Effects and mechanism of p53 gene deletion on energy metabolism during the pluripotent transformation of spermatogonial stem cells / 生理学报

Previous studies have shown that long-term spermatogonial stem cells (SSCs) have the potential to spontaneously transform into pluripotent stem cells, which is speculated to be related to the tumorigenesis of testicular germ cells, especially when p53 is deficient in SSCs which shows a significant increase in the spontaneous transformation efficiency. Energy metabolism has been proved to be strongly associated with the maintenance and acquisition of pluripotency. Recently, we compared the difference in chromatin accessibility and gene expression profiles between wild-type (p53+/+) and p53 deficient (p53-/-) mouse SSCs using the Assay for Targeting Accessible-Chromatin with high-throughput sequencing (ATAC-seq) and transcriptome sequencing (RNA-seq) techniques, and revealed that SMAD3 is a key transcription factor in the transformation of SSCs into pluripotent cells. In addition, we also observed significant changes in the expression levels of many genes related to energy metabolism after p53 deletion. To further reveal the role of p53 in the regulation of pluripotency and energy metabolism, this paper explored the effects and mechanism of p53 deletion on energy metabolism during the pluripotent transformation of SSCs. The results of ATAC-seq and RNA-seq from p53+/+ and p53-/- SSCs revealed that gene chromatin accessibility related to positive regulation of glycolysis and electron transfer and ATP synthesis was increased, and the transcription levels of genes encoding key glycolytic enzymes and regulating electron transport-related enzymes were markedly increased. Furthermore, transcription factors SMAD3 and SMAD4 promoted glycolysis and energy homeostasis by binding to the chromatin of the Prkag2 gene which encodes the AMPK subunit. These results suggest that p53 deficiency activates the key enzyme genes of glycolysis in SSCs and enhances the chromatin accessibility of genes associated with glycolysis activation to improve glycolysis activity and promote transformation to pluripotency. Moreover, SMAD3/SMAD4-mediated transcription of the Prkag2 gene ensures the energy demand of cells in the process of pluripotency transformation and maintains cell energy homeostasis by promoting AMPK activity. These results shed light on the importance of the crosstalk between energy metabolism and stem cell pluripotency transformation, which might be helpful for clinical research of gonadal tumors.

Proliferation and differentiation of mouse spermatogonial stem cells on a three-dimensional surface composed of PCL/gel nanofibers / Proliferación y diferenciación de células madre espermatogónicas de ratón en una superficie tridimensional compuesta de nanofibras PCL / gel

Spermatogonial stem cells (SSCs) have self-renewal and differentiation capacity essential for sperm production throughout the male reproductive life. The electrospun polycaprolactone/gelatin (PCL/Gel) nanofibrous scaffold mimics important features of the extracellular matrix (ECM), which can provide a promising technique for the proliferation and differentiation of SSCs in vitro. The goal of the present study was to investigate the effects of PCL/Gel nanofibrous scaffold on the propagation and differentiation of neonate mouse SSCs (mSSCs). mSSCs were enzymatically isolated, and the cells were purified by differential plating method and seeded on scaffold. After 2 weeks, viability, colony number and diameter, and expression of specific spermatogonial cell genes were investigated. After mSSCs propagation, the cells were cultivated in a differentiation medium on the scaffold for another 2 weeks, and differentiating cells were analyzed by real-time PCR. The number of mSSC colony (P<0.01) and expression levels of specific spermatogonial genes Plzf and Inga6 (P<0.01) and also differentiation genes c-Kit, Tp1 and Ptm1 (P<0.05) were higher in scaffold group compared with control during the culture period. We conclude that mSSCs can be expanded and can differentiate toward spermatid cells on PCL/Gel nanofibrous scaffold with improved developmental parameters.

Las células madre espermatogónicas (CME) tienen capacidad de auto renovación y diferenciación esenciales para la producción de esperma a lo largo de la vida reproductiva masculina. El «scaffold¼ nanofibroso de policaprolactona / gelatina (PCL / Gel) electrohilado imita características importantes de la matriz extracelular (MEC), que puede proporcionar una técnica prometedora para la proliferación y diferenciación de CME in vitro. El objetivo del presente estudio fue investigar los efectos del «scaffold¼ nanofibroso PCL / Gel en la propagación y diferenciación de CME de ratones neonatos (mSSC). Los mSSC se aislaron enzimáticamente y las células se purificaron mediante un método de siembra diferencial y se sembraron en un «scaffold¼. Después de 2 semanas, se investigaron la viabilidad, el número y el diámetro de las colonias y la expresión de genes específicos de células espermatogónicas. Después de la propagación de mSSC, las células se cultivaron en un medio de diferenciación en el «scaffold¼ durante otras 2 semanas, y las células se analizaron mediante PCR en tiempo real. El número de colonias mSSC (P <0,01) y los niveles de expresión de los genes espermatogónicos específicos Plzf e Inga6 (P <0,01) y también los genes de diferenciación c-Kit, Tp1 y Ptm1 (P <0,05) fueron mayores en el grupo de «scaffold¼ en comparación con el control durante el período de cultivo. Concluimos que los mSSC pueden expandirse y diferenciarse en células espermátidas en un «scaffold¼ de nanofibras PCL / Gel con parámetros de desarrollo mejorados.

Establishment of a pheasant (Phasianus colchicus) spermatogonial stem cell line for the production of interspecies germ line chimeras

Background Spermatogonial stem cells (SSCs) are important for the production of interspecies germ line chimeras. The interspecies germ cell transfer technique has been suggested as a way to conserve endangered birds. Our objective was to develop a technique for restoring endangered birds by developing interspecies germ line chimeras between pheasant (Phasianus colchicus) and chicken (Gallus gallus) with SSCs. Results SSCs were isolated from the surgically removed testis of a pheasant. Growth conditions for pheasant SSCs were established by co-culturing STO (SIM mouse embryo-derived thioguanine and ouabain resistant) cells and pheasant SSCs. The colony-forming cells divided and proliferated stably to yield an established SSC line. Pheasant SSCs showed strong reactivity for GDNF family receptor alpha1 (GFRa1) marker. Finally, production of germ line chimeras was attempted by transferring pheasant SSCs into recipient embryos. Although final embryo survival was 5.6% (20/354), the initial survival rate was 88% (312/354). To measure the percent transfer of donor SSC to gonads, the pheasant SSCs were labeled with PKH 26 fluorescent dye. We observed 30% donor cells and 9.48% c-kit/CD117-positive cells in the gonads of recipient chickens. Donor SSCs were thus stably engrafted in the recipient gonads. Conclusions This study showed that SSCs can be used as a tool for the conservation of endangered birds and the production of germ line chimeras. Our findings yield insights into how we may use the pheasant spermatogonial stem cell line for efficient production of interspecies germ line chimeras and ultimately, to the restoration of endangered birds.

Vascular differentiation of multipotent spermatogonial stem cells derived from neonatal mouse testis

We previously reported the successful establishment of embryonic stem cell (ESC)-like multipotent spermatogonial stem cells (mSSCs) from neonatal mouse testis. Here, we examined the ability of mSSCs to differentiate into vascular endothelial cells and smooth muscle cells, and compared to that of mouse ESCs. We used real-time reverse transcriptase polymerase chain reaction and immunohistochemistry to examine gene expression profiles of mSSCs and ESCs during in vitro vascular differentiation. Both mSSCs and ESCs exhibited substantial increase in the expression of mesodermal markers, such as Brachyury, Flk1, Mesp1, Nkx2.5, and Islet1, and a decrease in the expression of pluripotency markers, such as Oct3/4 and Nanog during the early stage of differentiation. The mRNA levels of vascular endothelial (VE)-cadherin and CD31 gradually increased in both differentiated mSSCs and ESCs. VE-cadherin- or CD31-positive cells formed sprouting branch-like structures, as observed during embryonic vascular development. At the same time, vascular smooth muscle cell-specific markers, such as myocardin and alpha-smooth muscle actin (SMA), were also highly expressed in differentiated mSSCs and ESCs. Immunocytochemical analysis revealed that the differentiated cells expressed both alpha-SMA and SM22-alpha proteins, and exhibited the intracellular fibril structure typical of smooth muscle cells. Overall, our findings showed that mSSCs have similar vascular differentiation abilities to those of ESCs, suggesting that mSSCs may be an alternative source of autologous pluripotent stem cells for vascular regeneration.

[ effect of laminin and gelatin extracellular matrix on short-term cultivation of neonate mouse spermatogonial stem cells]

To compare the effect of laminin and gelatin on short-term culture of spermatogonial stem cells [SSCs] from neonatal mouse testes. Cell suspension containing SSCs were isolated from testes of 6 day-old mice and cultured in the presence of Glial-derived neuroterophic factor [GDNF], Epidermal Growth Factor [EGF] and Basic Fibroblastic Growth Factor [bFGF] on laminin-and gelatin-coated plates for 9 days. Number and area of colonies were measured in 5th, 7th and 9th days after culturing. At 9th day Immunostaining was used to detect expression of SSC markers, alpha 6-Integrin and beta 1-Integrin. moreover, the colonies were harvested and the percentage of alpha 6-Integrin and beta 1-Integrin positive cells was assessed by flowcytometery in both groups. Immunostaining analysis showed that our culture system contained SSC colonies as they were positive for alpha 6-Integrin and beta 1-Integrin. Additionally, the number of colonies those were formed on laminin were significantly higher in comparison with those of other group. but colony area was higher on gelatin. There was no significant difference in percentage of cells that expressed alpha 6-Integrin, beta 1-Integrin detected by flowcytometry in both groups. laminin as extracellular matrix cause to increase the number of neonate spermatogonial colonies and decrease the area of them [P