Number 42                                                                                                                 December 1997

We spent another very enjoyable and productive summer at the Roscoff laboratories in Brittany. The station celebrated the re-opening of the totally remodelled dormitory and dining facilities. Charley worked on sperm surface proteases, sperm binding and penetration in Phallusia while Gretchen collected and freeze-dried many samples of Ascidiella aspersa and Phallusia eggs for a collaborative project with David Epel on egg UV absorbing material. We are grateful for much help from the Goudeau, Meijer and Potin laboratories. Bill Jeffery and Billie Swalla continued their exciting work with Molgula spp. at the Roscoff lab, while Marie and Henri Goudeau continue their electrophysiology experiments on Phallusia eggs. In October we attended The 2nd Intl. Symposium on the Molecular and Cell Biology of Egg and Embryo Coats in Sapporo, Japan, where we heard a great deal of exciting new research. We will be spending January at the Friday Harbor Labs before returning to Fullerton for Charley's last semester of teaching before he retires in June. We will continue with AN for a while after retirement but will most likely limit distribution to email and our web page. But do please continue to send us reprints of all your ascidian research. There are 114 new papers listed in the New Publications section of this newsletter; keep up the good work!

*Ascidian News is not part of the scientific literature and should not be cited as such.

1. Lambert reprints available: Charles is retiring next June and we will be moving to Seattle. We still have a large supply of reprints of most of our publications of the past 27 years. If you want any of them, now is the time to ask! In June many of them will probably be discarded. You can view our list of publications back to 1990 on our internet home page http://nsm.fullerton.edu/~lamberts/ascidian/ Many older papers are also available.

2. Microscopic Anatomy of Invertebrates. 1997. Vol. 15. Hemichordata, Chaetognatha, and the Invertebrate Chordates. Harrison, FW and Ruppert EE (Eds). 537 pp. Available from: John Wiley & Sons, Inc. Attn: N. English -- 9th Floor, 605 3rd Ave., New York, NY 10158-0012 or Fax: 212-850-8888. $185.00 (ouch!) Table of Contents: Ch. 1 - Introduction: Microscopic Anatomy of the Notochord, Heterochrony, and Chordate Evolution. E.E. Ruppert; Ch. 2 - Hemichordata. J. Benito and F. Pardos; Ch. 3 - Chaetognatha. G. L. Shinn; Ch. 4 - Urochordata: Ascidiacea. P. Burighel and R.A. Cloney; Ch. 5 - Cephalochordata (Acrania). E.E. Ruppert. The excellent 126 page chapter on ascidians by Burighel and Cloney covers nearly all aspects of the ultrastructure of ascidians and is illustrated by high quality light and electron micrographs from the work of many investigators. We have not seen the other chapters but are sure they are also of high calibre.

3. New book on Hawaiian ascidians: Reef and Shore Fauna of Hawaii, section 6B: Ascidians (Urochordata) by DP Abbott, AT Newberry and KM Morris, edited by G. Lambert. 64 pp. This work, which we believe will be very useful to ascidiologists, is a compilation of field information by DP Abbott over many years and prepared after his death by Todd Newberry with excellent drawings by Kendal Morris; $19.95 in paperback from the Bishop Museum Press, 1525 Bernice Street, P.O. Box 19000-A, Honolulu HI 96817-0916. (808) 848-4135, fax (808) 841-8968, or wilson@bishop.bishop.hawaii.org.

4. Dr. Laura Stocker, Inst. for Sci. and Technol. Policy, Murdoch Univ., Murdoch, Western Australia 6150 has a chapter on 'Chordata, Hemichordata and Chaetognatha' in a textbook entitled Invertebrate Zoology. A special feature of the chapter will be reproduction in colonial ascidians, especially didemnids. The book will be published by Oxford Univ. Press and edited by Prof. Donald Anderson, recently retired from the Univ. of Sydney. Predicted date of publication is March 1998. Most contributors and hence much of the material will be Australasian, filling a conspicuous gap in our downunder literature.

5. Ascidians featured as journal cover articles: There have been many important research articles on ascidians featured on journal covers, but we would like to mention two recent ones. The April 1997 issue of Zoological Science sports a beautiful color photo of four adult Molgula tectiformis, with an article on their direct development (elimination of the tadpole stage) by Tagawa, Jeffery and Satoh. Created by in situ hybridization, a gigantic Halocynthia roretzi tadpole on the cover of the Oct. 15, 1997 Dev. Biology advertises an article by Okada et al. on neuronal lineages as revealed by expression of a sodium channel gene.

6. New embryology text, Embryology: Constructing the Organism, published by Sinauer Assoc. Inc. contains a long chapter on the Tunicates by William Jeffery and Billie Swalla, and another on the Cephalochordates by J.R. Whittaker, as well as chapters on a wide variety of invertebrates, vertebrates and plants. Embryology: Constructing the Organism ed. by Gilbert and Raunio $ 69.95 ISBN 0-87893-737-2, 550 pp. email: orders@sinauer.com Fax (413) 549-1118

6. Dr. Nina Georgievna Vinogradova, a biography and tribute submitted by her son Georgyi (Egor) Vinogradov (egor@ecosys.sio.rssi.ru), also a marine biologist who works on amphipods. It is in part a translation of a tribute prepared by her colleagues at the Inst. of Oceanology in Moscow.
Nina Georgievna Vinogradova (May 30, 1928 -- March 10, 1997), Russian marine biologist and oceanologist, died full of creative plans and dreams of future works. She selflessly loved the Sea and devoted her whole life to the Sea. She was the favorite student and follower of one of the initiators of P. P. Shirshov Institute of Oceanology (RAS) academician Lev Alexandrovich Zenkevitch. She was an enthusiastic second year student of the Biology Dept. of Moscow State University when she visited for the first time the White Sea, the austere and beautiful Kandalaksha Gulf. Then she worked in the Caspian Sea, and, above all, took part in the first cruise of R/V "Vityaz'" in the Sea of Okhotsk in 1949. There she grew under the supervision of Lev Alexandrovich Zenkevitch into a marine benthos biologist enamored of her business. She graduated in 1951 and entered the post-graduate school of IO RAS. And, at last, she participated in the remarkable deep-sea cruise 14 of R/V "Vityaz'" in the region of Kurile-Kamchatka trench (1953). It was the beginning of her works dealing with fauna of the extreme ocean depths. The material was processed, the data of all the world expeditions concerning the deep-sea fauna was analyzed. The quantity of published information consulted could be measured not in papers and volumes, but in linear meters of library shelves. As a result information about hundreds of deep-living species was collected. Maps were constructed of the deep-living fauna as a whole, but not divided into taxonomic groups. Nina Georgievna discovered the fundamental global regularities of vertical and horizontal distribution of deep-living species and established the first zoogeographical map of ocean abyssal based on the huge amount of analyzed material. This map is not surpassed even now and is widely cited all over the world in reviews and courses for students. Undoubtedly, the Ph.D. thesis on the deep-sea biogeography was defended brilliantly. In 1955 she became a researcher at the Institute of Oceanology and took part in a dozen large-scale cruises, which covered the Pacific and Indian oceans, and the Antarctic. The regularities of life at the abyssal depths and in deep trenches remained the main point of her interest. It was good tradition in the Shirshov Inst. of Oceanol.: researcher dealing with general problem must also be expert in some group of animals. And Nina's group was ascidians. Of course, at first turn deep-sea ascidians. And here she found very interesting animals - like strange, sack-like Situla pelliculosa from the Kurile-Kamchatka Trench. Tens of new species, new genera, faunistic lists of ascidians from previously unvisited regions of the Ocean. She was really expert here - like in all fields of her interests. Nina was a participant of many international symposia and conferences, member of deep-sea ecology SCOR working group, Russian coordinator of the Deep-Sea Newsletter, author of more than 130 scientific works. She worked in the institute during her last days and took part in her last marine expedition - to the Norwegian Sea - in 1995. It is difficult to imagine how painful for us it will be to live without her bright eyes, without her recommendations, without her support and love --- in general, without her. Sincerely yours -The friends, the colleagues, and everybody who loved her.
A partial bibliography :
Vinogradova NG 1958. On the finding of a new ascidian species - Cnemidocarpa zenkevitchi in the fjord of the Banger Oasis (Antarctic). Zoologycheskyi Zhurnal, 37(9):1375-1379 (in Russian).
--- 1959. The geographical distribution of the deep-water bottom fauna in the abyssal zone of the ocean. Deep-Sea Research, 5:205-208.
--- 1962. Ascidiae simplices of the Indian part of the Antarctic. Exploration of the fauna of the seas I(IX): Biological results of the Soviet Antarctic expedition (1955-1958), 1. IzdatelstvoAN SSSR, Moscow & Leningrad. P. 196-215 (in Russian, many new species).
--- 1962. Vertical zonation in the distribution of deep-sea benthic fauna in the ocean. Deep-Sea Research, 8:245-250.
--- 1962. Some problems of the study of deep-sea bottom fauna. J. of the Oceanographic Soc.of Japan, 20th Anniv.Volume. P. 724-741.
--- 1969. On the finding of a new aberrant ascidian in the ultraabyssal of the Kurile-Kamchatka Trench. Byulleten Moskovskogo Obshchestva Ispytatelej Prirody, Seria Biologicheskaya, 74(3):27-43 (in Russian, Situla pelliculosa gen. et sp. nov.).
--- 1969. The geographical distribution of the deep-sea bottom fauna. In: L.A.Zenkevitch [Ed.]. The Pacific Ocean: Biology of Pacific Ocean. Book II: Deep-sea bottom fauna; pleuston. Nauka, Moscow. P. 154-181 (in Russian).
--- 1970. Deep-sea ascidians of the genus Culeolus from the Kurile-Kamchatka Trench. Trudy Instituta Okeanologii AN SSSR, 86:489-512 (in Russian).
--- 1975. On the discovery of two new species of an aberrant deep-water ascidian genus Situla in the South-Sandwich Trench. Trudy Instituta Okeanologii AN SSSR, 103:289-306 (in Russian).
--- 1976. Big face of the Ocean. Priroda, No 11. P. 94-105 (in Russian).
--- 1979. The geographical distribution of the abyssal and hadal (ultra-abyssal) fauna in relation to the vertical zonation of the Ocean. Sarsia. 64:41-50.
--- 1988. Tunicata, or Urochordata. In: R.K.Pasternak [Ed.]. Life of the Animals, Vol. 2. Prosveshchenie, Moscow. P. 256-285 (in Russian).
---, Gebruk A.V., Romanov V.N. 1993. Some new data on the Orkney Trench ultra abyssal fauna. In: Klekowski R.Z., Opalinsky K.W. [Eds.]. The 2nd Polish-Soviet Antarctic symposium. Dziekanow Lesny. P. 213-221.
--- 1997. Zoogeography of the abyssal and hadal zones of the ocean. Adv. in Mar. Biol. 32 (in press).

7. White Point BioMarine introduces a Light version of NAPIS (NAtural Products Information System), available for FREE download with optional Taxonomy lookup table data and sample data from: http://www.esri.com/base/users/conservation/whitepoint/napis.html
NAPIS supports researchers at the government, industry, and university levels by providing a link between the library, laboratory and field. In addition to sample tracking and bioactivity directed isolation, NAPIS integrates geographic information system (GIS) technology using ESRI's Map Objects. Researchers can query using the GIS map and drill down on chemical structures, or vice versa; query on chemistry/bioactivity; and show the results on a map. Natural products chemicals have played, and will continue to play, a key role in drug discovery. Screening natural products extracts, however, is expensive when compared with synthetic and combinatorial chemical libraries. Biological activity directed isolation studies and structural elucidation of unknown compounds adds to the expense. Acquiring and tracking natural products source organism collections have extended requirements for information handling. Combining these requirements within one system, NAPIS, will not only reduce their costs, but can provide the leverage to convert them to assets for guiding future efforts. The download file is large (9 mb), you may contact our office for a CD ($30 media fee). For more information, contact: White Point BioMarine, Inc., P.O. Box 2989, 180 First Street, Friday Harbor, WA 98250 USA. Phone: 360-378-7292, Fax: 360-378-7260, E-mail: napis@wpbm.com

1. From Drs. Tatjana Shaposhnikova and Olga Podgornaya: We are looking for help in getting information, for the amount of journals available in Russian libraries is very restricted. For several years we have investigated the protein composition of blood cells of the ascidian Styela rustica and particularly morula cells. These cells take part in tunic formation and repair and in defense reactions. Blood cell separation in a discontinuous Percoll gradient shows 4 fractions. The 4th fraction contains 90-100 % of morula cells. These cells possess two major proteins of mw. 47 and 26 kDa. Previous studies of our colleague had shown histochemically that morula cells contain phenoloxidase (PO), some cation proteins and some substrates for PO. It's very interesting for us if these proteins are similar to phenoloxidase. We produced polyclonal antiserum against these proteins. Each antiserum reacts with both proteins on the blot and stains the granules of morula cells. Our requests to everybody who can help are: a.We are very interested in reprints about PO system and associated proteins, its functions etc. and we will be very grateful to everybody who can help us. b. In what journal should our material be published? Please send us the Information for Contributors from the appropriate journal,or an address to which we can write. Thank you. Dr. T.Shaposhnikova, Dept. of Cytol. & Histol., Fac.of Biol. & Soil Sci., Tihoretsky pr.4, St. Petersburg State Univ., Universitetskaja nab. 7/9, St-Petersburg, 199034, Russia. email: shapa@histo.bio.pu.ru Dr.Podgornaya, Institute of Cytology RAS, 194064 St. Petersburg, Russia. email: podg@osint.stud.pu.ru Fax: 007 (812) 520 97 03

2. Dr. Chris Ireland (cireland@deans.pharm.utah.edu) writes: I am particularly interested in the chemistry of didemnid ascidians. My home page address is http://ocean.pharm.utah.edu ; it lists short summaries of the projects ongoing in the lab.

3. Charles and Gretchen Lambert continue to document new arrivals of nonindigenous ascidians into southern California harbors. Botryllus firmus was first noted in San Diego Bay this past September.

1. Feeding physiology of the cold water appendicularian Oikopleura vanhoeffeni (Tunicata). Alexander Bochdansky, Ocean Sciences Centre, Memorial University of Newfoundland, St. John's, Newfoundland, Canada A1C 5S7. July 1997 doctoral thesis abstract:
Clearance and ingestion rates, behavior, gut passage time, pigment degradation and assimilation efficiency were examined for Oikopleura vanhoeffeni (Appendicularia, Tunicata) in a gradient of concentrations of laboratory grown diatoms. Data from particle removal experiments and behavioral observations showed similar trends, although the magnitude of the responses varied depending on the technique used. Clearance rate decreased significantly with food concentration, although saturation was not reached for a particle concentration range representative of conditions in the field. No lower feeding threshold was found. Clearance rate also decreased with the age of the external filtering device (i.e. house). For an individual animal, a wide range of clearance rates can be encountered from 0 to a physiological upper limit given by the Morris and Deibel (1993) model. Using body size, tail beat frequency and proportion of time spent feeding from in situ observations it was possible to estimate clearance rate in the field. The means of these estimated clearance rates were within a threefold range of the means of five alternative methods. The mean gut passage time of O. vanhoeffeni was 0.8 h and was independent of trunk length and particle concentration, although it varied significantly among individuals. Studies with 68Ge incorporated into the silica frustules of diatoms as a conservative tracer, showed that chlorophyll a conversion (i.e. degradation into fluorescent and non-fluorescent breakdown products) was on average 79%. The chl a conversion was not related to the amount of food in the gut estimated by visual inspection, although it was inversely correlated with the amount of 68Ge and chl a recovered in the guts of animals. Assimilation efficiency of bulk diatom carbon was 67%. Extraction in various solvents allowed fractionation of food and feces into four main biochemical pools. Proteins and low molecular weight compounds were preferentially absorbed by the animals over lipids and polysaccharides. Predicted C:N ratios (by weight) for fecal pellets produced on a diatom diet ranged from 6.0 to 7.2, depending on the formula used and were consequently not much higher than the C:N ratios of the ingested diatoms (C:N = 5.2 - 6.3). The results of this thesis are relevant for the calculation of realistic population clearance rates as well as for the biochemical transformation of sinking material as appendicularians in general are major grazers in the world oceans. bochdan@morgan.ucs.mun.ca

2. Sequence analysis of five prospective manx alleles from the ascidian, Molgula oculata. Rebecca Jones - M.S. May 1996 with Dr. Billie Swalla, Vanderbilt University Biol. Sci.
Summary: there are at least 5 different alleles of Manx in the Molgula oculata population which show a high amount of polymorphisms when compared to other genes, such as actins.

1. Congress of the ISDCI at Williamsburg, VA July 1997.

Phenoloxidase, morula cells and cytotoxicity in the compound ascidian Botryllus schlosseri. L Ballarin, F Cima, A Sabbadin. Dpto. di Biol., Univ. di Padova, Viale G. Colombo, 3, 35121 Padova, Italy.
Morula cells (MC) of the colonial ascidian Botryllus schlosseri contain phenoloxidase (PO) inside their vacuoles. As the release of their vacuolar content at the border of contacting incompatible colonies entails the formation of necrotic masses, which characterize the rejection reaction, the role of PO in Botryllus cytotoxicity was investigated. When hemocytes are incubated with blood plasma from incompatible colonies, MC degranulate and after 60 min the cytotoxicity index becomes significantly greater than that observed in controls with autologous plasma. The rise in cell mortality is completely inhibited by the addition of the PO inhibitor Na benzoate 10 mM or of the serine protease inhibitors phenylmethylsulfonyl fluoride and benzamidine at 1 mM concentration, although MC regularly degranulate. In the presence of Na benzoate 10 mM there is a significant reduction in the number, size and color intensity of necrotic masses along the contact border of nonfusible colonies. These results strongly suggest that PO is the enzyme responsible of the cytotoxicity observed both in hemocyte cultures and rejection reactions.

2. 2nd Intl. Symp. on the Molec. & Cell Biol. of Egg- and Embryo Coats, Sapporo, Japan Oct. 12-17, 1997.

#8. Dual block to polyspermy in ascidian eggs: sperm-induced glycosidase release from follicle cells followed by depolarization of the egg plasma membrane. C. Lambert1,2, H. Goudeau 2, C. Franchet 2, G. Lambert1,2 and M. Goudeau2, 1Dept. Biol. Sci., Calif. State Univ., Fullerton, CA 92834. 2Sta. Biol., Place Georges Teissier, BP 74, 29682 Roscoff, France.
Ascidians, the most primitive members of the phylum Chordata, are hermaphroditic, live in dense assemblages, and usually release sperm before the eggs. Hence, eggs are often spawned into dense clouds of sperm. Because fertilization by more than a single sperm leads to chaotic cleavage and early embryonic death, ascidians have evolved at least two successive blocks to polyspermy: the rapid release of N-acetylglucosaminidase that inhibits sperm binding to the vitelline coat (VC) (Lambert 1989) and a subsequent change in membrane potential that prevents supernumerary sperm-egg fusion (Goudeau & Goudeau 1993; Goudeau et al. 1994; Lambert et al. 1997). Although these eggs do not produce a fertilization membrane, they undergo cortical contractions soon after fertilization (McDougall et al. 1995). The putative g-protein inhibitor suramin inhibits cortical contractions in Phallusia mammillata eggs in a dose-dependent manner but fails to affect sperm motility, sperm binding or glycosidase release (Lambert et al. 1997). Although suramin has been suggested to be specific for uncoupling g-proteins (Dasso & Taylor 1991), it actually blocks cell surface protease activity in Phallusia sperm. This blocks the fertilization current and entry of sperm into eggs as judged by DNA specific fluorescent probes (Lambert et al. 1997). Suramin treatment is completely reversible; intact eggs exhibit the electrical response after the drug is washed out. Thus suramin effectively uncouples the two polyspermy blocks. Sperm must contact the follicle cells before passing through the VC. Eggs with the VC removed and fertilized in the presence of 20 (M suramin show the electrical response and sperm entry 35% of the time; thus VC removal enhances sperm entry (Lambert et al. 1997). That suramin fails to block N-acetylglucosaminidase while preventing penetration of the egg coat suggests that sperm might interact directly with follicle cells to induce glycosidase release. Sperm of Phallusia collide with and bounce off isolated follicle cells without binding. Fertilization leading to cleavage is species specific in ascidians (Lambert et al. 1990). However, glycosidase release from Phallusia eggs can be induced by sperm from Ascidiella aspersa, Ascidia mentula or Ciona intestinalis. None of these sperm penetrate the vitelline coat of Phallusia eggs. Follicle cells isolated from Phallusia eggs release glycosidase in response to sperm. Thus these eggs have two blocks to polyspermy that operate in sequence: an early first block resulting from enzymatic modification of the VC by N-acetylglucosaminidase released primarily from follicle cells, and a second electrical block operating at the egg plasma membrane level and requiring sperm-egg fusion. The glycosidase is membrane bound before fertilization (Lambert & Goode 1992) and can be released by the tryrosine kinase activator dimethylbenzanthracene (DMBA) (Archuleta et al. 1993). Twenty mM DMBA causes glycosidase release without the contractions. Glycosidase release in response to DMBA is inhibited with 20 mM genistein or 200 mM tyrphostin A-23, both tyrosine kinase inhibitors (Moore & Kinsey 1995). Sperm-induced glycosidase release is inhibited by tyrphostin A-23 but not the genistein. Isolated follicle cells from Ascidia ceratodes eggs release glycosidase in response to sperm or DMBA. This release is inhibited by tryphostin A-23. Thus ascidian sperm activate a tryosine kinase system in follicle cells on contact. This causes early glycosidase release and the early block, while sperm must pentetrate the VC and fuse with the egg surface to induce the later electrical block.

#P17. Phallusia mammillata sperm have cell surface spermosin, acrosin and chymotrypsin activity and all three proteases may be involved in fertilization. CC Lambert, Dept. Biol. Sci., Calif. State Univ., Fullerton, CA 92834-6850.
Fertilization involves the sperm recognizing and binding to egg coats, penetration of the egg coats, and finally fusion of sperm and egg membranes (2). Proteases have been suggested as used in both binding to and penetration of the egg coats (2,4,9). In ascidians there is evidence for both glycosidases and proteases being operational in sperm binding (2). Ascidian eggs have complex coats with distinct non-cellular domains and two layers of cells (1). In order to fertilize the eggs, sperm must traverse these seemingly formidable barriers. They have a very simple sperm (3) that binds to and penetrates the egg coats by a combination of mechanical (5) and enzymatic processes (2, 4, 8). Previously three sperm surface proteases were shown to be necessary for penetration of the egg coats and fertilization in the most highly evolved stolidobranch ascidians (2,11,13). Fertilization of intact eggs is inhibited by fertilization of the eggs in the presence of protease inhibitors, but these have no effect upon eggs whose coats have been removed (2). The simpler phlebobranch ascidians also have three sperm surface proteases (6, 8). Although sperm from phlebobranch ascidians express all three proteases, only two seem essential for fertilization: the chymotrypsin-like enzyme (8) and spermosin (10). Chymostatin inhibits fertilization in Ciona intestinalis (8) as does an antibody to spermosin (10). Phallusia mammillata is a large phlebobranch ascidian abundant on the English Channel coast of France. Their sperm express acrosin (cleaves Phe-Ser-Arg-MCA), spermosin (cleaves Val-Pro-Arg-MCA), and chymotrypsin (cleaves N-Suc-Leu-Leu-Val-Tyr-MCA) activities on their surface as assayed by cleavage of the appropriate substrates (12) in sea water. Protease activity is inhibited by chymostatin, lima bean trypsin inhibitor and suramin. Cleavage is inhibited by fertilization in the presence of chymotrypsin substrate or chymostatin. This inhibition is dose-dependent with complete blockage at 16 uM chymostatin. Cleavage is delayed but not inhibited by spermosin or acrosin substrates and inhibitors. Protease substrates (30 uM) or inhibitors (50 ug/ml) have no effect on sperm binding to the vitelline coat scored at one minute after insemination. Ascidian eggs undergo cortical contractions leading to ooplasmic segregation and the resumption of meiosis following fertilization (7). Using cortical contractions as an indication of fertilization, we find that acrosin and spermosin substrates both inhibit cortical contractions scored at 3 min after insemination. However, this is a delay rather than an absolute block; lima bean trypsin inhibitor (50 ug/ml) causes a 4 min delay in eggs undergoing the first contraction. All three protease substrates and several inhibitors block sperm penetration as scored directly by counting the number of sperm within the vitelline coat at 2 min after insemination. Thus while chymotrypsin-like activity is essential for penetration of the phlebobranch vitelline coat (3, 8), spermosin and acrosin both function to increase the rate of penetration. That spermosin and acrosin are auxiliary proteases in phlebobranch fertilization but essential in stolidobranchs suggests that a crucial step in ascidian evolution involved these formerly auxiliary proteases becoming essential. (1)Eisenhut M & Honegger TG 1997. Mar. Biol. 128: 213-224. (2) Hoshi M et al. 1994. Essays in Dev. Biol. 5: 201-208. (3)Koch RA & Lambert CC 1990. J. E.M. Tech. 16: 115-154. (4)Koch RA et al. 1994. Dev. Biol. 162: 438-450. (5)Lambert CC & Epel D 1979. Dev. Biol. 69: 296-304. (6)Marino R et al. 1992. Mol. Repro. & Dev. 26: 383-388. (7)McDougall A et al. 1995. Zygote 3: 251-258. (8)Pinto MR et al. 1990. Mol. Repro. & Dev. 26: 319-323. (9)Sawada H et al. 1996. BBRC 222: 499-504. (10)Sawada H et al. 1997. J. Repro. & Dev. 43: Suppl. (11)Sawada H & Someno T 1996. Mol. Repro. & Dev. 45: 240-243. (12)Sawada H et al. 1984. J. Biol. Chem. 259: 2900-2904. (13)Takizawa S et al. 1993. J. Exp. Zool. 267: 86-91.

#P24. Role of cAMP-dependent phosphorylation of dynein light chain on the SAAF-dependent activation of sperm motility in the ascidian Ciona intestinalis. M. Nomura, K. Inaba, M. Morisawa Misaki Mar. Biol. Sta., Misaki Miura Kanagawa 238-02 Japan.
Spermatozoa of the ascidians Ciona intestinalis and C. savignyi initiate their motility by the factor derived from unfertilized egg, designated sperm activating and attracting factor (SAAF) which elevates intracellular cyclic-AMP (cAMP) level dependent on extracellular Ca2+ (6). From experiments using demembranated Ciona sperm, It was shown that cAMP is required prior to ATP to initiate and to activate axonemal movement (1, 2, 3, 4). It was also shown that many sperm flagellar proteins including dynein light chain are phosphorylated during incubation of demembranated sperm with ATP and cAMP (3, 5). However, there is no evidence of which proteins are phosphorylated during the activation of Ciona sperm by SAAF. In the present study, when the sperm of C. intestinalis which were immotile in the SW was demembranated in the extracting medium containing Triton X-100 and then reactivated in the reactivating medium containing ATP, axonemal movement did not occur, but axonemes became motile within 1 minute upon addition of cAMP, suggesting the requirement of cAMP for sperm activation (see also 1, 2, 3, 4, 6). Furthermore when sperm were activated by SAAF in the seawater and then demembranated, sperm axonemal movement occurred by ATP alone without cAMP. In Ca2+-free sea water (CFSW) sperm were immotile if SAAF is present, and the demembranated sperm prepared from the immotile sperm is still immotile in the presence of ATP and cAMP. These suggest that Ca2+ influx is required before cAMP-dependent activation of axonemal movement; SAAF causes hyperpolarization of the plasma membrane (see poster#29 by Izumi et al.) and influx of Ca2+ and then the both events cause the increase of cAMP level through or activation of adenylyl cyclase, resulting in activation of sperm motility in Ciona. Pharmacological experiments showed that calmodulin inhibitor, W-7, myosin light chain kinase inhibitor, ML-7, and protein kinase A inhibitor, H-89, inhibited SAAF-dependent activation of sperm motility. IBMX , a phosphodiesterase inhibitor is an activator of sperm motility through increase in cAMP. H-89 treated sperm did not became motile in the presence of IBMX, however W-7 or ML-7 treated sperm became motile by IBMX as well as SAAF. Furthermore, demembranated sperm prepared from W-7 treated immotile sperm require cAMP for reactivation. These suggest that W-7 sensitive process, e.g. calmodulin, regulates cAMP synthesis system and H-89 sensitive process, e.g. flagellar proteins phosphorylations dependent on cAMP is present in the downstream of the system. Autoradiographies of 32P incorporation into proteins of demembranated sperm showed the presence of many cAMP-dependent phosphoproteins in the sperm flagellar axoneme. From comparison of phosphoproteins in the demembranated sperm prepared from SAAF-treated or-untreated intact sperm (21kDa and 26 kDa proteins phosphorylated in SAAF dependent manner. CFSW, W-7 and ML-7 inhibited SAAF dependent phosphorylation of both proteins. These suggest that this 21kDa and 26kDa protein phosphorylation is related to the SAAF-dependent activation of sperm motility. The 32P-labeled 21kDa protein along with some the other axonemal proteins was extracted with a high salt buffer containing 0.6M KCl, but 26kDa protein remained in axonemal fraction. Subsequent sucrose density gradient centrifugation of the extract showed that the 21kDa protein sedimented at approximately 20 S and the 21kDa peak has ATPase activity, suggesting that the 21kDa phosphoprotein is a dynein light chain of which its phosphorylation corresponds to SAAF-dependent activation of sperm motility. Dynein heavy chain was also phosphorylated with motility initiation in cAMP-depending manner. SAAF-dependent activation of sperm motility in Ciona intestinalis will occur as follow: When SAAF contact with the surface of the sperm plasma membrane, extracellular Ca2+ enter into sperm and entered Ca2+ bind to calmodulin-like Ca2+-binding protein accelerate intracellular cAMP synthesis and/or decelerate cAMP destruction systems, resulting in intracellular cAMP increase. The cAMP activates protein kinase A which phosphorylates the dynein light chain resulting in activation of sperm motility through microtubule sliding. (1)Brokaw CJ 1985. Ann. NY Acad. Sci. 438, 132-141. (2)Chaudhry PS et al. 1994. Cell Motil. Cytoskeleton 32, 65-79. (3)Dey CS & Brokaw CJ 1991. J. Cell Sci. 100, 815-824. (4)Morisawa et al.1984. Zool. Sci.1, 237-244. (5)Opresko L & Brokaw CJ 1983. Gamete Res. 8, 201-218.(6)Yoshida M et al. 1994. Develop. Growth & Differ., 36: 589-595.

#P39. Hyperpolarization of sperm plasma membrane mediated by K+ efflux induces an increase in cAMP and initiation of sperm motility in the ascidians Ciona intestinalis and C. savignyi. H. Izumi1, T. Marian2, K. Inaba1, Y. Oka1, M. Morisawa1. 1 Misaki Mar. Biol. Sta., Misaki Miura Kanagawa 238-02, Japan; 2 Positron Emission Tomograph Center, Univ. Med. Sch. of Debrecen, Hungary.
Calcium and cAMP are widely known as the important factors for the regulation of sperm motility as well as acrosome reaction in invertebrates and vertebrates (15). The initiation of sperm motility triggered by the second messengers through activation of protein kinase and resulting phosphorylation of the proteins (7, 15, 12, 5, 8). In the ascidians Ciona intestinalis and C. savigyni, sperm -activating and -attracting factor (SAAF) released from the egg at fertilization stimulates both Ca2+ influx and a transient cAMP elevation of the sperm leading to the activation of sperm motility. SAAF requires Ca2+ for the activation but a phosphodiesterase inhibitor, theophylline, also elevates cAMP level and activates their motility even in the absence of extracellular Ca2+, suggesting that activation of cAMP producing system requires Ca2+ (16). Recently, much attention has been paid on the roles of membrane potential for modulation of cAMP synthesis and consequent regulation of sperm activation and acrosome reaction: Speract, a peptide from the egg jelly induces both hyperpolarization of the plasma membrane and elevation of the cAMP level in sea urchin sperm. Artificially induced membrane hyperpolarization of the sea urchin sperm also causes an increase in the level of cAMP (2). A decrease in external K+ surrounding sperm causes their membrane hyperpolarization (14) and triggers initiation of the sperm motility without an increase in intracellular pH (3) through synthesis of intracellular cAMP (Morisawa; the present meeting) in the trout. We show here that valinomycin, a potassium ionophore alone as well as SAAF activated sperm motility in Ciona. The activation of sperm motility by valinomycin or SAAF was suppressed by high concentration of extracellular K+. Measurement of membrane potential with a slow voltage-sensitive arbocyanine dye, DisC3 (5) showed that membrane potential did not change in the absence of SAAF in all concentration of external K+ examined, but SAAF or valinomycin induced membrane hyperpolarization and subsequent addition of external K+ caused depolarization of the plasma membrane. The amplitude of the SAAF-induced hyperpolarization of sperm plasma membrane was dependent on external K+ concentration despite in the presence or absence of external Ca2+. These results suggest that SAAF-induced membrane hyperpolarization depends on K+ permeability of the plasma membrane but is independent of external Ca2+. Intracellular K+ concentration of Ciona sperm was measured to be 576 mM by nigericin null point method and membrane potential without SAAF was calibrated to be -42mV by valinomycin null point method. After treatment of sperm with SAAF in ASW containing more than 20mM K+, membrane potential of sperm plasma membrane became almost equal to equilibrium potential of K+, suggesting that the membrane potential changes which induced by SAAF mainly occurs through an increase in K+ permeability of the plasma membrane. A voltage dependent K+ channel blocker, mast cell degranulating peptide (MCD-peptide), blocked SAAF-induced activation of sperm motility at 10mM. Membrane potential changes was also inhibited by 10mM MCD-peptide. These results suggested that changes of membrane potential through opening a kind of K+ channel is important to the activation of sperm motility in Ciona. SAAF induced a transient elevation of cAMP level in Ciona savignyi (16) and C. intestinalis only in the presence of Ca2+. We show here that cAMP level reaches a peak within 5 seconds after addition of SAAF. Valinomycin also increased cAMP level but the increase developed slowly and the cAMP level reached a plateau 30 second after addition of valinomycin. In the presence of MCD-peptide, SAAF failed to increase cAMP but valinomycin increased it, suggesting that membrane hyperpolarization produced by opening of voltage dependent potassium channels causes cAMP synthesis, resulting in increase in intracellular cAMP concentration. SAAF induced membrane hyperpolarization independently of external Ca2+ in the normal ASW (K+= 9.39mM) and the hyperpolarization regulates the cAMP synthesis. However without external Ca2+ SAAF fails to initiate sperm motility. A Ca2+ ionophore, A23187 did not induce sperm motility (not shown). These results suggest that both hyperpolarization of plasma membrane and Ca2+ influx is necessary for the synthesis of cAMP. (1)Cosson MP et al. 1989. Cell Motil. Cytoskeleton 14, 424-434. (2)Beltran C et al. 1996. Biochem. 35, 7591-7598. (3)Boitano S & Omoto CK 1991. 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3. 37th Amer. Soc. for Cell Biol. annual meeting Washington DC Dec. 13-17, 1997. Go protein stimulation activates sperm of the sea squirt (Ascidia ceratodes). H Girma, S Goel, and RA Koch, Dept. of Biol. Sci., Calif. State Univ. Fullerton, Fullerton, CA 92834-6850.
Sperm activation in the sea squirt Ascidia ceratodes is defined by mitochondrial translocation (MTL)--a Ca2+-triggered actin/myosin-based process. We have used a microscopic assay for MTL and a spectrofluorometric assay for [Ca2+]i using the Ca 2+-sensitive dye Fura PE3. In order to test the involvement of Go proteins in this process we used mastoparan, a peptide toxin from wasp venom known to mimic surface receptors by stimulating Go proteins, and its active derivative, mas7, and inactive derivative, mas17, as well as the Go protein blocker, pertussis toxin (PTX). In the MTL assay, samples were incubated for 30 min (RT) in a solution containing activators, attached to coverslips, fixed in formaldehyde (1%) and analyzed (300 sperm were counted per replicate using sperm cells from three different animals). The ability of sperm cells to be activated was determined by high pH (9.4) stimulation (positive control) and revealed that 77 + or -2.1% (mean + or - SD, n=900). Negative controls were exposed to the inactive peptide, mas17, and resulted in 10 + or - 3.6% activation. Using MTL assay, we found that either mastoparan (17 uM) or mas7 (3.5 uM) induced sperm activation at a level equal to 70% of positive controls. For spectrofluorometric assay, sperm cells were incubated with Fura PE3 for 20 min, centrifuged, resuspended in artificial sea water (pH 6.8), placed in cuvettes in the spectrofluorometer where ratiometric analysis was performed using the excitation wavelengths of 350 and 380 nm with emission at 550 nm. Spectrofluorometric analysis revealed that after 25 min of treatment by mas7 (7.0 uM) at 15o C, [Ca2+]i (as determined by 350/380 ratios) increased by 24% over baseline, an elevation to 72% of positive control values. This mas-dependent rise in [Ca2+]i decreased by 60% in the presence of PTX. Thus, we conclude that a mastoparan/mas7- and PTX-sensitive trimeric G protein (presumed to use ao subunit) initiates a signaling cascade which culminates in elevation of [Ca2+]i thus triggering sperm activation. Funded by NIH MBRS SO6-GM08258 and NIH AREA R15-HD28229.

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