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.
Ascidians are invertebrates from the phylum Chordata, subphylum Tunicata, class Ascidiacea, considered by many to be the closest living relatives of the vertebrates (Ruppert 2005; Delsuc et al. 2006; Jeffery 2007). Because of their evolutionary position, large numbers of easily fertilized eggs, invariant cleavage pattern, small genome size (16 000 protein coding genes) and known structure (Dehal et al. 2002), ascidians are highly studied in terms of molecular development (Satoh 2001; Satoh & Levine 2005). The opportunity to study ascidian oocytes is increasing rapidly as they are among the most invasive of marine organisms and have had a deleterious effect on aquaculture (Lambert 2007).
Ascidians are distinguished from other tunicates by being sessile as adults. They include three orders: Aplousobranchia, Phlebobranchia and Stolidobranchia (Lahille 1890). The aplousobranchs are all colonial with small zooids and ovaries either in the loop of the gut or posterior to the gut. All aplousobranchs have internal fertilization from large, complex broadcast-spawned sperm. Most phlebobranchs and stolidobranchs are large (>1 cm) solitary individuals with relatively small free spawned eggs, minute simple sperm and small uncomplicated larvae. In phlebobranchs such as Ascidia and Corella the ovary is confined to the surface of the stomach or in the loop of the gut. Stolidobranchs like Styela and Halocynthia have their ovaries in the body wall where there are usually multiple hermaphroditic gonads.
Within the ascidian ovary each oocyte is enclosed within a layer of outer follicle cells left behind at ovulation, a layer of inner follicle cells (usually referred to as follicle cells) (labeled outer follicle cells in Berrill 1929), a non-cellular vitelline coat and a layer of test cells (termed inner follicle cells in Berrill 1929). The test cells function in formation of the larval tunic (Cloney 1990; Cloney & Hansson 1996).With few exceptions inner follicle cells are larger than outer follicle cells. Figure 1A,B illustrates this organization. These structures together with the oocyte constitute an ovarian follicle (Fig. 1A). Since the ovulated oocyte lacks the outer follicle cells, in this review, when not otherwise specified I will refer to the inner follicle cells as follicle cells but recognize that this is only strictly correct after ovulation has removed the outer follicle cells (Fig. 2A–C).
“Extensive evidence, both direct and indirect, demonstrates that somatic follicle cells, particularly those enveloping and directly attached to the oocyte, execute multiple and variable functions throughout the life and at distinct stages of gamete and follicle development in most animals” (Schuetz 1985). The follicle cells are involved in all aspects of oogenesis but have specific functions during maturation and early development. This review is concerned mainly with these events and does not deal with other earlier functions of follicle cells during oogenesis.
Ovulation and structure of follicle cells in solitary ascidians
Contraction of ovarian cells overlying the oocyte results in ovulation throughout the animal kingdom. This has been studied in many vertebrates and invertebrates, but not in ascidians (Schroeder & Talbot 1985). Oocytes of Pyura haustor, Herdmania pallida and H. momus assume a dumbbell shape during ovulation (C. Lambert, unpubl. data, 1985) which is indicative of contraction by overlying cells (Schroeder & Talbot 1985). Furthermore the heavily pigmented, contracted outer follicle cells are clearly visible after ovulation in Herdmania momus (Fig. 1B). Therefore it is reasonable to conclude that contraction of the outer follicle cells causes ovulation with the expulsion of the oocyte with its remaining accessory cells into the lumen of the follicle stalk. The contracted outer follicle cells remain behind in the ovary (Fig. 1B). It is not known if they persist as a corpus luteum after ovulation. In most species the outer follicle cells are transparent and difficult to see in living ovarian follicles (Villa & Patricolo 2001) but in Herdmania momus the outer follicle cells are pigmented red, which makes them readily visible especially during and after contraction and ovulation (Fig. 1A,B). The epithelium and lumen of the follicle stalk is continuous with the epithelium and lumen of the ovary. Thus the ovulated oocyte covered by its inner follicle cells arrives in the lumen of the ovary. Ovulated oocytes may be stored in the lumen of the ovary or in the oviduct depending upon the species (Burighel & Cloney 1997). Ascidian oocytes are ovulated with intact germinal vesicles, a situation quite different from brachiopod (Stricker & Folsom 1997) and mammalian oocytes, which initiate germinal vesicle breakdown (GVBD) at ovulation. In the phlebobranchs Ascidia, Phallusia and Ciona mature eggs are stored in the oviduct; in Corella inflata they are stored in the ovary and pass into the ciliated oviduct during spawning (C. Lambert, unpub. data, 1967). Stolidobranchs store eggs in the ovary or multiple oviducts.
Structure and interspecific variation in follicle cells
Follicle cell size and shape varies between species especially in phlebobranchs. They are more uniform in stolidobranchs. Probably the greatest differences in size are seen in phlebobranchs where one species such as Ascidiella scabra or Corella eumyota produces eggs that sink in still water, while the sister species Ascidiella aspersa and Corella inflata produce floating eggs with large follicle cells (Fig. 3A–C,E) (Lambert et al. 1995). Follicle cells in the genus Ciona are coniform and of considerable length, an adaptation that retards sinking (Fig. 3F). Each Ciona follicle cell contains one or more refringent granules. The granules are terminal and single in Ciona intestinalis and Ciona roulei (Lambert et al. 1990) but sub-terminal and multiple in Ciona edwardsi and Ciona savignyi (Byrd & Lambert 2000).
Intraspecific variation: Incipient speciation
Currently there is a great deal of interest in speciation in the genus Ciona. Ciona intestinalis has historically been considered to have a world-wide distribution, but recently it has been shown that the species by which classical morphology has been identified as C. intestinalis actually includes at least two genetic clades: A and B (Nydam & Harrison 2007). In 2005 I examined oocytes of C. intestinalis from Gamagori Harbor in Ise Bay, Japan and found that their follicle cells were very different from those collected in California. Rather than the “normal” California organization with a single terminal refringent drop (Byrd & Lambert 2000), most had a single drop midway along the length of the follicle cell, while some individuals lacked the refringent drop. I call this difference to the attention of ascidiologists because differences in follicle cell structure easily seen in a conventional light microscope may very well mirror differences in molecular makeup.
Origin of follicle cells
The original of follicle cells is a contentious issue with well reasoned hypotheses supporting two quite divergent views. In one model the inner follicle cells are thought to have arisen by division of cells of the outer follicular envelope (Tucker 1942; Sugino et al. 1987; Manni et al. 1993). In the other model, undifferentiated hemoblasts (stem cells) infiltrate the outer follicular epithelium to take up their definitive positions and undergo differentiation (Mukai & Watanabe 1976; Gianguzza & Dolcemascolo 1978; Mancuso 1994; Cloney 1995). Thorough searches of sections have not been successful in either detecting mitosis of outer follicle cells or migration of hemoblasts into ovarian follicles (Cloney 1995). What is needed to clarify this long-standing issue is studies on cell surface markers, either naturally occurring glycosides or antibodies to cell surface markers. A monoclonal antibody that recognizes certain hemoblast cells of Halocynthia roretzi also binds to follicle cells (Arai et al. 2001). Moreover, Fabio Gasparini (pers. comm., 2008) isolated a monoclonal antibody to a cell surface marker that labeled one type of circulating blood cell in Botryllus schlosseri, which also labeled follicle cells. These data support the hemoblast origin of follicle cells, but leave us with the problem of trans-epithelial emigration of hemoblasts which has not been seen in gonads but occurs in other tissues of ascidians and many other organisms (Cloney & Grimm 1970).
Synthetic properties of follicle cells
Electron microscopy of ascidian follicle cells discloses multiple signs of protein synthesis including a well developed rough endoplasmic reticulum (Cloney 1995; Burighel & Cloney 1997) and secretory granules (Cloney 1995). In the colonial stolidobranch Botryllus schlosseri the outer follicle cells are much larger than the inner follicle cells; these outer follicle cells secrete protein yolk during oogenesis (Zaniolo et al. 1986; Martinucci et al. 1988) which is incorporated into growing oocytes. Direct autoradiographic data on synthesis by follicle cells by Jeffery (1980) demonstrated nuclear incorporation of radioactive uridine and cytoplasmic incorporation of radioactive leucine. He showed that the follicle cells synthesize a 42 000 MW polypeptide of unknown function. Isolated follicle cells release a protease (Lambert 2008) and a glycosidase (Lambert et al. 1997; Honegger & Koyanagi 2008), which were presumably synthesized by the follicle cells.
Role in GVBD
In stolidobranch ascidians, oocytes with intact germinal vesicles are stored in the ovary. Transfer of oocytes to ordinary sea water results in breakdown of the germinal vesicle in a few minutes. GVBD occurs very rapidly after placing oocytes of the stolidobranchs Halocynthia roretzi (Hirai 1941), Molgula manhattensis (Sawada & Schatten 1989), Herdmania pallida and Boltenia villosa (Lambert 2005, 2008) in pH 8.2 sea water (SW). Oocytes kept at low pH fail to undergo GVBD unless stimulated (Sakairi & Shirai 1991; Lambert 2005, 2008).
Removal of the follicle cells of Halocynthia roretzi oocytes blocks GVBD in response to normal pH 8.2 SW (Sakairi & Shirai 1991). The opposite was concluded in other studies with Halocynthia roretzi (Bates & Nishida 1998; Prodon et al. 2008) and Ciona intestinalis (Prodon et al. 2006), though it is possible that the follicle cells had already triggered GVBD before removal (Bates & Nishida 1998; Prodon et al. 2006, 2008). Boiling or addition of protease inhibitors to a supernatant obtained from Halocynthia oocytes undergoing GVBD suggested to Sakairi & Shirai (1991) that a protease was produced from the follicle cells that induced GVBD. Ascidians, like several other invertebrates, use an increase in cytoplasmic cAMP levels to induce GVBD (Lambert 2008). Removal of the follicle cells of Boltenia villosa oocytes abolished GVBD in response to pH but not to the adenylyl cyclase stimulator forskolin (Lambert 2008). In addition, soybean trypsin inhibitor inhibited GVBD in response to increased pH but not forskolin. I then isolated Boltenia villosa follicle cells in pH 4.0 SW, raised the pH to 8.2 and found that protease activity was released, supporting the Sakairi & Shirai (1991) hypothesis. However, other compounds such as serotonin and progesterone also trigger GVBD (Lambert 2008). Therefore, clearly the follicle cells are involved in GVBD in Ciona intestinalis (Prodon et al. 2006), Molgula manhattensis (Sawada & Schatten 1989), Halocynthia roretzi (Hirai 1941) and Boltenia villosa (Lambert 2008), and perhaps other species. Protease inhibitors block GVBD, and isolated follicle cells release protease activity, but the actual nature of the meiosis inducing substance remains to be elucidated.
In the colonial stolidobranch Botryllus schlosseri, follicle cells secrete a glue-like substance, which anchors the brooded embryo to its somatic attachment site (Zaniolo et al. 1986). Molgula pacifica is a direct developing solitary stolidobranch that frequents high current, wave swept surfaces (Bates 2002). Spawned eggs adhere to rock surfaces by means of a sticky substance secreted by the follicle cells (Young et al. 1988; Bates & Mallett 1991). A single large vacuole in the follicle cell secretes the sticky substance by holocrine secretion in response to lowered salinity (Young et al. 1988).
Several Corella species produce eggs with large follicle cells that float in still water (Fig. 3A,B). However, in the brooder C. eumyota (Brewin 1946; Lambert et al. 1995), the embryos adhere to one another and to the atrial wall in the vicinity of the gonoduct opening (Lambert 2004) by means of a sticky mucus secreted by the follicle cells (Fig. 3C,D). Eggs removed directly from the ovary into pH 6.0 sea water are not initially sticky but become sticky upon transfer to pH 8.0 sea water (Lambert et al. 1995). South African C. eumyota exhibit a high degree of self-fertilization (Dupont et al. 2007) as do those from New Zealand (Mackie G. pers. comm. quoted in Lambert 2004).
Density (sinking/floating rate) of ascidian eggs and embryos varies strikingly between species; some float in still water while most sink rapidly. Density of many species is determined by the follicle cells (Child 1927; Berrill 1929; Lambert & Lambert 1978; Epel et al. 1999). Some species such as Corella inflata (Fig. 3A,B), C. willmeriana, C. japonica, C. parallelogramma and Ascidiella aspersa (Fig. 3E) have large buoyant follicle cells that cause the eggs to float in still water (Lambert & Lambert 1978; Lambert et al. 1995) while other species such as Ascidia ceratodes have intermediate-sized follicle cells that slow the sinking rate (Epel et al. 1999). Also the elongate follicle cells of Ciona species (Fig. 3F) decrease the sinking rate by increasing the frictional drag. These decreases in egg sinking rate subject the eggs to increased danger of solar UV damage, which is to a major degree inhibited by UV absorbing mycosporine-like amino acids contained in the test cell/follicle cell layer in A. ceratodes, the only ascidian species so far examined for such molecules (Epel et al. 1999; Epel 2003). Flotation in C. inflata is the result of a decrease in follicle cell density by the accumulation of ammonium ions that replace more dense ones. This is an energy dependent process which uses glycolysis rather than oxidative phosphorylaton (Lambert & Lambert 1978). In C. inflata the floating eggs and embryos are held in a special atrial brood chamber until well after hatching (Lambert et al. 1995) which diminishes the dangers of UV present in free-spawned eggs.
Comparison with follicle cells in other animals
Other invertebrate chordates
While oogenesis in nearly all animals involves various functions of the follicle cells, in only a few species do they function in maturation and development. Little is known about the follicle cells of the pelagic tunicates. Doliolids have scattered follicle cells that persist through development while the follicular layer is so dense in salps that they obscure development (Godeaux 1990). Nothing is known about their function. In the Appendicularia, some species such as Oikopleura dioica produce afolliculate oocytes, while others such as Oikopleura fusiformis, O. longicauda and O. labradoriensis produce oocytes with a follicular envelope. Their function in the folliculate species remains a mystery (Ganot et al. 2006).
Other invertebrate phyla
With relatively recent re-alignments of invertebrate relationships, the only other common invertebrate deuterostomes besides tunicates are the cephalochordates, hemichordates and echinoderms (Swalla 2001; Winchell et al. 2002). Cephalochordates and hemichordates ovulate oocytes without follicle cells and there is nothing known about their possible functions during maturation.
In the phylum Echinodermata follicle cells are usually left behind in the ovary at ovulation (Smiley 1990) but are on the oocyte surface in artificially extracted oocytes. Asteroid oocytes are stimulated to undergo GVBD by radial nerve factor, which causes the follicle cells to release 1-methyladenine, the asteroid meiosis inducing substance (MIS) (Kanatani 1975). Unlike ascidians and several other invertebrates, cAMP inhibits GVBD in asteroid oocytes (Stricker & Smythe 2001 for review). In the closely related ophiuroids an increase in cytoplasmic cAMP induces GVBD without follicle cells (Yamashita 1988). Holothurian oocytes are also stimulated to undergo GVBD by an MIS released from follicle cells in response to radial nerve factor. The MIS appears to be 2-methyl 8-amino adenine or some other di-substituted adenine (Smiley 1990). Echinoids are quite different from asteroids, holothurians, crinoids and ophiuroids as they undergo ovulation and GVBD weeks or months before spawning (Smiley 1990). However, pigmented follicle cells of sand dollars produce an MIS that causes GVBD in asteroid oocytes (Burke & Bouland 1989).
In most other invertebrate phyla, follicle cells are important in oocyte growth in oogenesis, but with little evidence for involvement in GVDB or development. In many invertebrates, the function of the follicle cells is completed much before ovulation. Insect oocytes are surrounded by follicle cells during GVBD but their function in this process is unknown. Articulate brachiopod oocytes undergo maturation shortly after detachment of the follicle cells and ovulation (Stricker & Folsom 1997) but in an inarticulate brachiopod, Glottidea, detachment of the follicle cells does not lead to GVBD (Freeman 1994). Glottidea oocytes resume meiosis upon incubation in membrane-permeant forms of cAMP, suggesting that a rise in oocyte cAMP is instrumental in GVBD (Freeman 1994). Perhaps the follicle cells either trigger this process by releasing an MIS or directly release cAMP into the oocyte.
Generally vertebrate oocytes are enclosed by a follicular layer which like the follicle cells of ascidians and echinoderms secretes an MIS, in vertebrates usually designated a maturation inducing hormone (MIH) (Yamashita et al. 2000 for review). In fish the MIH is 17α, 20β-dihydroxy-4-pregnen-3-one (Nagahama et al. 1995). Most studies on vertebrate follicle cell function involve amphibians in which the natural MIH appears to be progesterone produced by the follicle cells, although insulin and growth factors can also induce GVBD (Ferrell 1999). Mammalian oocytes are stimulated to undergo GVBD by other steroids specific to each group (Byskov et al.1999). In most vertebrate and starfish oocytes, increased cAMP levels in the oocyte inhibits GVBD (Ferrell 1999). In contrast, several marine invertebrates including nemerteans, hydrozoans, molluscs and ascidians are stimulated to undergo GVBD by an increase in intracellular cAMP levels (Lambert 2008). Oocytes of a few mammals are also stimulated to undergo GVBD by an increase in intracellular cAMP (Stricker & Smythe 2001 for review).
Rabbit follicle cells and their matrix are responsible for ciliary transport on the fimbriae of the ampulla of the oviduct (Blandau 1969); adhesion of the oocyte complex to the oviductal cilia is mediated by the follicle cell secretions. Similarly, egg pickup in other mammals also requires follicle cell adhesion to oviductal cilia (Lam et al. 2000; Talbot et al. 2003 for review). Adhesion of the oocyte to the oviduct requires the follicular matrix to bind to the cilia. This is reminiscent of the situation in the ascidians Corella eumyota and Molgula pacifica where follicle cell secretions are responsible for adhering the eggs to the atrial chamber in C. eumyota (Lambert et al. 1995) or substrate in M. pacifica (Young et al. 1988; Bates & Mallett 1991). The ascidian lineage diverged from the lineage that led to mammals millions of years ago, yet when adhesion of oocytes was required in mammalian reproduction similar mechanisms were deployed.
Gretchen Lambert contributed to many of the experiments. I am also indebted to her for help with this manuscript and figures. My experiments were carried out in laboratories around the world; the directors and staffs gave considerable help with development of ideas, collecting, and carrying out experiments. I owe thanks to T. Honegger, and the International Journal of Developmental Biology for permission to use a micrograph from Honegger & Koyanagi 2008. Development, Growth & Differentiation and the Canadian Journal of Zoology also allowed use of previously published figures. R. A. Koch and J. S. Johnson provided the unpublished micrographs used in Fig. 2C–E.