Polygenic Sex Determination System in Zebrafish

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A 46,XY female DSD patient with bilateral gonadoblastoma, a novel SRY missense mutation combined with a WT1 KTS splice-site mutation

Patients with Disorders of Sex Development (DSD), especially those with gonadal dysgenesis and hypovirilization are at risk of developing malignant type II germ cell tumors/cancer (GCC) (seminoma/dysgerminoma and nonseminoma), with either carcinoma in situ (CIS) or gonadoblastoma (GB) as precursor lesion. In 10-15% of 46,XY g

Transcriptomic Analyses Reveal Novel Genes with Sexually Dimorphic Expression in the Zebrafish Gonad and Brain

Background Our knowledge on zebrafish reproduction is very limited. We generated a gonad-derived cDNA microarray from zebrafish and used it to analyze large-scale gene expression profiles in adult gonads and other organs. Methodology/Principal Findings We have identified 116638 gonad-derived zebrafish expressed sequence tags (ESTs), 21% of which were isolated in our lab. Following in silico normalization, we constructed a gonad-derived microarray comprising 6370 unique, full-length cDNAs from differentiating and adult gonads. Labeled targets from adult gonad, brain, kidney and ‘rest-of-body’ from both sexes were hybridized onto the microarray. Our analyses revealed 1366, 881 and 656 differentially expressed transcripts (34.7% novel) that showed highest expression in ovary, testis and both gonads respectively. Hierarchical clustering showed correlation of the two gonadal transcriptomes and their similarities to those of the brains. In addition, we have identified 276 genes showing sexually dimorphic expression both between the brains and between the gonads. By in situ hybridization, we showed that the gonadal transcripts with the strongest array signal intensities were germline-expressed. We found that five members of the GTP-binding septin gene family, from which only one member (septin 4) has previously been implicated in reproduction in mice, were all strongly expressed in the gonads. Conclusions/Significance We have generated a gonad-derived zebrafish cDNA microarray and demonstrated its usefulness in identifying genes with sexually dimorphic co-expression in both the gonads and the brains. We have also provided the first evidence of large-scale differential gene expression between female and male brains of a teleost. Our microarray would be useful for studying gonad development, differentiation and function not only in zebrafish but also in related teleosts via cross-species hybridizations. Since several genes have been shown to play similar roles in gonadogenesis in zebrafish and other vertebrates, our array may even provide information on genetic disorders affecting gonadal phenotypes and fertility in mammals

Sex ratios of offspring groups generated by repeated single pair crossings show close correlation.

<p>Nineteen randomly selected breeding pairs were crossed twice; eighteen of them are shown here. The high R² value indicates that sex ratios between 1st and 2nd crosses from the same breeding pair are very similar. Red circles indicate pairs producing offspring with female-biased sex ratio, orange diamond labels the pairs with unbiased sex ratio, whereas blue squares indicate pairs producing offspring with male-biased sex ratio.</p

Wide-ranging sex ratios were observed among 62 zebrafish families.

<p>We have crossed randomly picked zebrafish individuals, grown their offspring to sexual maturity and determined their sex ratio based on presence/absence of sexual dimorphic phenotypic markers.</p

The percentage of males from repeated single pair mating of 19 randomly selected zebrafish pairs.

*<p>Removed from further analysis.</p>+<p>Does not include mating pair 6.</p

PCR-based validation of aCGH results that showed apparent family specific sex-linked inheritance pattern confirms that none of the three CNVRs analyzed are sex-linked.

<p><b>A</b>) The lack of sex-linkage for CNV regions 2 and 5 as confirmed by PCR. Size of the amplified fragments for CNVR2 and CNVR5 are 157 bp and 183 bp, respectively. CNVR2 was present only in males from the Toh1 family (Father and Son 1 and 2), while CNVR5 was only seen in female samples from the AB2 family (Mother and Daughter 1 and 2). As they showed a family-specific, sex-linked pattern, additional offspring (one son and one daughter; red boxes) were used for the validation. Upon further validation, CNVR2 and CNVR5 were found not to be sex-linked. <b>B</b>) CNV region 3 could only be validated by real time qPCR. As the three female samples from Toh2 family used for aCGH showed a loss with reference to the father's genome, additional offspring (one son and one daughter; red bar) were used for validation. Further validation also showed that this is not a sex-linked CNVR.</p

Comparative analysis of CNVRs in four zebrafish families.

<p>Out of 255 CNVRs detected, only five were present in all four families tested, however, those common CNVRs have not shown any association with sex. The number of CNVRs detected for each family is indicated in the bracket.</p

Coefficients of variation for each generation family sex ratios show selection effect on sex ratio.

<p>Panels A & B: For both families, CV for the F₀ generation (unselected) was more than two-folds higher than those for the subsequent generations, which underwent selection. Panel C: In the control experiment, after selecting for pairs that produced high proportion of males at F₀ generation, CV for F₁ generation family sex ratio decreased by about three-folds. However, when selection pressure was removed at F₂ generation by performing a random mass cross, CV for F₃ generation family sex ratio returned to a level similar to that of unselected F₀ generation.</p