Mutant <i>sox9b</i>-expressing cells demonstrate a reduced cellular association.

<p>(A–F) The morphologies of wild-type (A and B) and mutant (C–F) medaka gonads. Ventral views of XX gonads at 8 dpf (A–D) and 10 dph (E and F) are shown. Green, <i>sox9b</i>-expressing cells were immunostained with anti-GFP; red, germ cells were stained with anti-OLVAS; blue, nuclei were counterstained with DAPI. Medaka germ cells are completely surrounded by <i>sox9b</i>-expressing supporting cells and demonstrate a smooth surface (B), whilst mutant <i>sox9b</i>-expressing cells have cytoplasmic protrusions (D, arrows). Some isolated germ cells were not completely surrounded by <i>sox9b</i>-expressing cells in the mutants (F, asterisks). This was not seen in wild-type animals. (G–K) Representative images of somatic chimera. Green, donor-derived <i>sox9b</i>-EGFP expressing cells; red, host <i>sox9b</i>-DsRed positive cells; blue, germ cells stained with anti-OLVAS. (L) Calculated ratio of donor-derived <i>sox9b</i>-EGFP expressing cells associated with germ cells (black) to those not associated with germ cells (white). Scale bar, 50 µm.</p

Both type I and II germ cells are eliminated by apoptosis in the <i>sox9b</i> mutant medaka.

<p>(A–H) Ventral views of 10 dph medaka gonads immunostained with anti-OLVAS (germ cell marker, purple) and anti-cleaved caspase 3 (blue). Merged images (A, C, E and G) and cleaved caspase 3 signals (B, D, F and H) are shown. Low levels of germ cell apoptosis only were evident in wild-type XY and XX medaka at 10 dph. However, type I and type II germ cells (type I, arrows; type II, a bracket) were eliminated by apoptosis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029982#pone-0029982-t002" target="_blank">Table 2</a>) in the homozygous (E and F) and heterozygous (G and H) mutants. Scale bar, 50 µm.</p

Early stage sex differentiation processes are unaffected in the <i>sox9b</i> mutant gonads.

<p>(A–L) <i>GSDF</i> (A–D), <i>DMRT1</i> (E–H) and <i>aromatase</i> (I–L) expression (blue) were detected by in situ hybridization analysis of st. 39 (A–D) and 10 dph (E–L) medaka embryos, respectively. Consistent with the pattern found in wild-type (WT) medaka gonads, <i>GSDF</i> and <i>DMRT1</i> were found to be expressed in XY gonads of the homozygous <i>sox9b</i> mutants, whilst <i>aromatase</i> was expressed in XX gonads of the homozygous <i>sox9b</i> mutants. More than five gonads were examined in each experiment. Dotted lines (A–L), gonadal outlines; asterisks in (A–D), germ cells. Scale bar, 10 µm (A–D); 20 µm (E–L).</p

Germ cell proliferation is impaired in the <i>sox9b</i> mutant medaka.

<p>(A) Medaka exhibit two modes of germ cell division during the early stages of gonadal differentiation. Intermittent divisions (type I) lead to germ cell maintenance and occur in both males and females, whilst synchronous and successive divisions (type II) form germ cell-cysts that are committed to gametogenesis. Type II divisions occur in developing female gonads and cause a female-specific rapid increase in germ cell number. Germ cells undergoing type I or II divisions are identifiable by the presence of isolated (arrows) or packed germ cells (brackets), respectively. (B) In the <i>sox9b</i> mutants, germ cells undergoing both type I and II divisions were reduced in number. Cysts containing more than two germ cells were counted as undergoing type II divisions. (C and D) Representative images of EdU labeling experiments in wild-type and mutant medaka gonads at stage 39 are shown. Note that the nuclei of type I germ cells are positively labeled by EdU (yellow) in wild-type (arrows) but not in mutant medaka. Germ cells were stained with an anti-OLVAS antibody (purple). (E) The percentage of EdU-positive type I germ cells was calculated, and type I divisions responsible for germ cell maintenance found to be significantly impaired in the mutants. All values are the mean ± SEM. *<i>P</i><0.05 student's <i>t</i> test (each value was compared with wild-type XY and XX, respectively). Scale bar, 50 µm.</p

<i>Sox9b</i> mutations lead to a discordance between the genetic and phenotypic sex in the medaka.

1<p>The terms ‘male’ or ‘female’ are defined as secondary sex characteristics and adult gonad morphology (testis or ovary). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029982#pone.0029982.s003" target="_blank">Figure S3</a>.</p>2<p>Sox9b tg; one additional copy of <i>sox9b</i> transgene.</p

Increased apoptotic activity in the germ cells of <i>sox9b</i> mutant medaka.

<p>Increased apoptotic activity in the germ cells of <i>sox9b</i> mutant medaka.</p

Identification and characterization of two <i>sox9b</i> mutant alleles in medaka.

<p>(A) Schematic representation of the sox9b medaka protein and the locations of mutations. (B) Genomic sequences of wild-type <i>sox9b</i>, and of the <i>sox9b^K16X</i> (−/−) and <i>sox9b^K136X</i> (−/−) mutants. (C) Western blotting analysis of sox9b and alpha-tubulin demonstrating the absence of expression in the homozygous mutants, confirming that both alleles are null.</p

Gonadal morphology and germ cell number in the <i>sox9b</i> mutant medaka.

<p>(A–D) Ventral images of the medaka gonad at 10 dph obtained by confocal microscopy. Germ cells and nuclei were stained with an anti-OLVAS antibody (purple) and DAPI (green), respectively. The tissue structures are formed but the germ cell numbers are reduced in the mutant gonads. (E) Number of germ cells in wild-type and <i>sox9b</i> heterozygous and homozygous mutant medaka during gonad differentiation. *<i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001, Student's <i>t</i> test. All values are the mean ± SEM. Scale bars, 50 µm.</p

Female to male sex reversal phenotype is rescued in <i>sox9b</i> and <i>hotei</i> compound heterozygous mutants.

<p>Female to male sex reversal phenotype is rescued in <i>sox9b</i> and <i>hotei</i> compound heterozygous mutants.</p

An Essential Role of the Arginine Vasotocin System in Mate-Guarding Behaviors in Triadic Relationships of Medaka Fish (<i>Oryzias latipes</i>)

<div><p>To increase individual male fitness, males of various species remain near a (potential) mating partner and repel their rivals (mate-guarding). Mate-guarding is assumed to be mediated by two different types of motivation: sexual motivation toward the opposite sex and competitive motivation toward the same sex. The genetic/molecular mechanisms underlying how mate presence affects male competitive motivation in a triadic relationship has remained largely unknown. Here we showed that male medaka fish prominently exhibit mate-guarding behavior. The presence of a female robustly triggers male-male competition for the female in a triadic relationship (2 males and 1 female). The male-male competition resulted in one male occupying a dominant position near the female while interfering with the other male's approach of the female. Paternity testing revealed that the dominant male had a significantly higher mating success rate than the other male in a triadic relationship. We next generated medaka mutants of arginine-vasotocin (<i>avt</i>) and its receptors (<i>V1a1</i>, <i>V1a2</i>) and revealed that two genes, <i>avt</i> and <i>V1a2</i>, are required for normal mate-guarding behavior. In addition, behavioral analysis of courtship behaviors in a dyadic relationship and aggressive behaviors within a male group revealed that <i>avt</i> mutant males displayed decreased sexual motivation but showed normal aggression. In contrast, heterozygote <i>V1a2</i> mutant males displayed decreased aggression, but normal mate-guarding and courtship behavior. Thus, impaired mate-guarding in <i>avt</i> and <i>V1a2</i> homozygote mutants may be due to the loss of sexual motivation toward the opposite sex, and not to the loss of competitive motivation toward rival males. The different behavioral phenotypes between <i>avt</i>, <i>V1a2</i> heterozygote, and <i>V1a2</i> homozygote mutants suggest that there are redundant systems to activate V1a2 and that endogenous ligands activating the receptor may differ according to the social context.</p></div