Figure 1.
Phylogenetic analysis of Tesk protein sequences.
A phylogenetic tree was constructed with the neighbor-joining algorithm in MEGA 4.0. Dendrogram graphically showed the relations for different organisms based on the amino acid sequence. The relative genetic distances are indicated by the scale bar and the branch lengths. Protein sequences used in this analysis: Pundamilia nyererei Tesk1 (XP_005745859), Maylandia zebra Tesk1 (XP_004565879), Oreochromis niloticus Tesk1 isoform1 (XP_003459044), Oreochromis niloticus Tesk1 isform2 (XP_005464337), Salmo salar Tesk1 (ACI33724), Danio rerio Tesk1 (NP_001083043), Gallus gallus Tesk1 (XP_003642315), Chrysemyspicta bellii Tesk1 (XP_005291483), Chelonia mydas Tesk1 (EMP299900), Columba livia Tesk1 (EMC88537), Anas platyrhynchos Tesk1 (EOB04897), Mus musculus Tesk1 (NP_035701), Homo sapiens Tesk1 isoform1 (NP_006276), Homo sapiens Tesk1 isoform2 (BAA09459), Rattus norvegicus Tesk2 (NP_596887), Homo sapiens Tesk2 (NP_009101).
Figure 2.
Real-Time quantitative PCR analysis of tesk1 of C.semilaevis.
(A) The expression of tesk1 in various tissues of tongue sole. H: heart, L: liver, K: kidney, I: intestine, SP: spleen, SK: skin, M: muscle, B: brain, G: gonad. (B) The expression of tesk1 in gonads of different genotypes. F: female, M: male, PM: pseudo-male, TM: triploid male. (C) The expression of tesk1 at different developmental stages of the gonads. The tesk1 mRNA amount was normalized to the β-actin transcript level. The data was analyzed by one-way ANOVA followed by Duncan comparison tests using SPSS 18.0. Bars represent the triplicate mean±SE from three separate individuals (n = 3). Bars with different letters differed with statistical significance (p<0.05).
Figure 3.
Identification of genotype of C.semilaevis.
F: female, M: male, PM: pseudo-male, TM: triploid male. One 206 bp band corresponding to diploid male (ZZ) and triploid male (ZZZ), two bands (206 bp and 218 bp) corresponding to diploid female (ZW) and diploid pseudo-male (ZW). Triploidy has been identified by flow cytometry analysis of DNA relative content and karyotyping.
Figure 4.
In situ localization of tesk1 mRNA in gonads of C. semilaevis.
Gonads in situ hybridization using antisense RNA probe of tesk1 performed in tongue sole. (A): testis of diploid male at8 months, (B): testis of diploid male at 1 year, (C): testis of diploid male at 2 years, (D): testis of diploidpseudo-male at 2 years, (E): testis of triploid male at 2 years, (F): ovary of diploid female at 8 months, (G): ovary of diploid female at 1 year, (H): ovary of diploid female at 2years. Abbreviation of the cells indicated by arrows. PSC: primary spermatocyte, SSC: secondary spermatocyte, SP: spermatid, SZ: spermatozoon, SE: Sertoli cells, LE: Leydig cells, OC: oocyte. Scale bars, 10 µm.
Figure 5.
Chromosomal localization of the C. semilaevis tesk1.
Tesk1-BAC FISH analysis of tongue sole chromosomes showing four signals in ZZ male (A) and two signals in ZW female (B). BAC clone Hind017D15, which contains the full length tesk1 gene was labeled and used to probe male (ZZ) and female (ZW) chromosome spreads. Arrows indicate the fluorescent signals. Arrowhead indicates the W chromosome which is identified by its largest size of all chromosomes. Scale bars, 5 µm.