Dynein axonemal intermediate chain 2 plays a role in gametogenesis by activation of Stat3.

We previously identified the mouse dynein axonemal intermediate chain 2 (Dnaic2) gene. This gene expresses a component of the axonemal dynein complex that functions in cilia or flagella. We found that overexpression of Dnaic2 results in female subfertility and male infertility. In this study, we generated Dnaic2 knockdown (KD) mice and identified the potential regulatory mechanisms involved in Dnaic2 function. For phenotype analysis, we found that body weight was lighter and size was smaller in Dnaic2 KD mice than in wild-type mice. A total of 45% of these Dnaic2 KD mice were infertile due to sperm abnormalities in males, or had oocyte abnormalities and pathological changes in the tunica mucosa in the oviduct of females. Moreover, Dnaic2 overexpression enhanced the expression of proliferating cell nuclear antigen (PCNA) in the ovaries, which suggested that Dnaic2 stimulated proliferation of cells in the ovaries. However, PCNA expression in the testis of Dnaic2-overexpressed mice was lower than that in controls. Additionally, the ratio of Bax/B-cell lymphoma-2(Bcl-2) in the testis of Dnaic2-overexpressed mice was higher than that in controls, which suggested that Dnaic2 inhibited cellular proliferation in the testis. To examine the molecular action of Dnaic2, immunoprecipitation analysis was used and showed that Dnaic2 protein interacted with signal transducer and activator of transcription 3 (Stat3). Molecular modelling analysis showed that Dnaic2 bound with the linker and SH2 domains of Stat3. Furthermore, overexpression of Dnaic2 promoted phosphorylation of Stat3. In conclusion, our study suggests that Dnaic2 plays a role in oogenesis and spermatogenesis by activation of Stat3.

Male germ cell apoptosis and epigenetic histone modification induced by Tripterygium wilfordii Hook F.

Multiglycosides of Tripterygium wilfordii Hook f (GTW), a Chinese herb-derived medicine used as a remedy for rheumatoid arthritis, are considered to be a reversible anti-fertility drug affecting the mammalian spermatids. However, the mechanism behind this effect is still unknown. To study the possible mechanism behind the impact of GTW on spermatogenesis, we administered 4 groups of 4-week-old male mice with different doses of GTW. We found a dose-dependent decrease in the number of germ cells after 40 days of GTW treatment, and an increase in apoptotic cells from the low-dose to the high-dose group. During this same period the dimethylated level of histone H3 lysine 9 (H3K9me2) in GTW-treated testes germ cells declined. Additionally, spermatogonial stem cells (SSCs) from 6-day-old mice were isolated to evaluate the possible effect of GTW or triptolide on development of SSCs. We found a significantly higher incidence of apoptosis and lower dimethylation level of H3K9me2 in the SSCs of GTW or triptolide treatment than in controls. Thus, these data suggest that the GTW-induced apoptosis might be responsible for the fertility impairment in mice. This damage could be traced back to the early stages of spermatogenesis. GTW also affected the epigenetic modification of H3K9 in spermatogenesis. Molecular dynamics simulation suggested that triptolide and dimethylated or trimethylated H3K9 might have similar interaction mechanisms with EED (embryonic ectoderm development). These candidate activation mechanisms provide the first glimpse into the pathway of GTW-induced gonad toxicity, which is crucial for further research and clinical application.

Production of transgenic mice by random recombination of targeted genes in female germline stem cells.

Oocyte production in most mammalian species is believed to cease before birth. However, this idea has been challenged with the finding that postnatal mouse ovaries possess mitotically active germ cells. A recent study showed that female germline stem cells (FGSCs) from adult mice were isolated, cultured long term and produced oocytes and progeny after transplantation into infertile mice. Here, we demonstrate the successful generation of transgenic or gene knock-down mice using FGSCs. The FGSCs from ovaries of 5-day-old and adult mice were isolated and either infected with recombinant viruses carrying green fluorescent protein, Oocyte-G1 or the mouse dynein axonemal intermediate chain 2 gene, or transfected with the Oocyte-G1 specific shRNA expression vector (pRS shOocyte-G1 vector), and then transplanted into infertile mice. Transplanted cells in the ovaries underwent oogenesis and produced heterozygous offspring after mating with wild-type male mice. The offspring were genetically characterized and the biological functions of the transferred or knock-down genes were investigated. Efficiency of gene-transfer or gene knock-down was 29%-37% and it took 2 months to produce transgenic offspring. Gene manipulation of FGSCs is a rapid and efficient method of animal transgenesis and may serve as a powerful tool for biomedical science and biotechnology.