 4AND T:,CONCENTRATIONS

IN LARVAL SEA LAMPREYS (Petromyzon marinus L.).

The majonty of information presented in this chapter has been modified fkorn:

"The effects of exogenous thyroxine (T4)or tniodothyronine (T3),in the presence and

absence of potassium perchlorate, on the incidence of metamorphosis and on serum

T.4 and T3concentrations in larval sea lampreys (Petromymn marinus L,)" by R.G-

Manzon and J.H. Youson, in General and Comparative Endocrinology, Volume 106,

21 1-220, copyright O 1997 by Academic Press, repnnted with permission from the

publisher. ABSTRACT

Larval sea Iampreys (Petromyzon mari.nus) measunng 100 - 119 mm in length were

exposed to either thyroxine (T4; 10 mgfiter) or 3,5,3' tniodothyronine c3;1 mgniter), in

the presence and absence of the goitrogen potassium perchlorate (RCL04; 0.01 %), for 4 - 24

weeks. Every four weeks, treated and untreated (control) groups of sea lampreys were

examined for extemal signs of metamorphosis and serum was assayed for T4and T3

concentrations. Precocious metamorphosis was observed foIlowing 8, 12, and 24 weeks of

KCl04 treatrnent; however, metamorphosis was not observed in any controI, or T4-, T3-, T4 +

KC104-, and T3 + KC104-treated larvae. In addition, serum T4 and T3 concentrations were 62

% and 72 % lower in KCI04-treated individuais than in control animds, respectively.

Treatment with exogenous thyroid hormones (TH), in the presence or absence of KCQ

resulted in semm T4 concentrations which were significantly greater (1.2 - 58 foId) than

those of the controls in a11 sampLing periods except one, but senun T3 concentrations were

not signif~cantlyelevated in more than 50 % of the cases. TH + KClo4 treatments produced

semm T3 concentrations which were significanly greater than those of KC104-treated

animals and never less than those of the controls. These data indicate that Iarval sea

lampreys have a tremendous capacity to take-up and store exogenous T4 in their serum, but

the uptake andor semm storage of T3 appeacs to be stringently regulated. Also, the absence of both metamorphosis and a decline in serurn TH concentrations in TH + KC104-treated animals suggests that a decline in serum TH concentrations may be an essentid factor contributing to the induction of metamorphosis by KCL04. INTRODUCTION

Amphibian metamorphosis has been extensively studied, and it is cIear that thyroid

hormones (TH) play a very important stimulatory role in the initiation of this process.

Tadpoles exposed to exogenous TH undergo precocious metamorphosis (for review see

Etkin, 1964, 1968; Gorbman, 1964; Dent, 1968; Kaltenbach, 1968)- These observations

reflect the observed changes in serum TH concentrations during the life cycle of amphibians.

Thyroxine (T4)and 3,5,3'-triiodothyronine f13) are first deteceable just prior to the onset of

prometamorphosis, increase rapidly, peak during metamorphic climax, and then decline

rapidly (Regard et aL, 1978). Changes in TH concentrations during metamorphosis of the

flounder and conger eel foliow a simiIar pattern to those observed in amphibians (Miwa et al., 1988; Tagawa et al., 1990; Yamano et aï., 1991a), and metamorphosis can be induced

with exogenous Tqand/or T3 treatmeots @nuiand Miwa, 1985; Miwa and Inui, 1987). The observed increase in senun TH concentrations of tadpoles and larval teleosts is essential for metamorphosis. The fact that tadpoles and larval teleosts fail to metamorphose following thyroidectomy or treatrnent with antithyroid agents (goitrogens) supports this belief (Aiien,

1929; Weber, 1967; Miwa and Inui, 1987).

The mechanisms that control lamprey metamorphosis have not been studied in great depth, and to date the roles of TH in this developmental event are unclear. Serum TH are detectable early in the larval Iife of sea lampreys (Pefromyzonmarinus), increase gradually throughout the 3-7 year land period, and peak just prior to the htextemal signs of metamorphosis (Wright and Youson, 1977; Lintlop and Youson, 1983; Youson et al., 1994).

Coincident with the first external metamorphic changes in aîi lamprey species studied, is a rapid decline in serum TH concentrations which never remto the high levels observed in larvae (Wright and Youson, 1977; ~intlopand Youson, 1983; LeatherIand et al., 1990;

Youson et al., 1994)-

Several attempts have been made to induce precocious metamorphosis in larval lampreys using goitrogens, but the success rates have been variable, Hoheisel and Sterba

(1963) were the fist to attempt an induction of metamorphosis in Lampetra planeri using potassium perchiorate (K(3104) and an incomplete metamorphosis was Observed,

Conversely, Suzuki (1986, 1987, 1989) claimed that a complete metamorphosis in Lampetra reissneri occurred foiiowing treatment with various goitrogens (KC104, propylthiouracil

CPTU], or thiouracii). A significant decline in serum T4 and T3concentrations was observed by Leatherland et al. (1990) during an unsuccessful attempt to induce metamorphosis in

Geotria austrais with PTU. KCL04 successfully induced precocious metamorphosis in larval sea lampreys fiom 3 size groups at a tirne of year when spontaneous metamorphosis does not occur, but metamorphosis did not proceed dong the normal developmental path leading to fûnctional juveniies (Holmes and Youson, 1993). In addition, KC104 treatment significantly depressed serum T3 concentrations in ail 3 size groups, and serum T4 concentrations in all but the largest size group (Youson et al., 1995).

In these earlier studies, it was not clear if the reduced serum TH concentrations in

KC104-treated larval sea lampreys were a consequence of the treatment and subsequently involved in the initiation of metamorphosis, if it was a consequence of KC104-induced metamorphosis, or if ie was independent of KC104-induced metamorphosis. In an attempt to understand the relationship between the observed deciine in serurn TK concentrations and

KCI04-induced metamorphosis, the present study was designed to determine the effects of exogenous TH, in the presence and absence of KCl04, on both semm TH concentrations and the incidence of KC104-induced metamorphosis of Iarval sea lampreys. The assumptions

were that larvae can take up exogenous TH and store it in their blood and that the

maintenance of high levels of serum TH will prevent the metamorphosis of KCL04-treated

Iarvae.

MATERIALS AND METHODS

AnidcolZection and mainfenance

Larval sea Iampreys (Petromyzon marinus) were collected fkom Fish Creek, NY

from Septernber 8 - 10 by electrofishing and transferred to a large aquarium with fiowing,

dechlorinated, areate tap water in KHI Youson's laboratory at the University of Toronto at

Scarborough. Ten lanral sea lampreys, LOO - 119 mm in length and 1.3 - 2.7 g in weight,

were selected from a plof 700 animals and randomly assigned to each of 42 aquaria at

room temperature (15 - 20 OC). Larval sea lampreys were housed in static (i.e.. without a

flow-through water supply), well-aerated 21 liter glass aquaria (40 1 X 20 h X 25 w cm) EUed

with 12 liters of dechlorinated tap water. Each aquarium was provided with 6 - 8 cm of clean

industrial sand for substrate and maintained on a photoperiod of 15 L :9 D. Aquarium water temperature was monitored twice daily. Mean water temperatures for the various aquaria varied fiom 17 - 19 OC,and the maximum and minimum temperatures recorded were 20 and

15 OC,respectively. Animals were fed a suspension of baker's yeast @leishmann's) once a week, equalling 1 g of yeast/anirnaUweek. Aquaria were cleaned, the water changed and fiesh treatments (see below) were added every 2 weeks. Sand was replaced every four to eight weeks. The animals were acclimated to this environment for 3 weeks pnor to treatrnent. The 42 aquaria were randomly divided into 7 experimental groups (6 treatments and 1control) containing 6 aquaria each, and each aquarium in a particular group was randornly assigned a sampling order 1through 6. Subsequently, each of the 7 experhental groups was assigned a treatment at random (see Table 1for treatrnents and concentrations).

Treahnent regime

Potassium perchlorate (KC104; Aldrich, Milwaukee, WI, USA) was added to aquaria from a stock solution (0.24 %; 17.28 mM) to achieve a final aquarium concentration ofO.01

% (0.72 mM). L-thyroxine (74; Sigma, St. Louis, MO, USA) was administered to the appropriate aquarium as a cloudy solution containing 120 mg of T4in 500 ml of dechlorinated water to achieve a nominal ambient concentration of 10 mghiter (1 1.2 pM). A fresh 500 ml solution of T4 (240 mgfiter) was prepared for each aquarium. Twenty miIliliters of a 3,5, 3' L-Eliiodothyronine p3;Sigma, St. Louis, MO, USA) stock solution (60 mg T3 dissolved in 100 ml of 0.1 M NaOH) was used to create a nominal ambient T3concentration of 1 mg/liter (1 -48 CIM). The T3 stock solution was made using 0.1 M NaOH due to the low solubility of 4 in water. To control for the effects of NaOH in the T3treatment groups, I used an NaOH control group which received 20 ml of a 0.1 M NaOH stock solution for a

Final concentration of 0.17 mM. The addition of NaOH did not alter the pH of the water relative to other experimental groups.

Treatment concentrations used in this study were based on the reports of previous investigators working on lampreys, teleosts, and amphibians. Holmes and Youson (1993) observed no si-cant difCerence in the incidence of metamorphosis beîween larvae treated with 0.01% and 0.05% KC104. K used a KC104 treatment concentration of 0.01 % because it was the lowest KC104 dose shown to induce metamorphosis in sea lampreys. A T3treatment concentration of 1 mg/liter was used because Leatherland et al. (1990) had previousiy shown that this concentration was effective at elevating semm T3 concentrations in larval G. australis. 1used a T4 treatment concentration wfilch was 10 fold greater than the T3 treatment concentration because exogenous T3 is a more potent stimuiator of metamorphic processes than T4. Exogenous T3was severai times more potent at inducing morphogenesis and settling in the Iapanese flounder, Paralichthys olivaceus, than Tq (Miwa and Inui, 1987).

In X. laevis, tadpoIe tail tissue is up to ten times more sensitive to T3than T4 (Robinson et al., 1977)-

Experimental sampling, serum collection, and radioimntunoussay (RIA)

One aquarium from each experhental group was sampled every four weeks for twenty-four weeks, beginning in October. The sampüng process involved anaesthetizing animais in 0.05 % triche methanesulfonate (MS-222, SyndeI Laboratories Ltd., Vancouver,

BC.), rneasuring lengths and weights, exarnining for external signs of metamorphosis according to Youson and Potter (1979), collecting blood by caudal severance, and sacrificing animals by decapitation. BIood was collected in heparinized haematocrit tubes (Fisher

Scientific, Unionville, ON, Canada), stored overnight at 4 OC (to allow for clotting), and centrifuged the next mornïng at 7000 g for 3-5 minutes. Senun was collected and stored at -

70 OC for future analysis.

Total senun T4 and T3 concentrations were measured using different Amersham

Amerlex RIA kits (Johnson and Johnson, Markham, ON) modified for serum volumes and concentrations of larval lampreys (Leatherland et al., 1990). AU samples were assayed in duplicate or triplicate, and T4 and T3 concentrations were measured for each sample. Semm samples from more then one animal were pledin some cases due to insufficient volumes. Inter- and intra-assay variance was c 12 9b for both assays, and assay sensîtivïties were 8.0 and 0.24 nmoVliter for T4 and T3,respectively. The Tqantisem used in the RIA'S has a cross reactivity of 1.5 % with 4,and the 4 antiserum had a cross reactivity of 0.3 % with

T4-

Data analysik

AU experimentai data was tested for homoscedasticity (Le., heterogeneity of variance) using the Cochran's Q test before analysis of variance (ANOVA) was performed.

Cochran's Q was calcuIated using Statistix for DOS (Andyticai Software)- Length and weight data met this assumption of ANOVA, and were subsequently andyzed for significant differences using a one-way ANOVA and Tukey-Kramer's pst-hoc multiple cornparison tests on Statistix for DOS and SuperAnova for the Macintosh (Gagnon, 1990). Hormone concentration data were transformed (logio) to meet the assumption of homoscedasticity

(Sokal and Rom, 198 1) pnor to statistical analysis with ANOVA and Tukey-Kramer's post- hoc multiple comparison tests. Ail one-way ANOVAs for hormone data were perfomed on logLotransformed data using Statistix for DOS and SuperAnova for the Macintosh- Means are presented as r 2SE (2 standard errors), and were accepted as signincantly different if P I

0.05.

RESULTS

Animal size

No significant differences in mean animal size (length and weight) were observed between sarnpiing periods within an experimental group at the onset of the study. Based on this finding, the length and weight data for aii sampling periods within an experimental group were pooled and examined for ciifferences between groups. No significant ciifferences in mean animal size were observed at the onset of the study. However, there were several simcant ciifferences in animal sUe between expenmentai groups at the thne of sampling.

Significant ciifferences in mean size were not observed between experimentd groups at the 4-week sampling period, but control larval sea lampreys fiom the 8-week sampling period (104.6 mm) were significantly shorter than animals in NaOH control- (1 L3.3 mm), T3-

(1 13 -3 mm), and T3+ KClo4- (1 13.5 mm) treatment groups (Fig. 3A). The weights of

KCL04-treated animals (1.46 g) in the 12-week sampling peGod were signifcantly lower than those of T4-treated larvae (2.01 g; Fig. 3B), and KCl04-treated animals (1.37 g) were signincantly Lighter than both WHcontrol(l.97 g ) and T3-treated larvae at the 16-week sampling (1.86 g; Fig. 3B). Sizes were signifïcantly different at the 20-week samphg period. Control larval sea lampreys (100.5 mm, 1.36 g) were shorter and lighter than T4- treated animais (109.0 mm, 1-85g), and Iighter than Iarvae treated with T4 + KC104 (1.88 g;

Figs, 3A and 3B). Potassium perchlorate-treated animais (1-41 g) were lighter than Te treated individuais (1.70 g; Fig. 3B). The KClO4-treated Iampreys (1.12g) of the 24-week sampling were lighter than T3-exposed larvae (1.70 g; Fig. 3B).

Metamorphosis

Precocious metamorphosis was observed in animais treated with KC104 alone, but no t in any other treatment or controL group. The first externat metamorphic changes in

KC104-treated larval sea lampreys were observed following 8 weeks of treatment (Fig. 4), at which time one sea lamprey was at stage 1of metamorphosis. By the 12th week of KC104 treatment (Fig. 4), 3/10 sea lampreys were at stage 1 of metamorphosis. Metamorphosis was not observed at the 16- or 20-week sampling periods; however, 6/10 Iarvae were clearly

undergoing metamorphosis foiiowing 24 weeks of treatment (Fig. 4).

The most developed metamorphosing sea lamprey was designated as stage 5 because

of its large eyes, visible pupil and Es, and somewhat oval branchiopores- However, the ord

hood was small and not fulIy fuseci, and other oral components (Le., teeth and piston) had not

developed; therefore, development was asynchronous relative to what is observed during

spontmeous metamorphosis. Of the remaining five metamorphosing individuals in this

sampling group, two were at stage 1, and one at each of stages 2,4, and 5 of metamorphosis.

Serurn T4 and T3concenîrafrbns

No significant ciifferences were seen in either serum Tq or T3concentrations when metamorphosing and non-metamorphosing animals withh a treatment were compared; thus the data were combined for aIl fuaher analyses. In addition, the semm TH concentrations of untreated control and NaOH control sea lampreys did not merfor all samplings. Thus, the use of the word control for the remainder of this paper refers to the combined untreated control and NaOH control serum TH data.

Sea lampreys in three of the KCI04-treatment groups undenvent precocious metamorphosis. Moreover, KClO4-treated sea lampreys maintained mean semm T4 (16 - 32 nmol/liter) and T3 (2 - 13 nmolfliter) concentrations that were significantly lower than ail other expenmental groups including the controls (40 - 60 molfiter and 21 - 29 nmoVLiter for T4 and T3, respectively) for each of the 6 sampling periods (Figs. 5 and 6). SemT4 and

T3concentrations of KC104-treated sea lamprey were 62 % and 72 % lower than controls, respectively. Serum T4 concentrations for T4, T3, T4 + KClO4, and T3 + KC104 groups (750 -

272 1,93 - 168,689 - 2054, and 78 - 203 mnoüliter, respectively) were significantly elevated relative to control concentrations for each sampling period with one exception (Fig. 5).

Serum Ta concentrations of the T3+ KCI04 group (70 nmollliter) were not significantly greater than those of control larvae (60 nmol/liter) at the 8-week sampling period. The serum

T4 concentrations of larvae exposed to T4*T3, T4 + KCI04, and T3 .+ KCI04 treatments represent increases ranging fiom 13 - 58, 1.8 - 3.6, L2 - 42, and 1.2 - 4.3 fold greater, respectively, than control concentrations- In addition, sea larnpreys treated with T4 or T4 +

KC104 had senun T4concentrations that were significantiy greater than control, T3,and T3+

KC104 groups for all sampling pends (Fig. 5)-

Severai other signincant differences in semm concentrations were observed. At the 12- and 20-week sampting periods, serum T4 concentrations of T3-treated animals (13 L nrnolkter and 155 nmoMïter, respectively) were significantly greater than those of the T3+

KC104-treated individuals (85 nmoYliter and 9 2 nmoVliter, respectively; Fig. 5). Animds treated with T4 + KCIOQhad serum levels (1063 nrnol/liter) which were significantly greater than those treated with only T4(750 nmolAiter) at the 12-week sampling period. Lampreys treated with only T4 had semm concentrations (2532 nmoVLiter) which were significantly greater than those treated with T4 + KCI04 (2054 nrnoVLiter) at the 20-week sampling period (Fig. 5).

The pattern of change in senun T3 concentrations following either T4 or T3treatment was more variable in cornparison to what was observed for senim T4 concentrations.

Exposure to T4,T3, Tq + KC104, or Ts+ KC104 elevated serum T3concentrations to levels that were significantiy greater than controls in some sarnpling periods, but not others (Fig. 6).

What follows is a description of the observed differences in serum T3concentrations between the various treatments and controls which appeared to follow a trend. Serum T3 concentrations of larvae exposed to exogenous Tq(37 - 43 nmol/liter) were significantly elevated relative to controls (21 - 29 nmoVLiter) for all sampling periods, except the 16- and 20-week samplings (24 and 30 nmollliter, respectively; Fig. 6). In contrast, following exogenous T3 treatment, senun T3 concentrations were significantly elevated (52 and 42 nmol/liter, respectively) relative to controIs (21 and 29 nmovliterrespectively; Fig, 6) only at the 16- and 20-week sampIing penods. Exposure to exogenous T4 + KC104 for 12- or

24-weeks resulted in serum T3concentrations (42 and 38 nmol/liter, respectively) which were significantly greater than controls (21 and 21 nmoVliter respectively; Fig. 6). Serum T3 concentrations in the T3 + KC104 treatment groups of the 4, 16-, and 20-week sampling penods (38,59,and 3 1 nmoYliter, respectively) were significantiy greater than their respective controis (27,28, and 21 mnoVliter, Fig. 6).

Generally, serum T4 and T3concentrations within an experimental group were not affected by treatment length; however, a few sporadic significant differences were observed within the KCI04 treatment group between samplings. Senun T4 and T3concentrations of larvae exposed to KC104 for 24 weeks were significantly lower than those fiom al1 other sampling periods (Fig. 5). Semm T4 concentrations for the 16- week sampling pend were significantly greater chan all but those of the 20-week sampling periods, and serum T3 concentrations for the 4- and 16-week samplings were significantly greater than all other sampling periods (Figs. 5 and 6)

DISCUSSION

At the onset of this study, significant differences in size (length and weight) were not observed between experimental groups, and al1 animals were 100 - 119 mm in length and 1.3

- 2.7 g in weight. The animals used in this investigation did not meet the minimum size requirement of presumptive metamorphosis (120 mm and 3.0 g) that has been determined for

landlocked sea lampreys (Youson et ai., 1993; Hoimes and Youson, 1994; Holmes et al.,

1994). The absence of differences in size between groups at the begianing of the study

suggests that any observed differences in the incidence of metamorphosis were noe due to

differences in size- Furthemore, the fact that control lamal sea Iampreys did not

metamorphose cm be accounted for by both the size of the mimals used and the time of year

the study was conducted (October - March); spontaneous metamorphosis is not initiated untiI

July (Potter, 1980)-

As described in the Results section, several significant differences in size were

observed between various experimental groups at the time of sampling. These differences

appeared to be random and did not follow any consistent pattern; thus, it is rny conclusion

that they did not affect the incidence of metamorphosis and are probably not due to the

effects of the assigned treatments. These differences in size may be related to laboratory

conditions, food availability or food nutritive value, However, this study was not designed to

control for, or examine, the effects of the aforementioned parameters and the various

treatments on size; therefore, a conclusive statement cannot be made.

Previous studies using year class III larvae of P. rnat5w.s (110 - 125 mm in length)

reported serum T4 concentrations of approximately 69 nmoyliter and senun T3

concentrations of 22 - 32 nmol/liter (Youson et al., 1994, 1995). The serum T3 concentrations in the control larvae of the present study were similar (21 - 29 nmomiter) to

those of the previous studies, but serum T4 concentrations were slightly lower (4Q - 60

nmoVliter). These discrepancies may be due to either the larger animals used by Youson et al. (1994, 1995). diierences between populations, or minor variations in assay methods between laboratories.

Although metamorphosis was not observed in control animals, it was observed in larvae exposed to KC104 for 4, 12-, and 24-weeks. This induced metamorphosis occurred at a time of year when spontaneous metamorphosis does not occur, and in animais that did not meet the size requirements of presumptive metamorphosis. Excluding for the moment the absence of metamorphosis in the 16- and 20-week sampling periods, the incidence of metamorphosis and the degree of development increased with the length of treatment (Fig.

4). However, metamorphosis did not reach completion, despite the fact that the treatment length (24 weeks) was longer than the time necessaty for the completion of spontaneous metamorphosis (12 - 16 weeks; Potter er al., 1978; Youson and Potter, 1979). The asynchronous metamorphic changes observed in thïs study were consistent with those described by HoImes and Youson (1993).

Holmes and Youson (1993) observed 22 and 55 % metamorphosis in larval sea lampreys 69 - 95 mm and 110 - 119 mm in length, respectively, following 117 days (16.7 weeks) of KC104 treatment. In the present study on 100 - 119 mm larvae, a 30 and 60 % incidence of KC104-induced metamorphosis was obsewed foilowing 12 and 24 weeks of treatment, respectively. Accounting for the variation in size groupings and treatment Iength between the two studies, these results appear consistent with those reported by Holmes and

Youson (1993).

It is unclear why metarnorphosis was not observed in the 16- and 20-week KC104- treatment groups, but examination of the main factors implicated in the metamorphosis of sea larnpreys, namely temperature, size, and serum TH concentrations (Holmes and Youson, 1993; Youson et al,, 1993; Holmes and Youson, 1994; Holmes et al., 1994; Youson, 1994),

revealed that differences in size or temperature were not involved, conclusion is based

on the fact that am-mals in the 16- and 20-week sampling periods were neither the smailest

KCI04-treated sea lampreys, nor were they exposed to different temperatures In addition,

the differences in serum TH concentrations between sampling perïods within the KCL04

treatrnent group were probably not invoived, since they were significantly lower than controls in all cases. However, it should be noted that the animals in these 2 sampIing perïods did appear to deviate fiom the other samplings with respect to serum T3 concentrations in the TH treatment groups, as weU as semm T4and T3concentrations in the

KCL04 treatment group.

Exposure of Iampreys to antithyroid agents such as ITU or KC104 has resulted in significant declines in serum TH concentrations and, in the case of KCL04, the induction of metamorphosis (Hoheisel and Sterba, 1963; SuzuIci, 1986, 1987, 1989; Leatherland et al,,

1990; Kolmes and Youson, 1993; Youson et al,, 1995)- The 62 % decfine in serum T4 concentrations of Iarvae exposed to KClo4 (Fig. 5) is close to the 72 % decline observed during spontaneous metamorphosis (Youson et al., 1994). However, a decline of only 27 -

3 1 % was reported by Youson et al. (1995) for sea lampreys of a similar Iength to the ones used in this study (1 10 - 119 mm). Conversely, the 72 % decline in serum T3concentration

(Fig. 6) was not as great as the 91 - 95 % decline reported by Youson et al. (1995) foilowing

KC104 treatment. This latter value is similar to the decline observed during spontaneous metamorphosis (Lintlop and Youson, 1983; Youson et al., 1994)- The size or condition of the animals, the populations fiom which the animals were obtainecl., or the temperatures at which the studies were conducted may have contributed to the variable results reported in these different experiments. For instance, the study conducted by Youson et al. (1995)

maintained aquarium water temperatures between 10 and 14 OC,while temperatures in this

study ranged from 15 - 20 OC. There is evidence that the senun Tq and T3concentrations of

lampreys are affected by temperature (Wright and Youson, 1977; Lintiop and Youson, 1983;

Youson et al., 1994)-

In accordance with severai earlier studies which were unsuccessful at inducing

lamprey metamorphosis using thyroid gIand stimdation or exogenous thyroid hormone

supplernents (for review see Youson, 1980, 1988,1994), neither exogenous T4 nor T3

initiated metamorphosis during the present study. The absence of metamorphosis following

exogenous TH treatment was not due to an inabiIity to absorb the honnone nom the water;

this is evidenced by the significantly elevated senun TH concentrations following exogenous

TH treatment, even in the presence of KClo4.

The serurn T4 concentrations of animals treated with Tq,T3, T4 + KCI04, OC T3 +

KCL04, demonstrate that sea lamprey larvae have a tremendous capacity to take-up and store this honnone and to maintain elevated serum T4concentrations. Serum T4 concentrations of

TH-treated anirnals were increased up to 58 fold (>2000 nmoIfiiter) greater than control values. Treatment with exogenous T4 Ga ingestion or intraperitoned injection has shown that teleosts are also capable of taking-up and storing Te(Blaschuk et al., 1982; Sweeting and Eales, 1992; Kuhn et aL, 1993). in one study conducted by Fok and Eales (1984), rainbow trout (Oncorhynchus mykiss) maintained semm T4concentrations 5 - 54 and 300 -

LOO0 times greater than control concentrations following injections of 9.4 - 69 and 330 -

1440 nglg body weight, respectively. Serum T3 concentrations were elevated significantly following -ment with T4,T3,

T4 + KCIOq or T3+ KClO4 in some sampiing periods, but not others, These results are sornewhat different fiom those of a study by Leattherland et ai. (1990) on G. australis where serum T3 concentrations of animals exposed to exogenous T3 were more than double the control concentrations, This variabfe response may reflect a variation between species, or may be a consequence of the smaller sample size (eight) used by LeatherIand et al. (1990),

Semm T3 concentrations of teleosts can also be significantly elevated following exogenous

T3 treatment (Sweeting and Eales, 1992; MacLatchy and Eaies, 1993). Can semm T3 concentrations in 1ma.I sea lampreys be elevated in a similar fashion to senun T4 concentrations? The mechanism for regulating serum T3concentrations in lampreys and preventing their increase in the presence of exogenous TH presents an interesting area for future research.

The increase in serum T4 concentrations but not senim T3concentrations following exogenous TH treatments suggests that serum T3 concentrations in larvai sea lampreys are regulated more stringently than semm T4 concentrations. Regulatory mechanisms could include a decrease in 5' (outer-ring) deiodination (ORD) of T4 to T3,OC increases in T3 degradative pathways and excretion rates (Eales, 1985; Kuhn et al,, 1993)- This finding concurs with results reported for teleosts (Blaschuk et al., 1982; Fok and Eales, 1984) and other vertebrates (Kuhn et al., 1993) exposed to exogenous Tq,in which significant elevations in serum T4concentrations were observed without any change in serum T3 concentrations. In the study conducted by Fok and Eales (1984), T4 injections as high as 69 ng/g body weight resulted in semm Tq elevations as high as 54 fold greater than controls, but did not alter serum T3concentrations. Injections of 330 and 1440 ng/g body weight resulted in semm T4 concentrations approximately 300 and 1000 tïmes control values but resulted in

only a 14 and 132 fold increase in serum T3concentrations, respectively (Fok and Eales,

1984). A similar phenornenon was observecl in mammdian studies; serum T4concentrations

of individuals exposed to exogenous & continued to rise, but semm T3 concentrations

plateau within the normal physiological range (Refetoff and Nicoloff, 1995). The apparent

stnngent regulation of serum T3 concentrations in lard sea lampreys may be largely a result

of its penpherd regulation as in other vertebrates (Eaies, 1985; Kuhn et al, 19931, and

agrees with the observations that T3 is the more biologically active TH (Jameson and

DeGroot, 1995).

The consistent elevation of serum T4 concentrations foiiowing exogenous Tg

treatment, even in the absence of an elevation of serwn T3 concentrations, suggests that in

larval sea lampreys serurn T4 acts as a source of T3, when it is required. This feature is also

seen in teleosts and other vertebrates where Tj is largely produced extrathyroidally fkom the

peripheral ORD of T4 (Eales, 1977, 1985; Kuhn et al., 1993; Refetoff and Nicoloff, 1995).

Perhaps exposure to exogenous T3in the present study acts to inhibit the ORD of &, thus

preventing both T3 production and T4 degradation, and creating a subsequent increase in

semm concentrations of T4, This view is based on the fact that decreases in T4 ORD have

been observed in teleosts following exposure to exogenous T4 and T3 (MacLatchy and Edes,

1993). However, this may not be the ody factor involved in the observed increase in

lamprey serum T4 concentrations following exogenous T3 exposure, since serum T4

concentrations also increase in response to T3 in the presence of KC104.

LarvaI sea lampreys treated with a TH and KClO4 maintained serum T4 and T3 concentrations greater than or equd to control animds, and significantly greater than KCiO4- treated animais, in al1 sampling periods. This result indicates that exogenous TH can compensate for the endogenous TH removed fkom the serum, presumably as a resuIt of

KC104 treatment. This compensation is correlated with the absence of metamorphosis and implies that KC104-induced metamorphosis can be blocked by administration of TH.

However, the concentrations of TH used were probably greater than those necessary to simpl y counteract the effects of KcIo4. Moreover, the incidence of induced metamorphosis was Iow in the smaller animais used in the present study- Future investigations should use

Iarger larvae to increase the incidence of KC104-induced metamorphosis (Hoimes and

Youson, 1993), and lower dosages of hormone to confirm whether or not exogenous TH can block KC104-induced metamorphosis.

In the present study I have shown that Iarvd sea iampreys have a tremendous capacity to take-up exogenous Tqin the presence and absence of KCI04, and maintain elevated semm Tqconcentrations over a 24-week period, whereas the limits of senun T3 concentrations appear to be stringently regulated, possibly via the controlled deiodination of

T4. In addition, the absence of metamorphosis in TH + KC104-treated larvae indicates that perhaps KC104-induced metamorphosis can be blocked by exogenous TH. There was no decline in serum T4 or T3concentrations of animais exposed to TH + KC104. This result suggests that the deciine in senun TH concentrations associated with KC104 treatment alone is in part involved in the inducement of metamorphosis by this goitrogen. TabIe 1, Experimental groups and nominal ambient aquarium concentrations to which

larval sea lampreys (Petromyzon us) were exposed for 4 - 24 weeks.

Experimental Groups Nominal ambient concentration

Conîrol

NaOH Control 0-17mM

Potassium perchlorate (KCLO4) 0.0 1 96 (0-72mM)

Triiodothyronine (T3) 1 mmter (1.48 CLM) + 0.17 rnM NaOH

T3 + KC104 1-48 pM T3 + 0.72 rnM KC104 + 0-17mM NaOH

Thyroxine (T4) 10 mg/liter (1 1.2 CLM)

T4 + KC104 11.2 pibf T4 + 0.72 mM KC104 Figure 3. Mean (k 2SE) lengths (A) and weights (B) of sea lamprey (Petromyzon marinus)

Iarvae exposed to various treatments for 4 - 24 weeks. Values which are significantly different (P < 0.05) fiom control (*) and KC104-treated (+) Iampreys within a sarnpling week are indicated- T4= thyroxine (10 mgIliter), Tj = triiodothyronine (1 mghter), KCL04 = potassium perchlorate (0.0 1 %) 4 8 12 16 20 24 Weeks of treament Figure 4. Number of metamorphosing larval sea lampreys (Petromyzon marinus) foIIowing exposure to potassium perchlorate (KC104; 0.01 46) for 4 - 24 weeks. AU lampreys were classifïed as being in stage 1 of metamorphosis, with the exception of animals in the final sampling period Following 24 weeks ofKC104 treatment I observed two stage 1, one stage

2, one stage 4, and two stage 5 metamorphosing sea lampreys. Sample size is ten for all treatment lengths, 4 8 12 16 20 24

Treatment length (weeks) Figure 5, Cornparison of the mean (k 2SE) serum thyroxine (T4) concentrations of sea larnprey (Petrumyzon mannus) Iarvae exposed to either Tq(10 mgIliter) or triïodothyronine

(T3; 1 mg/liter), in the presence or absence of potassium perchiorate (KCL04; 0.01 %), for 4 to 24 weeks. Semm T4concentrations for all five treatrnent groups were signincantly different (P < 0.05) than control concentrations within each of the sampling weeks, unless indicated otherwise (*), Note the scale on the Y-axis is intempted between 250 nmoi/liter and 500 nmoVLiter. The control represents data pooled fkom untreated control and NaOH- treated animals, Weeks of treatment Figure 6. Cornpan-son of the mean (k 2SE) serum triiodothyronine m3)concentrations of sea lamprey (Perromyzon mannus) larvae exposed to either thyroxine ('&; 10 mmter) or T3 (1 mg/liter), in the presence or absence of potassium perchlorate &CIO4; O.Ol%), for 4 to 24 weeks. Values which are significantly different (P c 0.05) frorn either control (*) or ail groups (+) within a sarnpling week are indicated. The control represents data pooled fiom untreated and NaOH-treated animals. Weeks of treatment BLOCKING OF KC104-INDUCED METAMORPHOSIS IN

PREMl3TAMORPHIC SEA LAMPREYS BY EXOGENOUS THYR!OID

HORMONES (TH); EFFECTS OF KcLo4 AND TH ON SERUM TH

CONCENTRATIONS ANI) INTESTINAL THlYROXLNE OUTER-RING

DEIODINATION.

Acknowledgment:

Deiodinase assays were performed by Dr. J.G. Edes and Audrey Waytiuk.

The majority of information presented in this chapter has been modïfied fiom:

"Blocking of KC104-induced metamorphosis in premetamorphic sea lampreys by

exogenous thyroid hormones (TH); effects of KCL04 and TH on serum TH

concentrations and intestinal thyroxine outer-ring deiodination" by R.G. Manzon,

J.G. Eales, and J.H. Youson, in Generai and Comparative Endocrinology, Volume

112,54-62, copyright O 1997 by Academic Press, reprinted with permission from the

publisher. ABSTRACT

Irnmediately premetamorphic- Iarvai sea larnprey (Petrornyzon mannus) (1120 mm in length) were treated for 4,8 or 16 weeks with one of two concentrations of either exogenous thyroxine (T4;1 mgfiter or 0.5 mgater) or 3,5,3'-triiodothyronine m3;1 mgfiter or 0.25 mg/liter) in the presence or absence of the goitrogen potassium perchlorate (KC104; 0.05 %) as well as with KC1O4 done. Larvae from a11 treatments were examined for signs of metamorphosis, changes in semm T4 and T3concentrations (senun T4 and semm T3), and changes in intestinal Tq outer-ring (5') deiodination to T3 (T40RD). KC104 depressed both serum T4 and T3, and induced metamorphosis in 80 % of larvae treated for 8 weeks or longer.

However, neither effect was observed in larvae exposed to KC104 combined with either thyroid hormone (TH)- These data confirm previous suggestions that exogenous TH blocks

KC104-induced metamorphosis by elevating serum TH concentrations, and provide evidence that declines in serum TH concentrations are mandatory for precocious metamorphosis.

Serum T4, but not senun T3, was elevated following exogenous Tq treatment in the presence or absence of KC104. This maintenance of serum T3at control levels, in the presence of a T4 challenge was not due to decreases in intestinal T40RD activity, since T40RD activity was not affected by any treatments in the study. Exogenous T3 elevated both serum T4and T3.

However, serum T3in T3-treated larvae decreased with time, suggesting a strïngent T3 regulation. Elevation of senun Tqfollowing T3treatrnent may have ken a result of either inhibition of T4 metabolism, or stimulation of T4 secretion by the endostyle. Based on these results, 1conclude: i) exogenous TH blocks KC1O4-induced metamorphosis in sea larnpreys and ii) serum T3 is maintained at control levels despite elevations in serum T4, its immediate precursor, but this does not involve any changes in intestinal T40RD activity. Spontaneous metamorphosis in Iarval sea Iarnpreys (Petromyzon rnan'nus) is reIated

in part to several endogenous factors. Firstly, metarnorphosis depends on body length,

weight and condition factor [CF = (weight (g)/length (mm)3) X 10~1,which are in tum related

to the accumulation of lipids. Throughout the larval period (3 - 7 years), larvae grow

gradually until they attain metamorphic Iength (120 mm) (Potter, 1980; Youson et ut-, 1993)-

At this time they begin to accumulate Lipids rapidly for the pending nontrophic

metarnorphosis (Lowe et al., L973; 07Boyleand Beamish, 1977). The lipid content nses

fiom about 4 % wet body weight in larvae, to 14 % in immediately premetamorphic larvae

(Lowe et al., 1973; OTBoyleand Beamish, 1977). This event causes body weight to increase

without changing body length, and hence CF increases as weU, Thus, in the fd, sea lamprey

Larvae must be a minimum of 120 mm in Iength. 3.0 g in weight and have a CF of 1.45 or greater to metamorphose the foliowing July (Youson et al,, 1993; Holmes and Youson, 1994,

1997).

Secondly, metarnorphosis depends on semm thyroid hormone concentrations.

There is a gradua1 rise in thyroxine (T4) and 3,5,3'-tniodothyronine (T3)semm concentrations as the larvae grow (Youson et d,1994)- In sea lampreys, serum TH concentrations peak pnor to the onset of metamorphosis, and decline sharply at approximately the sarne time (July) that the first extemal changes are observed (Wright and

Youson, 1977; Lintlop and Youson, 1983; Youson et al., 1994). This contrasts with the changes in serum TH concentrations of other vertebrates which undergo a true metarnorphosis, where there is a rapid nse in serum TH concentrations duhg prometamorphosis followed by a peak and decline dunng metamorphic climax (Regard et al-,1978; GaIton, 1988; ïnui et ai-, 1994)- Furthermore, TH administration to anuran tadpoles and flounder larvae initiates metamorphosis, but T3administration to larvd sea lampreys inhibits spontaneous metamorphosis (Youson et al., 1997). Goitrogens, whic h inhibit TH biosynthesis, prevent spontaneous metamorphosis in teleosts and amphibians and produce overgrown larvae (Miwa and hui, 1987; Galton, 1988). Contrary to this, the goitrogen, potassium perchlorate (KCI04). induces precocious metarnorphosis in lampreys

(Hoheisel and Sterba, 1963; Suzuki, 1986; Holmes and Youson, L993), and depresses serum

TH concentrations relative to values for untreated Iarvae (Youson et al., 1995; Manzon and

Youson, 1997).

KC104-induced metamorphosis in sea Iampreys, like spontaneous metamorphosis, is size-dependent (Holmes and Youson, 1993). In this latter study, 22,52 and 98 % metamorphosis was observed in KC104-treated larvae measuring 65-95, 110- 119 and >130 mm in length, respectively. In a recent shidy, Manzon and Youson (1997) suggested that the decline in serum TW concentrations associated with KC104 treatment was essential for

KC104-induced metamorphosis. This suggestion was based on evidence of me tamorphosis and a significant decline in serum TH concentrations in larvae exposed to KC104, and on the absence of either effect in larvae exposed to both KC104 and exogenous T4 or T3.However, only 17 % of the lampreys (10 out of 60) underwent metamorphosis when exposed to KClO4 alone. This low incidence of metamorphosis may have been related to using intermediate sized larvae (100 - 119 mm in length). Whether a decline in serum TH concentrations is essential in KC104-induced metamorphosis requires confirmation using immediately premetamorphic larvae- In an earlier study. Manzon and Youson (1997) suggested that KCi04 andor TH treatment might alter the extrathyroidal regdation of serum TH concentrations, TH deiodinase activities have recently been measured in various tissues of the sea Iamprey

(Eales et al., 1997). In larvae, the intestine is the principal site of T4 outer-ring (5') deiodination WRD)to T3, with no T3outer-ring (3') deiodhation (T30R.D) to 3,s- diiodothyronine m),T4 imer-ring (5) deiodination (Tm)to 3,3',5'-triiodothyronine

(reverse T3), or T3 inner-ring (5) deiodination (T31RD) to 3,3'-diiodothyronine (T2).In contrast, adult Tmand T31RD activities were detected in addition to T40RD activity.

Since TH administration affects deiodination in teleosts @ok and Eaies, 1984; Eales and

Finnson, 1991), it is of interest to see how intestinal T4OR.D in larvae relates to metamorphic stage and to changes in serum TH concentrations following a TH chalienge and EX104 treatment.

Therefore, in this study 1 have determined for immediately premetamorphic sea lampreys: i) the efficacy of TH in suppressing KC104-induced metamorphosis, ii) the extent to which serum TH concentrations are regulated during these TH challenges and iii) any changes in intestinal T40RD activity occurring under the above conditions.

MATERIALS AND METHODS

Larval sea lampreys (2 120 mm) were colleçted in September from the Salmon River near KeeseviiIe, New York using backpack electrofishers. They were transported to the

University of Toronto at Scarborough, held in large fiberglass tanks with 6-7 cm of industrial sand for substrate, and provided with continuously flowing, dechlorinated city tap water.

Two weeks before the study, 30 glass aquaria (40 cm X 20 cm X 25 cm; 21 liter) containing

12 liters of static (i.e., without a flow-through water supply) aerated, dechlorinated tap water and 6 - 7 cm of industriai sand, were arranged in three adjacent banks of 10 aquaria each, and

were allowed to adjust to the ambient room conditions. Nine days before the onset of the

study (November 27), larvae were randody removed in groups of 5 nom a pool of 3 10

individuals, measured for length and weight, and randomly assigned to 1of the 30 experimental aquaria, until each aquarium contained 10 Iarvae. Aquarïa were randomly assigned to 1 of 10 experimental groups (9 treatments and a control) fiable 2)- Each experimental group consisted of 3 aquaria randomly assigned a sampling penod representing

4, 8 or 16 weeks of treatment, Larvae were acclimated to their new environment before the onset of the study.

The expenment was started on December 6 by adding the appropriate chernicals to the aquaria (Table 2)- KC104 (Aidrich, Milwaukee, WI, USA) was added from a 1 % stock solution to achieve a nominal ambient concentratioa of 0.05 % (3.6 mM). A KC104 treatment concentration of 0.05% was used in an attempt to ensure a higher incidence of metamorphosis than observed in Chapter I (Manzon and Youson, 1997) with 0.01 8 (0.72 mM). Nominal Tq (Sigma, St. Louis, MO, USA) ambient concentrations of 1 mg/liter (1.12 pMJ and 0.5 mgIliter (0.56 pM) were achieved by adding a 150 ml solution containing 12 mg or 6 mg, respectively, of T4 to the appropnate aquarïa. T3 (Sigma, StLouis, MO, USA) stock solutions of 0.89 rnM and 0.22 mM were used to obtain nominal ambient T3 aquarium concentrations of 1 mgniter (1.48 pM) and 0.25 mg/liter (0.37 pM), respectively. TH treatment concentrations were selected based on my experience during a previous study

(Manzon and Youson, 1997). T3 stock solutions were prepared using 0.1 M NaOH because of the low solubility of T3in water. Previous studies indicated that the 20 ml of0-1 M NaOH added to the aquarium water does not signifïcantiy affect the animals, or the water pH

(Manzon and Youson, 1997)-

Water temperature was measured twice daily in one randomly chosen aquarium per

bank of 10 and ranged nom 16 - 18 OC. Dechiorinated city tap water was added to the

aquaria regularly to maintain the voIume at 12 liters- Animals were fed a suspension of

baker's yeast (Heishmann's) once a week, equaIling approximately 1g of

yeast/anirnal/week- Every two weeks the aquaria were cleaned, the water was changed and

fiesh treatment was added without removing the animals. Sand was replaced every four to

eight weeks.

After 4,8 or 16 weeks, one aquarium fiom each experimental group was sampled.

Al1 animais in the aquarium were anaesthetized (0.05 % aicaine rnethanesulfonate; Syndel

Laboratories, Ltd., Vancouver, British Columbia, Canada) and assigned a stage of

metamorphosis (1, eadiest to 7, latest) based on externai charactenstics (Youson and Potter,

1979). Lengths and weights were measured, blood was coiiected by caudal severance and

the animals were decapitated. Various tissues were harvested, flash frozen on liquid nitrogen

and stored at -70 OC for T40RD assays and other studies. The blood was alIowed to clot

overnight at 4 O C, was centrifuged at 7000 g and the senim was stored at -70 OC.

Semm Tqand T3 concentrations (semm Tqand serum T3)were determined (in dupiicate) using the Amersham Amerlex TT4 and 'ïT3 radioimmunoassay (RIA) kits, respectively (Johnson and Johnson, Marktiam, Ontario, Canada). The RIA kits were modified for Iamprey serum volumes and TH concentrations (Leatherland et ai., 1990). The cross-reactivities of the T4antibody with T3and the T3antibody with T4 were 1.5 % and 0.3 %, respectiveIy. Assay sensitivities were determined to be 7 and 0.2 nmoUliter for T4 and T3, respectively; the inter- and intra-assay variances were S 14 %.

TORD activity of intestinal microsomal fraçtions was determined for lampreys fiom al1 treatments at 4 and 16 weeks according to Shields and Eaies (1986) using a T4 substrate concentration of 0-1 nM, Microsornes were prepared using entire intestines usually pooled from two individuais fiom the same aquarium- The GORD activity was expressed in two ways. Firstiy, it was calculated as fmols T4 deiodinated/hrfmg microsomal protein- Ail values were then norrnalized to a protein concentration of 0.3 mghi because of the dependence of intestinal T@RD speciflç activity on microsomal protein concentration, and the tendency of T40RD activity to increase at microsornai protein concentrations below 0.15 mghl (Eaies et ai-,1997). Secondly, T&RD activity was expressed as fhols T4 deiodinated/hr/incubation tube. Since dl incubation tubes were prepared with a standardized dilution of the entire intestinal microsomd fiaction, this provides an index of the T&RD activity for the entire intestine regardless of microsomal protein concentration, Microsornai protein content was altered by some of the treatments, and this could be independent of deiodinase function, Therefore, 1considered the latter method to be more representative of deiodinating activity since microsomal protein concentration was not a denominator.

Size, CF, serum TH concentrations and VRDactivity were examined statistically using analysis of variance (ANOVA), and Tukey-Kramer's post-hoc test- Al1 data were tested for homoscedasticity of variances prior to ANOVA analysis. Data which did not meet this assumption were transformed ( log,,) to minirnize heteroscedasticity (Sokal and Rohlf.

198 1). Al1 statisticai analyses were performed on Statistix for DOS. Means are presented as k 2 SE (standard errors), AI1 differences between means in the data considered below were statisticaliy significant (PS 0-05)-

Semm TH concentrations were compared either between sampling periods within an experimental group, or between experimental groups within a samphng period. Since there were no differences in serum TH concentrations between metamorphosing individuais and

Iarvae within any single aquarium, ail individuais withïn an aquarium were represented by a single mean, When testing for differences between experimental groups 1fiused on cornparisons between: i) the control group and each of the nine treatments, iï) the KC104 and

TH+KC104 treatments, iii) the two TH concentrations used and iv) each TH treatment and its complementary TH+KC104 treatment,

Site and incidence of mefamorphosis

Length (137 k 1.2 mm), weight (3.54 + 0.09 g) and CF (1.36 + 0.01) recorded at the onset of the study did not ciiffer between tanks or experimentai groups (either within a sampling period or for al1 sampling periods pooled), Metamorphosis was not observed in any control, TH-treated or TH+KClO4-treated Iarvae throughout the study. However, metamorphosis was observed in 80 % of the larvae exposed to KC104 for 8 and 16 weeks

(Fig. 7). Specifically, following 8 weeks of KC104 treatment 1observed two unrnetamorphosed larvae, one sea larnprey at stage 1, three between stages 1 and 2, and four at stage 2 of metamorphosis. Following 16 weeks of KC104 treatment, 1obsewed two unmetamorphosed larvae, one sea lamprey between stages 1 and 2, four sea lampreys at stage

3, and three sea lampreys at stage 4 of metamorphosis. The extemal characteristics used to assign the above metamorphic stages clearly indicate that metamorphosis is 0ccUmng

(Youson and Potter, 1979); however, KC104-induced metamorphosis was abnormalLy

synchronized relative to spontaneous metamorphosis. The eyes fiequently developed more

rapidly and in advance of the developing oral disc and branchiopores, as well as changes in

pigmentation, For this reason some individuds were cIassined as king between two

metamorphic stages,

Contparkon of semm THcortcenbatr%nsbetween sarqdiigperiocs within an experimental group

Changes in semm Tq were obsewed over time, but these were inconsistent across experimental groups (Fig. 8). In control larvae, senun Tq decreased with sampling time. It was greater at 4 weeks (1 11 nmollliter) than at 8 weeks (84 nmoVLiter), md both these mean values were greater than at 16 weeks (46 nrnoVIiter). For LT4 Iarvae, serum T4 was greater at

8 weeks (628 nmoVliter) than at either 4 weeks (346 nmoVLiter) or 16 weeks (360 nmoyliter) which did not differ fiom one another. On the other hand, serurn T4 of HT4 larvae was greater at 16 weeks (646 nmolfiter) than at 4 weeks (402 nmoVLiter), but did not ciiffer fiom that at 8 weeks (537 moYliter) (Fig. 8).

Differences in serum T4 were not observed over tirne within the LT3, HT3, KI04or

HT3+KC104treatments (Fig. 8). SemT4 increased over the in LTQ+KC104larvae (270,

3 87 and 583 nmoMiter for 4,8 and 16 weeks, respectively), but decreased over time in the

HT4+ KC104 larvae where senun T4 at 4 weeks (1575 nmoVLiter) was greater than semT4 at 8 weeks (719 nmomiter) and 16 weeks (545 nmoVliter). Serum T4of LT3+KCI04larvae at 16 weeks (42 nmoyliter) was Iower than at both 4 and 8 weeks (78 nmollliter and 62 nrnoMiter, respectively). Semm T3did not change over time within the control, LT4, Eïï4 or LT4+KCL04 treatments (Fig, 9). In response to various treatments with T3 there was a trend toward Lower serum T3 in Iarvae that were treated longer- In LT3 Iarvae, serum Tîat 8 weeks (76 nrnolfiter), but not at 16 weeks (86 nmoVLiter) was lower than at 4 weeks (128 nmoUIiter).

Serurn T3of larvae did not merbetween 4 weeks (129 nmol/liter) and 8 weeks (132 nmol/liter), but feU at 16 weeks (38 nmoVLiter). In Ka04 Iarvae, serum T3 at 4 weeks (6.6 nrnoyliter) was greater than at both 8 weeks (1.5 ~noVliter)and 16 weeks (1.3 nmoWter),

Semm T3 for LT3+KCI04 Larvae decreased with time (58,30 and 17 nmoYliter for 4,8 and

16 weeks, respectively). Treatment of larvae with HT3+KC104 for 4 weeks produced a mean serum T3 of 284 nmolfiter which exceeded that observed at both 8 (100 nmoVLiter) and 16

(164 nmoVLiter) weeks (Fig. 9).

Compatiron of serum TH concenfrafrbnsbetween experimenfal groups within a sampling period

A cornparison of senun TH concentrations between the 9 treatrnents and the control showed signincant effects. haeexposed to only KCL04 for 48or 16 weeks maintained serum T4 and T3levels that were lower than those of control larvae or larvae kom any other treatments (Figs. 8 and 9). At al1 sampling periods, semm T4 in larvae exposed to either concentration of exogenous T4,in the presence or absence of KC104, was elevated (2.5 - 14 fold) relative to controls. Exposing Iarvae to either exogenous LT3 or HT3 alone, elevated serum T4 at ail 3 sampling periods, with the exception of the HT3 treatment at 4 weeks (Fig.

8)-

Treatrnent with either concentration of T4 in the presence orabsence of KC104 did not affect serum T3(Fig. 9). Conversely, exogenous T3alone generally resulted in 3- to 5- fold elevations of serum T3, except at 16 weeks for HT3 Iarvae. Larvae treated with

LT3+KClO4 for 4 weeks had elevated serum T3, but concentrations at 8 and 16 weeks did not differ from control values- Treatment of Iarvae with w3+KC1o4 increased semm T3 above control values at ail three sampling periods (Fig. 9).

In the absence of KClO4, both the high and low TH treatments had similar effects on serum TH concentrations wîthin a samplïng penod (Figs. 8 and 9). However, when administered with KC104, exogenous TH had dose-dependent effects on senun TH concentrations. Larvae exposed to the HT4+KCIO4 treatment for 4 and 8 weeks (1575 nmoVliter and 7 f 9 nmoVLiter, respectively) had greater serum T4 than larvae exposed to

LT4+KC104 for 4 and 8 weeks (270 nrnoVIiter and 387 nmoI/liter, respectively) (Fig, 8).

However, differences in serum T4 were not observed between Iarvae treated with LT4+KCL04 and HTQ+KC104for 16 weeks. Serum T3 did not differ between the LT4+KC104 and

HT4+KCL04treatments for any samphg period (Fig. 9). Semm T4 and serum T3 in

HT3+KC104 larvae were greater than for LT3+KC104 larvae at ali three sampling periods

(Figs. 8 and 9).

Generally, the effects of exogenous T4 (at either concentration) on serum TE3 concentrations were not influenced by KC104. However, for all three sarnpüng perïods,

Iarvae exposed to LT3 alone maintained higher serum and serum T3 than Iarvae exposed to

LT3+KC104 (Figs. 8 and 9). No consistent differences were observed in serum T4 or Sem

T3between EST3 and m3+KC1O4larvae (Figs. 8 and 9). TORD activa

Intestinal T40RD activity expressed either as fmols T4 deiodinated/hr/mg microsomd

protein (25.3 t 2.1; n = 94) or as hols T4 deiodinated/hr/incubation tube (3.84 f 0-15; n =

94) was unaltered by any of the 9 treatrnents at 4 or 16 weeks (Fig. 10).

DISCUSSION

Our treatment of immediately premetarnorphic sea Iampreys with the goitrogen

KCl O4 induced metamorphosis in 80 % of Larvae treated for 8 weeks or longer, at a time of year when spontaneous metamorphosis does not occur. This agrees with previous studies in which KC104 induced precocious metamorphosis of sea lampreys (Holmes and Youson,

1993; Manzon and Youson, 1997). KClO4 treatment in this study also depressed senun% and T3 to 20 and 13 % of control values, respectively, confhming previous reports (Youson et al., 1995; Manzon and Youson, 1997), and suggesting that this decline in serum TH concentrations is either invo1ved in inducing metamorphosis or is a consequence of induced metamorphosis. Further support cornes from a previous study in which larvae treated with both TH and KCL04 have neither depressed serum TH concentrations, nor do they metamorphose, suggesting that exogenous TH can block KCL04-induced metamorphosis

(Manzon and Youson, 1997). Unfortunately, this latter study was inconclusive due to the low incidence of KCIOs-induced metamorphosis (20 % after 8 - 24 weeks of treatment) in younger larvae. However, in the present study, using immediately premetamorphic larvae and a higher KC104 treatment concentration, 1obsewed 80 % metamorphosis after ml04 treatment for 8 or 16 weeks, and a complete absence of metamorphosis in any of the 120

Iarvae treated with KC104 and either exogenous T4 or T3.TheSe results confimi that exogenous TH do indeed block KC104-induced metamorphosis. Exogenous TH presumably prevent the decline in senim TH concentrations accompanying both KCI04-induced and spontaneous metamorphosis (Youson et al., 1997)-

KC104 treatments lowered serum T4 and senun T3, but did not change either of the

T40RD indices. Thus, intestinai T40RD activity was insensitive to treatrnent with KCL04 alone. However, even without a change in the level of T40RD activity as measured in this assay, KCL04 will depress T3 production in vivo by decreasing the availability of its substrate precursor, T4. While it is possible that changes in T40RD activity couid be occumng in other tissues, a recent survey of various tissues from sea tamprey larvae indicated that the intestine was the predominant site of deiodination (Eales et al., 1997).

T4 treatment with or without KCIO4 increased serum T4 above control levels to concentrations greater than normal physiological levels, but had no effect on senim T3- This agrees with my earlier study in which semm T3 was unaitered despite large increases in serum T4,its immediate precursor (Manzon and Youson, 1997). The stability of semm T3 concentrations in the presence of large elevations of its immediate precursor (T4) suggests serum T3may be regulated, to some degree, independently from semm T4, In this context, perhaps serum T3 is autoregulated (Le., regulated independentiy of serum Tq) as described by

Refetoff and Nicoloff (1995). In teleosts, autoregdation of circuIating T3 in response to a Tq challenge is achieved by down-regulating the Tmpathway which makes T3,and by up- regulating the T31RD pathway that degrades T3 to inactive T2(Eales and Brown, 1993). 1 found that in vivo T4 challenges had no effect on intestinal T40RD activity when assayed in virro at a constant substrate level. Therefore, T3autoregulation in sea lampreys could presumably be achieved either by increasing T31RD activity, or by other mechanism. Unfortunately, there was insufficient microsornai material to examine T31RD activïty-

Although T31RD activïty was undetectabIe in the intestine and severai other larval tissues, it was found in adults (Eales et al., 1997). Thus, the potentiai exists for induction of the T31RD pathway in Iarvae in response to a T4chaUenge.

Although exogenous T3was inconsistent in elevating serum T3 in my previous study

(Manzon and Youson, 1997), it did raise serum T3in the current study. This difference may be due to either an increased branchial uptake of T3 or a slower induction of putative serum

T3 regulatory mechanisms (potentialiy Tm)in larvae of irnmediately premetamorphic size in cornparison to smaller, younger larvae- Support for this latter suggestion is derived fiom the observation that both T3 and T3+KClO4 treatments became less effective over time at elevating serum T3 in the current study.

Consistent with rny earlier study (Manzon and Youson, 1997), T3 treatment raised serum T4. Examination of serum T4 and T3for individual treatments showed that this elevation in serum T4 was not due to the artifact of T3cross-reactivity with the T4 antibody used in the RIA. T3treatment may raise serurn T4 either by decreasing T4 degradation or stimulating endostylar Tq production and secretion. With regard to the latter possibitity, endostylar pituitary control by thyroid stimulating hormone (TSH;thyrotropin) has not ken established (Youson, 1994, 1997; Eales, 1997), but T3may stimulate the endostyle directly.

In this event, the semm T3 titer would be crucial in maintainhg high larval leveIs of both serum T4 and T3 via positive feedback. Furthemore, interruption of this feedback loop by lowenng serum T3 through the depression of T40RD activity andor stimulation of T31RD activity could facilitate the decline in semT4 and T3 at metamorphosis. Alternatively, the ambient T3 which potentiaily bathes the endostyles in the pharyngeal cavities of T3-treated larvae, may stimulate the endostyIe to secrete Tq directIy, irrespective of the serum T3tïter,

One of the go& of this study was to determine the changes in intestinal -RD activity in response to dtered serum TH concentrations orStates of metamorphosis induced by combinations of Kc104 and TH treatments. However, T&RD activîty as measured in the in vitro assay at a constant subnanomolar T4 substrate level was undtered by any treatment,

This does not necessady mean that there were no changes in T&RD activïty. Alterations in

T40RD activity might occur in vivo due to allosteric interactions (EaIes and Brown, 1993)-

It is of future interest to investigate this possibility and determine the extent of change in other larval deiodination pathways, in particuiar the conversion of T3 to inactive T2.

1conclude that in imrnediately premetamorphic larvae: i) KCI04-induced metamorphosis can be completely blocked by either exogenous T40r treatments, and ii) serum T3 concentrations appear to be stringently regulated during a major T4 challenge despite large elevations in its immediate precursor (T4), but this involves no change in

T40RD activity. Table 2. Treatment groups and nomina1 ambient aquarium concentrations to which larvai sea lampreys (Petromyzon marinus) were exposed for 4 8 or 16 weeks.

Treatment groups Nominaï ambient concentration

Control

' LT4 = Low concentration thyroxine; HT4 = high concentration thyroxine; LT3 = low concentration triiodothyronine; EE3 = high concentration triiodothyronine; KC104 = potassium perchlorate Figure 7. Number of metamorphosing larval sea lampreys following exposure to KI04

(0.05%) for either 4, 8, or 16 weeks. Sampfe size is equai to ten for each treatment ïength.

The nurnber of animais at each stage of metamorphosis (1, earliest to 7, latest; Youson and

Potter, 1979) are also indicated, Cornpanion groups of control larvae, TH- and TK+KClO4- treated Iarvae underwent no changes associated with metamorphosis (data not show). Note: sea Iamprey between stages of metamorphosis were assigned the earlier of the two stages. 8 16 treatment length (weeks) Figure 8. Mean (& 2SE) senun thyroxine fl4) concentrations in larval sea Iamprey foiiowing exposure to either a Low (L) or high (H) Tq (0.56 and 1.12 pM, respectively) or triiodothyronine (T3;0.37 and 1.48 pM, respectively) treatment in the presence (+) or absence (-) of the goitrogen potassium perchlorate (KC104; 0.05%) for 4,8 or 16 weeks.

Sample size is equal to ten for ail treatments and samplings except HT4+KC104 at 8 weeks where sample size is nine. An * indicates semm concentrations which differ significantly (P

I0.05) fiom controi (no hormone and no KC104) values within a sampling period. Within a treatment, serum T4concentrations are significantly different (P 10.05) between sampling periods, if they have different Letters (a, b, c).

Figure 9. Mean (& 2SE) serum triiodothyronine f13) concentrations in lacval sea Iamprey following exposure to either a low (L) or high (H) thyroxine a;0.56 and 1.12 pM, respectively) or T3(0.37 and 1-48 pM, respectively) treatment in the presence (+) or absence

(-) of the goitrogen potassium perchlorate (KCI04; 0.05%) for 4,8 or 16 weeks. Sample size is equal to ten for aiI treatments and samplings except HT4+KC1O4 at 8 weeks where sample size is nine. Actual values are provided in two cases where the mean or SE extends beyond the y mis. An * indicates serum concentrations which diEer significantly (P 5 0.05) fiom control (no hormone and no KCQ) values within a sampling penod- Within a treatment, serum T3 concentrations are significantiy different (P 10.05) between samplùig penods, if they have different letters (a, b, c).

Figure 10. Activity (fmols T4 deiodinated/hour/incubation tube) of intestinal T4 outer-ring

(5') deiodination to 4 (T40RD), in larval sea lamprey exposed to either a Iow a)or high

T4 (0.56 and 1.12 pM, respectively) or T3(0.37 and 1.48 pM, respectively) treatment in the presence (+) or absence (-) of potassium perchlorate (KClO4) for 4 or 16 weeks. Sample size

(N) is equal to five for all treatments and sampling periods with the exceptions of m104,

HT3, LT4+KCI04and HT4+KC104at 4 weeks, and El& and HT3 at 16 weeks, where N = 4.

T4 = thyroxine; T3 = 3,5,3'-triiodothyronine. Hormone - - LT4 HT4 LT3 El3 LT4 ET4 LT3 RT3 KClOq - + - - - - + + + + Treatment VARIABLE EFFECTS OF GOITROGENS IN INDUCING

PRECOCIOUS METAMORPHOSIS IN SEA L4'MP?EYS

(Petromyzon marinus).

Acknowledgment:

Dr. I.A. Holmes setup and rnaintained the two propylthiouracil experiments, but 1 perforrned

al1 thyroid hormone assays and data anaiysis. ABSTRACT

The ability of different goitrogens (anti-thyroid agents) to induce precocious metamorphosis in 1arva.i sea lampreys (Petromyzon M~~US)was assessed in four separate experiments, Two of these goitrogens (propyIthiouraci1 CPTU] and methimazde m)are inhibitors of thyroid peroxidase-catalyzed iodination and three (potassium perchlorate w104], potassium thiocyanate KSCW and sodium perchlorate [NaC104]) are anionic cornpetitors of iodide uptake. Since aII these goitrogens cm prevent thyroid hormone (TH) synthesis in vertebrates, 1dso measured their influence on serum concentrations of thyroxine and triiodothyronine- All goitrogens except PTU signincantly Lowered serum TH concentrations and induced metarnorphosis. The incidence of metamorphosis was correlated with these Iowered TH concentrations. KC104, NaC104, and MMI treatments resulted in the lowest serum TH concentrations and the highest incidence of metamorphosis in sea lampreys, while fewer Iarvae metamorphosed in the KSCN and Iow-KC104 treatment groups and their serum TH concentrations were greater than the values in the aforementioned groups. MMI treatment at the concentrations used (0.087 and 0.87 m.)was toxic to 55 % of the exposed sea Iampreys within six weeks. Potassium administered as KCl did not alter serum TH concentrations or induce metamorphosis. 1conclude: i) Goitrogens other than

PTU can induce metarnorphosis in larval sea lampreys and this induction is coincident with a decline in semm TH concentrations. ü) The rnethod by which a goitrogen prevents TH synthesis is not directly relevant to the induction of metamorphosis. iii) P'ïU has variable effects on TH synthesis and metamorphosis among Iamprey species. iv) Umein protochordates, potassium ions do not induce metamorphosis in sea lampreys and are not a factor in the stimulation of this event, The stimdatory role of the thyroid gland on the onset and progression of amphibian metamorphosis was £ktdescribed by Gudematsch (L9 12). Following this initiai report, several studies have shown that a rise in senun thyroid hormone (TH) titers (thyroxine [T4] and triiodothyronine ml) fiom prometamorphosis to their peak at metamorphic climax, is criticai to metamorphosis. Furthemore, treatment with exogenous TH stimulates precocious metamorphosis and anti-thyroid agents (goitrogens) can prevent or dramaticaiiy delay the onset of spontaneous metamorphosis in amphibians. The stimuiatory role of TH on metamorphic development has been observed in most amphibians (for review see Dodd and

Dodd, 1976; White and Nicoll, 1981; Galton, 1983) and bony fishes (ffat fishes, eels and tarpons) (Just et al,, 1981; Inui et al., 1994) studied to date, Within the vertebrate subphylum, the Petromyzontiformes (lampreys) represent the one exception to this trend

(Youson, 1997).

The nature of the involvement of TH in lamprey metamorphosis has yet to be resolved; however, some data are consistent with the idea that their function differs fiom that observed in other vertebrates. Senim TH concentrations in the sea lamprey (Petromyzon mrinus) increase graduaily throughout the 3 - 7 year larval pend, peak pnor to the onset of metamorphosis and decline sharpty concomitant with the tirst external signs of metamorphosis (for review see Youson, 1997)- Throughout metamorphosis and for the remainder of the sea lamprey Iife cycle serum TH concentrations remain low. The nature of this decline in serum TH concentrations at the onset of sea lamprey metamorphosis is supported by similar observations in other lamprey species. Significant declines in serum T4 and T3 concentrations fiom larvaI values were observed in the southern hemisphere lamprey,

Geotria austraLis (Leatherland et al-, 1990) and the American brook lamprey, Lampetru appendùr (Holmes et af., 1999) by stages 1 and 2 of metamorphosis, respectively.

Lampreys aIso ciiffer fiom other vertebrates in that goitrogens induce rather than inhibit metamorphosis. Hoheisel and Sterba (1963) first reported the induction of an incomplete metamorphosis in Lampetra pianen followhg exposure to the goitrogen potassium perchlorate (KCL04)- The induction of a complete metamorp hosis foilowing treatment with either KC104, sodium perchlorate (NaC104), propylthiomcil 0or thiourea was later reported in a series of prelirninary studies on Lampeîra reissneri (Suzuki,

1986, 1987, 1989). More recent studies conducted at a time of year when spontaneous metamorphosis does not occur resulted in the incomptete metamorphosis of P. marinus

(Holmes and Youson, 1993) and L appendur (Holmes et ai., 1999). In these studies, KClO4 treatment resulted in significant declines in senim TH concentrations, suggesting that declines in semTH concentrations may be permissive to the onset of metamorphosis

(Youson et al., 1995; Manzon et al., 1998; Holmes et al., 1999). In contrast, PTU did not induce metamorphosis in G. austrulis (Leatherland et al., 1990) or Lbappendix (ETolmes et al., 1999) despite its ability to significantly lower serum TH concentrations.

The correlation between lowered serum TH concentrations and the induction of metamorphosis in goitrogen-treated lampreys indicates that high TH titers may have an anti- metarnorphic effect, preventing the onset of metamorphosis. Altematively. very low TH titers may permit or even trigger the onset of metarnorphosis if the appropriate physiological and environmental conditions have been met (Youson, 1997; Manzon and Youson, 1999).

The absence of induced metamorphosis in PTU-treated lampreys of two different species, however, provides compelhg evidence to challenge these notions. Whether the induction of

precocious metamorphosis is due to a decline in semm TH concentrations or to a

phenornenon relateci to an uhown extrathyroidai effects of certain goitrogens (Le., KC104)

remains to elucidated, AIternatively, PTU may have extrathyroidal effects which are either

inhibitory to metamorphosis or toxic to Iampreysc Perhaps the ability of a particular

goitrogen to induce metamorphosis is reiated to the mechanism through which it inhibits TH

synthesis,

In the curent study, 1attempt to resoive some of the uncertainties surrounding goitrogen-induced metamorphosis in lampreys. 1 assessed the ability of various goitrogens to lower serum TH concentrations and to induce precocious metamorphosis in larval sea lampreys. This study consisted of four experiments in which larvai sea lampreys of different sizes were exposed to various goitrogens. The goitrogen treatments included three anionic cornpetitive inhibitors of iodide uptake (KCI04, NaCl04 and potassium thiocyanate, KSCN) and two inhibitors of thyroid peroxidase-catalyzed iodination (PTU and methimazole, MMI)

(Gentile et al., 1995). Since potassium ions induce metamorphosis in larval protochordates

(Degnan et al., 1997), 1conducted an experirnent designed to examine the effects of potassium ions 0,associated with KC1o4 and KSCN treatment, on the induction of metamorphosis in lamprey S.

MATERIALS AND METHODS

Experimental Protocols and AnimaCs

This study consisted of four experiments conducted in different years, at a time of year that spontaneous metamorphosis does not occur. Metamorphosis occurs in sea lampreys between Iuly and October (Potter et al., 1978). The foUowhg is a description of the procedures for animai cokction and experimentd setup, monitoring and maintenance used in al1 four experiments. Any variations from these procedures are provided in the detailed description of each individual experiment- Larval sea lampreys were collected fiom various streams in the Great Lakes drainage basin (Canada and USA) in the spring and summer months using electrofishing equipment. Sea lampreys were transported to the University of

Toronto at Scarborough and housed in large fiberglass aquaria supplied with substrate (7 - 10 cm of industriai sand) and continuously flowing, aerated, dechlorinated tap water. Larvae were maintained at seasonal water temperatures and fed a suspension of baker's yeast once weekly (1 g of yeast per animal).

Prior to the onset of each experiment, land sea lampreys were anaesthetized in a

0.05% solution of tricaine methanesulfonate (MS-222, Syndel Laboratones Ltd., Vancouver,

British Columbia, Canada). Their lengths and weights were recorded, and groups of IO larvae were randomiy assigned by lottery to 21 liter aquaria (40 X 20 X 25 cm). Each aquarium was supplied with 7 - 10 cm of substrate and 10 Liters of dechlonnated water.

Aquaria were maintained static (i.e., not on a flow-through system) on a 15 h light, 9 h dark light cycle and were continuously aerated. Water temperature varied with the ambient room temperature (14 - 22 OC). Routine monitoring and maintenance included: recording water temperatures twice daily; adding water as needed to account for evaporation; feeding larvae once weekly; cleaning aquaria, changing aquaria water and adding fiesh treatments to aquaria every two weeks; and changing the substrate every 1 - 2 months. AU chernicals used for experimental treatments were obtained fiom Sigma-Aldrich, Canada. nie onset of each experiment was designated as the fmt day that treatments were added to the appropriate aquaria and the experiment was terminated when the last sea lamprey was sampled.

Treatrnents were added to aquaria from stock solutions prepared in dechlorinated water unless otherwise indicated. Sampling of sea lampreys was cimied out as foilows: anaesthetizing in 0.05 % MS-222; recording animal lengths and weights; assigning a stage of metamorphosis (1 to 7) based on extemal morphology (Youson and Potter, 1979); and collecting serum. Animals which died dunng the experiment were promptly removed and replaced with Iarvae marked by latex dye injection into the caudal sinus- Marked larvae were used to maintain animal density within an aquarÏum but were excluded from all experimental and data analyses.

Experintent 1: Propylfhiouracil and tdfodohyronine

Larval sea lampreys 65 - 95 and 105 - 119 mm in Iength were collected fiom Fish

Creek, New York (September, 1993) and Pigeon River, Michigan (June, 1993), respectively.

Thirty larvae fiom each size group (10 larvae per aquarium and three replicate tanks per expenmental group) were randody assigned to each of the following expenrnental groups: control (untreated), PTLJ (6-n-propyl-2-thiouracil), T3, and PTU plus T3(PTU+T3); see Table

3 for nominal ambient aquarium concentrations. In addition, at the onset of the experiment 1 sampled 30 larvae 65 - 95 mm in length (Fish Creek) and 20 larvae 105 - 119 mm in length

(10 fiom each stream population) to serve as baseline estimates of larval size and TH status and as a control between the two populations- The experiment began in January, 1994 with the addition of the aforementioned treatments to the appropriate aquaria (Table 3).

Treatments were administered to each aquarium as 50 ml of a stock solution prepared in 0.1

M NaOH. NaOH was used to increase the solubility of PTU and T3 when making the stock solutions. NaOH did not have any effects on animal size, TH status or the incidence of metamorphosis in an experiment conducted on larvd sea lampreys £iom Fish Creek (Maozon and Youson, 1997), so an NaOH control treatment was omitted in this study- AU experimentd anirnals were sampled 23 weeks after the experiment began.

Experirnenf 2: Propy&hiouruciZ

Larvai sea lampreys were collected fiom Beaverdam Brook, New York, in May 1995.

Larnpreys were housed at various water temperatures and animal densities, throughout the spnng and summer months, to assess the effects these variables have on the incidence of spontaneous metamorphosis. In November, 1995 larvae fiom this population that were greater than 129 mm in length were pooled. Larvae fiom this pool were randody assigned to glass aquaria (10 Iarvae per aquarium) and three replicate aquaria were randomly assigned to each of the control (untreated), WH,and PTU experimental groups (Table 3).

Treatments were adminiktered following the procedures used in Experïment 1. The study began in November, 1995 and was tenninated following 18 weeks of treatment at which time al1 animals were sampled.

Experiment 3: Potassium perchlorate, potcrssium thiocyanate and methimazole

Larval sea lampreys were collected from the Platte River, Michigan (August, 1996).

Larvae 2 115 mm in length were randomly sorted into glass aquan'a (10 Iarvae per aquarium) and three replicate aquaria were randomly assigned to each of the following experimental groups: control (untreated); Iow-KC104 (L-KC1O4); high-KCLO4 (H-KC104); low-KSCN (L-

KSCN); high-KSCN (H-KSCN); low-MMI (2-mercap to- 1-methyIimidazole, L-MMI) ;and high-MM[ (H-MMI) (Table 3). In addition, 30 larval sea lampreys were sampled at the onset of the experiment to serve as baseline data for Lamprey size and TH stanis. The expenment

98 began in January, 1997 and was terminated 16 weeks later. However, due to the high mortality in both MMI treatment groups, dl surviving W-treated animais were sampled 6 weeks after the study began. Sea lampreys in one randomly chosen tank from the control, H-

KC104 and H-KSCN expenmental groups were assessed for any sigos of metamorphosis at the time that the MMI-treated lampreys were sampled-

Experinzent 4: Potassium perchlorate, sodium perchlorate and potassium chloriXe

Larval sea lampreys were coltected fiom Fish Creek, New York (May, 1998) and sorted into two size groups, 1 10 - 119 mm and > 119 mm in length. Larvae from each size group were randomly sorted into glas aquarïa (9 larvae per aquarium). One aquarium from each size group was randomly assigned to each of the following expenmental groups: control (untreated); KCU; Nam;low potassium chloride (L-KCI); and high potassium chloride (H-KC1) (Table 3). The experhnent began in March, 1999 and was terminated following 13 weeks of treatment, at which tirne alL suMving sea lampreys were sampled- At the staa of the expriment 1sampled nine larvae nom each size group to serve as baseline values for animal size and TH status.

Serum collection and TH rneasurement

Blood was collected from anaesthetized sea lampreys by caudal severance using heparhized haematocrit tubes and was allowed to dotovernight at 4 OC. The foliowing morning the blood was centrifhged at 7000 g for 3-5 minutes and sera were collected and stored at -70 OC until analyzed. Animals were sacnficed by decapitation immediately after blood collection. Total senun T1 and T3 concentrations were detemiined in duplicate using the

Amersham Amerlex ïT4 and TT3 radioimmunoassay @UA)kits. respectively (Johnson and

Johnson, Markham, Ontario, Canada), The RIA kits were used according to Leatherland et al. (1990) to accommodate for low serum volumes. ïntra- and inter-assay coefficients of variance for both TK RIAS were Iess than 9 and 14 %, respectively. Assay sensitivities ranged from 7 - 11 nmoVLiter for Tqand 0.15 - 0.4 nmoVLiter for T3. Serum Tq and T3 concentrations were detennined for Experiments 1,3 and 4 but not for Experiment 2 (serum samples were lost when the laboratory fieezer failed). In some instances, equai volumes of senun from two or more sea lampreys fiom the same expenmentd aquarium were pooled to produce a single sample of sufficieut volume to assay both TH.

Sfati'sîicalanalyses

The data from al1 four experiments were analyzed with analysis of variance

(ANOVA) and Tukey-Kramer's pst-hoc test using individual aquaria andlor expenmentai groups as independent variables. and animai length, weight and serum TE3 concentrations as dependent variables. Prior to these statistical analyses, al1 data were tested for homoscedasticity with Cochran's Q test; data which did not meet this assumption were transformed (loglo)to minùnize the heterogeneity of the variances (Sokal and Rohlf, 1980).

ALI statistical analyses were performed using Statistix for DOS. The data are presented as mean f 2 SE (standard errors) and differences were accepted as statistically significant if P c

0.05. Any deviations from these statistical analyses are indicated as they are presented in the results section. RESULTS

Experiment 1: PropyIthburd and tdinfothyronine

Aquaria water temperatures ranged from 14 - 18 OCwith a mean of 16 k 0.1 OC,

There was 50 % moaality in the PTU+T3 experimental group (65 - 95 mm size group); however, 67 % of these deaths occurred in a single aquarium in which al1 larvae died- Other deaths included one 105 - 119 mm sea Iarnprey in each of the control, PTU and PTU+T3 experimentai groups. Metamorphosis was not observed in any of the treated lacval sea lampreys, but one control animai was at stage 3 of metamorphosis at the the of sampiing.

At the start of the experhent, larval length and weight in the 65 - 95 mm size group did not cliffer between the four experimental groups; the overd mean size of these larvae was 84.4 k 1.4 mm and 0.89 t 0.04 g. Baseline larvae in this size group had a mean length and weight (93.1 + 1 mm and 1.12 t 0.05 g) which were significantly greater than those recorded for each of the experimentai groups. Significant differences in size between larvae - of the four experimental groups adorthe baseline group were not observed in the 105 - 119 mm size group at the onset of the experiment; the overail mean size of these larvae was 113.3

+ 0.7 mm and 2.20 + 0.05 g. At the end of the experiment, larvae within a size group tended to be smaller in size than at the onset and experimental larvae were significantly smaller than baseline larvae, indicating animal size decreased over the course of the experiment (Table 3).

Baseline larvae in the 65 - 95 mm size group had serum T4 concentrations (154 + 14.5 nmol/liter) which were significantiy greater than the values measured, at the end of the experiment, in the control, PTU, PTCT+T3, and T3 (40 f 6.2,35 f 6.6,48 + 11.2 and 113 +

19.7 timollliter, respectively) experirnental groups (Fig. 1 1A). Treatment of 65 - 95 mm larvae with exogenous T3 signif~canlyelevated semm Tqconcentrations relative to values in the control, PTU and PTU+T3 expenmentai groups (Fig. 1 LA).

In the 105 - 119 mm size group, serum Tq concentrations in baseiïne larvae (145 t

44-4 nmoVLiter) were significantly greater than values in the control and PTZI-treated animals

(40 + 4.9 and 41 + 4-4 nrnoI/Iiter, respectively), but did not ciiffer fiom vdues in the T3- OC

ETU+T3-treated (192 + 26 and 120 f 26.7 nmoYliter, respectively) animais (Fig. i 1C)-

Exogenous T3treatment, in the presence or absence of PTU, sigaificantly eIevated semm T4 concentrations relative to values in the control and PTU experimentai groups (Fig. 11C).

PTU did not alter senim Tq concentrations relative to control values in either size group

(Figs. 11A and 11C). Senun T3concentrations of animais In either size group did not mer significantly between any of the experimental groups (i-e., control, PTU, PTU+T3, or T3),but within each size group the serum T3 concentrations of baseline animals were significantly greater than those of the experimental groups (Figs, 1 LB and 1 ID).

Expen'ment 2: Propyiihiouracil

Aquarïa water temperatures rangeci fiom 15 - 21 OC with a mean value of 17 t 0.1 OC.

At the start of the experiment, significant differences in sea Iamprey length or weight were not observed between the various experimental groups; the overail mean animal size was 133

_+ 0.9 mm and 3.42 + 0.08 g. As was the case for Experiment 1, larvae tended to be smaller at the tirne of sampling than at the start of the expriment (Table 3). Metamorphosis was not observed and no deaths occurred in any experimental groups. Experiment 3: Potassium perchIorute, poîassiüm thiocyanate und methiinazoIe

Aquarium water temperature ranged fiom 18 - 22 OCwith a mean of 20 +. 0.1 OC.

Moctalities were not observed in the control, KCIO4 or KSCN expehental groups; however, a high incidence of mortality in the L-MMI and H-MMI treatment groups precipitated the sampiing of ail survïvlng sea lampreys fiom these groups 6 weeks after the experhent started. At this tirne 18/30 and 17/30 Iarvae had died in the L-MMI and H-MMI experimentai groups, respective@ Al1 cornparisons between experimental groups at the time of sampling were made using MMI Iarvae sampled 6 weeks after the experiment began and control, KC104 and KSCN larvae sampled 10 weeks later, unless otherwise indicated.

Significant differences in mean larval length or weight were not observed between the seven experimental groups andfor baseline larvae at the start of the experiment; the overall mean size was 130.3 t 1-6 mm and 3.23 +. 0.1 g. The mean size of all experimental animals at the time of sampling was 121.6 k 1.8 mm and 2-40 + 0.12 g (Table 3) indicating that animal size had decreased over the course of the experiment.

The three goitrogen treatments used in this experiment induced precocious metamorphosis in sea larnpreys; however, the number of metamorphosing Iampreys varied depending on the type and concentration of goitrogen used. Treatment of larval sea lampreys with L-MMI or H-MM1 for 6 weeks induced metamorphosis in 33 % and 46 % of survMng sea lampreys, respectively (Fig. 12A). When the MM-treated sea lampreys were sampled, 1 also examined 10 of the 30 larvae in the control, -104 and H-KSCN experimental groups and found that only 2 sea lampreys from the H-KC104 treatment group had cornmenced metamorphosis (Fig. 12A). Mter sixteen weeks of treatment, metamorphosis was not observed in the control

group, but 10,66,20 and 43 % oflampreys had begun to metamorphose in the L-KC104, H-

KC104, L-KSCN and H-KSCN experhnental groups, respectively (Fig. 12B). Sea lampreys

in each of the seven stages of metamorphosis were observed mg. 12B), and these goitrogen-

treated larnpreys were clearly undergoing external morphologïcai changes characteristic of spontaneous metamorphosis- However, in most instances the timing of these morphological changes was asynchronous compared to spontaneous metamorphosis-

To detennine the effects of the various treatments on semTH concentrations, 1 tested for statistically signiflcant differences between experimental groups and baseline anirnals (Fig. 13). In addition, 1tested for daerences between larvai and metarnorphosing lampreys within an experimental group (Table 4)- Semm T4 concentrations in baseiine sea lampreys were signincantly greater than those fkom the experimentai groups (Fig. 13A). Sea lampreys in the H-KSCN, L-KC104, H-Km4, L-MM, and H-MM1 experimental groups had semm T+concentrations which were significantly lower than control values, but treatment with L-KSCN did not alter semm T4 concentrations (Fig, 13A). Within the L-

KSCN and H-KSCN experimental groups, the serurn Tqconcentrations of metamorphosing lampreys were significantly lower than values for lampreys which did not metamorphose

(Table 4).

Serum T3concentrations in the H-KSCN,H-KC104, L-MMI, and H-MMI experimental groups were significantly lower than concentrations in the control and baseline groups (Fig. 13B). However, semm T3concentrations did not differ signincantly between the baseline, control, L-KSCN, and L-KC104 lampreys (Fig. 13B). Significant differences in serum T3 concentrations between larval and metamorphosing animais within an experimentd group were observed in the L-KSCN, H-KSCN and L-MMI expdmental groups (Table 4).

Surptisingly, serum T3concentrations in metamorphosing L-MMI sea lampreys were significantIy greater than the vaiues in larvd L-MMI sea lampreys; in both cases serum T3 concentrations were much lower than control vaiues (Table 4).

Experiment 4: Pofassium perchlorate, sodium perchlorafe, and potassium chloride

Aquaria water temperatures ranged from 17 - 22 OC with a mean value of 19.6 f 0.2

OC. With the exception of the H-KC1 experimental groups in which aii animals died within

4 days of the start of the experiment, no deaths occurred in either size group. There were no ciifferences in larval size between experimentai and baseline groups within either size group at the onset of the experiment. The mean animal size at this time was 127.7 f 2.1 mm and

2.77 + O. 18 g in the > Il9 mm size group, and 113.1 t 0.1 mm and 1.89 t 0.06 g in the 110

- 119 mm size group, Larvae of both size groups were smaIler in size at the time they were sarnpled than at the start of the experiment (Table 3)-

Metamorphosis was not observed in control or L-KC1-treated lampreys of either size group. However, in the 110 - 119 mm size group, 33 % of NaCL04- and KC104-treated sea lampreys were in either stage 1 or 2 of metamorphosis. Thevincidenceof metamorphosis in lampreys > 119 mm in length following KC104 or NaC104 treatment was 89 8, and these animals were in stages 1, 2, 3, and 4 of metamorphosis (Fig. 14).

In general, serum TH concentrations in baseline animals were significantly greater than those of experimental lampreys; serum T3in L-KCl sea lampreys (> 119 mm size group) was the only exception to this trend (Fig. 15). Semm T4and T3 concentrations did not differ significantly between control and L-KC1 sea lampreys within a size group (Fig. 15). The goitrogens KCIOI and NaC104 sÏ@ficantly lowered serum TEL concentrations in lampreys from both size groups relative to vaIues in the baseIine, control, and L-KCI groups (Fig. 15).

DISCUSSION

Since 1963, several Iaboratories have studied the effects of goitrogens on lamprey physiology and metamorphosis, with connicting results. The treatment of Iampreys with the goitrogen KC104 resulted in precocious metamorphosis in ail studies (Hoheisel and Sterba,

1963; Suzuki, 1986; Hoimes and Youson, 1993; Manzon and Youson, 1997,1999; Manzon et al., 1998; Holmes et al., 1999). SemTH titers in KC104-treated Iampreys were consistentIy lower than those of uatreated control lampreys, thus mimicking the low semm

TH concentrations associated with spontaneous metamorphosis (Leatherland et al., 1990;

Youson et al., 1994; Holmes et al., 1999)- These effects of KCL04 are consistent with the hypothesis that a decline in serum TH concentrations pennits the onset of metamorphosis in lampreys. Furthemore, high senun concentrations of TEX can inhibit or retard both spontaneous (Youson et al., 1997) and goitrogen-induced (Manzon and Youson, 1997;

Manzon et al., 1998) metamorphosis in lampreys. In contrast, the effects of PTU on lamprey serum TH concentrations and metamorphosis are not consistent and tend to challenge the results fkom these KC104 induction experiments.

The induction of metamorphosis foliowing treatment was fïrst reported in L reissneri by Suzdci (1987). Subsequently, both Leatherland et al. (1990) and Holmes et al.

(1999) reported a significant decline in the serum TH concentrations of G. australis and L- appendrjc, respectively, folIowing treatment with W. Despite these low serum TH concentrations, PTU treatment did not induce precocious metamorphosis. This absence of metamorphosis indicates that a decline in serurn TH concentrations may not be sufficient to induce metamorphosis, Akematively, FiW may have other effects on the physiology of some larnprey species which precludes the induction of metamorphosis, In this study 1 attempt to clare: i) whether severai goitrogens cmdepress semTH concentrations and induce sea lamprey metamorphosis, ii) whether this induction of metamorphosis is specific to

KC104, and iii) whether PTU is unique among goitrogens in its variabiiity at inducing metamorphosis in lampreys. The variable effects of ETU on larnprey metamorphosis may involve species differences, a possibïiity which cannot be assessed by this study.

In the current study, a total of 90 larvai sea lampreys of three different sizes groups

(65 - 95,105 - 119 and > 129 mm in length) were exposed to the goitrogen fTU for 18 - 23 weeks. Consistent with the frndings of Leatherland et al. (1990) and Holmes et al. (1999), but contrary to those of Suzuki (1987, 1989), there were no signs of metamorphosis in PTU- treated sea Iampreys. In the present experiment 1used the same PTU treatment concentration

(10 mgfiter) as Leatherland et al. (1990) and Hoimes et al. (1999)- Suzuki (1987, 1989) did not report the PTU concentration he administered to larval L. reissneri. Surpnsingly, PTU treatment did not significantly alter serum TH concentrations relative to the control group

(Fig. 11). AIthough it is tempting to suggest that the absence of metamorphosis in this experiment was due to the fact that PTU treatment did not lower senun TH concentrations, the studies by Leatherland et al. (1990) and Holmes et al. (1999) provide evidence which could refute this as a possibility. I cannot conclusively determine why PTU did not affect sea larnprey semm TH concentrations in Expriment 1, but this result may be related to the physiological condition of the sea lampreys in the current study or to dserences bekn species. Unfortunately, 1was unable to determine serum TH concentrations in PTU-treated larvae greater than 130 mm in Iength (Expriment 2). A change in the physiological condition of larvai sea lampreys over the course of the

experiment may have contributed to the absence of a decline in senun TH concentrations in

PTU-treated larvae. In Experiments 1 and 2.1 observed a decrease in mean larval length and

weight over the course of the experiment. In general, larvae withln a treatment group were

smaller in size at the theof sampling than they were when the study began. The cause of

this decrease in size is unknown. TH status aIso changed over tirne, Serum TH

concentrations in baseline lamal sea lampreys were significantiy greater than the values for

control larvae at the time of sampling in both size groups in Experiment 1 @ig. 11). Similar

trends towards a decrease in the mean animal size and semm TH concentrations over the

duration of an experiment were also observed in Experiments 3 and 4 (Figs. 13 and L5).

However, goitrogen treatment in each of these experiments depressed serum TH

concentrations and induced metamorphosis. The results kom Experiments 3 and 4 minimize

the likelihood that a decrease in size or a change in TH status fkom baseline values was responsible for the absence of metamorphosis or a decline in serum TH concentrations in

Experiments 1 and 2.

The treatment of larval sea lampreys 65 - 95 and 105 - 119 mm in length with exogenous T3 significantiy elevated serum T4 concentrations but had no significant effect on serum T3concentrations. These findings are similar to those observed by Manzon and

Youson (1997) with sea lampreys 110 - 119 mm in length. In this earlier snidy, exogenous

T3 significandy elevated serum T4concentrations in 6 of6 sampling periods ranging from 4 -

24 weeks of treatment. However, serum T3 concentrations increased in only 2 of these 6 sampling periods. In a second study using larval sea lampreys 2 120 mm in length, Manzon et al. (1998) found that exogenous T3consistently elevated both serum T4 and T3 concentrations. These data imply that large larvae (2 120 mm in length) can regulate TH

synthesis and metabolkm Merentiy than the smaiïer Iarvae used in the present study- Eales

et al. (1997,2000) have shown that the activity of the deiodinase pathways change during

and after metamorphosis. Simüarly, changes in TH deiodinase activity and/or metaboiism

may occur within the larval period. Semm TH concentrations increase throughout the larvd

period indicating the potential for a change in TH regulation. The ability of exogenous T3 to

elevate serum T4 without increasing semm T3 is an interesting phenornenon from a regulatory standpoint, The exact regulatory mechanism responsible for elevating serum T4 concentrations is unknown, but could involve changes in the regulation of TH synthesis, secretion, deiodination, anUor degradation.

AU sea lampreys in the control, KC104 and KSCN exprimental groups survived for the duration of Experiment 3. However, a large proportion (> 55 %) of MMI-treated larvae died earty in the experiment This high mortality rate led to the samphg ofail surviving

MMI-treated sea lampreys 6 weeks after the experiment began. The two concentrations of

MM1 used in the current study (0.087 and 0.87 mM) were lower than the concentration (1 mM) that Brown (personal communication, 1997) routinely uses to inhibit metamorphosis in

X. Laevis. In addition, LmM MMI was not toxic for Eleutherodactylus coqui (Callery and

Elinson, 2000). However, an unpredictable mortality rate was observed in zebrafish at MM1 concentrations greater than or equd to 0.5 mM,and 0.3 mM was the highest MM1 concentration which was not toxic (Brown, 1997). Although 1do not know the cause of

MM1 toxicity in lampreys, it appears to be consistent with the unpredictable effects of MM1 observed in zebrafish. As was the case in allexperïments, Experiment 3 was conducted at a time of year when sea lampreys do not metamorphose spontaneously. Thus, it was not surprising that larval sea larnpreys in the untreated control groups did not undergo metamorphosis; yet metamorphosis was observed in all goitrogen treatment groups in Experiment 3. Fotiowing a

6 week exposure, MMI was more successful at inducing precocious metamorphosis in tarvd sea lampreys than either KC104 or KSCN, At 6 weeks, 10/23 MMI-treated sea lampreys had commenced metamorphosis, as compared to 2/10 H-KC104-treated and 0/10 H-KSCN- treated sea lampreys (Fig. 12A)- These data differ fiom those of the PTU experiments and indicate that MMI, a thyroid peroxidase-Înhibitor, can induce metamorphosis in larvai sea lampreys. MM1 may also have the potential to induce metamorphosis in larval sea lampreys more rapidy than the other goitrogens tested to date. However, a cautious interpretation of these data is necessary because: oniy one third of the H-KC104- and H-KSCN-treated sea lampreys were examined at this 6 week point, metamorphosing sea lampreys in the MMI experimental groups were in the early stages of metamorphosis, and MM1 treatment was toxic to many individuals. Perhaps the rapid induction of mefamorphosis in MMI-treated larvae contributed to this observed toxicity. Further studies to establish a dose response curve for MMI are required to properly assess the capability of MMI to induce metamorphosis.

The observed differences in the incidence of induced metarnorphosis in the various goitrogen treatment groups (Fig- 12) can be correlated to the efficacy of each goitrogen at lowering semm TH concentrations. Following only 6 weeks oftreatment, L-MM1 and H-

MM1 induced proportionally more larval sea lampreys to enter metamorphosis than either the

H-KC104 or H-KSCN treatrnents. At the termination of Experiment 3, the H-KCI04 and H- KSCN treatments were the most successflll at inducing sea lamprey metamorphosis, The treatments (H-KCI04, H-KSCN, MMI) that resulted in a high incidence of induced metamorphosis, also resulted in the lowest serum TH concentrations (Fig. 13)-

Foilowing 6 weeks of treatment, MMI larvae had semTq concentrations which were as low or lower than larvae exposed to any other goitrogen treatment for 16 weeks (Fig,

13A)- Furthermore, 6 weeks of MMI treatment produced serum T3concentrations that were significantly lower than those in ail other expenmental groups except the H-KC104 experirnental group (Fig- 13B). H-KC104 treatment induced precocious metamorphosis in more sea lampreys than any other experimentai group, and serum TH concentrations in this and the MMI experimental groups were the lowest measured in Experiment 3 (Fig, 13).

Similarly, I measured higher senun TH concentrations in those expenmental groups in which a Iower incidence of metamorphosis was observed. The lowest incidence of metamorphosis occurred in the L-KCU and L-KSCN treatment groups. Larvae in these two experimental groups had mean senun T3 concentrations which did not ciiffer significantiy fiom control values (Fig. 13B). Moreover, L-KSCN-treated larvae had senun T4concentrations were also equivalent to values in the control group (Fig. 13A).

The results of Expenment 3 conFm that goitrogens other than KC104 cm induce precocious metamorphosis in larval sea lampreys. The observation that MMI, but not E'TU, could induce metamorphosis in sea lampreys is of particular interest, since both of these goitrogens act by inhibiting thyroid peroxidase-catalyzed iodination. Furtherrnore, the proportion of goitrogen-treated lampreys which were induced to metamorphose was correlated with the degree of the deciine in serum TH concentrations, This finding differs fiom that reported by Youson et al. (1995) for KC104-treated Iarval sea lampreys ofdifferent

sizes.

Potassium is a component of two of the goitrogens (KClo4 and KSCN) which induce

precocious metarnorphosis in lampreys. This ion induces metamorphosis in invertebrate

species from 5 different phyla including protochordate ascidians -1 et al., 1986; Pearce and Scheibling, 1994; Degnan et al., 1997). Modem protochordates and vertebrates are both mernbers of the phylum Chordata and are beiieved to s hare a common chordate-ike ancestor

(Whittaker, 1997)- One feature indicating a common ancestry is the presence of an endostyle in both larval Iampreys and protochordates. The endostyle of larval lampreys is a TH- producing, subpharyngeal gland that develops into typicd, foïlïcular, vertebrate thyroid tissue during metarnorphosis (Wright and Youson, 1980). In contrast, the endostyle of protochordates k prirndy involved in producing mucus to aid in filter feeding. Like the larval lamprey endostyle, the protochordate endostyle cm actively concentrate iodide and synthesize iodoproteins and TH (Eales, 1997). The induction metamorphosis by KC in protochordates necessitated elùninating the possibiiity that K', via KCl04 and KSCN, rnay induce lamprey metamorphosis. Expriment 4 was designed to ensure that goitrogen- induced metamorphosis in sea lampreys was due to the goitrogenic anion (Le., CIO4- or SCK

) and not to the effects of An NaC1O4 experimentai group was also included to verify that the observed effects were not related to an interaction between the effects of K+and the goitrogenic anion.

Treatment with 18 mM KCI resulted in the death of di sea lampreys within 4 days-

The high mortality rate within this treatment group rnay be reiated to an increase in the osmolarity of the aquarium water. In general, larval lampreys cannot osmoreguiate even in dilute sea water (Le., 10 "1, NaCl; Mathers and Beamish. 1974) and potassium ions may

exacerbate this osmoregulatory difficulty. Death was not observed in any other experimental

group over the course of Expriment 4. In the L-KCL experimental group larvae were

exposed to 3.6 mM KCI, the same concentration of K? that is present in a 0.05% KC104

treatment. At this concentration, KC1 had no effects on either the incidence of

metamorphosis or semai TH concentrations (Figs. 14 and 15)- Semm TE5 concentrations in

the KClO4 and NaCI04 experimentai groups were sigaificantiy lower than ail other experimentai groups (Fig- 15). Associated with these declines in serum TH concentrations

was an induction of metamorphosis. In the KC1a and NaCl04 experimental groups, 33 and

89 % of sea lampreys initiated metamorphosis in the 110 - 119 mm and > 119 mm size groups, respectively (Fig. 14). NaCIO4 has the same effects as KClOs on both senun TH concentrations and metamorphosis, whereas KC1 had neither of these effects. These results indicate that CL04- and not r,is the ion responsible for depressing serum TH concentrations and inducing metamorphosis in lampreys.

Zn summary, the present results show that the induction of precocious rnetamorphosis in larval sea lampreys is associated with a decline in serum TH concentrations following treatment with a goitrogen. The incidence of goiuogen-induced metamorphosis in sea lampreys is related to the ability of the goitrogen to lower serwn TH concentrations, mer supporting the claim by Manzon et al. (1998) that KCI04-induced metamorphosis requires a decline in serum TH concentrations. Despite the absence of metamorphosis foilowing FKJ treatment, 1 feel that the induction of metamorphosis is a response to goitrogens in general and is not dependent on the mechanism by which a goitrogen reduces the capacity to synthesize TH. This idea is supported by the observation that MMI, a thyroid peroxidase- inhibiting goitrogen, can induce Iamprey metamorphosis. Pï'U may be unique among goitrogens by having unlaiown effects that prevent Iamprey metamorphosis. Lastly. K+does not play a role in KCa-or KSCN-induced metamorphosis. Table 3. BaseIine and experimentai groups in four separate experiments, the nominai ambient aquarium concentration of the various experhentaî treatments, and mean sea lamprey (Petromyzon- &nus) size at the time of sampIing- Group Mean animal size at sampiîn& Treatment concentration

I-

Im Baseline Conuol PTU FïUtT3 T3

Baseline Control rn ETU +T3 -- T3 2 (> 129 mm) Control NaOH -- PTU Baseline Control L-KCL04 H-KC104 L-KSCN H-KSCN L-MM1 -- H-MMI 4 (110-119mm) Baseline Control KCIO~ NaCI04 L-KC1 E-KCL

(> 119 mm) Baseline Control KCIO4 NaCl04 L-KCL H-KC1 18 mM N, sample size at onset; SE, stai hyronine; NaOH, sodium hydroxide; L, low; H, high; KCI04, potassium perchlo&; KSCN, potassium thiocyanate; MMI. methimazole; NaC104, sodium perchlorate; KCI, potassium chloride * Animal sizes between groups within a size group are statistically different (P > 0.05) if labelled with different letters (a,b,c). Table 4. Cornparison of mean semm thyroxine and tniodothyronine concentrations in larval (A) and metamorphosing (M) sea lampreys (Petromyzon marinus) foUowing various goitrogen treatments (Expriment 3)-

Thyroxine (nmovliter) Group Baseline

Control

L-KCIO~~

H-KC104

L-KSCN

H-KSCN

L-MMP

H-MM1

AM, mean of combined larval and metamorphosing data; L, low; H, high; KClo4, potassium perchlorate; KSCN, potassium thiocyanate; MMI, methunazole * Values in parentheses are sample size and two standard errors of the mean, respectively. * Significantly different fiom larvd values within a group. Treatment length was 16 weeks for al1 gmups except the MMI groups which received only 6 weeks of treatment. Figure 11. Mean (f: 2 standard errors) serum thyroxine (T4;A and C) and tniodothyronine

(T3; B and D) concentrations in sea lampreys of two different groups (based on length).

Lampreys were either untreated as in the baseline (start of expriment) and control groups, or treated with propylthiouraciI(10 mglliter; FTU), PrCl and T3 (10 mg/liter + lmgniter;

PTUtT3), or T3 (lmg/'Iiter) for 23 weeks. Serum T4 and T3 concentrations are signincantly different (P < 0.05) if IabeIIed with different letters. Sample size for each group is iadicated in parentheses below the abscissa.

Figure 12. Stage and total number of metamorphosing sea Lampreys in untreated (control) and goitrogen-treated individuals foiiowing 6 weeks (A) or 16 weeks (B)- Goitrogen treatments included: methimazole (MM.9, potassium perchlorate (KC104), and potassium thiocyanate (KSCN); each goitrogen was administered at a high (H) and low (L) concentration (see Table 3). Sample size is equal to 30 unless otherwise indicated in parentheses below the abscissa. 1 stage 1 stage 2 stage 3 stage 4 I 1 stage 5 stage 6 stage 7 I

Control L-KCIO4 KKC104 L-KSCN H-KSCN Figure 13. Mean (* 2 standard errors) semm thyroxine (T4) and triiodothyronine f13) concentrations in sea lampreys. Lampreys were either untreated as in the baseiine (start of experiment) and control groups, or ueated with potassium perchlorate (KC104) or potassium thiocyanate (KSCN) for 16 weeks, or methimazole @dMI) for 6 weeks. Goitrogen treatment concentrations were either high (H) or low (L) as indicated on the abscissa (see Table 3).

Sample size is equal to 30 unless otherwise indicated in parentheses below the abscissa.

Concentrations labelled with different letters are signi£icantly different (P c 0.05).

Figure 14. Stage and total number of metarnorphosing sea lampreys in untreated (control) and treated individuds fkom two different size groups (based on length). Treatments included two goitrogen experimental groups (potassium perchlorate [KC104;3.6 mM] and sodium perchlorate maC104; 3.61nMJ), and a low potassium chloride (L-KCI; 3.6 mM) experimental group. Sample size for each group is nine. stage 1 stage 2 stage 3 11 stage 4

A 110-119 mm size group

Control KC104 NaC104 LXCl

B > 119 mm size group

Control KCIW NaC104 L-KCI Figure 15. Mean (k 2 standard errors) senun thyroxine (T.4) and tniodothyronine (T3) concentrations in untreated (control) and treated sea lampreys fÎom two different size groups

(based on length). Treamients ïncluded two goitrogen experimental groups (potassium perchlorate EC104; 3 -6 mM'J and sodium perchlorate WaClO4; 3.6 mm),and a Low potassium chloride (L-KCI; 3.6 mM) experimental group. Sample size for each group is nine. Concentrations are significantly daerent (P c 0.05) if Iabelled with different letters.

TEMPERATURE AND KCI04-INDUCED METAMORPHOSIS IN THE

SEA LAMPREY (Pefromyzon marinus).

The majority of information presented in this chapter has been modified from:

"Temperature and KC1O4-induced metamorphosis in the sea Lamprey (Peiromyzon

marinus)" by R.G. Manzon, and J.H. Youson, in Comparative Biochemistry and

Physiology, Volume 124C, 253-257, copyright O 1999 by Elsevier Science,

reproduced with permission fiom the publisher. LarvaI sea Iampreys (Petromyzon marinus) were exposed to either a warm (18 OC) or

a cold (3 OC) water temperature, either with (treated) or without (untreated) potassium perchlorate (KC1O4)- Foiiowing 23 weeks, larvae were examined for signs of metamorphosis and serum samples were coIIected to assay thyroxine m) and 3,5,3'-tniodothyronine (T3) concentrations, Water temperature did not significantly affect semm Tqor T3 concentrations in untreated Iarvae and metamorphosis did not occur in these groups, Semm Tq concentrations were not significantly diffierent between the tsvo temperature groups treated with KC104. However, serum T3concentrations were significantly higher in the cold water,

KC104-treated larvae (5-4 mgniter) than in the wann water, KC104-treated larvae (1.2 rngiliter). KC104 treatment at a warm water temperature induced metamorphosis in all larvae and resulted in senun T4 and concentrations which were 66 and 95 % lower, respectively, than untreated larvae in warm water. Despite having significantly lower serum T4 and T3 concentrations (73 and 80 %, respectively) than untreated cold water Iarvae, metamorphosis was not observai in cold water, KC104-treated larvae. The resuits of this study indicate that warm water is a requirement for the successful induction of metamorphosis with KClOa, and provide further evidence of water temperature as an important factor in the metamorphosis of lampreys. INTRODUCTION

The Iife cycle of the sea Iamprey (Petromyzon marinus) includes a sedentary, filter- feeding, larval period lasting 3 - 7 years and a true metamorphosis in which larvae undergo many morphologicd and physiological changes in preparation for a free-swimming, parasitic, juvenile penod (Youson. 1980, 1988). Numerous snidies (see Youson, 1994, 1997) have investigated the environmental and physiological factors which potentiaily influence the onset, rate, and incidence of metamorphosis in Iampreys- Included among these factors are temperature, lipid reserves, and thyroid hormones 0.

Temperature is the predominant environmental factor iduencing metamorphosis in larnpreys. Studies have indicated that temperature can affect the rate, the time of onset, and the incidence of spontaneous metamorphosis in Iarval sea larnpreys (Manion and Stauffer,

1970; Potter, 1970; Purvis, 1980; Youson et ai-,1993). In the aforementioned studies, warm water temperatures (21 OC) were more favourab1e for metamorphosis than cool water temperatures (13 OC).

Throughout the larval growth phase in sea larnpreys, serum thyroxine (T4)and 353'- triiodothyronine (T3)concentrations graduaily rise and reach their peak just prior to the onset of metamorphosis (Youson et al., 1994). Subsequent to a peak in serum TH concentrations and concomitant with early metamorphic change, serum TH concentrations decline rapidly

(Wright and Youson, 1977; Lintlop and Youson, 1983; Youson et al., 1994). The significance of this peak and decline is not Myunderstood, but several experiments suggest that they are important for metamorphosis, Treatment of larval sea larnpreys, including larvae smaller than the minimum size required for spontaneous metamorphosis, with potassium perchlorate (KCLO4) depressed semm TH concentrations and induced metamorphosis at a time of year when spontaneous metamorphosis does not occur (Holmes and Youson, 1993; Youson et al., 1995). Furthemore, KCL04-induced rnetamorphosis can be blocked with exogenous Tq or T3 treatments (Manzon and Youson, 1997; Manzon et al.,

1998), and T3retards spontaneous rnetamorphosis (Youson et al., 1997). These KC104 treatment studies were conducted at nonseasonai, warm, water temperatures (16 - 20 OC) in the winter months when spontaneous metamorphosis does not occur. The effects of temperature on the induction process have not been investigated.

The primary objective of the current study was to determine if wam water is required for KCL04 treatment to induce metamorphosis. Another goal was to investigate the effects of warm and cold water conditions in the presence and absence of El04on serum TH concentrations.

MATE,- AND METHODS

Larval sea lampreys (Petromyzon marinus) greater than 120 mm in length were collected fiom Puaiam Creek, New York in Iune 1995 and housed at a temperature of approximately 11 OC at the University of Toronto at Scarborough (Manzon et al., 1998). On

October 10, larvd sea lampreys were removed in groups of five nom a holding tank (without substrate) containhg 150 individuals. The lengths and masses of these larvae were recorded, and larvae were assigned to one of 4 experimental aquaria (21 liter; 40 X 20 X 25 cm) until each aquarium contained 10 larvae. Aquaria were maintained static (not on a flow-through system), and contained 12 liters of dechiorinated tap water and 6-7 cm of industrial sand for substrate, Water in the expenmentai aquaria was aerated and al1 aquaria were maintained on a 15 h light : 9 h dark photoperiod throughout the study. Each aquarium was assigned one of four experimentai groups: untreated cold water (untreated cold); untreated warm water (untreated warm); potassium perchlorate- (KClO4) treated cold water (cold KCIO); or

KC104-treated warm water (warm KCI04). Animds and experimenial groups were randody assigned to aquaria by lottery- Larvae were Ieft undisturbed in their new environment for 1 day prior to any change in temperature and 8 days @or to the addition of KC104, Water temperature in the cold water groups was graduaiiy lowered by approxïmately 2 OC per day until a temperature of 3 OC was attained (typicd winter temperatures for Putnam Creek [l - 4

OC];JE. Gersmehl, personal communication). CoId water temperatures were achieved using a circulating water chiller (Frigïd Units) to regulate the water temperature in a large insuIated tank which was used as a bath to maintain the temperature of the experimental aquaria.

Water temperature in the wami water groups was maintained at ambient room temperature

(16 - 20 OC). The experiment was initiated on October 17 when KCI04 was added to the appropriate aquaria. Potassium perchlorate was administered fiom a stock solution (86 mM) to achieve a find nominal ambient concentration of 3.6 mM (0.05%)-

The instantaneous water temperature in each of the untreated aquaria was recorded twice daily, larvae were fed 100 ml of a Fleischrnann's baker's yeast solution equd to 1 g of yeast,animai/week (Mallatt, 1983), and water was added to the aquaria as needed to keep the total volume at 12 liters. Aquaria were cleaned, the water was changed, and fresh treatments were added every two weeks without disturbing the burrowed larvae. Dead larvae were removed and replaced using animals marked with a latex dye injected into the caudal sinus; marked animals were excluded from all analyses, Eight weeks fiom the onset of the study al1 sea Iampreys were anesthetized in a solution of 0.05% tricaine methanesulfonate and examined for extemal signs of the seven stages (1 to 7) of metamorphosis (Youson and

Potter, 1979). The expriment was terminated 23 weeks fiom its onset at which thne sea lampreys were anesthetized, their Iengths, masses, and metamorphic stages were recorded and serum was coilected (Manzon and Youson, 1997)-

Total serum Te and T3 concentrations were measured using the Amersham AmerIex

TT4 and TM radioimmunoassay (RIA) kits, respectivefy, The kits were modified for sea lamprey serum TH concentrations and smdworking volumes (Leatherland et al*,1990), Ail serum sarnples were assayed in duplicate; assay sensitivities were 7 and 0.4 mg/liter for Tq and T3,respectively, and intra- and inter-assay variances were 9.4 and 11.8 % for Tq, and 7.2 and 12.9% for T3,respectively,

Differences in animal length and mass, and serurn Tgand T3 concentrations between aquaria (groups) were tested for statistical signiflcance using andysis of variance (ANOVA) and Tukey's HSD multiple cornparison test- Prior to ANOVA analyses, di data were tested for heteroscedasticity of variances using Cochran's Q test. Data which did not meet the assumption of homoscedasticity were transformed (logio);subsequent to transformation ail data satisfied this assumption (Sokal and Rohlf, 198 1). Al1 statistical calculations were performed using Statistix for Microsoft DOS, data are presented as mean + 2SE, and al1 differences were considered statisticaiiy significant if P < 0.05.

RESULTS

Mean water temperatures in warm and cold water aquaria were 18 t 0.17 OC and 3 +

0.07 OC, respectively- The mean length and mass of al1 larval sea lampreys at the onset of the study were 138 k 4 mm and 3.89 + 0.38 g, respectively, and these size parameters did not differ significandy among groups. Additionaüy, significant differences in animal length and mas were not observed among the groups at the termination of the expriment when the overalI mean animal size was 137 + 4 mm and 3-70 + 0.37 g.

Eight weeks fiom the onset of the study, metamorphosis was not observed in larvae from the untreated wann, untreated cold, or cold KCI.OQexperïmentai groups- However, at this time 80% of the warm KC1O4 larvae had commenced metamorphosis. Five animals were in stage 1 of metamorphosis, three in stage 2 of metamorphosis, and the remaining two individuals were larvae. At the end of the study (foliowing 23 weeks of treatment), metamorphosis was still not observed in any untreated or cold KC104 larvae. There had been some mortality of unknown causes in the warm KC104 experimental group but aiL surviving

(8/8) animais were in the process of metamorphosis (Fig. 16). Of these metamorphoshg individuals, one was at stage 1, two between stages 3 and 4, one at stage 4, three at stage 5, and one at stage 6 of metamorphosis. Warm KC104 larvae were clearly undergoing metamorphosis, but the timing and morphology of KCiû4-induced development was asynchronous. In several individuals the eyes, teeth, andior body coloration had undergone changes in advance of structures such as the oral disc and branchiopores. In spontaneous metamorphosis the changes in the morphology of these structures are highiy synchronized within and among individuals (Youson and Potter, 1979).

Serum T4 and T3concentrations of untreated larvae were not significantly affected by temperature. Mean semm concentrations were 92 and 100 nmoVliter for T4,and 23.7 and

27.6 nmoVliter for 4 in the untreated warm and cold larvae, respectively (Fig. 17).

Sirnilarly, serum Tq was not affected by temperature in the presence of KCI04; mean concentrations were 3 1 and 26 nmoVLiter for the warm and cold KCl04 groups, respectively

(Fig. 17A). KC104 treatment signincantly depressed semm T4 concentrations in both warm and cold KC104 Iarvae relative to untreated warm and coId Iarvae- Warm and cold KCI04 larvae had serum T4 concentrations 66 and 73 % Lower than their respective untreated warm and cold larvae. KCl04 treatmeot also signincantly depressed senun Tjconcentrations in both warm and cold KCI04 Iarvae relative to untreated wann and cold larvae, Serurn T3 concentrations in warm and cold KC104 larvae were 95 and 80 % lower than untreated warm and cold Iarvae, respectively. Furthemore, mean serum T3 concentrations in warm KC104 larvae (1.2 nmolfiter) were significantly lower than in cold KC104 larvae (5.4 nrnoVIiter;

Fig- 17B).

DISCUSSION

The goitrogen KC104 induces precocious metamorphosis and a concomitant deciine in serurn TH concentrations in larval sea lampreys (Holmes and Youson, 1993; Manzon and

Youson, 1997; Manzon et al., 1998; Youson et al-, 1995). Temperature and time of year were controlled in these studies, which were conducted during the winter months at warm

(summer) water temperatures (16 - 20 OC). In the current study, larval sea tampreys were exposed to either a warm or cold water temperature, equivalent to summer and winter conditions respectively, in the presence or absence of KClO4. The expenment was conducted at a time of year when spontaneous metamorphosis is not expected. Exposure of larvae to KC104 at warm water temperatures signincantly depressed serwn TH concentrations and induced metamorphosis in 100%of al1 surviving individuals. Mortality was due to causes seemingly unrelated to the treatment. These results with KCL04 treatment at a warm water temperature are similar to those of earlier studies (KoImes and Youson,

1993; Manzon and Youson, 1997; Manzon et al., 1998; Youson et al., 1995). However,

KC104 treatment at a cold water temperature did not induce metamorphosis in any larvae

134 despite significant declines in both semm T4 and T3 concentrations, The absence of

metamorphosis in cold KC104 larvae Meremphasizes the importance of water temperature

in the initiation of lamprey metamorphosis and îndicates that a decrease in serum TH

concentrations is no t sufncient to induce metamorphosis, G-induced metamorphosis in

amphibians is also temperature dependent Frieden et al- (1965) and Ashley et al. (1968)

found no change in fiog taii length or urea production, respectively, following T3 treatment at

5 O C but responses were detected at temperatures 2 7-5 O C.

Semm T3concentrations in warm KC104 lanrae were significantiy lower than serum

T3 in cold KC104 larvae, Furthemore, the magnitude of the declines in serum T3

concentrations fiom values in the respective untreated Iarvae were greater for warm KClo4

larvae (95 %) than coId KC104 larvae (80 96). Despite the differences in serum T3

concentrations between warm and cold larvae, it is not iikely that these differences

contributed to the absence of metamorphosis in cold KC104 larvae. In two previous studies,

metamorphosis was observed in larval sea lampreys treated with KC104 at a warm water

temperature in which serum T3 concentrations were onIy 72 to 77 % lower than values for untreated control larvae (Manzon and Youson, 1997; Manzon et al., 1998)- These declines in serum Tj levels are similar to those observed in cold KCL04 larvae of the cwrent study.

The absence of metamorphosis in cold KC104 larvae may have been a result of the effects of the cold water temperature on regulatory processes of metamorphosis other than serum TH concentrations.

Many biologicd processes essentid to life in poikilotherms are dependent either directly or indirectly on environmental temperature which has been shown to alter both basal rnetabolic rate and the rate at which developmental processes proceed (Brett, 1956)- In lampreys metabolic rate, measured as oxygen consumption, decreases with decreasing temperature (see Lewis, 1980; Randall, 1972)- Cold water temperatures may reduce the metabolic rate in lampreys below a minimum required for metamorphosis.

We have previously shown that Km4treatment at warm water temperatures does not affect intestinal Tqouter-ring (5') deiodination to T3(WRD), the primary site and deiodination pathway, in larval sea lampreys (Manzon et al-, 1998). However, changes in deiodinase activity coincide with changing developmental and physiologicd state in lampreys (Eales et al,,1997, 1999)- Although KC104 treatment at warm water temperatures did not alter intestinal T40RD activity (Manzon et al-, 1998), perhaps the activities of this or other deiodinase pathways were sufficiently altered in cold KC104 larvae of the curent study to prevent the induction of metamorphosis. Temperature affects TH deiodinase activity in rainbow trout (Johnston and Eales, 1995)- Furtherrnore, Kaltenbach (1996) posed the question: Does temperature alter the number of TH receptors in anurans? Could this question also apply to lampreys? Future research should be directed towards elucidating the function of the TH deiodinase pathways and TH receptors in metamorphosis and their responses to temperature change-

The initiation of lamprey metamorphosis occurs when a number of physiological and environmental requirements are met (Youson, 1994). KC104 depresses serum TH concentrations and can override some of these requirements such as size, condition factor, essential fat stores, and thyroid status resulting in the induction of metamorphosis (Youson,

1994). However, warm water temperature cannot be eliminated fiom the sequence of events involved in the initiation of lamprey metamorphosis. Figure 16. Stage and incidence of metamorphosis in untreated and potassium perchlorate-

(KC104; 0.05%) treated larval sea lampreys (Petromyzon marinus) at warm (18 OC) and cold

(3 OC) water temperatures after 23 weeks. Metamorphosing sea lampreys which were between stages were assigned the earlier of the two stages. Sample size is ten for ail groups with the exception of the warm KC104 group where N is eight. stage 1

Stage3

staga 4

Stage 5

stage 6

Untreated cold Cold KClO4 Untreated warm Wann KClOq

Groups Figure 17. Mean (k 2 standard errors) senun thyroxine (x;A) and 3,5,3'-tniodothyronine

(T3;B) concentrations in untreated and potassium perchlorate- (KC104; 0.05%) treated larval

sea lampreys at warm (18 OC) and cold (3 OC) water temperatures after 23 weeks.

Differences in serum Tq and T3 concentrations between groups are statistically significant (P

< 0.05) if labeled with different letters. Sample size (N) is ten for all groups with the exception of the warm KC104 group where N is eight.

KC104 INHIBITS THYROIDAL AClWITY IN THE LARVAL

LAMPREY ENDOSTYLE IN VITRO. ABSTRACT

An in vitro experimental system was devised to assess the direct effects of the goitrogen, potassium perchlorate (KCLO4), on radioiodide uptake and organification by the

Iarval lamprey endostyle- OrganZication refers to the incorporation of iodide into lamprey thyroglobuiin (Tg)- Histological and biochemical evidence indicated that the endostyles were viable at the termination of a four how in vitro incubation- A single iodoprotein, designated as lamprey Tg, was identified in the endostylar homogenates by polyacrylamide gel electrophoresis and Western blotting- Lamprey Tg was immunoreactive with a rabbit anti-human Tg antibody and had an electrophoretic mobility similar to that of reduced porcine Tg. When KClO4 was added to the incubation medium, both iodide uptake and organification by the endostyle were inhibited, as determined by gel-autoradiography and densitometry. Western blotting showed that KC104 lowered the total amount of lamprey Tg in the endostyle. Based on the results of this in vitro investigation, 1conclude that KC104 acts directly on the lmal lamprey endostyle to inhibit thyroidal activity, These data suppoa a previous supposition that KClOa acts directly on the endostyle to suppress the synthesis of thyroxine and triiodothyronine, resulting in a decrease in the senun levels of these two hormones. The larval lamprey endostyle, which gives rise to follicular thyroid tissue during

metamorphosis, is the site of thyroid hormone (TH) synthesis (ETardisty and Baker, 1982).

ExperimentaI, his toIogical, and biochemicd evidence support the idea that the mechanism of

TH synthesis in the lamprey endostyle is similar to that obsemed in foUicuiar thyroid tissue,

HistoIogicaI autoradiography at the light and eIectron microscope Ievels has shown that

radioiodide (LtS~ is concentrated and bound in several endostyle cell types. with cell types

2c and 3 being the primary iodide binding celis (Figs. 1B and 18; for review see Barrington

and Sage, 1972; Wright and Youson, 1976). These type 2c and type 3 cells are

immunoreactive with an anti-human thyroglobulin cg)antibody (Wright et al., 1978) and

also have peroxidase activity (Tsuneki et al., 1983). Biochemical studies have shown that

radioiodide, taken up by the endostyle, is organified (Le., incorporated) into the proteln

fraction and iodoproteins of different sizes and sedimentation coefficients have been

identified. ïncluded among these proteins are ones that are similar in size to marnmaiian Tg

(S uzuki anci Kondo, 1973; Monaco et al., 1978). Lastly, hydrolysis studies have confirmed

that these iodoproteins contain iodothyronines, TH, and the TH precursors monoiodotyrosine

(MIT) and diiodotyrosine (DIT) (Salvatore, 1969; Suzuki and Kondo, 1973). These data

indicate that the necessary components for TH synthesis, namely an iodide-concentrating

mechanism, a Tg-like molecule and peroxidase activity, can be localized to particular ce11 types in the larval lamprey endostyle.

The processes involved in the regulation of TH synthesis by the lamal lamprey endostyle are less clear. Studies conducted to date have not confirmed whether or not the larval endostyle is regulated by the hypothafamic-pituitary axis (Knowles, 1941; CIements- Merlini, 1962b; Barrington and Sage, 1963% b, 1966; Pickering, 1972). Anti-thyroid agents

(goitrogens), known to inhibit either iodide uptake or organification in follicular thyroid tissue, alter both the functional and morphological aspects of the larval endostyle. Overail, the data from these goitrogen studies are not consistent (Salvatore, 1969) and are difficult to interpret. Clements-Merlini (1962b) found that the goitrogens thiourea and thiocy anate inhibited radioiodide accumulation and that thiourea inhibited the formation of iodinated tyrosines, In addition, reports fiom several studies indicate that thiourea and thiouracil alter the functional morphology of ail endostyle celi types including those not involved in thyroidal activity (Banington and Sage, 1963%b; Barrington and Sage, 1966).

More recent studies have focused on the ability of goitrogens to lower semTH concentrations and induce precocious metamorphosis in lampreys (Hoheisel and Sterba,

1963; Suzuki, 1986, 1987; LeatherIand et ai., 1990; Holmes and Youson, 1993; Youson et al., 1995; Holmes et al., 1999; Chapter 3). These studies have suggested that the induction of metamorphosis is Linked to a decline in semm TH concentrations. Furthemore, Manzon et al. (1998) have shown that potassium perchlorate- (KC104) induced metamorphosis cm be blocked by elevating semm TH concentrations with exogenous TH. They concluded that a decline in semm TH concentrations is mandatory for the induction of precocious metamorphosis. However, whether goitrogens, such as KCI04, brhg about a decrease in serum TH concentrations and metamorphosis by: i) affecting some pmcess underlying both

TH titers and metamorphosis; ii) a direct effect on the endostyle; or iii) causing a generalized chronic debilitation has yet to be dete-ned. A partial solution to this problem can corne fiom determining if goitrogens can act directiy on the endostyle to reduce its ability to synthesize TH. Numerous studies have investigated the effects of goitrogens on endostyle

morphology, senun TA concentrations and metamorphosis in larval lampreys. Aowever,

Little is known about the direct effects of goitrogens on TH synthesis by the larvai endostyle.

One aim of this study was to develop an in vitro experimental system to investigate the mechanisms that goitrogens use to alter TA synthesis by the larval endostyle, resulting in the depression of senun TH concentrations rit vivo- My primary goal was to use this itt vitro system to determine the effects of KC104 on the uptake and organification of iodide by the larval endostyle. These data are valuable to our understanding of KC104-induced metamorphosis in lampreys (see Manzon and Youson, 1997; Maozon et al., 1998).

MATERIALS AND METEODS

Animais

Larval sea lampreys (Petromyton marinus) were coUected fiom the Harris River in

Port Perry, Ontario in May 1999- Larvae were transported to the University of Toronto at

Scarborough and were housed in fiberglass aquaria at seasonal water temperatures (Manzon et al., 1998). Sea lampreys fiom this population which did not metamorphose in the summer months, but were greater than 120 mm in length, were used in all in vitro expenments on this species except those conducted for light microscopy. Experirnents conducted for light microscopy were performed in the fall of 1998 on larval Amencan brmk lampreys,

Lampetra appendlx, because sea lampreys were not available. American brook lampreys greater than 130 mm in length were collected fkom Duffins Creek in Ajax, Ontario in

October 1998 and housed as describeci above. It has been shown that KClO* also depresses serum TH concentrations and induces metamorphosis in L appendix (Holmes et ai., 1999). In vitro expen'mentdprotocol

Five larvaï Iampreys were anaesthetized in 0.025 % tricaine rnethanesulfonate (MS-

222, Syndel Laboratories Ltd., Vancouver, British Columbia, Canada). The endostyfe

(subpharyngeal gland) from each lama was removed with minimal amounts of surrounding

connective tissue. To simpiiQ tissue processing, endostyles used for light microscopy were

removed with the ventral body wall attached. Each endostyle was rinsed in ice-cold

incubation buffer @) (1 10 mM NaCI, 1.9 mM KCl, 5 mM NaEKO3,lO mM HEPES, 5 mM glucose, 1.1 mM CaC12, 0.6 mM MgC12, 0.2 % bovine serum albumin, 0.006 % penicillïn G,

0-01% streptomycin sulfate; pH 7.4) (lto et al., L988), drained on a Kimw-ipe, weighed, and placed in a 25 ml Erlenmeyer flask containing 3 ml of ice-cold IB.

The in vitro experimental protocol used in this study was adapted from the protocol used by Ito et al. (1988) for the incubation of lamprey liver slices. In brief, 25 ml flasks containing five endostyles (4 - 6 mg each) and 3 ml IB were sealed with a rubber stopper containing an inlet and outlet made fiom syringe needIes (16 guage) to allow for continuous

Aushing with a 95 % 02: 5 % CO2 gas mixture during the incubation. The flasks were placed in a reciprocal water bath shaker at 25 OC and shaken at 70 strokes per minute.

Foilowing a 15 minute equiübration period, either 0.3, 3 or 30 pCi of carrier-& ~a'q

(NEN Life Science products, Boston, MA, USA) was added to the flask. The incubation was carried out for four hours either in the presence (treated) or absence (untreated control) of

KC104 (Table 5). A Iow (L-KC104, 0.72 mM) or high (H-KC104, 3.6 mM) dose of KC104 was added to the appropriate incubation flasks irnmediately prior to the addition of N~'~'I.

After the four hour incubation period, endostyles were washed for 45 minutes in 7 -

10 changes of IB containing 10 mM KI (to prevent türther incorporation of Pilot experhents indicated that this washiag protocol removed most of the unincorporated 12'r,

since the gamma radiation emitted by the wash buffer fiom the final two changes was

comparable to background levels. The gamma emission rate of each endostyle was then

rneasured in counts per minute (cpm) using a Beckman 4000 gamma counter to determine the

arnount of taken up by the endostyle. For each experiment, the mean cprn of KC104-

treated endostyles was expressed as the percentage of the mean of untreated control

endostyles, Subsequent to gamma counting, endostyles were either fixed in Bouin's fluid for

light microscopy or flash fiozen in liquid nitrogen and stored at -86 OC for electrophoretic

anaiysis.

Endostyles that were incubated with 3 pCi of N~'~'Iin the presence and absence of

L-KClO4 were fixed in Bouin's fluid for 24 hours, dehydrated in a graded series of ethanols, cleared in Histo-Clear @iaMed Lab Supplies Inc., Mississauga, Ontario, Canada) and embedded in Tissue-Prep Pisher Scientinc, Whitby, Ontario, Canada). Tissue was serially sectioned (6 pm) and mounted on chromic-sulfuric acid cleaned slides (Chromerge, VWR

Scientific, Mississauga, Ontario, Canada). Slides used for light microscopie autoradiography

(LM-autoradiography) were hydrated, oxidized in 0.5% aqueous periodic acid, rinsed in distilled water, air dried and dip-coated with Kodak NTB-2 nuclea.track emulsion (Kodak,

Rochester, NY, USA) (Kopriwa and Leblond, 1962). The emulsion was exposed at -20 OC for 7 - 14 days, developed in Kodak D-19 Developer, rinsed in water and fixed in Kodak

Fixer. Developed slides were stained with Liiiie' s "cold Schiff reagent (see Sheehan and

Hrapchak, 1980), counter-stained with Mayer's acid haemalum (modified by Lillie, L942) and 2 % aqueous orange G, and mounted with Pro-Texx mounting media (Baxter

Diagnostics Inc., Deefi~eld,IL, USA). The LM-autoradiography micrographs for endostyles

incubated with KCI04 are not presented because these tissue sections were coated with an

emulsion that was either contaminated or exposed to heat/light, The expense of replacing the

emulsion was prohibitive to repeating the LM-autoradiography experiments for these

sarnples. Experimental tissue sections that were not dip-coated were processed as above

except these slides were transferred fiom the distilled water rinse following oxidation

directly into Lillie's "cold Schiffreagent,

In addition, severai endostyles were fmed immediately after excision to serve as

unincubated controls and were prepared for Iight microscopy as described above. These endostyles were used for cornparison with incubated endostyles to determine if the four hour incubations had any adverse effects on endostyle morphology and cellular structure. Some of these unincubated control endostyIes were excised in the same manner as the endostyles used for homogenization (Le., without the ventral body wall attached) and were processed for

Light microscopy. This procedure was carried out to ensure that the endostyles used for electrophoretic analysis were intact and contained minimal extraneous tissue.

Protein samples were prepared by homogenizing 5 endostyles in 1 ml of lysis buffer

(20 rnM Tris-HC1 [pH 7.5],300 mM NaCl, 1 % Igepal CA-630 monidet P-401,O. 1 % sodium dodecyl sulfate [SDS], 0.5 % sodium deoxychoIate, 57 mM phenylmethylsulfonyl fluoride). The homogenate was centrifbged at 13,000 g for 30 minutes at 4 OC to remove particulate matter and the supernatant was coliected, The protein concentration of each sample was determined ushg the Bradford protein assay (Bradford, 1976). Samples were aiiquoted and stored at -80 OC until they were used for SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Samples were prepared for electrophoresis by mïxing an equd volume of the protein sample with sample buffer (LOO mM Tris-HCl, 200 mM dithiothreitol, 20 % glycerol, 4 % SDS, 0.002 % bromophenol blue) and immersing the mixture in boiling water for three minutes- When necessary, protein sampbs were diluted to ensure that the total amount of protein (IO or 15 pg) and the volume (40 or 50 pl) loaded onto a given gel were the same for different protein samples.

Sodium dodecyt sulfate - polyacrylamide gel electrophoresis was performed according to the method of Laemmli (1970) using 5 % stacking and 7.5 % resolving gels. In addition to the endostyle homogenates, broad-range molecular weight markers (Sigma, St.

Louis, MO) and 0.1 or 10 pg of porcine Tg (Sigma) were loaded onto each gel. The porcine

Tg used was electrophoreticaliy heterogenous, consisting of numerous fragments of porcine

Tg likely of proteolytic ongin, as supplied by the manufacturer. Further pwiiication of this porcine Tg was not pedormed. Foiiowing electrophoresis, gels were processed either for autoradiography (gel-autoradiography) to determine the amount of incorporated into thyroglobulin or for Westem bloning to determine the amount of thyroglobulin in KCIO4- treated endostyles relative to untreated controls. AU protein samples fiom an individuai in vitro experiment were always nui on the same gel. This process was repeated in quadruplicate for gel-autoradiography and in duplicate for Westem blotting for in viîm experiments designated 1 - 4 (Table 5).

Gels processed for autoradiography were stained with Coomassie brilliant blue G-

250, destained, dried onto filter paper and exposed to X-ray film (X-OMAT AR; Kodak) at -

86 OC for 6 - 72 hours. Film was developed in Kodak GBX Developer, nnsed in water, and fuced in Kodak Fixer, Gels processed for Western blotting were equilibrated in transfer

buffer (25 mM Tris, 192 mMglycine, 20 % methanol) for 1hour and the proteins were

electrophoreticaliy transfed to nitroceliulose flowbin, 1979). Nitroceliulose blots were

stained with Ponceau S to q~a~tatlvelyassess equal loading and transfer efficiency,

destained in distiIIed water, and blocked oveniight in a solution of 5 % dried skim milk in

Tween-Tris buffered saline (10 mM Tris-HCl, 250 mM NaCl, 0.05% Tween-20; pH 7.5).

Blots were incubated for 1 hour in a L:1000 dilution of an IgG-purified rabbit anti-human

thyroglobulin antibody (A 0251; Dako Diagnostics, Mississauga, Ontario, Canada) followed by a 1: 10diIution of horseradish peroxidase-conjugated secondary antibody (anti-rabbit

IgG, Amersham-Pharmacia, Piscataway, NJ, USA), Immunoreactive bands were visualized on X-ray film (Dupont Cronex MRF34; Marconi Medicai Systems, Brampton, Ontario,

Canada) using a cherniluminescence kit (ECL; Amersham-Pharmacia, Oakville, Ontario,

Canada) according to the manufacturer's instructions. Film was processed as indicated above.

Molecular mass was estimated by plotting the relative mobility (RF)of each Sigma molecuIar weight marker versus the logroof its molecular mass. The resultant graph produced a linear standard curve. The equation of this standard curve was detecmined using

Cricket Graph III for the MacIntosh and was used to estimate the molecular rnass of lamprey

Tg and the two porcine Tg bands based on their RFvalues. Molecular masses presented are the average value of the estimates obtained fiom seven different gels.

Densitometry was performed on gel-autoradiograms and Westem blot films using a

BioRad Imaging Densitometer (Mode1 GS 700) and Multi-Analyst software (Version 1.1, BioRad, Mksissauga, Ontario, Canada). uidividual bands on a single piece of nIm were selected and adjusted for differences in background and the denved value was expressed as the adjusted optical density X mm2 (OD). The ODs of bands fiom KCLOe-treated endostyles were expressed as a percentage of the ODs of the appropriate untreated controls. This process was repeated a total of eight times using two different pieces of film for each gel.

For each in vitro experiment 1electrophoresed four replicate gels for gel-autoradiography and two replicate gels for Western blotting. The mean values (+ 2 standard errors) from dl replicate gels fiom each in vitro experiment and the overd mean (& 2 standard errors) of the four in virro experiments are presented.

RESULTS

Following a four hour in vitro incubation with ~a'~'1,in the presence (data not shown) and absence of KC104, sections of L appendix endostyles examined by light rnicroscopy showed no morphological ciifferences fiom endostyles fixed immediately after excision. The organization of the anterior chamber, postenor medial chamber, posterior lateral chambers, and duct opening to the pharynx was sMarto that described by other researchers (for review see Barrington and Sage, 1972). As depicted in Figures 18A and

18C, the epithelial cells Lining the endostyle lumen were similar in morphology to those previously described and thus showed no visible effects fiom the incubation perïod. Routine light microscopy also confirmed that dissection, as performed for the electrophoretic sues, consistently removed the entire intact endostyle with very Little extraneous tissue.

Silver grains were primady localized over the type 2c and type 3 endostylar cells

(Figs. 18B and 18D) indicating that these were the primary ce11 types involved in the uptake and incorporation of '? in vitro. Type 2c and type 3 cells are also the prhary iodide- binding ceii types in vivo in sea Iampreys (Wright and Youson, 1976). Although some grains

are visible above the type 1ceus of the glanddar tracts and the connective tissue surrounding

the endostyle (Figs. 18B and 18D), these silver grains were considered background because

similar levels were visible in regions of the slide lacking tissue sections as weli as on non-

radiolabelled control slides (tissue sections fiom unincubated control endostyIes; data not

shown),

The uptake of by the larval Iamprey endostyle Nt vitro was determined by

rneasuring the rate of emission of gamma radiation by endostyles following a four hour

incubation with either 03,3, or 30 pCi of ~a'~'1-The levels of gamma radiation emitted by endostyles incubated in the presence of KCL@ were consistently less than those of untreated control endostyles. Overall, L-KC104- and H-KC104-treated endostyles emitted oniy 47 f 20

% and 39 f 7 %, respectively, of the radioactivity emitted by untreated controls (Fig. 19)-

Thus, KCLQ inhibited the uptake of LZS~-by the Iamprey endostyle in vitro. However, L-

KC104-treatedendostyles in replicate experiment number 4 emitted 86 % of the radioactivity emitted by untreated control endostyles (Fig. 19).

Polyacrylamide gel electrophoresis and autoradiography showed that taken up by the endostyle in vitro is incorporated into a high molecular mass protein with an electrophoretic mobiLity similar to reduced porcine Tg (Fig. 20)- Western blotting using a rabbit anti-human Tg antibody which cross-reacted with porcine Tg, detected ôn irnrnunoreactive protein with the same electrophoretic mobility as the protein visualized by gel-autoradiography (Fig. 21). These data are consistent with the identification of this protein as a lamprey Tg. Using SDS-PAGE under reducing conditions, the molecdar masses of Iamprey Tg, the primary porcine Tg band, and a less conspicuous, slower migrating porcine Tg band were estimated to be 226 kDa, 212 kDa and 236 kDa, respectively Fig.

20A).

KC104 treatment reduced the amount of incorporated into lamprey Tg by larvd lamprey endostyles. Densitometric anaiysis of gel-autoradiograms indicated that those endostyles incubated in the presence of L-KC104 or K-KC1OI incorporated an average of only 46 f 20 % (sample size M = 4) or 58 f 42 % (N = 2), respectively, of the incorporated by untreated control endostyles. The percent of incorporated by KC1O4- treated endostyles relative to control endostyles, for each replicate experiment, are presented in Figure 22A. OveraU, the data indicate that KCI04 inhibits the ability of the lamprey endostyle to organify iodide in vitro. However, in experiments 3 and 4, H-KCl04 and L-

KC104 ensdostyles, respectiveIy, did not show an appreciable decrease in 'Zr incorporation relative to the control endostyles (Fig. 22A).

The total amount of Tg in the larval lamprey endostyle decreased following a four hour incubation in the presence of KC104 (Fig. 22B). As determined by immunoreactivity with a Tg antibody and subsequent densitometry, endostyles incubated with in the presence of L-KC104 had 55 f 13 % of the Tg found in endostyles incubated with only

N~'~'I(Fig. 22B). High-KC104 treatment had similar effects on endostylar Tg levels; H-

KC104-treated endostyles had Tg levels that were 67 f 6 % of the levels in untreated controls

(Fig. 22B).

DISCUSSION

Morphological and biochernical observations have been presented which indicate that the larvaI lamprey endostyle is viable following a four hour incubation period in an in vitro experimental system. Endostyles incubated with N~'~'Iin the presence or absence of KC104

did not ciiffer in tissue or cellular organïzationfiom those endostyles examined immediately

after excision. At the level of light microscopy, the morphology of the endostyles was

comparable to that reported by other Iaboratories (see Barrington and Sage, 1972) (Fig. 18).

Moreover, the uptake of 12'r kmthe incubation medium, its binding to the appropriate ce11

types, and its incorporation into a Tg-Like molecule indicate that the endostyles incubated

with N~'~'Iwere biologicdly active. These observations were taken as evidence that endostyles incubated in vitro are performing biochemical processes, associated with TH

synthesis, which are similar to those occurring in vivo.

A single iodoprotein was identined in larval lamprey endostyle homogenates. This iodoprotein had an eIectrophoretic mobility simiIar to reduced porcine Tg and was

irnmunoreactive with an anti-human Tg antibody (Figs. 20 and 21); thus, this protein was designated as lamprey Tg. Several investigators have identified Tg-Like iodoproteins in the larval lamprey endostyle and adult lamprey thyroid tissue, but neither a definitive size nor sedimentation coefficient has been agreed upon. The collection of data f?om these studies suggests that lampreys possess a combination of iodoproteins andor Tg subunits similar to those found in higher vertebrates. Included among these iodoproteins are molecules with sedimentation coefficients of 3 - 8 S, 12 S, andior 17 - 19 S (Aloj et al., 1967; Roche et ai,.

1968; Suzuki and Kondo, 1973; Suzuki et al., 1975; Monaco et al., 1978). Aioj et ai. (1967) isolated a native subunit of lamprey Tg with a sedimentation coefficient of 11.7 S and determined its molecular mass to be 33 1 ma.

Early studies on mammalian Tg were also faced with difficufties in characterizing the sizes and molecular weights of Tg and its subunits. In the mid-1970s, it was confmned that the 19 S mammalian Tg was made up of two identical 12 S subunits with a moIecuIar mass of 330 kDa each (Ekholm, 1990). Other studies suggested that Tg molecules smailer than 12

S were a result of endogenous proteolytic activity (Ekholm, 1990). Dunn et al- (1983) showed that smail TH-rich iodoproteins (15 - 26 kDa) are formed when Tg is cleaved during the process of iodination and suggested that this may be a normal step in the production of TH,

In the present work, the molecular mass of lamprey Tg was estimated to be 226 kDa and that of porcine Tg was 212 - 236 kDa as determined by SDS-PAGE under reducing conditions. These molecular mas estimates are lower than expected and may be a resuit of either Tg cleavage or proteolytic activity, as discussed above, or they may be related to the anomalous electrophoretic rnobility of large glycoproteins. There was no evidence of any small iodoproteins or protein hgmentation in the gel autoradiograrns, but with the electrophoretic conditions 1employed, the separation of proteins or protein hgments smalIer than 25 kDa was not possible.

In a follicular thyroid gland, KC104 acts as a competitive inhibitor of iodide uptake into follicle cells (Wolff and Maurey, 1963). The perchlorate ion (Cl041 and similar anions compete with iodide for active transport by an iodide pump or transporter Located in the basolateral membrane of the follicle cells (Ekholm, 1990; Gentile et al., 1995)- The mode of action of these anionic competitive inhibitors diffen fkom those of other goitrogens such as the thioureylene dmgs (thiourea, thiouracil, propylthiouracil, methimazole) which inhibit thyroid peroxidase-catalyzed iodination of Tg (Gentile et al., 1995). The data presented in this study provides evidence that the goitrogen KCIOa acts directly on the larval lamprey endostyle to inhibit thyroidai activity. Relative to control endostyles, endostyles incubated in the presence of KC104 contained less radioactive iodide (Figg19),incorporated Iess iodide into Tg (Fig. 22) and had less Tg (Fig. 22)-

Potassium perchlorate administered to Iarval Iamprey endostyles in vitro lowered the total amount of Tg detected in endostyle hornogeaates as determuied by Westem blotting,

Endostyles incubated in the presence of KCLO4 had 55 - 67 % of the total Tg measured in control endostyles (Fig. 22B)- This level was similar to the decrease in iodide incorporation rneasured in response to KC104 treatment (46 - 58 % of untreated controls; Fig. 22A)-

Unfortunately, the porcine Tg sample 1used was electrophoreticaily heterogeneous and numerous fragments of porcine Tg were immunoreactive with the antibody used. Due to this heterogeneity, 1cou1d not use the porcine Tg as a standard with Western blotting to estimate the amount of lamprey Tg per milligram of endostyle protein for cornparison with other species. Western bIotting data were only used to make relative cornparisons between control and KCI04-treated endostyles-

The data coiiected fiom these in vitro experiments couid be interpreted in several ways depending on whether KCI04 affects the total Tg content in the larval endostyle directly or indirectly. The decrease in iodide organification in the presence of KC104 may be due to the inhibition of iodide uptake and the resultant absence of sufficient cellular iodide.

This decrease in iodide organification may subsequently result in a decrease in total endostylar thyroglobulin either by decreased synthesis, increased secretion or increased degradation. Alternatively, the decrease in iodide incorporation may be related to the observed decrease in totaI endostylar Tg. A decrease in endostylar Tg may be a result of either the direct effects that KC104 has on Tg synthesis or secretion, or more general toxicity effects of KC104 on the endostyle during a 4 hour in vitro incubation. Unfortunately, the results of this study do not aiiow me to determine whether the observed decrease in thyroidal activity following incubation with KC104 was a result of the direct inhibition of iodide uptake or organification, or due to the direct or indirect effects on total Tg. Further studies are necessary to clarify the mechanism through which KCL04 reduces thyroidal activity by endostyles N1 vitro, but speculation can be made on the implications of some of these possibilities.

The obsemed decrease in total Tg detected in Km4-treated larvai endostyles, relative to controls, may be a result of the direct effects of KCL04 on Tg synthesis or secretion, independent of the effects that it has on iodide uptake. If this hypothesis is tme, then the results of the current study should be interpreted to indicate that the observed decrease in iodide incorporation into Tg in response to KC104-treatment is due to the fact that KC104-treated endostyles were either synthesizing less or secreting more Tg than control endostyles. Altematively, the decreases in iodide incorporation into Tg and/or the total arnount of Tg may be secondary consequences of the effects that KC104 has on iodide uptake. This idea is more consistent with the known mode of action of KC104 in other vertebrates (i.e., inhibition of id*deuptake; Wolff and Maurey, 1963). A reduction in the uptake of iodide eventually decreases the amount of cellular iodide avaiiable for Tg iodination, resulting in an increase in the proportion of uniodinated or poorly-iodinated Tg.

In the Iarval endostyle, higher cellular concentrations of poorly iodinated Tg tnay in turn either decrease the rate of Tg synthesis or increase secretion and degradation rates, ultimately decreasing the amount of Tg in the endostyle. However, the notion that KC104 produces a decrease in Tg synthesis is contradictory to what is observed in the thyroid systems of other vertebrates (Wolff and Maurey, 1963)- In general, the treatment of vertebrates with a goitrogen results in the inhibition of Tg

iodination, either by preventing iodide uptake or by inhibiting the iodination reactions. In

this way the synthesis of TH is prevented and senun TH concentrations decrease (Cooper,

1990; Gentile et ai., 1995). Decreases in serum TH concentrations feed back to the

hypothalamic-pituitary axis and trigger the reIease of thyrotropin PSH; thyroid stimulating

hormone) (Wilber, 1995). Thyrotropin stimulates a number of factors involved in the

synthesis and secretion of TH, including Tg synthesis. In the presence of a goitrogen, the

iodination of Tg is prevented and large amounts of poorly-iodinated Tg can accumulate in

the follicular thyroid gland (Cooper, 1990)- This feedback system does not apply to this

study for several reasons: 1) The larval endostyle does not have the ability to store Tg in the

same manner as foüicular thyroid tissue, therefore, the regulation of Tg synthesis and

secretion in the endostyle may dBer fiom that of a foliicular thyroid gland. 2) In this study,

the endostyle was isolated from any hypothalamic-pituitary influences. 3) Conclusive evidence has not been presented that would indicate that the larval lamprey endostyle is regulated by the hypothalamic-pituitary axis in vivo. Thus, it is conceivable that a goitrogen such as KCI04 could depress Tg synthesis either directly or indirectly via its affects on iodide uptake.

In summary, 1have tested an in vin0 experimental system which will prove to be a usehil tool in studying TH synthesis by the larval lamprey endostyle and the factors involved in its regulation. The goitrogen KCIOI acts directly on the Iarval lamprey endostyle to inhibit thyroidal activity as evidenced by a decrease in the uptake and organification of iodide by the endostyle and the total amount of Tg in the endostyle, relative to control endostyles, three factors essential to the synthesis of TH. Table 5. Radioiodide (NaL% doses and potassium perchlorate (Kc104) treatment

concentrations administered to Iarval sea Iamprey (Petromyzon mannus) endostyles in vitro

and the data coilected from the endostyles in experïments 1 - 5.

Experiment ~a~1@Ci) Ka04 @Ml Data ~oflected 1 3 0-72 IU, IO and Tg content 0.72 IU, 10 and Tg content 0.72 IU, IO and Tg content 3 -6 IU, IO and Tg content 0.72 IU, 10 and Tg content 3 -6 iU, IO and Tg content 5 0.3 0.72 IU ' IU, Iodide uptake; IO, Iodide incorporation (organüïcation) into thyroglobuiin (Tg) Figure 18. Routine light microscopy (A and C) and autoradiography (B and D) of transverse

sections through the antenor portion of the lard lamprey (ulmpetra nppendlx) endostyle

following a four hour in vitro incubation with 3 pCi N~'=I. Endostyle ce11 types 1,2a, 2b,

2c, 3, and 5 are indicated. (A) The antenor endostyle consists of two straight, epithelium- lined chambers; the most prominent features are the four glandular tracts (Gt) which consist exclusively of type 1ceiis. Flanking the openings of the glandular tracts to the lumen of the endostyle are the type 2 celis. Continuous with the type 2c cells are the type 3 endostylar cells. (C) A high magaification of a glanddar tract and the surroundhg epithelia in the anterior portion of the larval lamprey endostyle. Note that the cells of the epithelia are intact with well-defined cytoplasm and nuclei, connrming that the four hour incubation with N~'~'I did not have my visible effects on cellular morphology. (B and D) Light microscopic autoradiography of sections similar to A and C, respectively. The type 2c and type 3 cells are the primary incorporating cell types. In all four micrographs the endostyles are onented with the ventral side on the left. Magnification is X165 (A and B) and X330 (C and

D). Blood vessels, Bv; Gills, Gi; Pigment, Pi.

Figure 19. The percentage of I2'I- uptake, as detemiuied by gamma radiation emissions, by potassium perchlorate- (KC104) treated endostyles relative to untreated (control) endostyles following a four hour in vitro incubation with ~a'~1.Endostyles were incubated with either

0.3 pCi (experiment 5), 3.0 pCi (experiments 1 ami 3) or 30 pCi (experiments 2 and 4) of

N~'~'I. KC104 treatrnent concentrations were either 0.72 mM (L-KCLO4) or 3.6 mM (H-

Kc104). 1 2 3 4 5 Replicate experiments Figure 20. (A) Autoradiogram to show radioiodide incorporation and organification into a lamprey thyroglobulin (Q) by Iarval lamprey endostyles incubated in vitro with 3 pCi of

~a'q.Protein samples (10 pg totai proteidlane) from untreated (control) endostyles (CON) and endostyles treated with either a Low (L-KC104; 0.72 mM) or high (H-KClO4; 3.6 mM) dose of potassium perchlorate (KCIOQ)are showa. The 205 kDa molecular weight marker and a porcine thyroglobulin sample (P-Tg; 10 ug) from the Coomassie blue-stained gel have been pasted on the left and right of the autoradiogram, respectively. The autoradiogram and

Coornassie blue-stained lanes are fiom in vitro experiment 3 (see Table 5). (B) Typical

Coomassie blue-stained gel used for autoradiography. This gel shows the protein profile of endostyle homogenates (10 pg total proteidlane) from in vitro experiment 4 (see Table 5), the molecular weight markers (Mt) and a P-Tg sample (right),

Figure 2 1. Irnmunodetection of thyroglobulin (Tg) in larval lamprey endostyles by Westem blot using a rabbit anti-human Tg antibody, foilowing an in vitro incubation with 30 pCi

N~'~'I.Protein samples (10 pg total proteinnane) nom untreated (control) endostyles (CON), endostyles treated in vitro with either a low (L--4; 0.72 mM) or high (H-KC104; 3.6 mM) dose of potassium perchlorate (KC104) and a porcine Tg sample (P-Tg; 0.1 pg) are showo. The porcine Tg sample used in this Westem blot was electrophoretically heterogeneous. The single band shown represents only a portion of the 0.1 pg of porcine Tg

Ioaded (see Materials and Methods). Due to thls heterogeneity porcine Tg could not be used to estimate the amount of Tg in the larval endostyle for cornparison with other species.

Figure 22. (A) The percentage of '%incorporation (organification) into Iamprey thyroglobulin (Tg) by endostyles treated with either low potassium perchlorate (L-KC104;

0.72 rnM) or high potassium perchlorate (H-KC1O4; 3.6 mM) relative to untreated (control) endostyles, following a four hour in vitro incubation wah ~a'?in four separate experiments. (B) The percentage of thyroglobului detected by Western blot in L-KC104- and H-KCI04-treated endostyles reIative to untreated (control) endostyles, foUowing a four hour in vitro incubation with N~'~'I.Data were obtained by dividing the adjusted optical densities (OD) of bands from KCIOfleated endostyles (pool of five) by those of control endostyles (pool of five) foilowing SDS-polyacrylamide electrophoresis and gel- autoradiography (A) or Western blotting (B). Data presented for gel-autoradiography and

Western blotting are the mean values (k 2 standard errors) fiom four and two replicate gels, respectively. Endostyles were incubated with either 3 pCï (experiments 1 and 3) or 30 pCi

(experiments 2 and 4) of N~'=I. Total thyroglobulin Percent of controls Percent of controls GENERAL DISCUSSION When compared to the metamorphosis of other vertebrates, lamprey metamorphosis

is unique. Its onset coïncides with a sharp decline in semthyroid hormone (TH) concentrations, rather than an increase. Furthemore, the precocious metamorphosis of

lampreys can be induced with antÏ-thyroid agents (goitrogens), and this induction is accompanied by a decline in serum TH concentrations. Prior to the commencement of this doctoral thesis, goitrogen-induced metamorphosis had been obsewed in three different larnprey species, and in two of these Instances a deciine in serum TH concentrations was reported (Hoheisel and Sterba, 1963; Suniki, 1986, L987; Hohes and Youson, 1993;

Youson et al., 1995)- However, the relationship of this decline in senun TH concentrations to spontaneous or induced metamorphosis had not been estabiished. 1s the observed decline in serum TH concentrations a consequence of metamorphosis, coincident with metamorphosis, or does it function to initiate or regulate metamorphosis? That is, does a decline in serum TH concentrations initiate metamorphosis or does it permit morphogenesis to proceed after another signal has caused its initiation? Leatherland et al. (1990) indicated that propylthiouracil (PTU) significantly lowers serum TH concentrations, but does not induce metamorphosis. This result supports the idea that a decline in serum TH is a consequence of metamorphosis, perhaps related to the transformation of the lamal endostyle into a follicular thyroid gland- One of the primary airns of my doctoral thesis was to provide an understanding of the role of reduced serum TH concentrations in goitrogen-induced and spontaneous larnprey metamorphosis and to provide insight into the mechanisms by which goitrogens induce metamorphosis in lampreys. Over the course of this thesis, 1conducted severai experiments to investigate goitrogen-induced metamorphosis in lampreys. Some of these experiments did not uiclude replicate tanks for each experimental group, thus a measurement of tank effect coulcl not be performed, The Iimiting factor was the number of anîmals available for use in these experiments- However, other studies on goitrogen-induced metamorphosis in lampreys have included replicate tanks and did not report variations between tanks (Chapter 3; Hoimes and

Youson, L 993; Holmes et al., 1999)- Coliectively, my experhnents provide conclusive evidence that the goitrogen potassium perchlorate (KClo4) induces precocious metamorphosis in lwaI sea lampreys at a time of year when metamophosis does not nonnaiIy occur, The treatment of Iarval sea lampreys with KC104 resulted in significant declines in serum thyroxine CT.4) and riiiodothyronine Cr3) concentrations in all experiments.

These data coafirm the results from previous studies (Hoheisel and Sterba, 1963; Suuki,

1986, 1987,1989; Holmes and Youson, 1993; Youson et al., 1995) and suggest that the onset of metamorphosis is correlated wÏth a decline in serum TH concentrations.

The more significant finding nom my experiments (Chapters 1 and 2) is that a decline in serum TH concentrations is essential for KCL04-induced metamorphosis. This conchsion is based on the observations that larval sea lampreys treated with KC104 and either exogenous TqOC T3 did not undergo precocious metamorphosis. KC104+TH-treated lard sea lampreys had serum TH concentrations that were greater than or equal to control values (Figs. 5,6, 8 and 9). Two hundred and forty Iarval sea lampreys were treated with

KC104 and either Tqor T3, and not one animai commenced metamorphosis (Figs. 4 and 7).

The results of these two experiments provide strong evidence that a decline in semm TH concentrations is essential for KCI04-induced lamprey metamorphosis. These results, however, do not indicate whether a decline in serum TH concentrations is involved in the

initiation of metamorphosis, or if elevated TH concentrations are involved in the inhibition of

metamorphosis. In other words, a decline may be permissive to metamorphosis. The idea

that one of these mechanisms rnay also be involved in spontaneous metamorphosis is further

supported by the observation that exogenous T3cm inhibit or retard spontaneous

metamorphosis (Youson et al,, 1997)-

Suzuki (1987, 1989) reported that the goitrogens ETU, thiourea, KC104 and sodium

perchlorate (NaClo.+) induced metamorphosis in Lmpetm rerSsneri; however, the

experimentai details and results were presented only for KC104- and NaC104-induced

metamorphosis, Conversely, PTU treatments failed to induce metamorphosis in Geotria

australis aeatherland et al*,1990) and Lampetra appendii (Holmes et al., 1999), despite a

significant decrease in semm TH concentrations relative to control values. These data for G, amiralis and L appendtjr suggest that the induction of metamorphosis in laryal lampreys

may be due to the effects of KC104 specifically rather than inhibited thyroidal activity. In contrast, 1have demonstrated that several goitrogens can induce metamorphosis in larval sea

lampreys and that the incidence of metamorphosis is related to the magnitude of the decline

in serum TH concentrations (Chapter 3, Appendix 1). These results provide evidence that the induction of metamorphosis is inextricably linked to a decline in serum TH concentrations, and not simpiy to the effects of the CIO4-anion.

The experiments presented in Chapter 3 showed that larval sea lampreys treated with either of KC104, NaClOs, methimazole (Mmor potassium thiocyanate (KSCN)underwent precocious metamorphosis. However, PTU failed to induce metamorphosis in any of the 90 larval sea lampreys fiom 3 different size groups that were treated with this goitrogen. The incidence of metamorphosis in al1 of these goitrogen treatment groups varied both between goitrogens and treatment concentrations Figs, L2 and 14) and was positively correlated with the magnitude of the decline in senun TH concentrations. Goitrogen treatments that resulted in the Iargest declines in semm T4 and T3 concentrations had the highest percentages of animals entering metamophosis (Table 6)- The goitrogens KClO4, NaCIO4 and MM1 were more effective at decreasing serum TH concentrations and inducing metarnorphosis than either KSCN or L-KClO4 (0.00 1%)(Chapter 3, Table 6).

The correlation that 1observed between the incidence of metamorphosis and the magnitude of the decline in serum TH concentrations mers fiom that reporteci in other studies. Holmes and Youson (1993) stated that KC104-induced metamorphosis in larval sea larnpreys was size-dependent and that significantly more large larvae commenced metamorphosis than smail Iarvae. These differences in the incidence of metamorphosis were not due to differences in the magnitude of the declïne in senun TH concentrations between larvae of different size groups (Holmes and Youson, 1993; Youson et al., 1995)- The lower incidence of metamorphosis in smaller larval sea lampreys may have been related to factors other than TH which are also important for metamorphosis, as discussed below.

Further evidence that depressed senun TH concentrations are essential for goitrogen- induced metamorphosis was provided by a preliminary experhent designed to examine the effects of KC104 treatment length on the incidence of induced metamorphosis (see Appendix

A). Briefly, larvae were either untreated (controls) or treated with KC104 (0.05 %; 3.6 mM) for 2,4,8, or 16 weeks. Twenty-eight weeks after the onset of the experhent, 1determined the stage of metamorphosis and coliected sera to assay T4 and T3concentrations. Al1 larvae treated with KCI04 for 16 weeks commenced metamorphosis in cornparison to a high of only 33 % foiiowing 8 weeks of treatment (Appendix A. Fig. 23). Senun T3concentrations in larval sea lampreys treated with KCI04 for 8 weeks or less were 36 - 46 % lower than control values; however, serum T3in the 16 week treatrnent group was 88 % Iower than control concentrations (Appendk A. Fig. 23). These results are consistent with those presented in

Chapter 3 and indicate that the incidence of goitrogen-induced metamorphosis is correIated to the magnitude of the decline in serum TH concentrations, particularly the decline in T3.

HoImes and Youson (1993) reported that a 0.05 % KC104 treatment did not induce significantly more larval sea lampreys to metamorphose than a 0.01 % Ka04treatment; nonetheless, 1showed that the goitrogenic anion Cl04 supplied at a treatment concentration of 0.05 % is the most effective inducer of metamorphosis in Iarval sea lampreys. Its effectiveness at inducing metamorphosis may be related to its ability to reduce thyroidal activiîy.

The results presented in this thesis indicate that the precocious metamorphosis of lampreys can be induced by most goitrogens and requires a large decline in serum TH concentrations. Additionaily, 1have deterrnined that KC104 acts directiy on the larval lamprey endostyle to inhibit thyroidal activity. The results presented in Chapter 5 suggest that the site of TK synthesis is also the site of KCL04 action and support the idea that KC104 induces metamorphosis in Iarval larnpreys by depressing thyroidal activity. However, reduced thyroidal activity or lowered serum TH concentrations are not sufficient to induce either a precocious metamorphosis or a fully metamorphosed juvenile. The importance of non-thyroidal factors in induced metamorphosis is demonstrated by the foliowing: i) Larval lampreys treated with KC104 at cold water temperatures (3 - 5 OC;Chapter 4) do not undergo metamorphosis despite large declines in semm TH concentrations. ii) Some species of larval Iampreys do not metamorphose foiiowing PTU treatment even though the declines in serum TH concentrations were similar to those observeci foIIowing KCL04 treatment (Chapter

3; LeatherIand et al-,1990; Holmes et al-,1999)- iii) The incidence of goitrogen-induced metamocphosis is Iower in srnaII iarvae than in Iarge Iarvae, and this is not related to differences in serurn TH concentrations (Chapters 1 and 2; Holmes and Youson, 1993). iv)

Only a few preliminary experhents on L reissneri have reported the induction of a compIete metamorphosis following goitrogen treatment. Clearly, factors other than a decline in semm

TH are important to ensure the successfûl induction of metamorphosis in lampreys, The most likely candidates include: temperature, physiologicai conditioning related to lipid accumulation and metabolisrn, and regdatoty secretions (protein or hormonal) from the hypothalamus, pituitary or pineal gland.

Of the environmental factors examined, the predominant factor influencing both spontaneous and induced Iamprey metamorphosis is temperature. Cool water temperatures cmdelay the onset of metamorphosis, decrease the incidence of metamorphosis, and affect the rate at which morphogenesis proceeds (Manion and Stauffer, 1970; PuMs, 1980; Youson et al., 1993; Holmes and Youson, 1994; Holmes et al., 1994). The importance of temperature in goitrogen-induced metamorphosis was established in Chapter 4. Although goitrogens can induce metamorphosis in Iarvae which have not met the minimum size requirements for metamorphosis, and can do so during the winter months when lampreys do not metamorphose spontaneously, it canot completely supersede the need for warm summer temperatures (Fig. 16). In Chapter 4, al1 KC104-treated larvae kept at summer water temperatures (1 8 OC) commenced metamorphosis, but KC104-treated larvae maintained at winter water temperatures (3 OC) did not display any extemal signs of morphogenesis despite reduced serum TH concentrations. This observed absence of metamorphosis at winter water temperatures confirms the importance of temperature to lamprey metamorphosis, However, the cold, winter water temperatures (3 OC) used may have kentw Iow to Myassess the role of temperature in goitrogen-induced metamorphosis. Frieden et al. (1965) and Ashley et al- (1968) found that tadpoles did not respond to exogenous T3 at 5 OC, but changes consistent with metamorphosis were detected at 7.5 OC. Future investigations are required to test whether KCL04 cmoverride the need for summer water temperatures and to determine if

KC104 can induce metamorphosis at a range of cooI water temperatures (5 - 18 OC)- One interesting experimeat wouId be to test whether KCI04 can increase the incidence of metamorphosis in immediately premetamorphic larvae maintained at temperatures (10 - 13

OC) shown to resuit in a decreased incidence of spontaneous metamorphosis (Youson et al.,

1993).

The accumuIation of iipids during the "arrested growth phase" (Potter, 1980; Youson,

1988) and lamprey size and condition factor (CF; see Generd Introduction) are excellent tools for predicting which lampreys wiil enter metamorphosis. Sea Iampreys must attain a size of 120 mm and 3.0 g and have a CF of 1.5 or greater prior to entering metamorphosis in

JuIy (Youson et al., 1993; Holmes et al., 1993; Holmes and Youson, 1994, 1998). Holmes and Youson (1993) and my work in this thesis have shown that goitrogens can ovemde these size and CF cntena to some extent and cminduce precocious metamorphosis in larval lampreys which are not immediately premetamorphic, that is, in individuals which have not met the minimum size and CF criteria, The incidence of induced metamorphosis is higher in those larvae that are closer to attaining the length and weight criteria than in smaIler Iarvae

(Holmes and Youson, 1993; Chapters 1and 2). The inabilit. to induce a normal and complete metamorphosis with goitrogens may be reiated to the lack of sufficient Lipid reserves, disruption of lipid metabolism, or dismption of signais that are essentid Iater in metamorphosis.

Kao (1997) described a two-phase pattern of changes in Lpid content, class and metabolism associated with lamprey metamorphosis, and he suggested that these phases were tissue-specific and under hormonal control (see Kao et al-, 1997a,b; Kao et al,, 1998; Kao er al,, 1999a, b). Phase 1 begins Iate in larval me, las& until stage 3 of metamorphosis and is characterized by increases in total Lipid, triacylglycerol content and lipogenesis as well as low Lipolytic activity. In phase 2, fiom stage 3 to stage 7 of metamorphosis, a dramatic switch in lipid metabolism is observed. There is a reduction in lipogenesis and an increase in lipolysis. This increase in lipid cataboiism and decrease in lipid deposition resuits in a decrease in total body Lipid. Insulin, somatostatin-14 (SS-14) and TH are thought to play a role in the regulation of Lipid metabolism in Iampreys (Plisetskaya et al., 1983; Kao et ai.,

1998; Kao et al., L999a, b). Coincident with the change in lipid metabolism from phase 1 to phase 2 is an increase in pancreatic-intestinal somatostatin44 (SS-14) concentrations

(Elliott and Youson, 1991). Other studies have shown that SS-14 injections increase lipid rnobilization as detennined by elevations in plasma fatty acid concentrations and enhanced lipolytic enzyme activity (Kao et al., 1998). Nternatively, treatment with exogenous TH tended to stimulate Lipogenesis and iipid deposition in contrasi to the lipolysis-promoting action of KC104 (Kao et al., 1999a). Although the injection of larval lampreys with insulin results in an increase in lipogenesis and lipid deposition, the serum concentrations of insulin in untreated Iampreys are relatively constant until their increase at stage 7 of metamorphosis

(Youson et al., 1994; Kao et al,,1999b). The switch from lipogenesis to Iipolysis that occurs at stage 3 of metamorphosis may be involved in processes other than providing a source of energy for survival and the completion of morphogenesis. Stage 3 may be a crucial the in metamorphosis when numerous metabolic and regulatory signais are interacting to ensure that metamorphosis proceeds synchronously. Other major physiological changes occur during stage 3 that may be involved in the regulation of metamorphosis. They are: a switch in intestinal deiodinase activity (see below, Edes et al,, 1997,2000), an increase in intestinal-pancreatic SS-14 levels (Eiliott and Youson, 1991; Kao et ai,, 1998), and increased synthetic and secretory activity of an unknown product in the caudal pars distalis of the pituitary (Wright, 1989).

These factors are likely to be essential for the progression of metamorphosis, and any disruption of their timing or occurrence may dramaticaüy alter the outcome of this developmental event, Perturbations of this nature may contribute to the asynchronous and incornplete development observed with goitrogen-induced metamorphosis.

Edes et al. (1997,2000) have shown that the intestine is the primary site of deiodinase activity in lampreys and that there is a dramatic change from outer-ring (ORD) to imer-ring (IRD) deiodinase activity at stage 3 of metamorphosis. The levels of T4 outer- ring deiodination (T40RD), which result in the production of the more biologically active T3, increase from the larval period until stage 2 of metamorphosis. During this tirne, the imer- ring deiodination of T4 (Tm)and T3(T3IRD) to produce the inactive reverse T3(r'ï3) and diiodothyronine (T2),respectively, are negligible. At stage 3 of metamorphosis, T40R.û activity is negligible and an increase in the inactivating Tmis observed, This increase in

TJRD activity between stages 2 and 3 of spontaneous metamorphosis may help explain the observed decline in serum TH concentrations early in spontaneous metamorphosis, but whether this increase has any additional regdatory roIe has not been determined. Although

the role of deidinases in goitrogen-induced metamorphosis has not ken fuily examined, the

treatrnent of larval sea lampreys with KC104 did not alter &ORD activity relative to control

values (Chapter 2). Inner-ring deiodinase activity was not determhed-

Joss (1985) showed that the rernoval of the caudal pars distalis of the pituitary did not

affect the initiation of metamorphosis, but resufted in an incomplete metamorphosis that is

arrested at stage 3. Wight (1989) showed that the synthetic and secretory activity of the caudal pars distaiis cells increased during stage 3 of metamorphosis- Perhaps the inabiiity of goitrogens to induce a complete metamorphosis is related to either the absence of a signai from the caudal pars distalis, or the disruption of the normal two-phase cycle of Lipid metabolism, two events which occur at stage 3 of metamorphosis. Overd, the treatment of lampreys with KCL04 had a tendency to increase lipolysis and decrease lipogenesis, which is consistent with phase 2 of the lipid cycle (Kao et al., L999a); however, the increase in lipogenesis associated with the early stages of metamorphosis was not detected. The change fiom phase 1 to phase 2 of this lipid cycle may be an essentiai feature of metamorphosis.

Metamorphosis in lampreys, as with amphibians, probably involves the coordination of several signals. Thyroid hormones are essential to both systerns; however, their role in lamprey metamorphosis appears to be unique. 1s the role of TH in Iamprey metamorphosis similar, or fundamentaily different fkom, its stimulatory role in other vertebrates? The answer to this question is not simple. In the following section, 1make two conjectures in an attempt to explain the nature of the decline in serum TH concentrations and its function in larnprey metamorphosis, whiIe bearing in mind the function of TH in the metamorphoses of other vertebrates. These conjectures are based on data collecteci fkom studies of spontaneous and induced metamorphosis. but our knowledge is far fiom complete. The first conje&re describes a scenario where lampreys are fundamentaily different nom other vertebrates in their utilkation of TH for development/metamorphosis. The second conjecture explains our observations from the perspective that iampreys are simifar to other vertebrates with respect to TH and metamorphosis.

Conjecture 1

The ptimary function of TH in the Iamprey Iife cycle may be to prornote growth and lipid accumulation in the Iarvai period for survival during the protracted, non-trophic phase of metamorphosis. The graduai increase in senun TH concentrations throughout the larval growth phase inhibits metamorphosis and encourages growth. The peak of serum TH titers prior to onset of metamorphosis may trigger the "arrested growth phase" and the rapid accumulation of lipids essential for metamorphosis. In this regard, TH would have an anti- metamorphic role in lampreys, antagonizing or inhibiting spontaneous metamorphosis

(Youson, 1997). This is analogous to the postulated role of prolactin in amphibian (see

Denver, 1996; Tata, 1996) and flounder (de Jesus et al., 1994) metamorphosis. Whether prolactin has a juvenilizing or anti-metamorphic effect in anurans has kenstrongly debated

(Buckbinder and Brown, 1993; Huang and Brown, 2000a,b). Once lampreys have attained the critical, minimum size for metamorphosis, some exogenous or endogenous signal(s) may trigger the onset of metamorphosis. Possible endogenous signals include secretions from the rostral par distalis (Joss, 1985) or pineaI gland (Eddy and Strahan, 1968; Eddy, 1969; Cole and Youson, 198 1), or the decline in semm TH concentrations. Once the program for metamorphosis has been initiated, the decline in serum TH concentrations rnay either permit morphogenesis, or may be essentiai for morphogenesis to proceed. This notion is supported by several lines of evidence- Fit, a decline in serum TH concentrations coincides with the onset of metamorphosis. Second, goitrogens act directly on the larval lamprey endostyle to inhibit thyroidd activity, depress senun TH concentrations and induce me ta morpho sis^

Third, exogenous TH treatrnents block goitrogen-induced metamorphosis. Fourth, T3cm inhibit or retard spontaneous metamorphosis. The inability to induce a complete metamorphosis despite this reduction in thyroidal activity may be related to the disruption of other regulatory signals important later in metamorphosis. The numerous changes associated with stage 3 may have important functions in the compIetion of a normal metamorphosis.

The signal that initiates metamorphosis in lampreys is not different from other vertebrates, but the timing during development differs. Perhaps the peak in semm TH concentrations associated with the end of the lamprey larvai perïod prepares the animal for metamorphosis. This peak may trigger an increase in responsivity to TH, similar to the autoinduction of the thyroid hormone nuclear receptor (TR) in amphibians (Tata, 1996).

Increases in TR synthesis or activity, cellular TH concentrations, or nuclear TH concentrations could facilitate a greater response to TH. Once the program to metamorphose has been set, the Iow (relative to larval levels) serum TH concentrations dunng metamorphosis may be suffîcient to drive morphogenesis. Senun TH concentrations in

Imal sea lampreys can be more than 10 fold greater than the climax values for anurans.

Moreover, serum TH concentrations during lamprey metamorphosis are at Ieast comparable to, if not greater than, levels during rnetamorphic climax in anurans (For data on anurans see

White and Nicoll, 198 1; Rosenkilde, 1985. For data on lampreys see Wright and Youson,

1977; Lintlop and Youson, 1983; Leatherland et al-, 1990; Youson et al., 1994; Chapters 1 - 4). ALthough this conjecture is more consistent wiai the role of TH in amphibian

metamorphosis, it is not as consistent as my first conjecture with respect to what is known of goitrogen-induced rnetamorphosis in lampreys. In conjecture 2, the abiiity of goitrogens to

induce metamorphosis may be related to the sudden drop in senun TH concentrations that disrupts cellular homeostasis enough to start morphogenesis at the tissue and cellular levei, despite the absence of an initial trigger. Once morphogenesis begins, rnetamorphosis proceeds, but not to completion because the nomal cascade of events has been disrupted. in amphibians, semm TH concentrations begin to decline after theu peak at metamorphic climax, but prior to the completion of morphogenesis. Perhaps metamorphosis in lampreys is similar to amphibians in this regard, but the amine of the deche in senun TH in lampreys has shifted so that most of the morphological changes occur following this event. Perhaps cells and tissues depend on hÏgh TH concentrations and rnorphogenesis ensues once TH levels drop and homeostasis is disrupted.

Surnmary and ConcZusions

The results presented in this thesis suggest that the goitrogen EX104 acts directiy on the larval lamprey endostyle to inhibit thyroidal activity, as evidenced by a decrease in iodide uptake and organifcation and a decrease in total endostylar thyroglobulin. This reduced capacity to synthesize TH results in a dramatic decrease in serum TH concentrations, an event that is inextricably linked to the induction of precocious metamorphosis. Several goitrogens induce rnetamorphosis in larval sea lampreys, and the incidence of induced metarnorphosis is correlated to the magnitude of the decline in semTH concentrations.

When this decline is prevented with exogenous TH, goiirogen-induced metamorphosis is blocked. Based on these data, and the resuits of investigations on spontaneous metamorphosis, I conclude that a decline in senun TH concentrations is essentiai, but not the only critical criterion, for the initiation of metamorphosis in Iarval sea lampreys. Whether the regulatory role of TH ciiffers fiindarnentaüy fiom that of other vertebrates at the cellular

Ievel remains to be elucidated. To determine the regdatory role of TH in lamprey metamorphosis, future studies should focus on the TH nuclear receptors, potential cytosolic

TH receptors and binding proteins, and cytosolic and nuclear TH concentrations to provide a better understanding of the local, cellular action that TH may have in Iampreys, This information will help determine whether TR cmregulate gene expression in lampreys and will provide a vaiuable tool for the studying the role of TH in the up- or down-regulation of genes related to morphogenesis. Future investigations of lamprey metamorphosis must also include a search for other regulatory factors which may modulate its initiation or progression in conjunction with, or independent of, TH. PotentialLy fniitful avenues of investigation include the study of the pineal gland, the hypothalamic-pituitary axis, the interrend axis, and hormones involved in the regulation of lipid metabolism. Table 6. Summary of the incidence of metamorphosis following treatment with one of severai goitrogens at different concentrations. The incidence of metamorphosis is reIated to the percent decrease in serum thyroxine (T4)and triiodothyronine (T3)concentrations fiom control values,

Thesis Goitrogen Treatment % decrease % decrease Incidence of chapter concentration in T4 in T3 metamorphosis (mM) (%) 1 KCLO~' 0.72 62 72 60

3 L-KC104 0.072 66 20* 10 H-KI04 0.72 69 84 66 L-MMI 0.087 68 90 33' H-MMI 0.87 66 88 46* L-KSCN 0.051 1* 25* 20 H-KSCN 0.51 52 43 43

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279: 145- 1%- APPENDM Figure 23. (A) Stage and number of metamorphosing sea Iampreys at 28 weeks following O

(controls), 2,4,8, or 16 weeks of treatment with 3.6 mM (0.05%) potassium perchlorate

(KCL04).(B and C) Mean (+ 2 standard errors) senun thyroxine (T4;B) and trüodothyronine

(T3; C) concentrations at 28 weeks following O, 2,4,8, or 16 weeks of treatment with 3.6 mM KC104. Analysis of variance and Tukey-Krarner's post-hoc test were used to examine for statistically significant merences in serum T4 and T3 concentrations berneen lengths of

KC104 treatment. DBerences were accepted as statisticdy signincant if P < 0.05.

Concentrations 1abelIed with different Letters are statistically significant. Sample size (N) is equal to 10 unless othenivise indicated below the abscissa. Experimental treatments were administered to sea lampreys greater than 120 mm in length coilected from Putnam Creek,

New York as described in Chapter 4.