Comparative genomic survey and functional analysis of DKKL1 during spermatogenesis in the Chinese soft-shelled turtle (Pelodiscus sinensis)

A feature of the Chinese soft-shelled turtle (Pelodiscus sinensis) is seasonal spermatogenesis; however, the underlying molecular mechanism is not well clarified. Here, we firstly cloned and characterized P. sinensis DKKL1, and then performed comparative genomic studies, expression analysis, and functional validation. P. sinensis DKKL1 had 2 putative N-glycosylation sites and 16 phosphorylation sites. DKKL1 also had classic transmembrane structures that were extracellularly localized. DKKL1's genetic distance was close to turtles, followed by amphibians and mammals, but its genetic distance was far from fishes. DKKL1 genes from different species shared distinct genomic characteristics. Meanwhile, they were also relatively conserved among themselves, at least from the perspective of classes. Notably, the transcription factors associated with spermatogenesis were also identified, containing CTCF, EWSR1, and FOXL2. DKKL1 exhibited sexually dimorphic expression only in adult gonads, which was significantly higher than that in other somatic tissues (P < 0.001), and was barely expressed in embryonic gonads. DKKL1 transcripts showed a strong signal in sperm, while faint signals were detected in other male germ cells. DKKL1 in adult testes progressively increased per month (P < 0.05), displaying a seasonal expression trait. DKKL1 was significantly downregulated in testes cells after the sex hormones (17 beta-estradiol and 17 alpha-methyltestosterone) and Wnt/beta-catenin inhibitor treatment (P < 0.05). Likewise, the Wnt/beta-catenin inhibitor treatment dramatically repressed CTCF, EWSR1, and FOXL2 expression. Conversely, they were markedly upregulated after the 17 beta-estradiol and 17 alpha-methyltestosterone treatment, suggesting that the three transcription factors might bind to different promoter regions, thereby negatively regulating DKKL1 transcription in response to the changes in the estrogen and androgen pathways, and positively controlling DKKL1 transcription in answer to the alterations in the Wnt/beta-catenin pathway. Knockdown of DKKL1 significantly reduced the relative expression of HMGB2 and SPATS1 (P < 0.01), suggesting that it may be involved in seasonal spermatogenesis of P. sinensis through a positive regulatory interaction with these two genes. Overall, our findings provide novel insights into the genome evolution and potential functions of seasonal spermatogenesis of P. sinensis DKKL1.