INVESTIGATION OF HISTAMINE IN ECHINODERMS

by

Shana L. Smith

A thesis submitted in partial fulfillaent of the requi rements for the degree of Kaster of Science in the Departaent of Marine Science in the University of South Florida

August 1992

Major Professorsr Noraan Blake, Ph.D. and Joseph Torres, Ph.D . Graduate Council

University of South Florida

Tampa, Florida

CERTIFICATE OF APPROVAL

Master's Thesis

This is to certify that the Master's Thesis of

Shana L. Smith

with a maj o r in Marine Science has been approved by the Examining Committee on June 23, 1992 as satisfactory for the Thesis requirement for the Master of Science degree.

Examining Committee: Major Professor: Norman Blake, Ph.D.

Major Professor: Joseph Torres, Ph.D.

Member: Richard Coulson, Ph.D.

Member: Kent Fanning, Ph.D.

Member: John Lawrence, Ph . D. ACJCNOWLEDGBHEif'l'S

I would like to express sincerest thanks to the members of my thesis committee, Drs. Norman Blake, Joseph

Torres (co-major professors), Richard Coulson, Kent Fanning, and John Lawrence, for their advice, help, and enthusiasm throughout the duration of this study. Thanks also to Ben

MacLaughlin, Department of Natural Resources, St. Petersburg,

Florida, and the crew of the R/V Suncoaster, Florida Institute of Oceanography, for supplying me with starfish. Host of all,

I extend my deepest gratitude to the ones I lovea my father,

Dr. Albert c. Smith; my mother, Mrs. Deborah J. Smith; my sister, Ms. Chelsea F. Smith; and my husband-to-be, Hr. Rick

R. Tucker, for constant support and understanding through some of my trickier moments. TABLE OF CONTENTS

LIST OF TABLES iv

LIST OF FIGURES v

LIST OF ABBREVIATIONS vi

ABSTRACT vii

CHAPTER l INTRODUCTION 1

CHAPTER 2 LITERATURE REVIEW 5 Histamine 5 Asteroid Reproduction 15

CHAPTER 3 DEVELOPMENT OF THE HISTAMINE ASSAY 29 Introduction 29 Materials and Methods 34 Discussion 39

CHAPTER 4 INCIDENCE OF HISTAMINE WITH REPRODUCTIVE 42 INDEX IN THE ASTEROID LUIDIA CLATHRATA Introduction 42 Materials and Methods 44 Results 48 Discussion 64

CHAPTER 5 DISCUSSION AND CONCLUSIONS 70

LIST OF REFERENCES 76

iii LIST OF TABLES

TABLE 1. Histamine receptor types and their 8 effects upon activation.

TABLE 2. Relative fluorescence readings of 37 L-histamine, L-histidine, spermidine and urea at 0.1, 0.5, and 1.0 mg/ml concentrations.

TABLE 3 . Histamine concentrations in 36 tissues/ organs of the echinoid Hellita tenuis, and the asteroids Luidia clathrata and Astropecten duplicatus .

TABLE 4. Reproductive index (total gonad weight / 63 total wet body weight) values per gonad developmental stage.

iv LIST OF FIGURES

FIGURE 1. Standard curve for histamine . 38

FIGURE 2. Early developmental stage of (a) female 50 and (b) male gonads of Luidia clathrata. Hematoxylin/eosin stain .

FIGURE 3. Late developmental stage of (a) female 51 and (b) male gonads of Luidia clathrata. Hematoxylin/eosin stain.

FIGURE 4. Ripe stage of (a) female and (b) male gonads 52 of Luidia clathrata. Hematoxylin/eosin stain.

FIGURE 5. Histamine concentration in the gonads of 53 female Luidia clathrata versus reproductive index .

FIGURE 6. Histamine concentration in the pyloric 54 caeca of female Luidia clathrata versus reproductive index.

FIGURE 7. Histamine concentration in the gonads of 55 male Luidia clathrata versus reproductive index.

FIGURE 8. Histamine concentration in the pyloric 56 caeca of male Luidia clathrata versus reproductive index.

FIGURE 9. Histamine immunostain of the (a) gonad 57 and (b) pyloric caecum of a female Luidia clathrata with a reproductive index value of 0.013.

FIGURE 10. Histamine immunostain of the (a) gonad 58 and (b) pyloric caecum of a female Luidia clathrata with a reproductive index value of 0.070 .

FIGURE 11. Histamine immunostain of the (a) gonad 59 and (b) pyloric caecum of a female Luidia clathrata with a reproductive index value of 0.15.

FIGURE 12. Histamine immunostain of the (A) gonad 60 and (b) pyloric caecum of a male Luidia clathrata with a reproductive index value of 0.018.

v FIGURE 13. Histamine immunostain of the (a) gonad 61 and (b) pyloric caecum of a male Luidia clathrata witp a reproductive index value of 0.07186. FIGURE 14 . Histamine immunostain of the (a) gonad 62 and (b) pyloric caecum of a male Luidia clathrata with a reproductive index value of 0.1652.

vi LIST OF SYMB OLS, ABBREVIATIONS AND NOMENCLATURE

HA------Histamine

CNS------Central Nervous System

HSF------HA Suppressor Facto r

HPF------HA Proliferative Factor

AI DS ------Acquired Immune Deficiency Syndrome

LH------Luteinizing Hormone

GSS------Gonad Stimulating Substance

"RNF------Radial Nerve Factor

HI S------Maturation Inducing Substance

1-HA------1- Hethyladenine

HPF------Haturation Promotin g Fac t or

HPL C------High-Precision Liquid Chromatography

HSLC------High-Speed Liquid Chromatography

PBS------Phosphate-Buffered Saline

vii INVESTIGATION OF HISTAMINE IN ECHINODERMS

by

Shana L . Smith

An Abstract

o f a thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in the Department of Marine Science in the University of South Florida

August 1992

Major Professors: Norman Blake, Ph . D. and Joseph Torres, Ph.D.

viii Histamine [2-(4-imidazolyl) ethylamine] (HA) was •easured in the gonads and digestive organs of the echinoid Kellita tenuis and the asteroids Luidia clathrata and Astropecten duplicatus. The gonads and pyloric caeca of k· clathrata were examined in detail, and their HA le"vels were related to reproductive status. Two procedures were used to detect HAa (1) a standard o­ pthaldialdehyde (OPT)-based fluorometric histamine assay, modified to inc 1 ude high-performance liquid chromatography, and

(2) on ~· clathrata only, a histological, histamine-specific immunostain. The OPT assay quantified HA in the whole organ, while the immunostain showed the cellular localization of HA. Individuals of L.clathrata displayed all phases of reproductive development from November through April, 1992. Reproductive index correlated inversely with whole gonad HA concentrations, but there was no correlation between reproductive index and pyloric caeca HA concentrations. Photomicrographs of HA-stained sections indicated that HA was concentrated in the peripheral germinal layer during early developmental stages, becoming aore diffuse in later developmental stages. The overall HA concentration does not change as the gametes grow. The results of this investigation show that (1) HPLC used

ix in c o mbination with the OPT-based HA assay is a reliable HA assay that accounts for any interfering substances , ( 2 ) HA exists i n measurable levels in the gonads and pyloric caeca of three species of echinoderms, and (3) the presence of HA in two organs in echinoderms parallels the presence of HA in mu ltiple organs in mammals . Hence, HA is probab 1 y an impo rtant physiological component of both vertebrate and inv ertebrate physiological systems.

Ab stract app r oved: ______Co -Ma jor Professo r, J ose To rres Professor, Department of Marine Science

Co-Ma j o r Professo r, No rm a n Blak e Pro fesso r , Deptartment of Ma rin e Science

Da te o f Approval

X 1

CHAPTER 1

Histamine [2-(4-imidazolyl) ethylamine) (HA) is a potent biochemical commonly associated with allergic and inflammatory reactions in mammals (Turner Associates, 1976;

Gillet al., 1987; Lorenz et al., 1987; Hammar et al.,

1990) . It has profound effects on several physiological systems . In the cardiovascular system, increased HA levels cause vasodilation, lowered blood pressure, increased capillary permeability, oedema, and heart arrhythmia

(Parcells, 1988; Schwartz et al . , 1991). In the central nervous system (CNS), HA affects behaviors, endocrine controls, and physiological responses such as the stress response, motor activity, drinking and eating, aggressive behaviors, and learning (Schwartz et al . , 1990; Schwartz et al., 1991). In the digestive system, HA stimulates gastric acid production (Popielsky, 1920; Wollin, 1989; Lonroth et al., 1990; Bado et al., 1991). In the iamune system, HA causes suppression of lymphocyte proliferation, T cell­ mediated cytotoxicity, lymphokine production, MK cell cytotoxicity, and antibody production by a-lymphocytes, lectin responsiveness, DNA synthesis, and tumour growth

(Bach, 1985; Rocklin, 1985; Sablovic, 1990). In the reproductive system, HA influences and may even help to 2 regulate ovarian blood flow (Schayer, 1962; Varga et al.,

1967 ; Piacsek and Huth, 1971; Krishna et al . , 1988).

Despite a considerable literature on its physiological effects in vertebrates, the physiological function of HA is not well understood.

Several methods are available to aeasure HA in animal tissues. The original technique is the o-pthaldialdehyde

(OPT)-based fluorometric procedure developed by Shore et al.

(1959 ) and modified to account for interfering fluorometric substances such as spermidine, histidine, and other biogenic amines and amino acids {Green and Erickson, 1964; Anton and

Sayre, 1968; Kownatski et al . , 1987). Various researchers have experimented with OPT fluorescence coupled with high performance liquid chromatography, and have come up with viable results (Skofitsch et al., 1981; Ronnberg et al . ,1983; Arakawa and Tachibana, 1986; Houdi et al.,

1986;Jarofke, 1987; Kasziba et al., 1988).

Non-fluorometric HA assays have also been described.

The radioenzymatic or enzyme isotopic assay, which uses HA methyl-transferase and a radioisotope, is a highly sensitive and reliable method, but painstaking to perform

(Snyder,1971). The most recent and specific aethod is the immunoassay, which utilizes a HA-specific •onoclonal antibody (Morel and Oelaage, 1988;Hammar et al., 1990).

This method has not been available until very recently, due to the difficulty in producing an antibody exclusively 3 specific to HA (Hita et al., 1984) .

Little information about HA is available in non­ mammalian systems . HA is present in several invertebrates

(Hettrick and Telford, 1963, 1965 ; Turner Associates, 1976 ) , including the echinoderms (Hettrick and Telford, 1963, 1965;

Smith and Smith, 1985). Smith and Saith (1985) reported that HA is released from coelomocytes of the echinoid,

Mellita tenuis, following exposure to foreign protein (human serum). Echinoderms have measurable levels of HA in both their gonads and digestive organs (Hettrick and Telford,

1963, 1965; unpublished observations) . In most echinoderms, there is an active exchange of nutrients, energy, and hormones between the pyloric caeca and the gonads (Oudejans,

1979a). It is conceivable that HA is translocated as well .

Echinoderms reproduce seasonally; gametic development in spec ies such as Luidia clathrata responds to exogenous factors such as changes in food availability and temperature

(Dehn, 1982). Gametogenesis itself is endocrine-controlled

(Smiley, 1990) . The presence of HA in the reproductive organs of echinoderms, co•bined with the knowledge that HA is a potent mediator of aaaaalian reproductive processes, suggests the possibility that HA is involved in echinoderm reproductive development .

The present study quantified HA in the organs and tissues of the echinoid Hellita tenuis and the asteroids

Luidia clathrata and Astropecten duplicatus ~~ing high- 4 performance liquid chromatography (HPLC) coupled with the

OPT-fluorometric reaction. HA concentrations were determined for the gonads and pyloric caeca of aale and female ~· clathrata, and the levels were coapared to reproductive status. Using a HA-specific aonoclonal antibody stain (Hilab, 1988), the cellular location of HA in

~· clathrata was identified . In light of the ubiquitous presence and potent nature of HA, as well as the lack of informati on about its role in non- mammalian physiology, thi s information should give insight about the physiological r o le(s) of HA in the echinoderms, and possibly in higher animals as well . 5

CHAPTER 2 LITERATURE REVIEW

Histamine

Histamine [2-(4-iaidazolyl) ethylamine] is a potent

amino acid derivative which has cardiovascular, nervous,

digestive, immunomodulatory, and reproductive effects in mammals. It is especially implicated in allergic and

inflammatory reactions (Turner Associates, 1976; Hammar et

al., 1990). It was first synthesized by Windaus and Vogt in

1907; Dale and Laidlaw (1911) first associated it with human anaphylaxis (Rafferty and Holgate, 1989). It is a small, pentamerous compound, formed from the decarboxylation of the amino acid histidine, and easily metabolized (Roitt et al.,

1989).

Typically, HA is stored in the granular aatrix of tissue mast cells and blood basophils of most aammals

(White, 1990). Riley and West (1953) proved that HA is contained in mast cells. The classical pathway of aast cell activation is the IgE response, in which HA and other immunoactive compounds are released when an antigen reacts with specific IgE FcR1 receptors on the aast cell surface

(Dvorak et al., 1983; Roitt et al., 1989). The subsequent local and systemic effects of histamine are those most commonly associated with allergy symptoms (Hammar et al., 6

1990). However, the distribution and differentiation of mast cells indicate that their role in HA release aay have function other than allergy. For example, Purcell and

Hanahoe (1991) discovered that HA in the aast cells of the human placenta exerts vasoconstriction on the placental vasculature, thereby possibly helping to expel the placenta during afterbirth. Tainsh et al. (1991) suggested that mast cells have gone through evolutionary differentiation, as indicated by the morphological differences seen between human uterine, skin, and lung aast cells. Such differentiation in mast cell morphology might indicate a differentiation in histamine effect/function as well. There are also non-immunologic histamine releasers, including endogenous peptides such as substance P and bradykinin, exogenous peptides such as mastoparan and peptide 401, and polyamines such as compound 48/80 and spermine. This release appears to be facilitated by G-proteins (Mousli et al., 1991) .

HA affects the cardiovascular system, the c~ntral nervous system (CNS), the digestive system, the immune system, and the reproductive •ystem (Schwartz et al., 1991;

Lonroth et al., 1990; Timmerman, 1990). Its effects are numerous, and some are pathological. There are three receptor types for HA that have been identified1 H1, H2, and

H3 (Schwartz et al., 1991). H1 and H2 receptors are located systemically; H3 receptors are noticeably most 7

abundant in the brain (Timmerman, 1990). Each receptor type

is implicated in a gamut of HA-related responses (Table 1) .

The effect of HA on the aammalian cardiovascular system

is perhaps the most widely known. In humans, the normal,

non-hypersensitive blood HA concentration is 0.3 to 1.0

ng/ml . If this concentration exceeds 2 ng/ml, vasodilation with accompanying skin flushing, lowering of blood pressure,

increased capillary permeability, oedema, and heart

arrhythmia occur (Parcells, 1988) . Intracerebro-ventricular

administration of HA increases the blood pressure and causes

bradycardia in conscious animals and tachycardia in

anaesthetized animals. This neurally-administered response

is not attributable to leakage of HA into the general

circulation, since HA has a hypotensive effect when it is

administered into the peripheral circulation (Schwartz et

al . , 1991). It is likely that different receptors are at

play. Within the blood, HA is an intracellular messenger

that mediates platelet aggregation . (Saxena et al . , 1989).

The source of this blood HA aay be from histaminergic neurons which, when exposed to platelet-liberated free radicals, are incited to release HA (Mannaioni et al . ,

1991). In the CNS , HA affects behaviors, endocrine controls, and physiological responses . Schwartz et al. (1990) gives two major sources of HA in the brain, mast cells and 9 histaminergic neurons. The latter have axons that form long fiber networks, enervating auch of the brain. He iaputed HA released from these mast cells and histaminergic neurons in such diverse effects as antidiuresis (aaintained through the release of vasopressin), induction of ACTH and prolactin secretion, increase in blood pressure and heart rate, and control of sleep arousal. Schwartz et al. (1980, 1991) also asso ciated HA with the stress response , aotor activity, drinking and eating, aggressive behaviors, and learning.

He further ascribed Hl and H2 receptors to specific hormone secretions, such as vasopressin, oxytocin, growth hormone,

ACTH (Hl), prolactin, thyrotropin, and gonadotropin (H2).

Studi es in the rat show that HA can regulate the release and / or inhibition of adenohypophysal hormones from the hypo thalamus via H3 receptors (Netti et al., 1991).

Timmerman (1990) said that HA is involved with the control of hormonal secretions, energy production, sleeping and waking, cerebral circulation, and control of muscle activity. Fujimoto et al. (1989) did a study on the adaptive behavior of rats from 21 C to 31 C, in which a parallel increase in hypothalamic HA with temperature is believed to have suppressed food intake and increased water intake . In such a way, hypothalamic HA seems to affect adaptive behavior to te•perature.

It has been known for a long time that HA stimulates gastric acid production (Popielsky, 1920; Wollin, 1989). In 10 the human gastric mucosa, HA occurs in three separate cell populationsa endocrine cells that are restricted to the pyloric oxyntic gland area, aast cells which are disseminated throuqhout the wall of the antrum and the body of the stomach, and nerves (Lonroth et al., 1990; Bado et al . , 1991). HA interacts with acetylcholine and 9astrin to control the digestive acid output via one of two hypothetical means. In the •permission hypothesis,• acetylcholine, gastrin, and HA all interact with individual recepto rs, but the full effects of gastrin and acetylcholine are only manifested if the H2 receptors are filled (Grossman and Konturek, 1974). In the •transmission hypothesis," acetylcholine and gastrin induce HA release from the qastric mast cells; HA then directly causes acid secretion

(Code,1965). Knowledge of how HA, acetylcholine and gastrin operate to elicit qastric acid secretion has revolutionized the treatment of peptic ulcer disease

(Lonroth et al., 1990).

HA also has numerous immunomodulatory effects, most of which seem to operate throuqh the H2 receptors. For example, HA causes suppression of lymphocyte proliferation,

T cell-mediated cytotoxicity, lymphokine production, NK cell cytotoxicity, and antibody production by B-lyaphocytes, lectin responsiveness, DNA synthesis, tumour 9rowth, as well as tumour formation and activation of suppressor T­ lymphocytes through the intermediate production of a 11 lymphokine called Histamine Suppressor Factor (HSF)

(Bach,1985; Rocklin, 1985; Sablovic, 1990).

The interaction of HA with H2 receptors on T-cells from the mouse spleen generates both a suppressor factor (HSF) and an enhancing factor, Histamine Proliferating Factor

(HPF) (Eckert and Repke, 1986). Therefore, it appears that

HA acts as a bimodal immunoaodulator on the humoral immune response in mice. HA acting via monocyte H2 receptors regulates natural killer (NK) cell activity by •odulating the interaction of NK cells with interleukin-2 (IL-2)

(Hellstrand and Hermodsson,1990). Rezai et al. (1990) stated that HA seems to exert immunomodulatory effects on

IL-2 receptor expression, especially in patients who have the acquired immune deficiency syndrome (AIDS) . The incidence of HA in patients with AIDS is also addressed by

Burtin et al. (1990), who show that blood HA levels in HIV­ infected infants and children decreased in symptomatic patients. This decrease was associated with the decrease in the number of blood basophils .

HA has a relatively high concentration in the aammalian ovary, with a range of 1 to 5 ug/g in haasters .to 3 to

10ug/g in humans. Varga et al. (1967) showed that HA influences ovarian blood flow, with possible biphasic effects. Free HA aay regulate ovarian hyperaeaia induced by the luteinizing hormone (LH) (Schayer, 1962). A study by

Piacsek and Huth (1971) showed that the Hl blocker 12 promethazine hydrochloride prevented an LH-induced increase of ovarian blood flow; Lipner (1971) and Krishna et al.

(1988) confirmed these results. Murdoch (1991) showed that

HA is acutely liberated from the ovary in response to LH, and proposed that HA (1) acts as an inflammatory aediator of ovulation and (2) causes ovarian contractility and progesterone synthesis. HA is indeed probably involved in ovulation in mammals, including rats, rabbits and humans.

Several HA studies in the past two decades have utilized b o th HA and various antihistamines on the ovary to elucidate its function(s) (Wallach et al . , 1978; Espey et al . , 1982;

Halterman and Murdoch, 1986) . HA increases the contractility of whole ovary at the time of ovulation, causes post-junctional and pre-junctional action on the neuromuscular complex in the follicle wall, causes accumulation of CAMP and subsequent progesterone synthesis, and causes vasodilation of the ovarian artery, which may reduce resistance and increase permeability (Schmidt et al.,

1990) .

Of course, HA is aost faaous for its profound pathological actions in the allergic response. HA plays a major role in such allergic diseases as allergic rhinitis, urticaria, anaphylaxis, and asthma. The cardinal signs of these diseases are iaplicit in their involvement with HA (West, 1990; White, 1990). HA may also contribute to the pathogenesis of atherosclerosis via increasing endothelial 13

permeability to large molecules such as cholesterol, growth

factors, and mononuclear cells,all of which are implicated

in early atherosclerosis (Gillet al., 1987). HA is an

important mediator for cell growth, including cancerous

growth, (Kahlson, 1960; Kahlson et al., 1963). Tills et al.

(1990) also showed that HA aay have a pathologic .role in the

migration and proliferation of H1 receptor-bearing tuaour

cells. However, Burtin et al. (1987) indicated that HA aay

clinically improve the condition of patients with advanced

cancer when given in conjunction with H2-antihistaminics.

Due to its immunomodulatory characteristics, HA is being

considered for other therapeutic purposes as well. In its

natural state, HA could never be used therapeutically,

because of its numerous and unpleasant systemic side

effects. However, new and desirable drugs could be made

which have the immunomodulatory effects of HA, and are

tissue-selective, by derivatizing HA through substitution of

small functional groups (Khan et al., 1987).

While HA is largely responsible for the symptoms of mammalian allergy, little is known about the phylogenetic

origins of the aammalian allergic response. It is thought

that allergic-type responses observed in lower animals, and even some of those in mammals, are primitive and protective.

For example, strong auscular contractions are a aeans to expel gut parasites (Baldo and Fletcher, 1975; Urban and

Gamble, 1989). This response could be aediated by HA, since 14

HA causes smooth .muscle contraction in mammals (Beaven,

1976; West, 1984; Rang and Dale, 1987). Allergy-like conditions have also been reported in fish (Fletcher and

Baldo, 1974; Baldo and Fletcher, 1975) and echinoderms (Smith, 1980).

HA is known to exist in the invertebrate phyla

(Hettrick and Telford, 1963, 1965; Turner Associates, 1976).

HA was isolated from the giant siliceous sponge Geodia by

Ackermann and List (1957). Later, Mettrick and Telford

(1963, 1965) found HA in the tissues and organs of twenty out of twenty-seven marine invertebrate species examined in the West Indies. Smith and Smith (1985) reported that HA is released from coelomocytes of the sand dollar, Mellita tenuis , following exposure to foreign protein (human serum).

HA is an important neurotransmitter in the marine mollusc, Aplysia californica (Schwartz, et al., 1980), and in barnacles (Callaway and Stuart, 1989). Hassel et al.

(1988) showed that interneurons in the brain and visual system of the fly have HA-iamunoreactivity,suggesting that

HA may be an important neurotransmitter/neuromodulator.

Their study is the first known example of a histaminergic system in insects. The distribution of histaainergic neurons in the cyclostomes, which diverged very early from the main vertebrate line, show similarities to corresponding systems in the CNS of amphibians and aammals. Such similarities 15 indicate that histaminergic neuronal systems are phylogenetically old, and have been conserved during evolution (Brodin et al., 1990).

HA has diverse physiological functions in the aamaalian system; it is necessary for complex cardiovascular, nervous, reproductive, and immune functions. It is also upbraided for its pathological effects, and looked upon hopefully as a potential therapeutic agent. HA is ubiquitous in the animal kingdom . Its activity in the invertebrates is quite complex, and is perhaps analogous to that in mammals .

Asteroid Reproduction

Starfish belong to the class Stelleroidea and the subclass Asteroidea in the phylum Echinodermata

(Barnes,1980) . They are commonly referred to as the asteroids. Asteroids reproduce via several means. By far, most asteroids reproduce sexually. They are dioecious; for most species, the male to female ratio approaches 1a1 .

While sexual dimorphism has been suggested, it has not been verified (Chia and Walker, 1991). Out of 1600 known asteroid species, only 26 accomplish reproduction via fission or autotomy (Chaet, 1962; Eason and Wilkie, 1980).

There is a much lesser, variable incidence of hermaphrodism

(Barnes, 1980; Lawrence,1987). Only one asteroid species, 16

Ophiadaster granifer, is known to reproduce via parthenogenesis (Yamaguchi and Lucas, 1984). Most echino derms accomplish fertilization via broadcast spawning, in which the developed eggs and sperm are expelled through the gonoduct and the gonopore, and shed into the surrounding seawater (Barnes, 1980). A smaller p~rcentage of e c hinoderms does not broadcast larvae, but rather broods them (Chia, 1976; Lawrence, 1987).

As with mo st organisms that employ external fertilization, echinoderm reproductive morphology is rather basic (Walker, 1982) . Echinoderm gonads are sac-like single or multiple organs with no elaborate internal structures .

All g o nads lie in contact with the other major organs, and open t o the exterior via gonopores (Walker, 1974a,b , 1976,

1979 ) . Gonads of both male and female asteroids can be mo rpho logically arranged in one of three ways, depending on the species: (1) as a single tuft with a single gonoduct and gonop o re , (2) as a series of tufts along the axis of each arm, or (3) as elongated, branching sacs which enlarge in each arm during gametogenesis (Ludwig and Hamann, 1899; cited in Chia and Walker, 1991).

Echinoderm gonads have distinctive components when examined histologically. In asteroids, ophiuroids, and echinoids, an outer sac exists which determines the overall shape of the gonad and contains muscles which contract during spawning (Walker, 1982). It is composed of three 17 tissues. The outermost of these is the external limiting peritoneum, which borders the perivisceral coelom. A connective tissue compartment separates this layer from the internal limiting epithelium. The outer sac surrounds an inner sac, which also contains ausculature that contracts during gamete release. Its priaary function is that it contains the germ cells for qametogenesis (Walker, 1982).

The inner sac is comprised of three similar, centrally arranged tissues. The outermost layer, called the limiting epithelium, consists of peritoneum that is aade up of myoepithelial cells. In the asteroids, ophiuroids, and echinoids, the limiting epithelium is called the inner schistocoelic muscle layer, and is comprised of peritoneum which borders the gonadal schistocoelic space. The limiting epithelium of the ovary is separated from a connective tissue compartment by a germinative inner epithelium. The germinal epithelium is made up of g~rminal and somatic cells, which are poised to differentiate into sperm and ova

(Davis, 1971; Walker, 1974, 1975, 1976, 1979, 1980;

Schoenmakers et al., 1981).

Sperm and ova have different biocheaical compositions

(Lawrence and Lane, 1982). These compositions can vary annually and with location (Turner and Lawrence, 1979).

Generally, the aain lipid coaponent of ova is neutral lipid, while the main lipid component of sperm is phospholipid

(Allen and Giese, 1966; Lawrence et al., 1966; Lawrence, 18 1973; Oudejans and Van der Sluis, 1979a,b; Turner and

· Lawrence, 1979). Furthermore, there tends to be aore

overall lipid in the ovaries, and hence a larger size, than

the testes of non-brooding asteroids (Fer9uson 1974, 1975a; Lawrence and Lane, 1982).

Both spermatogenesis and oogenesis involve an orderly

progression of germinal cells through differentiation

(Walker, 1982) . Cellular differentiation is orderly and

precise; Holland and Dan (1975) pointed out that for such

morphologically simple reproductive organs, mechanisms of

· development are under careful control, and that gross

morphology does not explain the entire picture.

Oogenesis is well-documented in asteroids (Chia and

Walker, 1991) . Oocytes arise from stem cells in the ovaries

called primordial germ cells. These germ cells produce

oogonia through mitosis, which in turn divide repeatedly to

form primary oocytes. Oocytes at all stages of development

are found adjacent to one another within the inner

epithelium of the same ovary acinus (Smiley, 1990). Oocytes

have a few distinctive organelles. The aost obvious is the

germinal vesicle, which is an enlarged nucleus (Kishiaoto,

1990; Smiley, 1990). The germinal vesicle is positioned

closer to the animal pole of the oocyte, indicating

prefertilization polarization (Smiley and Cloney, 1985).

Cortical granules are found within vesicles; these

organelles contribute to the formation of the fertilization 19 envelope upon·exocytosis (Kay and Shapiro, 1985; cited in Smiley, 1990). All echinoderm oocytes except those of holothurians can secrete three distinct extracellular layers at the plasma membrane at different pre-fertilization stages (Smiley, 1990) . The first of these is called the oocyte basal lamina, or oolamina in holothurians, and is laid down just after the primary oocyte& form from oogonia. The second, called the vitelline layer, is not laid down by holothurians, and is especially thick in asteroids. It is composed of a meshwork of about twenty-five extracellular proteins that are secreted by the oocyte. One of these proteins is most likely the receptor for the bindin protein which coats the sperm acrosome. The outermost layer is called the jelly coat; details of its formation are not well known . The main components of the jelly coat are hydrated glycoprotein& (Chandler and Heuser,1980) and sperm chemoattractants such as speract and resact (Santella et al . , 1983). There is also evidence for sperm surface receptors in the egg jelly (Keller, thesis proposal, 1991). There are a series of interconnected events which ultimately lead to fertilization (Sailey, 1990). These include the commitment of the oocyte to •eiosis, the acquisition of cytoplasmic fertilizability, the formation of the jelly coat, ovulation of the oocyte& into the ovarian lumen, broadcast spawning, and breakdown of the germinal vesicle. 20

A series of .hormones controls the above events. The three major mediators that have been described are the

Gonad Stimulating Substance (GSS), also known as the Radial

Nerve Factor (RNF), or peptide releasing hormone, the

Maturation-Inducing Substance (MIS), and the Maturation­

Promoting Factor (MPF) (Kishimoto, 1990). GSS is a heat stable, protease-sensitive peptide that was discovered by

Chaet and MaConnaughy (1959) in radial nerves of Asterias forbesi. It stimulates the follicle cells which surround each oocyte to prevent them from moving, and promotes the follicular production of Maturation Inducing Substance (MIS)

(Kanatani and Shirai, 1969; Shirai, 1986). MIS is an oocyte maturation hormone, identified as 1-methyladenine (1-MA), which acts on a naked oocyte and initiates the resumption of meiosis (Shirai et al., 1981). It also activates MPF, which acts in the oocyte cytoplasm to drive many of the events leading to fertilizationt nuclear envelope dissolution, acquisition of cytoplasmic fertilizability, germinal vesicle breakdown, events leading to ovulation and spawning, and the production of electrical charges necessary to prevent polyspermy (Kishimoto and Kanatani, 1976; Smiley,

1990). MPF also signals the production of cyclin, which in turn controls the amount of MPF activity propagated (Smiley,

1990). In its purified form, MPF can reinitiate aeiosis I in the oocytes of any asteroid (Kishimoto, 1986, cited in

Chia and Walker, 1991). 21

Meijer et al. {1986) denoted five events as evidence of oocyte maturation in asteroids1 {1) increased protein phosphorylation (see below), (2) appearance of cytoplasmic

MPF, (3) germinal vesicle breakdown, (4) eaission of the two polar bodies, and {5) formation of the female pronucleus.

Several biochemical systems involve phosphorylation during oocyte maturation (Smiley, 1990). The first is a decrease in CAMP-dependent protein kinase synthesis.

Protein kinase, along with cyclin, may hold MPF in its

inactivated phosphorylated state; CAMP seems to facilitate its formation . Raising CAMP levels delays subsequent steps

in oocyte maturation, but does not inhibit them; therefore, other yet-to~be-identified second messengers are involved.

The second biochemical system is the regulation of adenylate cyclase activity, and hence CAMP, by guanine nucleotide binding proteins (G-Proteins). G~Proteins are also implicated in other receptor-mediated events, including control over the third system1 synthesis and utilization of protein kinase C, which is a calcium-dependent kinase that phosphorylates several key proteins. Calmodulin, a calcium­ dependent regulator and universal calcium binding protein, activates enzymes such as phosphodiesterase, protein kinase,

NAD-kinase, and adenylate cyclase; it aay be an interaediate between 1-MA-induced calcium release and enzyme activation during oogenesis (Meijer and Wallace, 1979).

Before ova can be spawned, they must be ovulated. 22

Ovulation is initiated when follicle cells disjoin from the oocyte, retract,· and create a small annulus. The jelly coat becomes swollen, and the oocyte squeezes through the enlarging annulus. As it presses through, its shape becomes distorted into an hourglass form; the oocyte resumes its spherical shape once it is free in the ovarian lumen. The collapsed follicle, meanwhile, remains continuous with the parietal inner epithelium (Smiley, 1990). Neither GSS nor

MIS can stimulate actual ovarian wall contraction to induce spawning (Shirai et al., 1981). Shirai (1986) said that

"some active substance closely associated with maturing oocytes seems to act on the ovarian walls to stimulate contraction of ovarian muscles." This substance is apparently localized in the jelly layers of mature ova, and is characterized as being heat-labile, proteinase­ resistant,and of low molecular weight (Shirai and Kanatani,

1982; Shirai, 1986) .

The oocyte is called an ovum once complete maturation has taken place, the germinal vesicle has broken down, and two polar bodies are formed. Germinal vesicle breakdown is accomplished by a series of morphological and biochemical events, including phosphorylation (Stricker and Schatten,

1989). Fertilization often occurs prior to the formation of the two polar bodies. Spermatogenesis is an annual, predictable event, and there are striking similarities seen between 8pecies (Chia 23 and Walker, 1991). In the very early stages of spermatogenesis, after the previous year's aature sperm have been spent, the spermatogonia enter an asperaatogenic phase for several months . During this phase, they are suspended in Gl of the mitotic cell cycle, and only a ainimal amount of maintenance-type of mitosis is observed (Walker, 1980; van der Plas and Voogt, 1982; Smith and Walker, 1986). The spermatogenic phase is recognized by the formation of columns in the germinal epithelium. The germinal epithelium of the testis is comprised of nutritive somatic accessory cells and immature spermatogonia. It is from the base of these columns that spermatogonia develop via meiosis into primary spermatocytes, and eventually move towards the large central lumen of the testis (Walker, 1980, 1982, 1986 ) .

In holothurians and asteroids, 1-MA seems to be the h o rmone responsible for inducing sperm spawning. While 1-MA is acknowledged as the MIS in echinoderm oocytes, it does not appear to affect spermatocyte meiosis. Therefore, the mechanisms coordinating oocyte maturation, ovulation, and spawning are different from those controlling sperm release, and substituted adenine derivatives such as 1-KA may have multiple roles (Kanatani and Shirai, 1972; Shirai et al,1981; Meijer and Guerrier, 1985, cited in Chia and

Walker,1991; Shirai, 1986; Eckberg, 1988).

Fertilization is dependent upon the acrosome reaction, which is the exocytosis of the acrosome granule to expose 24 bindin and the polymerization of actin to form the acrosomal process. Bindin is the adhesive protein on the acrosomal process which attaches the sperm head to the vitelline layer of the ovum. When the sperm head contacts the jelly coat of the ovum, ligands in the jelly coat activate the sperm via the opening of ion channels, increasing respiration, activating cyclic nucleotide metabolism, and increasing activity of lipases , kinases, and phosphatases (Keller, thesis proposal, 1991). The amount of ova or sperm produced by an animal is called the reproductive output, while the amount of gametes produced per unit body weight is called the reproductive index (Lawrence, 1987). Gonad indices are used extensively in studies of invertebrate reproduction, often as the sole means of determining reproductive status (Grant and Tyler,1983). Change in reproductive index over time has been used to describe echinoderm reproductive cycles (Boolootian,1966) . Farmanfarmaian et al. (1958) used reproductive index to define the reproductive status of four asteroid species. In the asteroid Asterias rubens, the reproductive index increases rectilinearly with gonadal development (Oudejans et al., 1979). Reproductive index is a valuable tool for evaluating gross reproductive status of large sample populations (Chia and Walker, 1991). In bivalves, reproductive index is rendered less useful, since the weight of the whole gonad is affected by factors such as 25 the weight of the somatic cells or the existence of parasites, in addition to the developaental stage of the gametes. However, in echinoderas, reproductive index is more useful, since the allocation of somatic nutrients to the gonads reaches a aaximum threshold, after which it does not increase with increase in gamete size (Lawrence and Lane, 1982). Either way, it is always beneficial to support reproductive index values with histological observations (Grant and Tyler, 1983) . In echinoderms, there is an inverse relationship between the reproductive index and pyloric caeca index. The pyloric caeca contain reserve energy, and endocrine materials that are transferred to the gonads during development (Greenfield et al., 1958; Boolootian, 1966; Nimitz, 1971; Jangoux and Van Impe, 1977; Oudejans, 1979). Attaining the maximum reproductive index is dependent on these reserves in the pyloric caeca (Lawrence and Lane, 1982). Watts and Lawrence (1987) described the effects of estradiol and estrone on the interaediary aetabolism of the asteroid Luidia clathrata. !hey reported that estradiol increases the activity of several biosynthetic aetabolic pathways, which then facilitate the growth of the pyloric caecum. Estrone, on the other hand, negatively influences these biochemical pathways and aay help regulate the translocation of nutrients from the pyloric caecum to the gonad. Xu and Barker (1990) used radioimmunoassay 26 techniques to find estradiol-17B, estrone, and progesterone

· in the ovaries and pyloric caeca of Sclerasterias mollis

throughout its reproductive cycle. !heir findings strongly

suggest a transfer of those hormones from the pyloric caeca

to the ovaries during oogenesis. Once in the ovaries, the

estrogens likely promote oogenesis and protein synthesis~

progesterone may have an inhibitory function (Schoenmakers

et al., 1981~ Voogt and Dieleman, 1984~ Xu and Barker,

1990). Voogt et al. (1991) defined steroid metabolism and

transfer in detail in both ovaries and testes of Asterias

rubens; their findings were parallel to those described

above.

Endogenous endocrine factors are important in

regulating the progress of reproductive development, but

exogenous environmental factors are important in regulating

the level and timing of development (Watts and Lawrence,

1987; Smiley, 1990), A number of exogenous factors affect

reproductive development, such as food, temperature, light,

age, parasitism, and tidal flow. Chia and Walker (1991)

noted the difficulties in isolating individual factors to

determine their effects upon gametogenesis. However,

seasonal correlations do show cause-and-effect

relationships.

!emperature, food supply, and photoperiod are the three most important exogenous factors affecting asteroid

reproduction. Historically, food supply and temperature 27 have been considered the most important (Orton, 1920; Dehn,

1982; Voogt et al., 1991). When temperature is high, catabolism increases and insufficient nutrients are available for reproduction and growth; if food supply can be kept plentiful, this temperature effect is attenuated. Dehn

(1982) reported that while individual populations of L. clathrata have synchronous gametogenic cycles, well-fed laboratory animals have asynchronous cycles year-round.

Temperature, then, becomes important only when food supply is short. In ~· clathrata, there appears to be a synergistic effect between food levels and temperature, and the gametic cycle is cyclic only in terms of the environmental conditions it is exposed to. Echinoderms also respond to photoperiod; again, if food supply is consistent, this response is attenuated (Giese, 1959, cited in Voogt et al., 1991; Pearse, 1981, Pearse and Eernisse, 1982, Voogt et al., 1991). Pearse and his co-workers have shown the influence of photoperiod on gametogenesis in the echinoid

Strongylocentrotus purpuratus and in several asteroid species (Pearse et al., 1986b; Pearse and Beauchamp, 1986;

Pearse and Walker, 1986). Voogt et al. (1991) showed that photoperiod has a significant effect on the timing of reproduction in the asteroid Asterias rubens. The general findings show that long daylengths tend to suppress gametogenesis, and short daylengths tend to promote gametogenesis. Moreover, it is the change in photoperiod 28 which the animals are responsive to; there is no visible difference between animals accliaated to fixed daylengths. 29

CHAPTER 3 DEVELOPKBRT or ~HE HIS~AKI.E ASSAY

Introduction

Histamine (HA) was first synthesized by Windaus and

Voogt in 1907 . B~cause of its potent characteristics, there is a strong interest in accurately quantifying HA in animal tissues. Several techniques were employed initially

(Barsoum and Gaddum, 1935; Rosenthal and Tabor, 1948;

Mcintire et al., 1950; Lowry et al., 1954); Shore et al.

(1959) concluded that they were both laborious and unreliable. The fluorometric assay put forth by Shore et al. (1959) has since been modified, but the basic reaction he employed remains the most popular for modern HA quantifications. His method is to extract HA from tissue homogenates with n-butanol, and to couple the extracted HA to the compound o-phthaldialdehyde (OPT) to form a fluorescent product, which can then be read fluorometrically.

Contained are flaws to the oriqinal procedure by

Shore (1959). First, the HA extraction step does not remove all of the HA in the tissue (Anton and Sayre, 1968; Lorenz 30 et al., 1970). Second, the OPT reacts with coapounds other than HA, such as spermidine, histidine and other bio;enic amines and amino acids (Green and Erickson, 1964; Anton and Sayre, 1968; Kownatski et al., 1987; Lorenz et al., 1987). pH, temperature, dilution, and extraction aodifications to the original procedure of Shore et al. (1959) occurred throughout the next two decades, in ·an attempt to validate the specificity of the OPT reaction (Kreazner and Wilson, 1961 ; Udenfriend, 1962, 1969; Von Redlich and Glick, 1965; Anton and Sayre, 1968; Shore, 1971; Hakanson et al., 1972; Hakanson and Ronnberg, 1974 ; Siraganian, 1974; Turner Associates, 1976). It was not until the late 1970's that hi;h-speed liquid chromatography (HSLC) was considered in lieu of a laborious HA extraction procedure (Tsuruta et al. , 1978). HA can be separated chromatographically and then fluoresced with OPT, or first condensed with OPT and then passed throu;h a high­ performance liquid chromatograph (HPLC) reversed-phase column. Use of the HPLC also has an advantage in that no HA is believed to be lost from the tissue sample (Skofitsch et al., 1981). Various researchers have experiaented with OPT fluorescence detection coupled with HPLC, and have coae up with viable results (Ronnberg et al., 1983; Arakawa and Tachibana, 1986; Houdi et al., 1986; Jarofke, 1987; Xasziba et al . , 1988). Non-fluorometric HA assays have also been described. 31

The radioenzymatic or enzyme isotopic assay, which uses HA methyl-transferase and a radioisotope, is a highly sensitive and reliable method, but painstaking to perform

(Snyder,1971). The most hi9hly specific aethod is the

immunoassay, which utilizes a HA-specific monoclonal

antibody (Morel and Delaage, 1988; Hammar et al., 1990).

This method was not available until recently, due to the

difficulty in producing an antibody exclusively specific to

HA (Mita et al., 1984). The immunoassay is the most

specific, but the OPT/HPLC method requires less effort, and

utilizes standard laboratory reagents and equipment.

HA in echinoderms has been rarely described (Hettrick

and Telford, 1963, 1965; Smith and Smith, 1985), although

its presence in the echinoderms, and invertebrates in

general, is intriguing. There have been no determinations

of HA made in the echinoderms that utilize some of the

newer, HPLC-based methods. This study describes the use of

a modified HA assay, based on the HPLC method described by

Skofitsch et al. (1981), to measure HA levels in various

echinoderm species. 32

Materials and Methods

Experimental animals. Collections were made from

No v ember 15, 1989 through January 22, 1990. The echinoid

Hellita tenuis was collected on the sandbar off of Madeira

Beach, Florida , in depths from one to five meters . The asteroids Luidia clathrata and Astropecten duplicatus were c o llected in the Gulf of Mexico (27 35 ' N; 83 15 ' Wand 27

35' N and 82 50 ' W) and Tampa Bay, by Capetown Dredge and

SCUBA hand collection, in depths of five to thirty meters.

The animals were brought to the laboratory, weighed, measured, and dissected immediately . A portion of each organ or tissue (oesophagus / mouth, intestine, pyloric caecum, rectal sac, tube feet, mucus, gonad) was removed, wet-weighed, and either assayed at once or frozen at minus eighty degrees Celsius .

HPLC system and mobile phase . The HPLC system consisted of an LDC/Milton Roy ConstaMetric IIIG pump, a

Synchropak AX100 anion-exchange column, and a Perkin-Elmer

LS-5 fluorescence spectrophotometer. The chromatography was controlled byE-Lab (TM) (OMS Tech, Inc., Miami, FL) a PC-based chromatographic control and data acquisition system. Chromatogram recordings were charted on an IBM 33 pentane-sulfonic acid in 0.1N acetic acid, and 5\ acetonitrile. This concentration provided a polar elution medium which maximized the separation of HA from interfering substances. All solvents were filtered and thoroughly degassed before use. Flow pressure was aaintained at about

160 psi. The retention time of the HA fluorophore was 1.65 minutes; each analysis was carried out for 4 ainutes. After every six to eight runs, the •ystem was injected with 20 ul of pure acetonitrile to clean the column .

Histamine determination. The HA assay used in this study is a modification of the HPLC-based fluorometric procedure used by Skofitsch et al. (1981). Standards of L­ histamine, L-histidine, spermidine and urea in concentrations of 1 mg / ml, 0 . 5 mg / ml, and 0.1 mg/ml were run in order to assess the impact of the latter "interfering substances." Standards of HA in concentrations ranging from 0.010 ug/ml to 10 ug/ml were assayed to construct a standard curve. Because HA is known to degrade, only six samples were assayed at a time. Sample tissue was suspended in 1.0 ml

0 . 2N HC104 and homogenized with a Wheaton Overhead Stirrer at speed 4. The homogenate was centrifuged at 1000G for five minutes; supernatants were poured into labelled 16 X

100mm Dispotubes and set on ice. 0.4 al lN NaOH and 0.3 ml

1\ OPT were added rapidly in sequence. The tubes were aixed and allowed to react for five minutes. 0.1 ml 3N Hcl was 34 added to stabilize the reaction, and tubes were centrifuged for one minute. 20 ul of the supernatant was injected into the HPLC/spectrofluorometer system, at 360nm excitation and

450nrn emission wavelengths and SX slit setting. HA concentrations were computer-calculated by the E~Lab system, based on the entered standard values.

Results

Analysis of interfering substances . While L-histidine gave a noticeable peak, its retention time was close but not identical to that of HA; furthermore, HA fluoresced approximately twenty-five times more than histidine at 0.1rng / rnl (Table 2). While HA fluorescence increased with concentration, histidine and spermidine fluorescence was inconsistent. The fluorescence of spermidine and urea were both considered negligible. 35

TABLE 2

Relative fluorescence readings of L-histamine , L-histidine, spermidine and urea at 0.1, 0.5, and 1.0 mg/ml concentrations

Concentration (mg/ml)

0.1 0 . 5 1.0

Histamine 606.4 999.9 fluorescence limit

Histidine 24.8 14 . 1 23.4

Spermidine 2 . 5 0.9 0.9

Urea 0 36

Analysis of Standards. HA yielded a reproducible standard curve (Figure 1).

Analysis of echinoderm tissues/organs. Table 3 shows the HA determinations made for H· tenu~s, ~· clathrata, and !· duplicatus. The results indicate that of all the tissue/organ types and species evaluated, HA levels are highest in the gonads and pyloric caeca of Luidia clathrata. 37

TABLE 3

Histamine concentrations in various tissues/organs of the echinoid Mellita tenuis, and the asteroids Luidia clathrata and Astropecten duplicatus

November, 1990

Tissue/Organ Histamine Concentration Cuq/q)

Mellita Luidia Astropecten tenuis clathrata duplicatus (n•7) (n•7) (n•6)

Oesophagus/Mouth 0.493:t0.0814 0 ND

Intestine 1 . 62+0.0523 HA HA

Pyloric Caecum HA 4 . 53:t0 .. 124 0.0731+0.0231

Rectal Sac NA 0 0.0494:t0 . 0195

Tube Feet HA 0 0.0235+0.0104

Mucus HD 4.23:t0.799 HD

Testes 2. 22:t0. 0792 HD HD

Ovaries 9. 76:tl. 04 8.00+0.377 HD

Key: NA • tissue/organ type is not applicable to species ND • no data collected 38

Histamine Standard Curve 6X tl\f; 6* euttonitrilf t.O 0.9 v

/ v 0.8 v~ / 0.7 [7 -. t 0.6 / bJ~ v ~~ ·~ 0 / 0.5 / 't'~I! ... v ~ 0 v ~f 1) .4 / ..... v / 0.3 v 0.2 v v v 0.1 v 0.0 v o.o o.t o.4 o.6 o.s 1.0 1.t 1.4 #.6 '·' t.o Bilfclmint Ufl f

FIGURE 1 Histamine standard curve 39

Discussion

The HA determinations made by the modified HA assay

ar-pear to be accurate. The results show HA effectively

s~parated on the HPLC column, had a reproducible standard

curve, and had a much higher fluorescence than other OPT­

reGctive interfering substances. The method is superior to

pre~iously -described HPLC methods in that interfering

~u t stances are not problematic under the assay conditions.

and determinations can be made quickly and routinely.

Utilization of this assay on echinoderm tissues/organs

op~ns up s om e interesting questions about the function of HA

1n th1s phylum. HA levels were highest i n the gonads,

intestine, and pyloric caeca of the three species examined.

In mammals, HA plays a role in both ovulation and digestion

!Lonroth et al . , 1990; Schwartz et al . , 1991) . Its parallel

existence in echinoderm reproductive and digestive organs is

intriguing, and may reflect a similar function. If so, some of the broad physiological effects of HA may have been conserved through evolution . In echinoderms, hormones such as estrone, estradiol and progesterone, as well as nutrients and energy, are translocated from the pyloric caeca to the gonads as

reproductive development progresses (Oudejans, 1989; Watts and Lawrence, 1987). If in fact HA has a function related 40 to the reproductive and digestive physiologies of these

animals, the possibility that HA concentrations may also

change in these organs merits further investigation.

The role of HA as a gamete-shedding hormone is also a

possibility. Kanatani (1964) reported the presence of a

hormone called gamete-shedding substance, or GSS. This

hormone was later renamed gonad-stimulating substance

(Kanatani, 1969a, cited in Giese and Kanatani, 1987), and

its identity contains a species-variable number of different

amino acid residues (Kanatani et al., 1971) . Shirai (1986)

noted that neither GSS nor another reproductive maturation

hormone, called maturation-inducing substance (MIS) can

induce the ovarian musculature contractions neccessary for

broadcast spawning, and that some other unidentified, active

substance is involved. It is possible that HA, which causes

smooth muscle contractions in mammals (Turner

Associates,1976) may have some sort of spawning-induction

function .

The presence of HA in the intestine and pyloric caecum

is also interesting. There are several possibilities as to the existence of HA in digestive organs. Studies by

Hakanson et al. (1986) report that in vertebrates ranging from teleosts to mammals, HA is in all cases located in the gastric epithelial cells. Beaven (1976) states that through evolution, HA first becomes associated with the intestine, and only later with the mast cell. In the echinoderms, the 41 intestine and oesophaqus are the primary visceral tissues that have contact with both the external and internal environment (Barnes, 1980). HA in these tissues mav result in a change in permeability to seawater. since it affects permeability in vertebrate tissues (Beaven. 1976: Turner

Associates, 1976). Another possible role for HA in the intestine miqht be to expel intestinal parasites (Baldo and

Fletcher, 1975). It is interestinq to note that HA was not very concentrated in the pyloric caeca of A. duplicatus.

Its function in echinoderms is as vet unclear, and the minimal levels found in A. duplicatus as compared to L. clathrata and H. q~inquiesperforata suqqest that there may be species-related functional differences .

In conclusion. the HPLC fluorometric HA assav utilized in this study effectively measured HA in certain echinoderm organs. The data obtained from this assay suqqests that HA could be playinq one or several important roles in echinoderm phvsioloqy, which remain to be elucidated. 42

CHAPTER 4 INCIDENCE OF HISTAMINE THROUGHOUT THE REPRODUCTIVE CYCLE OF THE ASTEROID LUIDIA CLATHRATA

Introduction

There is a noticeable lack of information about the potent biochemical histamine [2-(4-imidazolyl} ethylamine]

(HA) in non-mammalian physiology. HA is found in the invertebrates (Mettrick and Telford, 1963, 1965; Turner

Assoc iates, 1976}; in the echinoderms, it has been measured in the reproductive and digestive tissues, and in the coelomic fluid (Mettrick and Telford, 1963, 1965; Smith and

Smith, 1985; unpublished observations}. Echinoderms have measurable levels of HA in both their gonads and digestive organs (Mettrick and Telford, 1963, 1965; unpublished observations). Smith and Smith (1985) reported the release of HA from coelomocytes exposed to a foreign protein (human serum).

In mammals, HA has potent effects on the cardiovascular system, the central nervous system (CNS), the digestive system, the immune system, and the reproductive system

(Varga et al., 1967; Bach, 1985; Wollin, 1989; Lonroth et al . , 1990; Timmerman, 1990; Bado et al., 1991; Murdoch,

1991; Schwartz et al. ,1991). The presence of HA in echinoderm reproductive and digestive organs, and the 43 knowledge that HA is a potent mediator of mammalian reproductive and other phvsiological processes, suggest the possibility that HA is involved in echinoderm reproductive dev elopment, and possibly in other functions.

Walker (1982) defined four broad reproductive stages in the echinoderms: (1) the agametogenic stage, which occurs between consecutive gametogenic events , and which is scme times absent in species with overlapping or continuous gamet1 c cycles , (2) the proliferative stage, in which there is gon1 al mitosis, (3) thP rl1fferentiative stage, during

\l hlch gon1a are differentiated and stored f or later spa\lning. and ( 4 ) and e va c uative stage ( spawn ing ).

Ec hinoderms generally reproduce seasonally. In the astero1d Luidia clathrata, the onset of ga metogenesis i s tr1gg ered by exogenous .fac t ors such as changes in temperature and food supply (Dehn, 1982 ). Echinoderm gametogenesis is endogenously controlled by endoc r1ne fac tors (G iese and Pearse, 1974 ; Smiley, 1990 ).

The weight of the gonad s in an anima l divided by its total body weight is called its reproductive index

(Lawrence, 1987 ) . It reflects the proliferative and differentiative reproductive stages defined by Walker (1982) above . Oudejans et al. (1979) showed that the reproductive index increases rectilinearly with gametic development in t he asteroid Asterias rubens . This relationship has not been similarly determined f or k · clathrata. Histological 44 determination of the relationship between reproductive index

and reproductive developmental stage would be a helpful tool

f e r bi ochemical analyses of echinoderm gonads.

The purpose of this study was to compare HA levels in

the gonads and pyloric caeca of ~· clathrata with

re~roductive indices, in order to infer if/how HA

conce ntration changes ~lith reproductive development, and to

determine the cellular location of HA in these organs . HA

~en be measured quantitatively in animal tissues using an o­

p ~ ha l d ialdehyde IOPT) fluorometr1c reaction (Shore, 1959 )

combined with high-performance liquid chromatography

(Skofitsch et al . , 1981). The actual location of HA at the

cellular l FvF l can be determined using aHA-specific

rncn oclonal antibody labeled to a histol ogical peroxidase­

an~lFercxida se (PAF ) sta in (Hilab , 1968 ; Hammar et al.,

1 ~~0· . It is hoped that this information will shed light on

the possibl e physiological function of HA in echinoderms,

an d possibly in higher animals as well.

Materials and Methods

Experimental animals. One-hundred ~· clathrata were

collected from Tampa Bay, Florida in the vicinity of Demen 's Landing, St . Petersburg (27 47.60 Nand 82 35.45 W) , in

February, 1992 . Animals lived at a depth of three t o five 45 meters. The animals were brought to the laboratory, weighed, measured, and dissected. One-tenth of the total gonads an~ one-tenth of the total pyloric caeca (half of the content~ of one arm) were separately removed, weighed, and frozen at -80 C until ready to be assayed. Reproductive index (wet body weight of gonads divided total body weight) and _sex were recorded for each animal.

Fifty ~· clathrata were collected from Tampa Bay, Flcrida at the same coordinates in Demen's Landing, St.

I~tersburg, in April, 1992. Animals were brought to the laboratory, weighed, measured and dissected. The total

~onads and tctal pyloric caeca were separately removed,

~e1ghed, and treated for histological preparation as d~~~r1r~d belo''· Reproductive index and sex were recorded f~r each animal. Histamine determination. The HA assay used in this study is ~ derived from the original fl~orometric procedure done by Shore et al. (1959), and is a modification of the HPLC-based fluorometric procedure used by Skofitsch et al.

(1981). Because HA is known to degrade, only six samples were assayed at a time. The HPLC system consisted of an LDC/Milton Roy

ConstaMetric IIIG pump, a Synchropak AX100 anion exchange column, and a Perkin-Elmer LS-5 fluorescence spectrophotometer. The chromatography was controlled by

E-Lab (TM} (OHS Tech. Inc., Miami, FL} a PC-based 46 chrom6tographic control and data acquisition system.

Chromatogram recordings were charted on an IBM Proprinter

II.

The mobile phase consisted of 0.1\ pentane-sulfonic acid in 0 . 1N acetic acid, and 5% acetonitrile. This concentration provided a polar elution medium which maximized the separ~tjon of HA from interfering substances.

All solvents were filtered and thoroughly degassed before t: .:· <:: • F 1 o \.' pre s sure "'as maintained at about 1 6 0 psi . The r~tent1on time of the HA fluorophore was 1.65 minutes; each

3~2lys1s w~s carried out for 4 minutes. After every six to eight runs, the system was injected with 20 ul cf pure acetonitrile to clean the column.

Sample tissue was suspended in 1.0 ml 0.2N HC104 and homogenized with a Wheaton Overhead Stirrer at speed 4. The

~omogenate was centrifuged at 1000G for five minutes; the supernatants were poured into labelled 16 X 100 mm

Dispotubes and set on ice. 0.4 ml lN NaOH and 0 . 3 ml 1% OPT were added rapidly in sequence. The tubes were mixed and allowed to react for five minutes. 0 . 1 ml 3N HCl was added to stabilize the reaction, and tubes were centrifuged for one minute. 20ul of the supernatant was injected into the

E-Lab HPLC/spectrofluorometer system, at 360nm excitation and 450nm emission wavelengths and SX slit setting.

Standards of HA in concentrations ranging from 0.010 ug/ml to 10 ug/ml were assayed to construct a standard curve. HA 47 concentrations are computer-calculated by the E-Lab system, based on the entered standard values.

Histology. The organs from fifty specimens were placed in Helly's (Zenker-formalin) fixative (Yevich and Barszsz,

1977b) - ~or eighteen to twenty-four hours. The organs were then put into individually labelled cassettes and washed with tap water for another eighteen to twenty-four hours.

Washed cassettes were stored in S-29 dehydrant until ready for processing. Processing was done in an Autotechnicon

(TM), which sequentially immerses the tissues in six changes of S-29 alcohol dehydrant followed by three changes of uc-

670 clearing agent. Organs were then embedded in Ameraffin

Tissue Embedding Medium (TM), using a Tissue-Tek II Tissue

Embedding Center (TM). Seven micron~thick sections were cut in duplicate sets on an American Optical (TM) rotary microtome~ these sections were placed on glass slides and incubated at 37 c in a moisture chamber until ready for staining. Staining. One set of thin sections was stained with

Harris's heaatoxylin and eosin (Yevich and Barszcz, 1977b).

These slides were used to visually stage the gonadal

~ development of k· clathrata, and to coapar~ the stages with the calculated reproductive indices. A duplicate set of thin sections was peroxidase-stained with the HA monoclonal antibody (Milab, 1988), in order to visualize the cellular location of HA at each reproductive 48 stage. Sections were deparaffinized in a series of alcohol, rehydrated to water, and then immersed in phosphate-buffered saline (PBS) . . one drop of HA antiserum was placed on the sections for twelve to twenty-four hours at four degrees celsius in a moisture chamber. After rinsing in PBS for thirty minutes, the sections were immersed sequentially with properly diluted rabbit IgG antiserum for thirty minutes at room temperature, a PBS rinse for thirty minutes, peroxidase-antiperoxidase (PAP) complex for thirty minutes at room temperature, and a fresh solution of 3.3' diaminobenzidine (60mg/ml) and hydrogen peroxide (0.01%) in

0.05M Tris buffer (Ph 7 . 6) for thirty minutes at room temperature. The sections were then dehydrated through a series of alcohol dilutions, and preserved for storage by the application of Permount (TM) and a coverslip. The site of the HA antigen-antibody reaction showed a brown peroxidase reaction product.

Results Reproductive stages. Based on the hematoxylin-eosin histological staining, both male and female gonads could be classified into the following three stagesr (1) early developmental stage (Figures 2A, 2B), (2) late developmental stage (Figures 3A, 3B), and (3) ripe stage (Figures 4A, 4B).

Figures A and B represent female and male stages, 49

respectively. Table 2 shows the characteristics and the

range of reproductive indices associated with each stage.

Histamine determination. Figures 5-8 show the HA concentration vs. reproductive index (RI) for female gonads

(r-squared = 80.9%), female pyloric caeca (r-squared=

15.81%), male gonads (r-squared 3 87.2%), and male pyloric caeca (r-squared=0 . 17%), respectively. The r-squared values

for Figures 4-7 were calculated by the multiplicative b interpretation (y=AX ) . Histamine values were, overall, higher in females than in males. Furthermore, females displayed more general HA-unrelated fluorescence (as seen in

HPLC profiles) than did males .

Immunostaining. Twelve immunostains were successfully performed. The results of the HA immunostaining are shown in Figures 9-14. Figures 9A, 10A, and 11A are photomicrographs of female gonads at reproductive index values of 0.01333, 0 . 0702, and 0.15939, respectively. These figures show the decrease in small oogonia with increasing gonad index. Figures 98, 108 and 118 are photomicrographs of the associated pyloric caeca. Figures 12A, 13A and 14A are photomicrographs of male gonads at reproductive index values of 0.01878, 0.07186, and 0.1652, respectively. These figures show the increase in mature spermatocytes in the lumen of the gonad. Figures 128, 138, and 148 are photomicrographs of the associated pyloric caeca. 5 0

FIGURE 2 Ea rly developmenta l stage o f ( a) fema l e and ( b ) ma le g oP.ads o f Luidia c lathrata. ~ ~ m a t o x y lin / e o s i n s t ai n. 51

FIGURE 3 Late developmental stage of (a) female and (b) male gonads of Luidia clathrata. Hematoxylin / eosin stain. 5 2

FIGURE 4 Ripe stage o f ( a) female and (b) male go n a ds of Luidia clathrata. Hematox ylin/ e o sin stain. 53

HISTAA11NE CONCENTRATION VS. Rl Ttma.~ Cona.ds, Luictia cl4thra.ta 7,------~

6 -

5- ..... ~. \ ~ ..-' '- 4-

~ ...~ 0 ~ 3- ~ · ~ ~ 2 - 0 0 D

f - D 0 Cl [J 0 0 0 D 0 [J Cl 0 0 Dr 0 04-~, --~~~1 --r-1 ~, --~, ~~r-~1 --r-1 ~1 --r-l~lr-T, --r-1 ~1 --~l ~lr-~T~ 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18

R~produc~v~ ln~z

FIGURE 5 Histamine concentration in the gonads of female Luidia clathrata versus reproductive index. 54

HISTAMINE CONCENTRATION VS. RI ffm4~ Pyloric Ccucum. Lu.icfid cla.th:ra.td 5.------~c~------. 0

4-

0 0 0 p 0

0 1 - 0 0 0 8 oo [

0~-~~~~~~~1 --r-1 1,--~,l,r-T,--r-,1,--r-,l,r-T,--r-,1,--r-,l,r-l,~ 0 0.02 0.04 0.06 0.08 O. t O.t! O.t4 O.t6 O.t8 Jlf'P1'0d:cJ.Ctivf /ntU:r

FIGURE 6 Histamine concentration in the pyloric caeca of female Luidia clathrata versus reproductive index. 55

HISTAl.1IflE CONCENTRATION VS. RI Jla.lt Cona.d.!, Luidia cl«tkra.ta. 2. 2 2. 1 - 0 2 - f .9 - t .8 - I. 7 - 0 I .6 - 0 Cl "' t .5 - ~ D, 1.4 - ;s ...... f .3-

~ 1.2 - ~ 0 ·.; f. f - E t! f - 0 0 ~ 0.9- 0 · ~ tz: 0.8 - 0 0.7 - 0.6 - 0 0 0 0. 5 - oo

0.4 - 0 0 0.3 - 0 [ oD 0 0 0.2 - 0

0. f I I I I I I I I I I I I I I I I 0.01 0.03 0.05 0.07 0.09 0.11 0.13 0.15 0.17

R~rod:u.ctiv~ ln

FIGURE 7 Histamine concentration in the gonads of male ~uidia clathrata versus reproductive index. 56

HISTAMINE CONCENTRATION vs. RI Jla.lt Con.a.d.!, Luid:ia. cl4th1-ata .f.f [J 2. f - f- I .9- 1.8 - I . 7 - 0

1.6 - D CJ ..... ~ f .5- \ D, 1.4 - ~ ..... 1.8- '\' 1.2 - ~ 0 ... f ' f - ~ f - [J 0 ~ 0.9 - 0 ·~ 0 ~ 0.8 - 0.7- 0.6- [J 0 0 0.5 - Do 0.4 - 0 0 0.3 - 0 oo 0 0 c 0.£ - 0

0.1 I I I I T T T I I I I I I I I I 0.0 I 0.08 0.05 0.07 0.09 O.tl 0.13 0.15 0. I 7

Rtprod:u..ctiv~ JniU:r

FIGURE 8 Histamine concentration i n the pyloric caeca of male Luidia clathrata versus reproducti ve index . FIGURE 9 Histamine immunostain o f the ( a ) gonad and (b) pyloric cecum o f a female Luidia clathrata with a reproductive index value of 0.01333. FIGURE 10 Histamine immunostain o f the (a) gonad and (b) pyloric caecum o f a female Luidia clathrata with a reproductive index value of 0.0702. 59

FIGURE 11 Histamine immunostain of the (a) gonad and (b) pyloric caecum o f a female Luidia c lathrata with a reproductive index value of 0. 15939. FIGURE 1 2 Histamine immuno stain of the (a) gonad and (b) pyloric caec um o f a male Luidia c lathrata with a repro duc tiv e index v alue o f 0 . 0 1878 . 61

FIGURE 13 Histamine immunostain o f the (a) gonad and (b) pyloric caecum of a male Luidia clathrata with a reproducti ve index v alue of 0 .07186. 62 A

FIGURE 14 Histamine immunostain of the ( a ) gonad and (b) pyloric caecum of a male Luidia clathrata with a reproductive index value of 0 . 1652 . 63

TABLE 4 Reproductive index (total gonad weight/total wet body weight) values per gonad developmental stage•

Stage Description Reproductive Index

early developmental; germ­ inal layer is thickest, immature gametes loogonia 1 and spermatogonia) are 0.01-0.05 prolific: existence of s permatogenic columns

late developmental; follicles have expanded to accommodate increasing numbers o t aametes; some oocytes have broken free 2 of follicle wall and are in 0.05-0 .1 lumen ; at least half of the male foll1cles contain a thicker center core of matu~e spermatozoa; germinal wall is thinner

ripe; follicle walls are thin, and follicles themselves have fully expanded; follicles con- tain almost all mature gametes; 0 . 1-0.16 thin germinal layer; absence of spermatogenic columns

*n = five animals per stage, male and female 64

Discussion

Dehn (1980) described five stages of gametic growth in the gonads of h· clathrata: Stage I is the developing gonad, in which the germinal layer is thick, developing oocytes are small and located on the periphery of the follicle, the lumen of the testis is filling with mature sperm and spermatogenic columns are present; stage II is the growing gonad, in which the germinal layer has become thinner, the lumen of the ovary is full of intermediate and large oocytes, and the lumen of the testis is filled with m~ture sperm; stage III is the mature gonad in which the germinal layer is thinner, few small oocytes are present, and the lumens of the ovary and testis are filled with mature gametes; stage IV is the relict gonad, which is the residual reproductive material left after spawning; stage V is the resorbed gonad, in which only a genital rachis, the peritoneum of the body cavity, is visible in ray cross­ section. The three stages observed in this study in the gonads of h· clathrata (Figures 2-4) are parallel to stages I-III described in Dehn (1980). Stages IV and V are post-spawning stages; hence, they are not encountered in the present study. Table 4 associates the histologically-defined 65

reproductive stages with a reproductive index value range.

As stated by Chia and Walker (1991), reproductive index

values appear to be a good way to grossly define the

r~ crn rluctive status of an individual. It is an effective measurement for studies of this sort, in which biochemical

trends are observed over the general span of reproductive development.

F1gures 5 and 7 show that HA tends to decrease predictably in the gonads with an increase in reproductive

index. It is interesting to note that the maximum HA levels and overall, non-HA fluorescence were highest in females.

In mammals. the reproductive association of HA has primarily been in females . HA has a high concentration in the mammalian ovary, with a range of 1 to 5 ug/g in hamsters to

3 to 10ug/g in humans. Varga et al. ( 196 7} showed that HA influences ovarian blood flow, with possible biphasic effects. Free HA may regulate ovarian hyperaemia induced by the 1 u te i ni zing hormone ( LH} ( Schayer, 196 2} . Piacsek and

Huth ( 1971), Lipner (.1971} and Krishna et al. ( 1986} showed that the Hl blocker promethazine hydrochloride prevented an

LH-induced increase of ovarian blood flow. Murdoch (1991) showed that HA is acutely liberated from the ovary in response to LH, and proposed that HA (1) acts as an inflammatory mediator of ovulation and (2) causes ovarian contractility and progesterone synthesis . HA increases the contractility of whole ovary at the time of ovulation, 66 causes post-iunctional and pre-iunctional action on the neuromuscular complex in the follicle wall. causes accumulation of CAMP and subsequent proqesterone synthesis, and causes vasodilation of the ovarian artery. which may reduce resistance and increase permeability (Schmidt et al .•

1990). It is possible that similar chemical messenqers are actinq with HA to influence qametoqenesis and ovulation in the female starfish.

Althouqh qonads show a noticeable decrease in HA with increasinq reproductive index. pyloric caeca show no trend related to reproductive index whatsoever. HA still seems to be a prominent, albeit variable. component of these orqans, since its concentrations encompass a considerable, unpredictable ranqe.

The HA - specific immunostain technique was successful with only minor modifications required: namely, alcohol dehydration steps need to be careful and numerous, and more hydroqen peroxide is required to display the peroxidase­ antiperoxidase staininq with the best resolution.

Figures 9-14 are especially intriquinq, because they show the qualitative localization of HA from a cellular viewpoint. It is very evident from the Photomicroqraphs that HA decreases with increasinq reproductive index in the gonads of both males and females, and has no visible trend in the pyloric caeca. These results are in aqreement with the assay results (Fiqures 5-8). The Photomicroqraphs also 67 reveal what appears to be a particular localization of HA in the germinal epithelium at early reproductive stages; this localization becomes considerably diffuse through development .

These results, combined with information about the role of HA in as an intracellular messenger in mammals for the same chemi ca l mediators involved in asteroid reproduction, suggest that HA may be playing an active role in the early ge rminati o n steps of gametogenesis . In mammals, HA is invo lved with such intracellular messengers as CAMP, cyclin ,

G proteins, reproductive, vasodilatory and growth hormones, and c alcium release (Schmidt et al., 1990; Timmerman, 1990 ;

Murdoch. 1 991 ; Schwartz et al., 1991). All of these mes senge r s are required for asteroid oogenesis. In a steroids , protein kinase, along with cyclin, holds the

Haturatio n Promoting Factor (MPF), a hormonal mediator of early oogenesis, in its inactivated phosphorylated state;

CAIIP seems t o facilitate its formation . Raising CAMP levels delays subsequent steps in oocyte maturation, but does not inhibit them; therefore, other yet-to-be-identified second messengers are involved. The regulation of adenylate cyclase activity, and hence CAMP, by guanine nucleotide binding proteins (G-Proteins), is also important during asteroid oogenesis. Calmodulin, a calcium-dependent regulator and universal calcium binding protein, activates enzymes such as phosphodiesterase, protein kinase , NAD- 68

. ki~ase, and adenylate cyclase and is a probable intermediate betueen 1-methyladenine (1-MA)-induced calcium release and enzyme activation during asteroid oogenesis (Meijer and Wallace, 1979) .

The variable presence of HA in the pyloric caeca of ~· clathrata is more enigmatic. Concentrations of endocrine compounds such as estrogen, estrone, estradiol, progesterone and synthesized steroids are known to change with gametic grc\ith; it is posE i ble that they are translocated from the p~ · lcr i c caeca to the: gonads (Oudejans et al.. 1979; Voogt a~~ Schoenmakers, 1979; Xu and Barker, 1989). If HA is Flaying a role in the reproductive phvsiology of k· cl a thrata, it may be similarly synthesized and/or tr a n~ l ocated with gametic development. Many speculations

Cl ~ b ~ mad e based upon the translocative relationship b€-t \I O:E:n t ht: pyloric caeca and the gonads (Greenfield et al.,

Boolootian, 1966 ; Nimitz, 1971 ; Jangoux and van Impe,

1~ 77 ; 0udejans, 1979; Lawrence, 1987; Barker, 1990) and the role of Hh in mammalian digestion (Lonroth et al., 1990:

Wollin, 1990; Bado et al., 1991) . It is possible that HA is playing an important role in both of these respects, but further investigation ·is needed . From a phylogenetic perspective, HA in echinoderms is both enigmatic and intriguing . A study by Delgadillo­ Reynoso et al. ( 1989) reports that the chordates share a more recent common ancestor with the echinoderms than with 69 any other i nvertebrate phyla. As such, the echinoderms may serve as a more primitive model of our own physiological systems. As in mammals, HA may have multiple roles (i.e . repro duction , digestion, immune responses) in echinoderms . The results of this study suggest that HA may display a common intracellular behavior in animal systems. 70

CHAPTER 5 DISCUSSION AND CONCLUSIONS

The results of this study successfully quantified HA

in the organs and tissues of the echinoid Hellita tenuis,

and the asteroids Luidia clathrata and Astropecten duplicatus, and hjstnlogically located HA in the gonads and

~ylori c caeca of k· clathrata. Techniques. Two techniques were utilized: (1)

m~a~~rement of HA with a modified assay, which utilizes

nig~-performance liquid chromatography (HPLC) coupled with

the CPT- flucrometric reaction {Shore et al., 1959; Skofitsch et al . , 1981 ) , and (2) histological staining of k· clathrata t isEues with a stain labelled with a HA-specific monoclonal

ant ibody (lliLab, 1988 ) . Both techniques are recommended for investigations of this type.

Non-fluorometric HA assays have also been described .

The radioenzymatic or enzyme isotopic assay, which uses HA

methyl-transferase and a radioisotope, is a highly sensitive

and reliable method, but difficult to perform (Snyder,1971) .

81 11989) showed that HA could be measured with a two-barrel

organic ion-sensitive microelectrode. The most recent and

specific method is the immunoassay, which utilizes a HA­

specific monoclonal antibody (Horel and Delaage, 1988; Hammar et al., 1990 ). This method has not been available

un~1l very recently, due to the difficulty in producing an 71 antibody exclusively specific to HA (Mita et al., 198 4).

While the latter assay is the most specific , the OPT / HPLC method utilized in this study is in many ways advantageous in that requires less effort, and utilizes standard laboratory reagents and equipment.

The HA-spec~fic immunostain is a new procedure i ntroduced by MiLab (1988). It had not been applied to e chinoderm t1~s u ~s previously. The technique was successful

':ith onlv minor modi fications required: namely, alcohol dehydration steps need to be careful and superfluous, and m(re hyd r ogen peroxide 1s required t o display the p~ro):id ase -antiperoxidase staining with the best reso lution .

Hlstamin e 1n echinod erm tissues a nd organs. The

~e su lts of this study verify the e >: Jstence of histamine ( HA ) in three species of echinoderms . Utilization of the fluorometric HA assay on echinoderm tissues /organs opens up scme i nteresting questicns about the functi on of HA in this phyla . HA levels were by far generally highest in the gonads, intestine, and pyloric caeca of the three species examined . In mammals, HA seems to play a role in both ovulation and digestion (Lonroth et al., 1990; Schwartz et al., 1991 ). Its parallel existence in echinoderm reproductive and digestive organs is intriguing, and may reflect a similar function. If so, some of the broad physiolog1cal effects of HA may have been conserved through 72 ev.:·l uti on.

Histamine in Luidia clathrata.

HA levels in the gonads and pyloric caeca of ~·

clathrata were compared with reproductive indices in order

to infer if/how HA concentration changes with reproductive d~v~lopment. The HA-specific histological immunostain was used to pinpoint the location of HA in gametic and digestive cell~ .

I ndividuals of ~· clathrata displayed all phases of

re~rod~ctiv~ development from November through April, 1992.

~~p~ o du c t1ve index correlat~d inversely with whole gonad HA con=entrations, but there was no correlation between re~r o ductive index and pyloric caeca HA concentrations.

The maximum HA levels and overall, non-HA fluorescence were hlghest in femal~s. This latter result is particularly interesting in light of the presence of HA in female man:mals. HA has a relatively high concentration in the m~mmal1an ovary, with a range of 1 to 5 ug/g in hamsters to

3 to 10ug/ g in humans. Varga et al. (1967) showed that HA influences ovarian blood flow, with possible biphasic effects. Free HA may regulate ovarian hyperaemia induced by the luteinizing hormone (LH) (Schayer, 1962). A study by

Piacsek and Huth (1971) showed that the H1 blocker promethazine hydrochloride prevented an LH-induced increase of ovarian blood flow; Lipner (1971) and Krishna et al. r 1986) confirmed these results. Hurdoch ( 1991) showed 73 that HA is acutely liberated from the ovary in response to LH, and proposed that HA (1) acts as an inflammatory mediator of ovulation and (2) causes ovarian contractility and progesterone synthesis. HA increases the contractility of vhole ovary at the time of ovulation, causes post­

)un~ tional and pre-junctional action on the neuromuscular c o mpl~ x in the follicle wall, causes accumulation of CAMP and subsequent progesterone synthesis, and causes va!odil ~t i o n o f th~ ovarian artery, which may reduc e r~ s J s tan c e and increase permeability (Schmidt et al., 1990 ) .

lt 1s possitle that similar chemical messengers are acting Hith HA to influence gametogenesis and ovulation in th ~ female starfish. Echinoderm gametogenesis is a carefu l ly controlled sequence of endogenously-controlled eve nts. There are thre e primary endocrine c omp ound s in v o l v~ d : ga m et~-shedding substance (GSS) , maturation­ i nduc ing substa nce (MIS). and maturation-promoting factor

( 1t F'r ) . It P F i s r e g u 1 ate d by the am ount o f c y c 1 i n p r e s e n t in the intercellular environment (Smiley, 1990). In mammals, HA plays a role in cyclin regulation (Schmidt at al., 1990). HA ma y have a similar regulatory effect of cyclin in starfish.

The variable presence of HA in the pyloric caeca of ~· clathrata is more enigmatic. Concentrations of endocrine comp ounds suc h a s estrogen, estrone, estradiol, progesterone and s ynthesized steroids are known to change with gametic 74 growth; it is possible that they are translocated from the pyloric caeca to the gonads (Oudejans et al., 1979; Voogt and Schoenmakers, 1979; Xu and Barker, 1989). If HA is playing a role in the reproductive physiology of ~. clathrata, it may be similarly synthesized and/or translocated with gametic development. Many speculations can be made based upon the relationship between the pyloric caeca and the gonads (Greenfield et al., 1958; Boolootian,

1966; Nimitz, 1971; Jangoux and van Impe, 1977; Oudejans,

1979 ; Lawrence, 1987; Barker, 1990) and the role of HA in mammalian digestion (Lonroth et al., 1990; Wollin, 1990 ;

Bado et al., 1991) . It is possible that HA is playing an important role in both of these respects, but further investigation is needed.

Photomicrographs of HA-stained sections indicate that

HA is concentrated in the peripheral germinal layer during early developmental stages, and becomes more diffuse in later developmental stages . It appears that the overall HA concentration is not changing as the gametes grow.

The results of this investigation show that (1) HPLC used in combination with the OPT-based fluorometric assay is a reliable HA assay, which accounts for any interfering substances which might be present, (2) HA exists in measurable levels in the gonads and pyloric caeca of three species of echinoderms, and (3) the physiological function of HA in these organs remains to be elucidated, but the 75 dlEtinct presence of HA in two organs in echinoderms parallels the presence of HA in multiple organs in mammals. Hen c e , HA is probably an important physiological component of both vertebrate and invertebrate physiological systems. It may be playing a regulatory role of such intercellular

~~s ~ o ~ ~rs as cyclin in echinoderms as well as in mammals, thereby functioning as an intercellular messenger itself during gam~togenesis .

!t i£ hop ~ d that the results of this study have shed

ll ~ h ~ on t he possible physi ological function of HA in

e c h : n~ de= m s. Further research, which should strive for an u r : d ~ r 5tonding of specific functions, would contribute to an

un d ~ r s t an d ing c f echinoderm physiology, and could give

f~ ~ t h er insight into the r c! ! of HA in higher animals . 76

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