| Spermatogonial stem cells(SSCs) are located in the niche of the basement membrane in the male testis, surrounded by the Sertoli cells and other somatic cells. Like other stem cells, SSCs also have the typical characteristics: self-renewal and differentiation. While maintaining a relatively stable number, SSCs continuously go through spermatogenesis to produce a large amount of sperms. Spermatogonial stem cells are the only adult stem cells exist in adult male mammal, which transfer of genetic and epigenetic information to the next generation. With the increasing age, the homeostasis of the niche of SSCs is disturbed. Combined with the accumulation of damages from oxidative stress, the self-renewal and differentiation functions of SSCs gradually deteriorate. SSCs pool is depleted and sperm quantity and quality are also decreasing, causing infertility of male animals.Resveratrol(Res) is a polyphenolic phytoalexin, it can mimic calorie restriction to extend the life span of yeast, c. elegans, fruit flies and other species. When adding Res in mouse food, SIRT1 protein was activated, the functional degeneration caused by aging and high-fat diet was effectively alleviated. Res plays strict role of tissue and dose-specific. It can also be used as chemotherapeutic drug, which can induce apoptosis of liver cancer, stomach cancer, colon cancer cells by mitochondria, p53 or BCL2 other pathways. Res inhibit the tumorigenesis, development and metastasis of cancers. Before trying to apply Res to alleviate damage of aging on SSCs, the effective concentration of Res should be determined firstly and explore whether Res has side effects and its related mechanisms on SSCs to avoid secondary damage.Busulfan is the most common myeloablative drugs used prior to bone marrow transplantation, and it is also a chemotherapy drug of cancer treatment. As an alkylating agent, busulfan distroy the stability of genetic material and induce apoptosis mainly depend on the increasing ROS levels and the crosslinkings between DNA-DNA, DNA-protein or protein-proterin. Similar to the machenism of aging, busulfan has a strong reproductive toxicity found during clinical application. Based on this phenomenon, azoospermia mouse model was established as the research platform of SSCs transplantation and the mechanisms of self-renewal, differentiation and aging of SSCs.This study identified the biological characteristics of in vitro cultured mouse SSCs C18-4 at first, and then examined the effect of different concentrations of Res on cell viability, proliferation and apoptosis levels. Further studies were executed to explore the mechanism of various concentrations of Res by testing the mitochondrial membrane potential, SIRT1-FOXO1 signaling and DNA integrity experiments. Taking this as a reference, we primarily tried to use Res to rescue the SSCs damage induced by busulfan. This study provided the foundation to apply Res to protect male fertility against the chemotherapy or aging process.1. The effects of Res on the mouse SSCs C18-4By morphological and immunofluorescence staining, C18-4 cells were observed to obtain typical characteristics of type A SSCs, like shape in round or oval, high nuclear-cytoplasmic ratio, expressing PLZF, NANOS2, VASA, SSEA1, CD49 f proteins and being negative of STRA8. 2 μM Res promotes C18-4 cell viability, but with concentration rising, cell viability was decreased. 20 μM Res arrested C18-4 proliferation in S phase, when the concentration was raised to 200 μM, C18-4 nucleus shrinkage, cell vacuolated, proliferation was inhibited and apoptosis rate increased significantly.2. Preliminary explore the mechanism of different concentrations of Res on mouse SSCsMitochondrial membrane potential assay showed 2 μM Res could protect the integrity of mitochondria, promoting energy metabolism. 20 μM and 200 μM doses of Res broke mitochondrial function. Double-stranded DNA breakage was found after treated by 200 μM Res, exacerbating C18-4 cell apoptosis. With the increased concentration of Res, SIRT1 protein expression gradually up-regulated to reduce the level of acetylated FOXO1, causing FOXO1 protein translocated into nuclear of mouse SSCs. The expressed pattern of apoptosis-related protein p38 was positively correlated with the concentration of Res. While anti-apoptotic BCL2 protein expression was increased after 2 μM Res stimulated, its expression was inhibited in 200 μM group to promote apoptosis.3. Res promote the recovery of SSCs in azoospermia mouse model4 weeks after busulfan injected in abdomina, the original positions of SSCs in the seminiferous tubules of mice testis were vacuolated, sperms were completely disappeared in epididymal. Res intervention could promote the recovery of spermatogonial stem cells, while 100 mg/kg/d dose was more effective, at this time sperms were visible in epididymis. Real-time qPCR analysis showed that Res could inhibit apoptosis related genes p53, c-Myc and FoxO1 transcription. The expressions of SSCs marker gene Thy1, Plzf were relatively increased, while the expression of differentiation marker genes c-Kit and support marker Sox9 gene expression were declined. Cell proliferation marker gene Pcna was also up-regulated. Res could inhibit the SSCs apoptosis and promote proliferation, thereby enhancing their ability to recover from damaged of busulfan. Western-Blotting analysis showed that the quantity of SIRT1 protein was significant increased after Res treated and deacetylated FOXO1 protein in the downstream. |
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