Download A new male-specific gene “OTOKOGI” in Pleodorina starrii

Biologia 63/6: 772—777, 2008
Section Botany
DOI: 10.2478/s11756-008-0097-9
Review
A new male-specific gene “OTOKOGI ” in Pleodorina starrii
(Volvocaceae, Chlorophyta) unveils the origin of male and female*
Hisayoshi Nozaki
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033,
Japan; e-mail: [email protected]
Abstract: Eukaryotic sex was initially isogametic and it is assumed that anisogamy/oogamy evolved independently in many
lineages including animals, land plants and volvocine green algae. The exact evolutionary mechanisms that were responsible
for the evolution of oogamy from isogamy were poorly understood until Nozaki et al. (2006) introduced the use of moleculargenetic data in elucidating the evolutionary origin of oogamy from isogamy in the colonial volvocacean Pleodorina starrii. In
the close relative Chlamydomonas reinhardtii, sexual reproduction is isogametic with mating-types plus and minus. Mating
type minus represents a “dominant sex” because the MID (“minus-dominance”) gene of C. reinhardtii is both necessary
and sufficient to cause the cells to differentiate as isogametes of the minus mating type. No sex-specific genes had been
identified in the volvocine green algae until Nozaki et al. (2006a) successfully cloned the MID gene of P. starrii. This
“OTOKOGI ” (PlestMID) gene is present only in the male genome, and encodes a protein localized abundantly in the nuclei
of mature sperm. Thus, P. starrii maleness evolved from the dominant sex (mating type minus) of its isogamous ancestor.
This breakthrough provides an opportunity to address various extremely interesting questions regarding the evolution of
oogamy and the male-female dichotomy.
Key words: evolution; maleness; mating type; oogamy; sex; Volvocales
Discovery of the male-specific gene from the
anisogamous/oogamous Volvocaceae
Both animals and land plants exhibit “oogamy,” that
is, they produce male and female gametes (sperm and
eggs) that differ markedly in size and motility. In
contrast, many primitive organisms exhibit “isogamy,”
having gametes of different mating types that are very
similar in size and appearance. The evolution of oogamy
from isogamy (and with it the evolution of male/female
differences) is surely one of the most interesting biological issues. However, no extant isogamous organisms are
known that are closely related to animals or land plants
(Karol et al. 2001; Rokas et al. 2005), and so there is
no molecular-genetic data that would indicate what the
evolutionary relationship may be between the two sexes
of oogamous organisms and the mating types of isogamous organisms until our recent study (Nozaki et al.
2006b).
The anisogamous/oogamous multicellular green algae in the family Volvocaceae provide an ideal model
for exploring such evolutionary relationships, because
evolutionary progression from isogamy to oogamy can
be observed within the closely related lineage (Nozaki
& Ito 1994; Nozaki et al. 2000; Nozaki 2003; Kirk
2006) and several mating type-specific genes have been
identified in the closely related isogamous, unicellular alga Chlamydomonas reinhardtii (Ferris & Goodenough 1994, 1997). However, no mating type-specific
genes had been isolated previously from the Volvocaceae until the recent study of Nozaki et al. (2006a).
Although modes of uniparental inheritance of the plastid and mitochondrial genomes have been characterized
in Chlamydomonas reinhardtii/smithii (Kuroiwa et al.
1982; Boynton et al. 1987) and the oogamous volvocacean Volvox carteri (Adams et al. 1990; Kuroiwa et
al. 1993), they are essentially different between these
two organisms. In Chlamydomonas, the plastid or mitochondrial genome is inherited from the plus or minus
mating type of the parents, respectively (Kuroiwa et al.
1982; Boynton et al. 1987). In contast, both the plastid and mitochondrial genomes are inherited from the
female parental strains of Volvox (Adams et al. 1990;
Kuroiwa et al. 1993). Thus, evolutionary relationships
between isogamous mating types in Chlamydomonas
and female/male in the anisogamous/oogamous volvocaceans can not be deduced based on comparison
of modes of uniparental inheritance of the organelle
genomes, and orthologues of the mating type-specific
genes of Chlamydomonas reinhardtii (Ferris & Goodenough 1994; Ferrris et al. 2002) are badly needed.
The difficulties of isolating mating type- or sex-
* Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007,
Slovakia.
c 2008 Institute of Botany, Slovak Academy of Sciences
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A new male-specific gene “OTOKOGI ” from Pleodorina starrii
specific genes from the colonial Volvocales appear to
have two causes. Firstly, evolution of the sex-related
genes is rapid. Ferris et al. (1997) tried to isolate homologues of the C. reinhardtii dominance (minus) mating type-determining (MID) gene from Gonium pectorale and Volvox carteri by Southern blotting, but
could not obtain the genes which they attributed to the
rapid evolution of sex-related genes. Secondly, the ability to induce sexual reproduction in the colonial Volvocales generally decreases during the long-term maintenance of the cultures (Coleman 1975). Especially in the
anisogamous/oogamous members of the Volvocaceae,
the decrease in sexual induction is rapid: male strains
of the anisogamous/oogamous volvocacean alga Pleodorina starrii (Fig. 1A) did not produce sperm packets
(bundles of male gametes) five years after their isolation
(Nozaki 2008). This problem may be resolved by cryopreservation of the colonial Volvocales, but favourable
results of cryopreservation treatments have not yet been
obtained (Mori et al. 2002).
In order to isolate sex-related genes from the colonial Volvocales, Nozaki et al. (2006a) used the newly
established, heterothallic strains of Pleodorina starrii
(Nozaki et al. 2006b), and degenerate PCR primers.
They eventually succeeded in isolating a Pleodorina
orthologue of the MID gene of Chlamydomonas reinhardtii that causes cells to develop as mating type minus
gametes. In the following chapters, I report details and
the biological significance of the Pleodorina ortholog of
MID (PlestMID, Nozaki et al. 2006a) that is here called
“OTOKOGI ” (meaning “Japanese male chivalry”).
A new male-specific gene “OTOKOGI ”
Nitrogen-starvation of Pleodorina starrii males induces
the formation of sperm packets that later dissociate
into individual sperm capable of fertilizing female gametes, or eggs (Nozaki et al. 2006b). Nozaki et al.
(2006a) used differential cDNA screening of nitrogenstarved male and female cultures to identify a cDNA
that was present only in nitrogen-starved, sexuallyinduced males. Like products of the MID genes of two
species of Chlamydomonas (CrMID and CiMID from
C. reinhardtii and C. incerta, respectively, Ferris et
al. 1997), “OTOKOGI ” has a median G+C content
of 48.4% in the coding region, and encodes a short
ORF (163aa) containing an RWP-RK domain and a
leucine-zipper in the C-terminal region (Fig. 1B) (Ferris & Goodenough 1997; Ferris et al. 1997; Nozaki et
al. 2006a). We established the presence of four introns,
three of which are in positions very similar to those of
the three introns that are present in the CrMID and
CiMID genes (Fig. 1B) (Ferris et al. 1997, Nozaki et al.
2006a). A second MID-like sequence, PsPlestMID, was
found in the male genome, but is probably a pseudogene of “OTOKOGI ” (Nozaki et al. 2006a). A Southern blot of restriction-digested male and female DNA
probed with a PCR-fragment of “OTOKOGI” that covered the second exon indicated that both “OTOKOGI ”
and PsPlestMID are present in the genome of the
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male strain, but neither is present in the female strain
(Fig. 1C).
RT-PCR experiments using “OTOKOGI ”-specific
primers demonstrated that the “OTOKOGI ” gene is
expressed in nitrogen-starved, gamete-producing males,
but neither in males that had not been nitrogenstarved to induce gametogenesis, nor in females regardless whether or not those females had been starved for
nitrogen (Fig. 1D). Nor was any PsPlestMID expression
detected when RT-PCR was performed with PsPlestMID-specific primers in conjunction with RNA from
either control or nitrogen-starved male or female cultures (Fig. 1D).
When sperm packets were stained with an antibody directed against the “OTOKOGI” protein, an
immunofluorescent signal was observed in the vicinity
of the nuclei, and the nuclear location of this signal
was much clearer after the sperm packets had dissociated to release individual sperm (Fig. 1E). The signal was prominent especially in the peripheral region
of the male nuclei (Fig. 1E). No immunofluorescent signal above background was detected in vegetative males,
even when they had been cultured in nitrogen-deficient
medium. These findings parallel those obtained for CrMID, which is expressed only in nitrogen-starved cells
that are actually producing minus gametes (Ferris et al.
2002). Because the CrMID protein is necessary for differentiation of minus gametes in C. reinhardtii (Ferris
& Goodenough 1997), and because CrMID, CiMID and
“OTOKOGI” (Fig. 1B) all encode similar RWP-RK domains and leucine zippers that might be involved in
DNA binding and protein dimerization (Schauser et al.
2005), our working hypothesis is that the “OTOKOGI ”
gene product plays an important role in male gametogenesis in P. starrii (Nozaki et al. 2006a).
Evolution of “OTOKOGI ” and female/male differentiation in the Volvocaceae
A phylogenetic analysis was performed with a
53-residue region spanning the RWP-RK domains
of the three MID proteins (CrmMID, CiMID and
“OTOKOGI”) and 67 other sequences containing this
domain, including 33 from chlorophytes (Chlamydomonas, Volvox, Ostreococcus and various land plants).
This group of proteins included a number of nodule
inception (NIN) and NIN-like proteins (Schauser et
al. 2005). Our analysis revealed clearly that the three
MID/“OTOKOGI” proteins formed a monophyletic
group with moderate bootstrap values, whereas all of
the other RWP-RK proteins of Chlamydomonas and
Volvox were separate from the MID/“OTOKOGI” lineage (Fig. 2). This outcome suggests that MID probably
evolved early in volvocalean evolution as a gene regulating the dominant mating type, and was conserved
for such a function in “OTOKOGI ” of the anisogamous/oogamous Volvocaceae. Thus, males in oogamous
volvocaceans such as P. starrii are homologous to the
dominant, minus mating type of C. reinhardtii. Therefore, evolution of sperm-producing males in the famUnauthenticated
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774
H. Nozaki
Fig. 1. The anisogamous/oogamous colonial green alga Pleodorina starrii and its male specific genes “OTOKOGI ” (PlestMID) and
PsPlestMID. A – Vegetative colonies, female gamete (egg, asterisk) and male gamete (sperm, arrow). B – Alignment of the sequences
of “OTOKOGI” and two Chlamydomonas MID proteins (CrMID and CiMID). Blue shading: intron locations; red box: an RWPRK motif; arrows: a leucine-zipper; asterisks: identical amino acids. C – Southern hybridization of “OTOKOGI ” probe with BamHI
(B) and SalI (S) fragments of DNA from female (F) and male (M) P. starrii strains. D – Semiquantitative RT-PCR analyses of
“OTOKOGI ” and PsPlestMID genes in female (F) and male (M) strains that were either nitrogen deprived to induce gametogenesis
(N) or nitrogen fed (V). The EF-1 like gene was used as an internal control. E – Visualization of “OTOKOGI” protein in mature
sperm after release from sperm packets. “OTOKOGI ” expression is obvious in nucleus (arrowheads in DAPI images). Specimens were
double-stained with 4’6–diamidino-2–phenylindole (DAPI) and either anti-“OTOKOGI ” antibodies. All panels show identical cells.
DAPI (pseudo-coloured) and immunofluorescence images are merged (rightmost panels). Scale bar represents 5 µm (from Nozaki et
al. 2006; reproduced by permission of Elsevier Ltd., Licence No. 1961691484312).
ily Volvocaceae appears to be based on the genetic
system controlled by the dominant, mating type minus locus of the family’s isogamous, Chlamydomonaslike ancestor. This system bears some resemblance
to the X/Y sex-determining chromosomal system of
mammals, in which the male is the dominant phenotype. But whereas the X/Y system that controls
male/female differentiation in the diploid mammals is
thought to have evolved 240–300 MYA (Lahn & Page
1999) after the establishment of oogamy in the origin of Metazoa (animals) (∼600 MYA, Rokas et al.
2005), isogamous C. reinhardtii and oogamous volvocaceans such as Pleodorina appear to have shared a
common ancestor as recently as 50 ±20 MYA (Kirk
2006) (Fig. 3). Thus, it appears that male/female differentiation evolved very rapidly in these green algae
based on the MID-regulated, sex-determination system
in the haploid cells. Such rapid evolution of gamete diUnauthenticated
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A new male-specific gene “OTOKOGI ” from Pleodorina starrii
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Fig. 2. Phylogenetic analyses suggesting a relationship between sex-specific MID/“OTOKOGI” proteins and other RWP-RK domaincontaining proteins. Green, red and yellow colors represent green plants, red algae and oomycetes, respectively. Numbers without or
within parentheses indicate bootstrap values of 50% or more, based on 500 or 100 replicates by Maximum Likelihood (based on WAG
model of protein evolution) or Minimum Evolution (using p-distances), respectively. Abbreviations used by Schauser et al. (2005),
accession numbers, or other forms of gene identification are given after the species names (from Nozaki et al. 2006; reproduced by
permission of Elsevier Ltd., Licence No. 1961691484312).
morphism may also have occurred in the origin of the
animals and land plants based on the dominant mating
type-controlled, sex-determination system of the haploid ancestral organisms.
In Chlamydomonas reinhardtii, CrMID is located
in the minus MT-locus, which consists of an ∼1 Mb
region of recombinational suppression; it is not found
in the mating type plus locus (Ferris & Goodenough
1994). Since both “OTOKOGI ” and PsPlestMID are
found only in the male genome (Fig. 1C), we assume that they are located in the male mating locus
of Pleodorina starrii. Ferris and Goodenough (1997)
demonstrated that the CrMID gene is necessary and
sufficient to convert a plus cell into a minus cell,
and suggested that all other genes that encode minusspecific gametic traits are carried by both mating type
plus and munus cells. Those genes can be considered
“autosomal” genes whose expression is controlled by
the CrMID gene product. However, the marked expression of “OTOKOGI” protein in the individual sperm
that have been released from sperm packets (Fig. 1E)
may indicate that in addition to its presumed role
in gametogenesis, this protein may also contribute to
maintenance or mating behaviour of the male gametes.
The male-specific gametic traits in the oogamous volvocaceans are more complex than those of mating type
minus gametes of Chlamydomonas, especially before
syngamy (Nozaki 1996). The differentiation of male
gametes of Pleodorina starrii involves the formation
of specialized male organs called sperm packets that
move to the female colony and dissociate into individual, small, spindle-shaped male gametes that penetrate
the female colony in search of fertilizable eggs (Nozaki
et al. 2006b). Therefore, essential genes encoding the
mating type minus/male-specific gametic traits probably have increased in number during the evolution of
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776
H. Nozaki
Fig. 3. Diagram showing comparison of evolution of male/female differentiation and sex-determination system between volvocaleans
(green algae) and mammals (animals).
oogamy in the family Volvocaceae. Some of those genes
may be localized in the male mating type locus, where
(assuming a behaviour similar to that of the C. reinhardtii mating type loci [Ferrris & Goodenough 1994;
Ferris et al. 1997]) they may have undergone extensive
rearrangement and rapid evolution. Indeed, the existence of a second male-specific gene, PsPlestMID, in
P. starrii indicates that there has been a duplication
of at least a part of the mating type locus. The malespecific gene “OTOKOGI ”, first identified by Nozaki et
al. (2006a), provides a powerful first step toward cloning
the male mating type locus and determining its gene
composition. Comparison of other mating type-linked
loci between Chlamydomonas and Pleodorina may reveal which other genes have been instrumental in the
evolution of volvocacean “maleness.” Studies of such
evolved genes should shed light on the mechanisms underlying the evolution of male and female gametes that
are differentiated in size and motility.
Acknowledgements
This work was supported by Grant-in-Aid for Creative Scientific Research No. 16GS0304 to HN and by Grant-in-Aid
for Scientific Research No. 17370087 to HN from the Ministry of Education, Culture, Sports, Science and Technology,
Japan.
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Received September 1, 2007
Accepted March 17, 2008
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