Obtaining gametes and embryos of ascidians.

Ascidians are basal chordates that have become increasingly important for understanding chordate evolution. They comprise three orders. In the orders Phlebobranchia and Stolidobranchia, most species freely spawn eggs and sperm, whereas members of the order Aplousobranchia form colonies that brood their eggs and broadcast sperm. In the two free spawning orders, eggs and sperm are easily obtained for in vitro fertilizations. In the third order, slices of colonies yield gametes and embryos of all stages. Methods are described for obtaining gametes, performing fertilizations, and culturing embryos. Also included are methods for removing follicle cells and vitelline coats from oocytes.

Signaling pathways in ascidian oocyte maturation: the roles of cAMP/Epac, intracellular calcium levels, and calmodulin kinase in regulating GVBD.

Most mature ascidian oocytes undergo germinal vesicle breakdown (GVBD) when released by the ovary into sea water (SW). Acidic SW blocks this but they can be stimulated by raising the pH, increasing intracellular cAMP levels by cell permeant forms, inhibiting its breakdown or causing synthesis. Boltenia villosa oocytes undergo GVBD in response to these drugs. However, the cAMP receptor protein kinase A (PKA) does not appear to be involved, as oocytes are not affected by the kinase inhibitor H-89. Also, the PKA independent Epac agonist 8CPT-2Me-cAMP stimulates GVBD in acidic SW. GVBD is inhibited in calcium free sea water (CaFSW). The intracellular calcium chelator BAPTA-AM blocks GVBD at 10 µM. GVBD is also inhibited when the ryanodine receptors (RYR) are blocked by tetracaine or ruthenium red but not by the IP(3) inhibitor D-609. However, dimethylbenzanthracene (DMBA), a protein kinase activator, stimulates GVBD in BAPTA, tetracaine or ruthenium red blocked oocytes. The calmodulin kinase inhibitor KN-93 blocks GVBD at 10 µM. This and preceding papers support the hypothesis that the maturation inducing substance (MIS) produced by the follicle cells in response to increased pH causes activation of a G protein which triggers cAMP synthesis. The cAMP then activates an Epac molecule, which causes an increase in intracellular calcium from the endoplasmic reticulum ryanodine receptor. The increased intracellular calcium subsequently activates calmodulin kinase, which causes an increase in cdc25 phosphatase activity, activating MPF and the progression of the oocyte into meiosis.

Ascidian follicle cells: Multifunctional adjuncts to maturation and development.

Ascidians are primitive chordates, subphylum Tunicata, that are sessile filter-feeding hermaphrodites as adults. Released oocytes are enclosed within a monolayer of follicle cells, a non-cellular vitelline coat and a monolayer of test cells that cover the egg membrane. Follicle cell structure is distinctive in different groups. They originate from circulating hemoblasts with functional nuclei. They are necessary for germinal vesicle breakdown in several species and may secrete a meiosis-inducing substance to the oocyte. In some families the follicle cells are necessary for fertilization. Although all ascidians are hermaphrodites, many are not capable of self fertilization. The follicle cells seem to be involved in self, non-self discrimination. Attachment of sperm to egg involves a sperm surface glycosidase binding to an egg surface glycoside. The primary block to polyspermy involves a glycosidase released by the follicle cells. In one species with direct development, the follicle cells secrete a sticky substance that anchors the embryos in a wave-swept rocky area; a brooding solitary ascidian with a tadpole larva uses a sticky substance secreted by follicle cells to attach the brood to the atrial chamber. Several species have floating eggs due to buoyancy of their follicle cells, a result of ammonia sequestration in at least one species. Many other marine invertebrates release eggs with attached follicle cells, and all vertebrates ovulate oocytes covered with follicle cells. Comparisons are discussed between these groups and ascidians.

Signaling pathways in ascidian oocyte maturation: the role of cyclic AMP and follicle cells in germinal vesicle breakdown.

Many ascidian oocytes undergo 'spontaneous' germinal vesicle breakdown (GVBD) when transferred from the ovary to normal pH 8.2 sea water (SW); however, low pH inhibits GVBD, which can then be stimulated while remaining in the low pH SW. Oocytes of Boltenia villosa blocked from GVBD by pH 4 SW undergo GVBD in response to permeant cyclic AMP (8-bromo-cyclic AMP), phosphodiesterase inhibitors (isobutylmethylxanthine and theophylline) or the adenylyl cyclase activator forskolin. This suggests that cAMP increases during GVBD. Removal of the follicle cells or addition of a protease inhibitor inhibits GVBD in response to raised pH but not to forskolin, theophylline or 8 bromo-cAMP. Isolated follicle cells in low pH SW release protease activity in response to an increase in pH. These studies imply that the follicle cells release protease activity, which either itself stimulates an increase in oocyte cAMP level or reacts with other molecules to stimulate this process. Studies with the mitogen-activated protein (MAP) kinase inhibitors U0126 and CI 1040 suggest that MAP kinase is not involved in GVBD. The Cdc25 inhibitor NSC 95397 inhibits GVBD at 200 nm in a reversible manner.

Signaling pathways in ascidian oocyte maturation: effects of various inhibitors and activators on germinal vesicle breakdown.

The Ascidiacea, the invertebrate chordates, includes three orders; the Stolidobranchia is the most complex. Until the present study, the onset of oocyte maturation (germinal vesicle breakdown) had been investigated in only a single pyurid (Halocynthia roretzi), in which germinal vesicle breakdown (GVBD) begins when the oocyte contacts seawater (SW); nothing was known about internal events. This study strongly suggests the importance of protein phosphorylation in this process. Herdmania pallida (Pyuridae) functions like H. roretzi; GVBD occurs in SW. Oocytes of Cnemidocarpa irene (Styelidae) do not spontaneously undergo GVBD in SW but must be activated. Herdmania oocytes are inhibited from GVBD by pH 4 SW and subsequently activated by mastoparan (G-protein activator), A23187 (Ca2+ ionophore) or dimethylbenzanthracene (tyrosine kinase activator). This requires maturation promoting factor (MPF) activity; cyclin-dependent kinase inhibitors roscovitine and olomoucine are inhibitory. It also entails dephosphorylation as demonstrated by the ability of the phosphatase inhibitor vitamin K3 to inhibit GVBD. GVBD is also inhibited by the tyrosine kinase inhibitors tyrphostin A23 and genistein, and LY-294002, a phosphatidylinositol-3-kinase inhibitor previously shown to inhibit starfish GVBD. LY-294002 inhibits strongly when activation is by mastoparan or ionophore but not when activated by dimethylbenzanthracene (DMBA). The DMBA is hypothesized to phosphorylate a phosphatase directly or indirectly causing secondary activation, bypassing inhibition.

Sperm surface proteases in ascidian fertilization.

Ascidian eggs are surrounded by a noncellular layer and two cellular layers, which are penetrated by sperm. Three sperm surface proteases are essential for fertilization of eggs from the stolidobranch ascidian Halocynthia: spermosin, acrosin, and the proteasome. In the phlebobranch Ciona, a chymotrypsin-like protease and the proteasome are essential in fertilization. Sperm from the phlebobranch ascidians Phallusia mammillata, Ascidia (=Phallusia) nigra, and Ascidia columbiana, all express spermosin, acrosin, and the proteasomal chymotrypsin activities on their surfaces. Chymostatin blocks cleavage in phlebobranchs, but inhibitors of spermosin and acrosin only delay it by several minutes. Protease inhibitors have little effect upon sperm binding in Phallusia but strongly affect the rate of sperm passage through the vitelline coat. Peptide substrates and inhibitors to spermosin and acrosin cause a significant decline in the number of eggs undergoing pre-meiotic contractions at 3 min after fertilization. Thus while chymotrypsin activity is essential for penetration of the vitelline coat, spermosin and acrosin both function to increase the rate of fertilization. A crucial step in the divergence of the phlebobranchs and stolidobranchs may have been the conversion of spermosin and acrosin to essential proteases in the stolidobranchs, or, perhaps, their essential function was lost in the evolution of phlebobranchs. Aplousobranch ascidians are all colonial with very small zooids. Sperm from Aplidium californicum, Aplidium solidum (Polyclinidae), and Distaplia occidentalis (Holozoidae) have acrosin and chymotrypsin activities but lack spermosin activity. This enzyme is also missing from sperm of colonial phlebobranch and stolidobranch ascidians, suggesting that spermosin is not necessary for small zooids with internal fertilization.

Independent Initiation of Calcium Dependent Glycosidase Release and Cortical Contractions during the Activation of Ascidian Eggs: (Ascidian, Ryanodine, Thimerosal, Thapsigargin, N-acetylglucosaminidase).

During fertilization or ionophore induced activation, ascidian eggs rapidly release cell surface N-acetylglucosaminidase activity used in the block against polyspermy and undergo cortical contractions before they re-initiate meiosis. To better understand the activation process, we probed the relationship between these two processes in Ascidia ceratodes eggs by activating with different agents that increase intracellular Ca levels and under different ionic conditions. Glycosidase activity release was followed by the use of a fluorogenic substrate, and cortical contractions were followed by examining changes in cell shape with light microscopy. Ionomycin (2.7 µM) and thimerosal (1 mM) initiate glycosidase release and cortical contractions when administered in complete sea water (SW) but only the contractions in low Ca SW. Ryanodine (0.67 mM), known to raise free intracellular Ca in a number of cell types by release from the endoplasmic reticulum, causes glycosidase release but fails to initiate cortical contractions in complete SW. Thapsigargin (10 µM), which inhibits Ca dependent ATPase in the ER, causes glycosidase release but induces the contractions only about 50% of the time. These experiments show that, although glycosidase release normally precedes the ooplasmic shape changes that accompany the resumption of meiosis in ascidian eggs, they are not obligately coupled. That both processes can be induced by treatments known to raise intracellular Ca in other systems but under different conditions indicates that there may be a multiplicity of Ca requiring but functionally independent events during egg activation.

Spicule formation in the solitary ascidian, herdmania momus.

Two types of calcareous spicules occur abundantly in Herdmania momus, a solitary pyurid ascidian with a worldwide warm water distribution. The large spindle-shaped body spicules are 1.5-2.5-mm long and are located primarily in the mantle, siphons, and branchial basket. Each body spicule possesses 100 or more rows of overlapping, unidirectional fringing spines. Numerous body spicules occur regularly spaced within a long common sheath of complex structure, and there are many sheaths per animal. Between neighboring body spicules and overlying the fringing spines are the tightly connected pseudopodial sclerocytes. Spine formation is hypothesized to occur within these cells. The body spicules apparently continue to increase in size throughout the animal's life. The tunic spicules are about one tenth the length of the body spicules. They have 20-40 rows of unidirectional nonoverlapping fringing spines and a mace-shaped spiny base that anchors them at the tunic surface. They form quickly in individual spicular envelopes inside the tunic blood vessels over a 4-5-day period. Each tunic spicule then leaves its surrounding envelope and blood vessel, passes into the tunic, and ultimately protrudes through the outer surface of the tunic. An organic covering inside the envelope closely adheres to the tunic spicules and stains with toluidine blue. Dissolution of the CaCO3 mineral phase by EDTA or EDTA-cetylpyridinium chloride-formaldehyde reveals an intricately patterned organic matrix within or upon which the spicules develop.

THE EFFECT OF LIGHT ON THE SPAWNING OF CIONA INTESTINALIS.

1. The spawning of Ciona intestinalis with respect to light was studied, using both white light and monochromatic light. 2. A one-hour dark-adaption period followed by exposure to light resulted in spawning by 66.6% of the 884 animals tested. 3. Spawning occurs an average of 27.3 minutes after the onset of illumination. 4. Illumination need not be continuous until spawning occurs; the animals spawn when returned to the dark after a short illumination period, provided they have received enough energy. 5. The action spectrum for spawning suggests cytochrome c as a chromophore.