Overview of the Volvox
The spheroidal chlorophycean Volvox and its close relatives are suitable model organisms for addressing fundamental issues in the evolution of multicellularity and the development of a germ-soma dichotomy (Kirk 1998; Nozaki et al. 2000; Kirk and Nishii 2001; Nozaki 2003; Hallmann 2011). They form a group of genera closely related to the genus Volvox within the order Volvocales. This group ranges in complexity from unicellular organisms, such as Ch. Reinhardtii, to homocytic colonial organisms, such as Gonium pectorale, to heterocytic multicellular organisms with different cell types, as is found in Volvox, which is the most highly developed genus. Several patterns of sexual reproduction are exhibited by the different species of the genus. Some species are monoclonic, while others are diclonic. Monoclonic species are either monoecious or dioecious. All diclonic species are dioecious and produce male and female spheroids in separate clones. Volvox mostly reproduces asexually, although it is able to switch to the sexual pathway.
The involvement of sex pheromones in switching was first described by Darden who showed that asexual V. aureus (a monoclonic, monoecious species) cultures could be induced to change to the sexual pathway by supplementing the cell-free cultured medium of mature male spheroids (Darden 1966). The same phenomena were subsequently reported for other species of Volvox (Kochert 1981).
Life cycles of Volvox carteri
V. carteri, a dioecious species, is the most intensively studied Volvox species (Kirk and Nishii 2001). It is composed of only two cell types, 2000–4000 biflagellate Chlamydomonas-like somatic cells, which form a monolayer at the surface of a hollow sphere and 16 reproductive cells (gonidia) that lie just below the sheet of somatic cells (Starr 1969). During embryogenesis, the embryos first cleave symmetrically five times to form a 32-cell embryo with identical cells, and then 16 cells divide asymmetrically to each produce one large gonidial cell precursor and one small somatic cell precursor (Kirk and Kirk 2004). These gonidial precursors divide asymmetrically two more times and produce additional somatic precursors at each division. The gonidial precursors then temporarily stop cell division, while the somatic precursors divide symmetrically about three more times. At the end of embryogenesis, the volume of the gonidial precursors expands to about
30-times that of their somatic precursors.
Sexual reproduction is initiated by a mutation-like switch with a probability of 2 × 10–4, which leads to the formation of the first sexual male colony (Weisshaar et al. 1984). A pheromone, named “sex inducer” or “sex-inducing pheromone”, has been the focus of considerable attention (Starr 1970). The sex-inducing pheromone is produced and released by this sexual male colony and acts on the asexual gonidia of both sexes. It alters their developmental pathway such that sexual forms (egg- or sperm-bearing forms) are produced in the next generation. In sexually induced male embryos, asymmetric cell division is postponed from the sixth to the eighth division cycle (Starr 1969, 1970; Hallmann et al. 1998). At this point, somatic cell precursors no longer divide and the large gonidial precursors each symmetrically divide seven times to form sperm bundles containing up to 128 sperm cells. Thus, the male ends up with 128 sperm bundles and 128 somatic cells. In female embryos, the first asymmetrical cell division is also postponed from the sixth to the seventh division cycle. Asymmetrical cell division then occurs and the somatic cell precursors continue to further cleave, with the large gonidial precursors developing as about 32 eggs and about 2000 somatic cells. Sperm bundles generated on the male colony contact the female colony by chance rather than by directed swimming (Coggin et al. 1979; Kirk 1998), after which a specific transient binding to somatic cells occurs. The sperm bundles break up into individual sperm cells and the sperm penetrate the extracellular matrix (ECM) of the female to reach the eggs inside the spheroids. The fusion of gametes results in the formation of a dormant diploid zygote that survives the drought. Under favorable environmental conditions, the germination of the zygote occurs with meiosis to form only a single viable germling and three nonviable polar bodies. The germling will produce a haploid female or male and then reproduce asexually (Starr 1975; Fig below).
The Volvox carteri Sex-inducing Pheromone
Because the production of pheromone could not be detected in sexual females or the somatic cells of sexual males, it was believed that expression of the gene encoding the pheromone was tightly linked to sperm development (Starr 1970; Gilles et al. 1981). However, both asexual females and asexual males were able to produce the pheromone following exposure to a heat shock (Kirk and Kirk 1986). The participation of reactive oxygen species in both the production of the pheromone and its activity in triggering sexual development in gonidia has been suggested (Nedelcu and Michod 2003). Sex-inducing pheromones were independently purified from two isolates of V. carteri: one from Japan (V. carteri f. nagarensis) and the other from the United States (V. carteri f. weismannia) (Kochert and Yates 1974; Starr and Jaenicke 1974). Both were glycoproteins of about 30-kDa. The inducer of the V. carteri f. nagarensis was strictly competent for its own gonidia whereas that of V. carteri f. weismannia induced sexuality of both of them (Al-Hasani and Jaenicke 1992). The pheromone of V. carteri f. nagarensis is one of the most potent biological effector molecules known, exhibiting its full effectiveness below 10–16 M. As a result of successful largescale production, partial amino acid sequences were obtained, allowing the cloning of a genomic clone encoding the pheromone (Tschochner et al. 1987) and the cDNA (Mages et al. 1988).
Possible Mode of Action of the Pheromone
The pheromone seems to have at least two modes of action. One is that the pheromone molecules act directly on the receptors of the gonidial cells. For this, the pheromone molecules must pass through the ECM. The other is that the pheromone molecules exert the effect by binding to the receptors on somatic cells, surrounding the spheroid. The binding triggers the synthesis of extracellular proteins, generating signal amplification. Because the very first experimentally detectable cellular responses to the sex pheromone come from the somatic cells at the surface, but not from the gonidial cells, the latter mode of action seems to be more plausible (Hallmann 2003).
The pheromone induces the synthesis of the deep zone hydroxyprolinerich glycoprotein (DZ-HRGP; Ender et al. 1999), chitinase/lysozyme, chitin-binding protein (Amon et al. 1998), and metalloproteinases (VMPs; Hallmann et al. 2001). Some of the ECM glycoproteins that are inducible by the sex pheromone are also inducible by mechanical wounding (Amon et al. 1998; Ender et al. 1999) but wounding does not cause the production of the sex pheromone itself. The majority of proteins synthesized shortly after the pheromone treatment in the ECM are part of a single family of glycoproteins: the pherophorins (Sumper et al. 1993; Godl et al. 1995, 1997). At least 34 different pherophorins occur in Volvox (Hallmann 2003, 2006; Prochnik et al. 2010). The carboxy-terminal domains of all pherophorins are similar to the pheromone. Pherophorin-II is considered to be responsible for the signal amplification mechanism of the pheromone (Sumper et al. 1993; Godl et al.
1995). Pherophorin-II is a glycoprotein that consists of three domains: the N-terminal domain, whose sequence is related to a motif of another ECM protein, SSG185; the polyproline spacer; and the carboxy-terminal domain, which is 30% identical to the sex-inducing pheromone. The carboxy-terminal domain is proteolytically liberated from the parent glycoprotein, after its pheromone induces synthesis. Because the inhibition of processing by protease inhibitors coincides with a suppression of sexual induction, and the induction of the gene expression of pherophorin-II by the pheromone could not be observed in all three independently isolated sterile mutants, the liberated domain may potentially act as an analog of the sex-inducing pheromone (Sumper et al. 1993; Godl et al. 1995). Transformed V. carteri expressing recombinant pherophorin-II, in which the carboxy-terminus had been fused with green fluorescent protein (GFP), indicated that the carboxy-terminal domain including GFP was cleaved proteolytically, as in the native protein (Ishida 2007). The GFP signal of the transformant was located at the ECM directly surrounding the gonidium, the final target of the sexual-induction signal. However, no sex-inducing activity of the domain has been experimentally demonstrated. Genes showing similarity with pherophorins have also been identified in other Volvocales (Ch. Reinhardtii, G. pectorale and Pandorina morum), although information regarding their expression conditions is not available (Hallmann 2006). These pherophorins contain a (hydroxyl-) prolinerich (HR) rod-like domain and are abundant within the extracellular compartment, in a similar manner to the extensins of higher plants. In addition, pherophorins show a striking general structural similarity with a special class of extensin: the solanaceous lectins. Pherophorins have been suggested to be used as the versatile building blocks for the ECM architecture. In view of the large number of pherophorins, the pheromone is considered to be a pherophorin paralog and might have evolved a new function over time (Hallmann 2006).
A small cysteine-rich extracellular protein, named VCRP, which was quickly synthesized by somatic cells in response to the pheromone, has been identified (Hallmann 2007). In addition, a VCRP-related protein, VCRP2, has also been found using genome information from V. carteri (Hallmann 2008). Both VCRPs are speculated to be candidates for the extracellular second messenger from somatic cells to gonidial cells.
Mating-type Loci of Volvox carteri
As indicated previously, sexual differentiation in Ch. Reinhardtii is largely controlled by the MID gene, encoding the RWP-RK family putative transcription factor. Nozaki et al. (2006) isolated an orthologous MID gene from the oogaomous volvocacean Pleodorina starrii. The gene, named PlestMID, is only present in the male genome and the protein is abundantly present in sperm nuclei. This finding strongly suggests that maleness was probably established from the minus mating type of its isogamous unicellular ancestor during the evolution of oogamy. The MID homolog has been identified in other volvocaceans, e.g., G. pectorale (GpMID in mt– genome; Hamaji et al. 2008). Recently, both alleles of the Volvox MT were sequenced. Only two sex-limited genes, MID and MTD1, located on the MT loci of Ch. Reinhardtii have recognizable homologs in the Volvox MT, and both are in the male MT. However, both Volvox MID (VcMID) and Volvox MTD1 (VcMTD1) are expressed constitutively (Ferris et al. 2010). The retinoblastoma-related protein1 (RBR1) gene, a homolog of Ch. Reinhardtii MATING-TYPE LINKED3 (MAT3), is located on the Volvox female MT and has a very different structure from the male MAT3 homolog (Kianianmomeni et al. 2008; Ferris et al. 2010). Both MAT3 homologs display sexually regulated alternative splicing and sex-specific selection. The predominant MAT3 splicing variant in sexual males includes an early termination codon. The downregulation of MAT3 in Volvox males may be linked to the production of small-celled sperm because mat3 mutants in Ch. Reinhardtii are known to produce tiny gametes (Umen and Goodenough 2001). However, MAT3 homologs from the five colonial species examined (isogamous G. pectoral and Yamagishiella unicocca, anisogamous Eudorina sp. and P. starrii, and oogamous Volvox africanus) had almost identical nucleotide sequences between the two sexes. The extreme gender-based MAT3 divergence observed in V. carteri species may not be directly related to the evolution of male and female dimorphism within the colonial Volvocales as a whole (Hiraide et al. 2013).