Calcium Influences on the Activity of Shedding Substance in the Starfish Patiria Miniata

MASTER'S THESIS M-894 MECKLENBURG, Theodor Anthony. CALCIUM INFLUENCES ON THE ACTIVITY OF SHEDDING SUBSTANCE IN THE STARFISH PATIRIA MINIATA. The American University M.S., 1965 Physiology

University Microfilms, Inc., Ann Arbor, Michigan Copyright by

THEODOR ANTHONY MECKLENBURG

1966 CALCIUM INFLUENCES ON THE ACTIVITY

OF SHEDDING SUBSTANCE IN THE

STARFISH PATIRIA MINIATA

Theodor Anthony Mecklenburg

Submitted to the

Faculty of the College of Arts and Sciences

of The American University

in Partial Fulfillment of

the Requirements for the Degree

Master of Science

Signatures of Committee :

Dean of the College

Date Date ;

1965 AMERICAN UNIVERSITY The American University LIBRARY. Washington, D.C. JAN 4 1966 WASHINGTON, D. C. Ill

ACKNOWLE DGEMENT

I wish to thank Alfred B. Chaet, Professor of

Biology, The American University, for his constant guidance and assistance throughout this investiga tion and Dennis H. Fox, Professor of Marine Bio chemistry, Scripps Institution of Oceanography, who provided for the use of the Scripps facilities. TABLE OF CONTENTS TABLE OF CONTENTS

Page

ACKNOWLEDGEMENT...... Ill

LIST OF FIGURES...... VII

INTRODUCTION ...... 1

LITERATURE REVIEW...... 3

MATERIALS AND METHODS

A. Collection of A n i m a l s ...... 9

B. Isolation and Preparation of Nerve Ex tracts Containing Shedding Substance. . . 10

C. Preparation of Sea Water Solutions . . . 11

D. Preparation of Ovarian Tissue ...... 12

E. Transfer Procedure and Description of S p a w n i n g ...... 13

RESULTS...... 15

DISCUSSIONS...... 20

SUMMARY...... 26

BIBLIOGRAPHY...... 27 LIST OF FIGURES VII

APPENDIX A LIST OP FIGURES

Figure 1 Ovarian fragments exposed to sea water containing shedding substance for nine hours. Maximum shedding (5 mg%) was equated to 100% shedding index.

Figure 2 Ovarian fragments washed both in sea water and calcium-free sea water and transferred to sea water containing calcium for a period of two hours.

Figure 3 Ovarian shedding while exposed to a 5 mg% solution of shedding substance (S.S.) and concentrations made up of either sea water, calcium-deficient sea water, calcium and magnesium-deficient sea water and magnesium-deficient sea water. Results demonstrate amount of shedding over 30 minute period and are based on five experiments.

Figure 4 Ovarian fragments constantly exposed to sea water or shedding substance (5 mg%) .

Figure 5 Ovarian fragments exposed for two to three seconds up to 34 minutes to calcium- free sea water containing shedding sub stance and then transferred to sea water.

Figure 6A,6B, Ovarian fragments of a starfish exposed 6C,6D for two to three seconds to calcium-free sea water containing shedding substance and then transferred to sea water. Each set of curves illustrates results obtained from one starfish.

Figure 7A,7B, Ovarian fragments of a starfish exposed 7C for four minutes to calcium-free sea water containing shedding substance and then transferred to sea water. Each set of curves illustrates results obtained from one starfish. VIII

Figure 8A,8B, Ovarian fragments of a starfish exposed 8C,8D for eight minutes to calcium-free sea water containing shedding substance and then transferred to sea water. Each set of curves illustrates results obtained from one starfish.

Figure 9A,9B, Ovarian fragments of a starfish exposed 9C,9D for 12 minutes to calcium-free sea water containing shedding substance and then transferred to sea water. Each set of curves illustrates results obtained from one starfish.

Figure 10A,10B, Ovarian fragments of a starfish exposed 10C,10D for 16 minutes to calcium-free sea water containing shedding substance and then transferred to sea water. Each set of curves illustrates results obtained from one starfish.

Figure llA,IIB, Ovarian fragments of a starfish exposed 11C,11D for 20 minutes to calcium—free sea water containing shedding substance and then transferred to sea water. Each set of curves illustrates results obtained from one starfish.

Figure 12A,12B, Ovarian fragments of a starfish exposed 12C,12D for 24 minutes to calcium—free sea water containing shedding substance and then transferred to sea water. Each set of curves illustrates results obtained from one starfish.

Figure 13A,13B, Ovarian fragments of a starfish exposed 13C.13D for 28 minutes to calcium-free sea water containing shedding substance and then transferred to sea water. Each set of curves illustrates results obtained from one starfish. IX

F igure 14A,14B, Ovarian fragments of a starfish exposed 14C,14D for 32 minutes to calcium-free sea water containing shedding substance and then transferred to sea water. Each set of curves illustrates results obtained from one starfish.

Figure ISA,15B, Ovarian fragments of a starfish exposed 15C,15D for 34 minutes to calcium-free sea water containing shedding substance and then transferred to sea water. Each set of curves illustrates results obtained from one starfish. INTRODUCTION INTRODUCTION

Since the discovery of gamete-shedding substance in

radial nerves of the starfish Asterias forbesi (Chaet and

McConnaughy, 1959) various investigators have become in terested in the physical and chemical nature of this sub

stance. The shedding substance from Asterias forbesi was

shown to induce the release of eggs or sperm when injected

into mature starfish of the same species (Chaet and Musick,

1960). Recent experiments demonstrated that isolated

ovarian fragments released eggs when immersed in sea water

containing shedding substance (Chaet, Andrews and Smith,

1964). Physiological and chemical investigations by

scientists both in the United States and in Japan estab

lished that the shedding substance was a polypeptide

(Chaet, 1964; Noumura and Kanatani, 1962).

Chaet, Andrews and Smith (1964) indicated that the

shedding substance possessed dual activities. They re

ported that this physiologically active peptide not only

initiated ovarian contraction but induced the conversion

of immature gametes to mature gametes. Myographic ex

periments were also designed by the above researchers to focus attention on ovarian contraction. Their results showed that the ovaries reached a contracted state several minutes prior to the release of eggs.

Other than a few preliminary experiments which in volved the influence of calcium on the shedding sub stance (Chaet and Smith, personal communication) no ex periments relating the biochemistry of ovarian muscle contraction and the precise role of calcium in the res ponse of starfish ovaries to shedding substance were known. Thus, this present research was concerned with investigations on the influence of the shedding sub stance activity and its coordinated response with calcium and magnesium ions as they pertain to the release of gametes from the starfish ovary. LITERATURE REVIEW LITERATURE REVIEW

Sensations and living processes of all animal organisms

are coordinated by nervous tissue. Electrical and/or chem

ical messages are relayed over a communication system from

cell to cell via a neurological network which terminates at

motor end plates of muscle cells or releases chemical agents

from neurosecretory cells at neurohumoral organs. In the

latter case, specialized nerve cells synthesize a chemical

agent while still others store and release this substance,

its terminating activity being metabolic control over target

organs.

Vertebrate mechanisms of neurosecretion have in the

past been extensively investigated, but more recently many

of the invertebrate systems have also opened new fields of

investigation. Researchers engaged in work with vast numbers

of marine invertebrates have greatly extended the physiologi

cal and chemical knowledge of biological systems. Initially,

publications were concerned with the effects of exogenous

factors on oceanic creatures. Light, temperature and salinity were shown to control feeding mechanisms, migrations and

reproductive cycles and were given initial consideration

(Giese, 1957). Recently, however, scientists have been concerned with understanding endogenous control over these activities (Giese, 1959). Boolootian (1963), in reviewing the field of echinoderm physiology, directed attention to environmental influence on the biochemistry and physiology of reproductive mechanisms. Knowledge concerning reproduc tive physiology in starfish was increased by the identifica tion of neural components inducing gamete-shedding (Chaet and

McConnaughy, 1959; Chaet and Musick, 1960). A physiologically active substance extracted in sea water from the radial nerves of Asterias f orbe si induced the release of eggs or sperm from the cuiimal's gonads (Chaet and Rose, 1961; Chaet and Smith,

1962).

Once extracted from the radial nerves, these neural com ponents were subjected to chemical analysis and demonstrated a polypeptide nature. Shedding substance of the Japanese starfish Asterias amurensis (Noumura and Kanatani, 1962) exhibited chemical and physiological properties similar to those shown by Asterias forbesi (Chaet, 1963). Non-specificity of the shedding substance was shown when nerve extracts from

Asterias forbesi injected into Henricia sanquinolenta resulted in shedding (Hartman and Chaet, 1962) . Histochemical techniques provided considerable evidence that neurosecretory cells permeated the radial nerves of starfish (Unger, 1962 ;

Noumura and Kanatani, 1962). Imlay and Chaet (1965) attached greater significance to these neurosecretory cells. These authors searched for the seasonal level of neurosecretory substance within the radial nerve and at tempted to relate the histochemical picture with periods of active gamete-shedding. Kanatani (1964) reported that star fish ovarian tension was released when the shedding substance liquified connectives between gametes and ovarian walls.

Chaet, Andrews and Smith (1964) proposed that the mechanisms involved in shedding were muscle contractions.

In marine invertebrates as well as in higher forms, two types of contractile tissue may be differentiated: voluntary or skeletal muscle which controls the movements of limbs, and smooth muscle which controls peristaltic movements around hollow organs. Individual skeletal muscle fibers show a striated pattern due to the numerous myofibrils embedded in the sarcoplasm. Smooth muscle fibers are masses of spindle- shaped cells functioning as a syncytium. Both types of muscle fibers are surrounded by a limiting membrane, the

sarcolemma (Bennet and Porter, 1953). Within the muscle fibers a well-ordered arrangement of tubular structures contacting individual myofibrils was given the name sarcoplasmic reticulum (Porter and Palade, 1958). This arrangement of muscular components in the starfish Asterias forbesi and Patiria miniata conforms to the muscle systems described above (Hyman, 1955; Hoyle, 1957; Ghiradella, 1965).

Within the resolution obtained by the electron micro scope, the myofibrils were shown to be composed of myofila ments (Bennett and Porter, 1959). These myofilaments con sisted of bundles of long-chain proteins which were extract- able from muscle tissue and were identified as a globulin- type protein, myosin (Jakus and Hall, 1947). As a result of Szent-Gyorgyi's studies (1953) actomyosin was first shown to be the unit of contractile mechanism in muscle. He demonstrated that actomyosin filaments can be made to contract in vitro by exposure to adenosine triphosphate and calcium ions. These filaments did not contract with ATP alone; cal cium ions were essential for the muscle filaments to contract and magnesium ions were found to be antagonistic to calcium.

The significance of calcium was emphasized by Heilbrunn and

Wiercinski (1947) who injected various ions into the interior of the muscle fibers; if calcium was omitted there was rib contraction. Frank (1950), by soaking out the calcium from muscle tissue in a calcium-free Ringer, inhibited contraction and was able to show that the time-course of inhibition was the determining factor for the diffusion of an ion from an extracellular space.

Various techniques have shown the distribution of cal cium ions on membranes and within muscle protoplasm. Gilbert and Fern (1957) showed that increases and decreases in extra cellular calcium caused proportional changes in muscle cal cium. Studies with a radioactive isotope of calcium (ca^^) showed that the influx of this ion increased during an elec trical stimulus (Harris, 1957; Bianchi and Shanes, 1960).

Acetylcholine applied to denervated smooth muscle increased permeability to monovalent ions; moreover, calcium influx was increased two-fold ( Jenkinson and Nicholls, 1964) . The absence of calcium did not prevent these permeability changes.

Smooth muscle completely depolarized in KCl contracted and then relaxed ; it contracted again in response to acetylcholine but not in the absence of calcium (Robertson, 1960) . 8

Although chemical substances such as acetycholine, serotonin or adrenalin caused the intact cells of smooth muscles to contract, Woolley (1963) concluded that they do not activate the actomyosin machinery directly. The effect of chemical agents or hormones must be an indirect one and may be related to calcium ions. MATERIALS AND METHODS MATERIALS AND METHODS

A. Collection of Animals

The West Coast starfish, Patiria miniata. was used in these investigations as Giese (1959) showed that ripe animals

could be found from June through late September. Prior to natural spawning of gametes, the gonads of Patiria miniata were greatly enlarged. It was determined that animals show ing aboral swelling had reached gonadal maturity and would possess larger gonads. Such "ripe" starfish could readily be detected underwater with the use of self-contained under water breathing apparatus (Scuba). Starfish beds containing mature animals were located in San Diego Mission Bay en trance channel and New Hope Rock (Northwest of Point Loma),

San Diego. Animals containing large mature gonads were

found in Mission Bay from June to August in ocean tempera

tures ranging between 19° and 22°C due to the horizontal

thermocline below 60 feet.

As the animals were brought to the surface, they were

immediately placed in iced sea water and transported to the

laboratory where they were transferred to and maintained in

running refrigerated sea water at 16°C. 10

B. Isolation and Preparation of Nerve Extracts Containing Shedding Substance

Since previous work by Chaet and Smith (1962) demon strated that the radial nerves of Asteroids contained the physiologically active peptide, their method with a few modifications was used in the isolation of Patiria nerves.

A medial-aboral incision was made along the longitudinal axis of the ray which exposed the coelomic cavity. An in cision along the dorsal surface of the exposed ambulacral groove separated right and left borders and esqjosed the radial nerve. Upon removal with forceps, the nerves were suspended in iced sea water, washed several times in sea water, pooled and lyophilized. The dehydrated nerves were pulverized into a fine powder. This lyophilized nerve powder was extracted three times in 10 cc of distilled water each. Each supernatant was collected after centri fugation and the entire 30 cc of supernatant was pooled and

lyophilized. Concentrations of nerve powder were redissolved

in sea water, calcium-deficient sea water, calcium and mag nesium-deficient sea water, and magnesium-deficient sea water. 11

c. Preparation of Sea Water Solutions

In working with isolated ovarian fragments, it was necessary to bathe the tissue in ionic solutions similar

to the animal's environment. Standardized sea water

frybmulae as designated in Formulae and Methods IV of the

Marine Biological Laboratories, Woods Hole, Massachusetts

fulfilled the ionic specifications. Solutions deficient

in magnesium and calcium ions maintained equal isosmotic

conditions by replacing magnesium and calcium cation de

ficiencies with NaCl. Prepared solutions were tested

against sea water on Patiria eggs, with both solutions maintaining osmotic equilibrium with the eggs.

Sea water media were prepared as follows:

a. Mq++ and Ca++ deficient sea water 480.71 ml of 1.0 M NaCl 9.00 ml of 1.0 M KCL 2.15 ml of 1.0 M NaHCO^ pH - 8.8 b . Ca++ deficient sea water 432.27 ml of 1.0 M NaCl 9.00 ml of 1.0 M KCL 22.94 ml of 1.0 M MgCl^ ' GH^O 25.50 ml of 1.0 M MgSO^ • 78^0 2.15 ml of 1.0 M NaHCO^ pH - 8.4 12

c. Mq++ deficient sea water 471.44 ml of 1.0 M NaCl 9.00 ml of 1.0 M KCL 9.27 ml of 1.0 M CaClg * ZHgO 2.15 ml of 1.0 M NaHCOg pH — 8.4

The pH of Scripps sea water was 8.35. The above quanti ties of each salt solution containing deficiencies of mag nesium and calcium cations were diluted to a total volume of one liter. Trace amounts of calcium ions, if present, were precipitated out of cation-deficient solutions with sodium carbonate as calcium carbonate which was then removed by fil tration. The pH-of each solution was checked prior to each experiment.

D. Preparation of Ovarian Tissue

Mature ovaries from the starfish Patiria miniata were used in these investigations. Patiria males were found un suitable for shedding investigations since mechanical dis turbances induced nonhormonal shedding (Chaet, 1964; Kanatani and Noumura, 1964).

Ripe ovaries were excised from starfish by making a longitudinal incision along the medial-aboral plane of each ray. This incision was extended from the disk center to the tip of each ray. The partition between two severed rays was 13

carefully folded back, the internal horizontal skeletal column was cut (running perpendicular to the main axis of the ray) and the intact ovaries exposed. By severing the gonoduct at the site of attachment to the arm the ovaries were lifted free from the animal.

Isolated ovaries were then cut into fragments (5.0 -

10.0 mm) while suspended in sea water. Repeated sea water washings removed free eggs released from the cut surfaces.

Calcium traces were removed from surface membranes by washing in calcium-deficient sea water and ovarian segments were maintained in magnesium and calcium-deficient sea water until use.

E. Transfer Procedure and Description of Spawning Index

The various solutions to be tested were placed in 96- hole disposable plastic spot plates. Ovarian fragments of approximately the same size were placed in each horizontal row of depressions. Each depression contained approximately

0.6 cc of test solutions. In certain experiments, at pre determined times, ovarian fragments were exposed to a test

solution and then removed to another solution. When tissues were transferred from one solution to another, excess fluid was absorbed on filter paper before the fragment was placed

in a new solution. 14

The final analysis of shedding activity was described

as a quantity of eggs released from each ovarian fragment

and was called the shedding index. This was designed to

give the shedding ovaries a numerical value according to

the amount of eggs released. The greatest response to

the shedding substance was expressed as a number five (#5),

and the weakest response as a number one (#1). Eight pieces

of ovarian tissue were exposed to each test solution. The

final analysis of shedding was made between ovarian frag ments of approximately the same size. Their results were

averaged and expressed as the total shedding index. The

numerical values were graphically illustrated as a percent

age shedding index based on the maximum shedding of the con

trol solution or the solution which gave the maximum response.

Each individual point on the accompanying diagrams represents

these numerical values which are based on an average of eight

separate measures. RESULTS 15

RESULTS

Although the radial nerve extracts of Patiria miniata were shown to evoke shedding response in the mature ovaries of Patiria (Chaet, 1964), it was necessary for the present study to establish the optimum concentrations of shedding substance that induced maximum shedding. Stored radial nerve extracts were prepared in 10 mg%, 5 mg% and 1 mg% concentrations of sea water. The response of ovarian frag ments to various concentrations of shedding substance was determined and graphically plotted (Figure 1) as the per-

- %- centage of shedding index against a function of time.

Heilbrunn and Wiercinski (1947) clearly demonstrated that the physiological contraction of muscle was affected by specific cations. Thus in order to determine any res ponse ovarian tissue might have in calcium ion deficiency, ovarian fragments were washed in calcium-deficient sea water and then returned to calcium-free sea water or sea water containing calcium ions. Figure 2 illustrates that the induction of shedding by removing calcium ions gave a shedding index of 12%. 16

Shedding activity, as induced by a 5 mg% solution of shedding substance, was shown to be dependent upon calcium at some stage in the shedding response (Figure 3). As can be seen from Figure 3, maximum shedding resulted when

Patiria ovaries were exposed to sea water containing cal cium and shedding substance. Physiological activity of ovarian tissue as influenced by shedding substance was appreciably diminished when calcium was removed. Shedding substance was also assayed in magnesium-deficient sea water with and without calcium. Shedding results were graphed as a percentage of the activity induced by shedding substance in normal sea water. As can be seen in Figure 3, magnesium and calcium-deficient sea water containing shedding sub stance induced shedding after 30 minutes. The shedding re sulting from ovarian fragments in calcium-deficient sea water containing magnesium and shedding substance was found to be

15%. On the other hand the response induced by magnesium- deficient sea water containing calcium and shedding substance was found to be 95% (maximum).

It was interesting to see when shedding substance actually triggered the ovarian tissue. Thus the experiments plotted in Figure 4 showed that when Patiria ovaries were continually 17

exposed to a 5 mg% concentration of shedding substance (in sea water) minimal shedding activity was observed before 30 minutes. It should be noted, however, that the rate of shed ding rapidly increased after 30 minutes and reached a 70% shedding index within 70 minutes. Although it was evident from Figure 4 that the shedding response was a delayed pro cess and that the greatest response followed constant exposure to shedding substance for 30 minutes, it left the question of how short an exposure to shedding substance was required to result in shedding activity.

In order to answer this, it was necessary to determine the minimum time (pulse) needed for shedding substance to initiate a physiological, although unobserved, response prior to the release of gametes, and if this response was dependent upon calcium. Ovarian fragments were exposed to shedding substance solutions of calcium-free sea water for a time interval ranging from two to three seconds up to 34 minutes and then transferred to sea water containing calcium but no shedding substance. These results were illustrated first as the greatest percentage shedding index for each time exposure (Figure 5) and second, as the rise in percent age shedding index for each exposure (Figures 6 to 15) . 18

Figure 5 summarizes the exposure curves and shows that

shedding activity increases with longer exposure to shed ding substance. Two to three second exposures are plotted

in Figures 6A, B, C and D. After 30 minutes the shedding

index increased to 80% (Figure 5A) but only minimum response

(5 to 20%) showed after two hours (Figures 6B, C and D ).

As the e3q>osure to shedding substance was increased up to

34 minutes the ovarian tissue continued to show heightened activity (Figures 7 to 14). It should be noted that in a

four minute exposure experiment (Figure 7A) the onset of

shedding began sooner than in the constant exposure control.

Increased pulses of eight minutes (Figure 8D) and twelve minutes (Figure 9B) produced shedding as soon as transferred

into sea water whereas the other eight and twelve minute pulse experiments (Figures 8A, B and C, and 9A, C and D)

showed only slight gains in activity after two hours in sea water.

With increased exposure times there was also a cor

responding increase in the maximum shedding index (Figures

10, 11, 12, 13, 14 and 15). Twenty minute exposures (Figure 11)

gave results that were similar to those shown in Figure 10 but shorter delaying time was evident in the onset of shed

ding after transfer. The delayed activity after transfer was 19

more negligible after a 24 minute exposure in that shedding activity began almost immediately after the ovaries were placed into sea water containing calcium. In Figures 13 through 15, which represent exposure pulses of 28, 32 and

36 minutes respectively, shedding activity began to express itself immediately upon transferring the ovarian fragments to sea water. DISCUSSIONS 20

DISCUSSION

It can be seen from these experiments that extracts

from the radial nerves of Patiria miniata exhibited phys

iological properties similar to those noted in Asterias

forbesi (Chaet and McConnaughy, 1959), Asterias vulgaris and Henricia sanguinolenta (Hartman and Chaet, 1962), and

Pisaster giganteus and Patiria miniata (Chaet, 1964).

When injected with shedding substance intracoelomically these mature starfish released viable gametes.

In addition, isolated ovaries of both Asterias (Chaet,

Andrews and Smith, 1964) and Patiria when cut into frag ments still exhibited vigorous shedding upon immersion in

shedding substance. Obviously, since shedding substance

activity was still pronounced even through the Patiria

ovaries had been structurally altered by segmentation,

the expelling properties must be able to continue at the

tissue level and in all probability at the cellular level.

In a recent paper by Chaet (1964) evidence was pre

sented on the possible existence of an inhibitor substance

(shedhibin). He theorized that at "iTigh concentrations

shedhibin prevents the shedding substance from inducing a

shedding response. In the present tests, the curves in 21

Figure 1 show that a 5 mg% concentration of shedding sub

stance induced the optimum response of shedding in the ovarian fragments. This fact, together with the minimal response of shedding produced by a 10 mg% concentration of the radial nerve extract could be interpreted as the

inhibitor substance being present in sufficient concen tration only in the 10 mg% solution. It is also note worthy that a 1 mg% concentration was insufficient to either induce or inhibit shedding. Since there was no

substantial increase in shedding index after two hours,

subsequent experiments were terminated at that time.

Magnesium ions play important roles in the inhibition

of muscle actomyosin systems in some vertebrates and in

vertebrates (Kishimoto, 1961; de Villafranca, 1959) . The

results described in Figure 3 fail to implicate magnesium

as either enhancing or inhibiting the shedding activity.

Magnesium-deficient sea water containing shedding substance

showed considerably more shedding (95%) while calcium-

deficient sea water containing shedding substance indicated

a shedding index of only 15%. However, when calcium-free

sea water containing shedding substance was replaced by sea water containing calcium, the ovarian tissue subsequently began to shed. 22

Ovarian contraction in Asterias forbesi recorded by

Chaet, Andrews and Smith (1964) began five minutes prior to release of gametes. In Patiria miniata it was not certain when the initial contraction of ovarian tissue occurred; it was theorized that shedding substance was needed to induce the influx of calcium from the beginning of exposure. Thus, shedding would begin when the excited ovarian membrane reached the contraction threshold.

Figure 4 illustrates the rise in percent shedding index after 30 minutes, this index increasing as a function of time up to 120 minutes. This led to the question of why there existed a delay before the onset of shedding. Evidence con cerning this problem was first presented with the findings of Kanatani (1964). He theorized that shedding substance induced the release of gametes by diffusing into the ovary and by liquifying connecting elements between gametes. Whether or not the shedding substance affected the contractile mech anism is not commented on by the above author.

If a mechanism does exist which dissolves adhering forces within the ovary, then the following suggested activity may be hypothesized. First, shedding substance may initiate an action potential on the sarcolemma depolarizing the muscle 23

and thus Increasing its permeability. Second, the per meability increase would allow an influx of extracellular

calcium and possible shedding substance. The calcium would pass through the muscle and dissolve the connecting forces between the gametes. The contraction of muscle may occur

first and any gametes to be released from gonadal tissue would

await the arrival of the shedding substance. On the basis of this theory the picture would be altered when calcium ions

are removed. It might then follow that tissue in calcium-free

sea water would depolarize when exposed to shedding substance,

thus allowing shedding substance to permeate through the de polarized membrane. But even though the gametes were freed

from each other, shedding could not occur until the calcium had activated the actomyosin filaments.

The curves plotted in Figures 7B and C denote ovarian

tissue which received a four minute exposure to shedding sub

stance before being placed into sea water containing calcium

ions. These results suggest that a four minute exposure was

sufficient to depolarize the membrane, but insufficient to

allow shedding substance to pass through the ovarian wall

and to dissolve adhering forces between eggs. Similarly,

ovarian tissue exposed to shedding substance up to 24 minutes 24

(Figure 12) was in some cases less-than-adequate time for the shedding substance to complete its job.

Essentially then, the mechanical response of contraction required extracellular calcium. Permeability changes were not prevented by the absence of calcium. Therefore a time gradient must exist in which shedding substance not only in duces permeability but also reaches the gametes within the ovaries.

Increasing exposure rates of ovaries to a time greater than 24 minutes resulted in a distinct change. A 24 minute exposure to shedding substance was followed by immersion into sea water containing calcium, the ovaries then demon strating an immediate release of gametes from several open ings in the tissue. It is theorized that at this point the critical concentration of shedding substance needed to de polarize the tissue and pass into ovarian walls, thus dis solving egg connectives, was reached. Increasing the time exposures to shedding substance beyond 24 minutes before transfer had no further effect on the magnitude of shedding

(Figures 12, 13, 14 and 15). 25

By predisposing ovaries to shedding substance before calcium, it was shown that longer exposures were sufficient for gametes to be affected. The ovaries showed maximum shedding when calcium was added.

This situation in which shedding substance exerted a dual role could be analogous to the roles played by epine phrine. On the one hand it has a stimulatory effect on smooth muscle and, on the other, it has the ability to accelerate the breakdown of repeating chains of hexose molecules. Although new theories on the ovarian mechanisms and shedding substance have been presented, undoubtedly more investigations are needed to elucidate if, and indeed, shedding substance, like so many hormones and chemical me diators, induces the depolarization of membranes. SUMMARY 26

SUMMARY

1. Ripe ovaries from the starfish Patiria miniata were cut into small fragments (5-10 mm) and immersed in salt water extracts (1-5%) of Patiria lyophilized radial nerves.

2. Ovarian contraction resulting in the release of eggs occurred within 40 to 60 minutes after immersion in nerve extract solutions. Control solutions failed to release any eggs.

3. If calcium ions were removed from the nerve extract solutions shedding did not occur. Substitution of magnesium for calcium was unsuccessful.

4. It was demonstrated that ovarian fragments released eggs within minutes after transfer from calcium-free sea water containing shedding substance to sea water contain ing calcium.

5. These experiments also showed that short exposures (from four to twelve minutes) to shedding substance were more effective in inducing shedding than when the tissue remained continuously in the presence of shedding substance and sea water containing calcium.

6. Although short exposures (from four to twelve minutes) to shedding substance were more effective in inducing initial shedding, the longer exposures (from 14 to 38 minutes) would cause greater shedding when the tissue was transferred to sea water containing calcium. BIBLIOGRAPHY 27

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36. Villafranca, G.W. de, Scheinblum, T.S. and Philpott, D.E. 1959. A study on the localization of contractile pro teins in the muscle of the horseshoe crab (Limulus polvpmus) Biochem. Biophys. Acta. 34:147-157 APPENDIX A FIGURES O O H § 5mg% a O M O'- 60 a •rl C5 o TJ iz; oo- TS M <1) Q XI A ID H o W IN~ s CO a G O *H VO X H Rj Ü 8 10mg% < o FH ir— a RO 8 T3 « o — <1> -d- ID A (ti w o

o nj- lmg% O r-t

3 ^ 3 6 TIME (Hours)

FIGURE 1

OVARIAN FRAGMENTS EXPOSED TO SEAWATER CONTAINING SHEDDING SUBSTANCE FOR 9 HOURS. MAXIMUM SHEDDING (5 mg%) WAS EQUATED TO 100% SHEDDING INDEX. lO

X o111 z (9 z o o UJ z lU o ■a g z w 111u a; 111 0.

FIGURE 2

FIGURE 2 ILLUSTRATES OVARIAN FRAGMENTS WASHED BOTH IN SEAWATER AND CALCIUM FREE SEAWATER AND TRANS FERRED TO SEAWATER (containing calcium) FOR A PERIOD OF TWO HOURS. ( Five Experiments) SEA WATER (Containing Mg & Ca'^^) (S.D. 0 )

SEA WATER (Containing Mg^tCa'^t & S.S.) (S.D. 15.28)

Ca Deficient Sea Water (Containing Mg^ ) (S.D.7 .0 )

Ca^^Deficient Sea Water (Containing & S.S.) ( S.D. 6.30)

Mg Deficient Sea Water (Containing Ca^^) ( S.D. 1 0 .0 )

Mg^^ Deficient Sea Water (Containing Ca'*"*’& S.S. )(S.D.8.3O)

Mg'*’^ a n d Ca'*’^ Deficient Sea Water ( S.D. 8.9)

Mg^^and Ca^^ Deficient Sea Water plus S.S, ( S.D. 16.70)

I I I I I I I I I I______T Ü 30 50 70 90 PERCENTAGE SHEDDING INDEX

FIGURE 3 ILLUSTRATES OVARIAN SHEDDING WHILE EXPOSED TO A 5 mg% SOLUTION OF SHEDDING SUBSTANCE (S.S.) AND CONCEN TRATIONS MADE UP OF EITHER SEA WATER, CALCIUM DEFICIENT SEA WATER, CALCIUM AND MAGNESIUM DEFICIENT SEA WATER AND MAGNESIUM DEFICIENT SEA WATER. RESULTS DEMONSTRATE AMOUNT OF SHEDDING OVER 30 MINUTE. PERIOD AND ARE BASED ON 5 EXPERIMENTS.

FIGURE 3 H

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