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 by 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 of 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).