The Divergent and Conserved Expression Profile of Turtle Nanog Gene Comparing with Fish and Mammals.

Nanog is a homeodomain-containing transcription factor, and it plays a vital role in maintaining the pluripotency of embryonic stem cells. Nanog's function has been well studied in many species. However, there is lack of reporting on the Nanog gene in reptile. Here, we identified a 1032 bp cDNA sequence of a Nanog gene in Pelidiscus sinensis, known as PsNanog. PsNanog has a highly conserved HD domain and shares a high identity with that of Chelonia mydas and the lowest identity with Oryzias latipes. Similarly, PsNanog presented a tight cluster with C. mydas Nanog, but was far from those of teleosts. Additionally, we cloned a length of 1870 bp PsNanog promoter. Dual luciferase assay showed that the DNA fragment of -1560 to +1 exhibited a high promoter activity. The RT-PCR and RT-qPCR results showed that PsNanog was predominantly expressed in ovary, and then in testis. The in situ hybridization and immunohistochemical analysis showed that PsNanog was expressed in the early primary oocytes and the cytoplasm of the cortical region of stage VIII oocytes in ovary, and distributed in most stages of germ cells in testis. Collectively, the results imply that PsNanog probably has the conserved function in regulating germ cell development across phyla and is also a pluripotent cell gene and expressed in germ cells, which is similar to that in teleosts and mammals.

High Prevalence of a Monogenic Cause in Han Chinese Diagnosed With Type 1 Diabetes, Partly Driven by Nonsyndromic Recessive WFS1 Mutations.

It is estimated that ∼1% of European ancestry patients clinically diagnosed with type 1 diabetes (T1D) actually have monogenic forms of the disease. Because of the much lower incidence of true T1D in East Asians, we hypothesized that the percentage would be much higher. To test this, we sequenced the exome of 82 Chinese Han patients clinically diagnosed with T1D but negative for three autoantibodies. Analysis focused on established or proposed monogenic diabetes genes. We found credible mutations in 18 of the 82 autoantibody-negative patients (22%). All mutations had consensus pathogenicity support by five algorithms. As in Europeans, the most common gene was HNF1A (MODY3), in 6 of 18 cases. Surprisingly, almost as frequent were diallelic mutations in WFS1, known to cause Wolfram syndrome but also described in nonsyndromic cases. Fasting C-peptide varied widely and was not predictive. Given the 27.4% autoantibody negativity in Chinese and 22% mutation rate, we estimate that ∼6% of Chinese with a clinical T1D diagnosis have monogenic diabetes. Our findings support universal sequencing of autoantibody-negative cases as standard of care in East Asian patients with a clinical T1D diagnosis. Nonsyndromic diabetes with WSF1 mutations is not rare in Chinese. Its response to alternative treatments should be investigated.

Identification and characterization of DAZ family genes in Chinese soft-shell turtle (Pelodiscus sinensis).

The DAZ family genes, including boule, dazl, and daz, play pivotal roles in germ cell development and differentiation during gametogenesis in organisms, which have been widely studied in mammals, reptiles, or fishes. Dazl was bisexual expressed in both mitotic and meiotic germ cells, daz was male premeiotic expressed, whereas boule exhibits largely in unisexual meiotic germ cells but bisexual expression in several fishes, however, there is lack of report on boule gene and the evolutionary conservation and divergence of dazl and boule in reptile. Here, both boule and dazl genes were characterized in Pelodiscus sinensis. The quantitative real-time polymerase chain reaction analysis showed that boule and dazl were abundantly expressed in adult ovary and testis but barely in somatic tissues, such as heart, brain, liver, spleen, and kidney. Moreover, through fluorescent in situ hybridization, bisexual and germline-specific expression profiles of boule and dazl messenger RNAs (mRNAs) were demonstrated. Boule mRNA exhibited a maximal meiotic expression in spermatocytes, and a relatively low, but distinct expression in oocytes at meiotic stages in P. sinensis, similar to the expression profile of human boule in ovary. However, dazl mRNA was richly distributed in male germ cells at almost all stages during spermatogenesis, and predominantly expressed in most of stages of oocytes including premeiotic and meiotic stages. These findings imply that boule and dazl would play distinct roles in the sexual differentiation of germ cells during turtle gametogenesis, and the major functions of daz family members involved in germ cell differentiation would be conserved across species including P. sinensis.

Isolation and in vitro culture of ovarian stem cells in Chinese soft-shell turtle (Pelodiscus sinensis).

Gonadal cell lines provide valuable tools for studying gametogenesis, sex differentiation, and manipulating germ cells in reproductive biology. Female germline stem cells have been characterized and isolated from ovaries of mammalian species, including mice and human, but there has been very few studies on female germline stem cells in reptiles. Here, we described an ovarian stem cell-like line isolated and cultured from the Chinese soft-shell turtle (Pelodiscus sinensis), designated as PSO1. The cells showed high alkaline phosphatase activity with a normal diploid karyotype. As shown by reverse transcription-polymerase chain reaction, the cells were positive for the expression of germ cell-specific genes, vasa and dazl, as well as a stem cell marker, nanog, but negative for the expression of the folliculogenesis-specific gene, figla. Likewise, through fluorescent immunostaining analyses, both the Dazl and Vasa proteins were detected abundantly in the cytoplasm of perinuclear region, whereas Nanog and PCNA were dominantly observed in the nuclei in PSO1 cells. Moreover, PSO1 cells transfected with pCS2:h2b-egfp could properly express the fusion protein in the nuclei. Taken together, the findings suggested that the germline stem cells exist in the ovary of juvenile Chinese soft-shell turtle and these cells can be isolated for a long-term in vitro culture under experimental conditions. This study has provided a valuable basis for further investigations on the molecular mechanisms whereby the germline stem cells develop and differentiate into gametes in turtles. Also, it has paved the way for studies on oogenesis in turtles, even in the other reptiles.