Zuping He

Zearalenone induces apoptosis and necrosis in porcine granulosa cells via a caspase-3- and caspase-9-dependent mitochondrial signaling pathway

Zearalenone is a mycotoxin produced mainly by Fusarium. There are numerous incidences of mycotoxicosis in laboratory and domestic animals, especially in pigs. However, little is known about molecular mechanisms of zearalenone toxicity. Granulosa cells are the maximal cell population in follicles, and they play an essential role in the development and maturation of follicles. The objective of this study was to explore the effect of zearalenone at high concentrations on proliferation and apoptosis of porcine granulosa cells and uncover signaling pathway underlying the cytotoxicity of zearalenone. We found that zearalenone reduced the proliferation of porcine granulosa cells in a dose‐dependent manner as shown by the MTT assay and zearalenone resulted in an obvious apoptosis and necrosis in porcine granulosa cells as determined by the TUNEL analysis and flow cytometry. In addition, TUNEL assay with caspase inhibitors showed that zearalenone triggered a caspase‐3‐ and caspase‐9‐dependent apoptotic process in porcine granulosa cells. Fluorescence spectrophotometer displayed that zearalenone led to a loss of mitochondrial transmembrane potential of porcine granulosa cells but enhanced reactive oxygen species (ROS) levels of the cells. Notably, Western blots revealed that caspase‐3 and caspase‐9 were activated by zearalenone in porcine granulosa cells. Collectively, our results suggest that zearalenone induces apoptosis and necrosis of porcine granulosa cells in a dose‐dependent manner via a caspase‐3‐ and caspase‐9‐dependent mitochondrial pathway. This study thus offers a novel insight into molecular mechanisms by which zearalenone has adverse cytotoxicity on reproduction. J. Cell. Physiol. 227: 1814–1820, 2012.

The roles and regulation of Sertoli cells in fate determinations of spermatogonial stem cells and spermatogenesis

Spermatogenesis is a complex process by which spermatogonial stem cells (SSCs) self-renew and differentiate into spermatozoa under the elaborate coordination of testicular microenvironment, namely, niche. Sertoli cells, which locate around male germ cells, are the most critical component of the niche. Significant progress has recently been made by peers and us on uncovering the effects of Sertoli cells on regulating fate determinations of SSCs. Here we addressed the roles and regulation of Sertoli cells in normal and abnormal spermatogenesis. Specifically, we summarized the biological characteristics of Sertoli cells, and we emphasized the roles of Sertoli cells in mediating the self-renewal, differentiation, apoptosis, de-differentiation, and trans-differentiation of SSCs. The association between abnormal function of Sertoli cells and impaired spermatogenesis was discussed. Finally, we highlighted several issues to be addressed for further investigation on the effects and mechanisms of Sertoli cells in spermatogenesis. Since Sertoli cells are the key supportive cells for SSCs and they are very receptive to modification, a better understanding of the roles and regulation of Sertoli cells in SSC biology and spermatogenesis would make it feasible to identify novel targets for gene therapy of male infertility as well as seek more efficient and safer strategies for male contraception.

Generation, Characterization and Potential Therapeutic Applications of Mature and Functional Hepatocytes from Stem Cells

Liver cancer is the sixth most common tumor in the world and the majority of patients with this disease usually die within 1 year. The effective treatment for end‐stage liver disease (also known as liver failure), including liver cancer or cirrhosis, is liver transplantation. However, there is a severe shortage of liver donors worldwide, which is the major handicap for the treatment of patients with liver failure. Scarcity of liver donors underscores the urgent need of using stem cell therapy to the end‐stage liver disease. Notably, hepatocytes have recently been generated from hepatic and extra‐hepatic stem cells. We have obtained mature and functional hepatocytes from rat hepatic stem cells. Here, we review the advancements on hepatic differentiation from various stem cells, including hepatic stem cells, embryonic stem cells, the induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, and probably spermatogonial stem cells. The advantages, disadvantages, and concerns on differentiation of these stem cells into hepatic cells are highlighted. We further address the methodologies, phenotypes, and functional characterization on the differentiation of numerous stem cells into hepatic cells. Differentiation of stem cells into mature and functional hepatocytes, especially from an extra‐hepatic stem cell source, would circumvent the scarcity of liver donors and human hepatocytes, and most importantly it would offer an ideal and promising source of hepatocytes for cell therapy and tissue engineering in treating liver disease. J. Cell. Physiol. 228: 298–305, 2013.

Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study.

Recent studies have reported that induced pluripotent stem (iPS) cells from mice and humans can differentiate into primordial germ cells. However, whether iPS cells are capable of producing male germ cells is not known. The objective of this study was to investigate the differentiation potential of mouse iPS cells into spermatogonial stem cells and late-stage male germ cells. We used an approach that combines in vitro differentiation and in vivo transplantation. Embryoid bodies (EBs) were obtained from iPS cells using leukaemia inhibitor factor (LIF)-free medium. Quantitative PCR revealed a decrease in Oct4 expression and an increase in Stra8 and Vasa mRNA in the EBs derived from iPS cells. iPS cell-derived EBs were induced by retinoic acid to differentiate into spermatogonial stem cells (SSCs), as evidenced by their expression of VASA, as well as CDH1 and GFRα1, which are markers of SSCs. Furthermore, these germ cells derived from iPS cells were transplanted into recipient testes of mice that had been pre-treated with busulfan. Notably, iPS cell-derived SSCs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids, as shown by VASA and SCP3 expression. This study demonstrates that iPS cells have the potential to differentiate into late-stage male germ cells. The derivation of male germ cells from iPS cells has potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development.

Generation of Haploid Spermatids with Fertilization and Development Capacity from Human Spermatogonial Stem Cells of Cryptorchid Patients

Summary Generation of functional spermatids from azoospermia patients is of unusual significance in the treatment of male infertility. Here, we report an efficient approach to obtain human functional spermatids from cryptorchid patients. Spermatogonia remained whereas meiotic germ cells were rare in cryptorchid patients. Expression of numerous markers for meiotic and postmeiotic male germ cells was enhanced in human spermatogonial stem cells (SSCs) of cryptorchidism patients by retinoic acid (RA) and stem cell factor (SCF) treatment. Meiotic spreads and DNA content assays revealed that RA and SCF induced a remarkable increase of SCP3-, MLH1-, and CREST-positive cells and haploid cells. Single-cell RNA sequencing analysis reflected distinct global gene profiles in embryos derived from round spermatids and nuclei of somatic cells. Significantly, haploid spermatids generated from human SSCs of cryptorchid patients possessed fertilization and development capacity. This study thus provides an invaluable source of autologous male gametes for treating male infertility in azoospermia patients.

Fractionation of human spermatogenic cells using STA-PUT gravity sedimentation and their miRNA profiling

Human spermatogenic cells have not yet been isolated, and notably, their global miRNA profiles remain unknown. Here we have effectively isolated human spermatogonia, pachytene spermatocytes and round spermatids using STA-PUT velocity sedimentation. RT-PCR, immunocytochemistry and meiosis spread assays revealed that the purities of isolated human spermatogonia, pachytene spermatocytes, and round spermatids were 90%, and the viability of these isolated cells was over 98%. MiRNA microarrays showed distinct global miRNA profiles among human spermatogonia, pachytene spermatocytes, and round spermatids. Thirty-two miRNAs were significantly up-regulated whereas 78 miRNAs were down-regulated between human spermatogonia and pachytene spermatocytes, suggesting that these miRNAs are involved in the meiosis and mitosis, respectively. In total, 144 miRNAs were significantly up-regulated while 29 miRNAs were down-regulated between pachytene spermatocytes and round spermatids, reflecting potential roles of these miRNAs in mediating spermiogenesis. A number of novel binding targets of miRNAs were further identified using various softwares and verified by real-time PCR. Our ability of isolating human spermatogonia, pachytene spermatocytes and round spermatids and unveiling their distinct global miRNA signatures and novel targets could provide novel small RNA regulatory mechanisms mediating three phases of human spermatogenesis and offer new targets for the treatment of male infertility.

Melamine causes apoptosis of rat kidney epithelial cell line (NRK-52e cells) via excessive intracellular ROS (reactive oxygen species) and the activation of p38 MAPK pathway.

There was an outbreak of urinary stones associated with consumption of melamine‐tainted milk products in 2008 in China, leading to serious illness of many infants and even death. We have recently demonstrated that melamine causes oxidative damage on the NRK (normal rat kidney)‐52e cells. The objective of this study was to explore the cellular signalling pathway that mediates the cell apoptosis induced by melamine in the NRK‐52e cells. Fluorescence microscope showed that melamine enhanced intracellular ROS (reactive oxygen species) levels of the NRK‐52e cells. AO/EB (acridine orange/ethidium bromide) staining and flow cytometry revealed that melamine increased apoptotic and necrotic percentages of the NRK‐52e cells in a dose‐dependent manner. Notably, MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide] assays and flow cytometry displayed that SB203580, an inhibitor for p38 MAPK (mitogen‐activated protein kinase) pathway, increased the proliferation of the NRK‐52e cells and reduced the apoptotic and necrotic percentages of the NRK‐52e cells. Western blots further demonstrated that p38 phosphorylation was activated by melamine in the NRK‐52e cells and inhibitor SB203580 blocked the increase of p38 phosphorylation induced by melamine. Together, these results suggested that melamine causes apoptosis of the NRK‐52e cells via excessive intracellular ROS and the activation of p38 MAPK pathway. This study thus offers a novel insight into molecular mechanisms by which melamine has adverse cytotoxicity on renal tubular epithelial cells.

Direct transdifferentiation of spermatogonial stem cells to morphological, phenotypic and functional hepatocyte-like cells via the ERK1/2 and Smad2/3 signaling pathways and the inactivation of cyclin A, cyclin B and cyclin E

BackgroundSevere shortage of liver donors and hepatocytes highlights urgent requirement of extra-liver and stem cell source of hepatocytes for treating liver-related diseases. Here we hypothesized that spermatogonial stem cells (SSCs) can directly transdifferentiate to hepatic stem-like cells capable of differentiating into mature hepatocyte-like cells in vitro without an intervening pluripotent state.ResultsSSCs first changed into hepatic stem-like cells since they resembled hepatic oval cells in morphology and expressed Ck8, Ck18, Ck7, Ck19, OV6, and albumin. Importantly, they co-expressed CK8 and CK19 but not ES cell markers. Hepatic stem-like cells derived from SSCs could differentiate into small hepatocytes based upon their morphological features and expression of numerous hepatic cell markers but lacking of bile epithelial cell hallmarks. Small hepatocytes were further coaxed to differentiate into mature hepatocyte-like cells, as identified by their morphological traits and strong expression of Ck8, Ck18, Cyp7a1, Hnf3b, Alb, Ta t, Ttr, albumin, and CYP1A2 but not Ck7 or CK19. Notably, these differentiated cells acquired functional attributes of hepatocyte-like cells because they secreted albumin, synthesized urea, and uptake and released indocyanine green. Moreover, phosphorylation of ERK1/2 and Smad2/3 rather than Akt was activated in hepatic stem cells and mature hepatocytes. Additionally, cyclin A, cyclin B and cyclin E transcripts and proteins but not cyclin D1 or CDK1 and CDK2 transcripts or proteins were reduced in mature hepatocyte-like cells or hepatic stem-like cells derived from SSCs compared to SSCs.ConclusionsSSCs can transdifferentiate to hepatic stem-like cells capable of differentiating into cells with morphological, phenotypic and functional characteristics of mature hepatocytes via the activation of ERK1/2 and Smad2/3 signaling pathways and the inactivation of cyclin A, cyclin B and cyclin E. This study thus provides an invaluable source of mature hepatocytes for treating liver-related diseases and drug toxicity screening and offers novel insights into mechanisms of liver development and cell reprogramming.

Characterization, Isolation, and Culture of Mouse and Human Spermatogonial Stem Cells

Spermatogenesis is a special process by which spermatogonial stem cells (SSCs) divide and differentiate to male gametes called mature spermatozoa. SSCs are the unique cells because they are adult stem cells that transmit genetic information to subsequent generations. Accumulating evidence has demonstrated that SSCs can be reprogrammed to acquire pluripotency to become embryonic stem‐like cells that differentiate into all cell lineages of the three germ layers, highlighting potential important applications of SSCs for regenerative medicine. Recent studies from peers and us have made great achievements on the characterization, isolation, and culture of mouse and human SSCs, which could lead to better understanding the biology of SSCs and the applications of SSCs in both reproductive and regenerative medicine. In this review, we first compared the cell identity and biochemical phenotypes between mouse SSCs and human SSCs. Notably, the cell types of mouse and human SSCs are distinct, and human SSCs share some but not all phenotypes with mouse SSCs. The approaches for isolating SSCs as well as short‐ and long‐term culture of mouse SSCs and short‐period culture of human SSCs were also discussed. We further addressed the new advances on the self‐renewal of SSCs with an aim to establish the long‐term culture of human SSCs which has not yet been achieved. J. Cell. Physiol. 229: 407–413, 2014.

Cytoprotective effect of trolox against oxidative damage and apoptosis in the NRK-52e cells induced by melamine.

An outbreak of urinary stones associated with consumption of melamine‐tainted milk products occurred in 2008 in China, leading to serious illness of many infants and even death. However, the toxicity of melamine in kidney epithelial cells remains unclear. We have explored the effects of melamine and trolox on renal NRK‐52e (normal rat kidney 52e) cells. The IC50 of melamine was measured by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide] assay. Total SOD (superoxide dismutase) was determined by NBT (Nitro Blue Tetrazolium) staining method. GSH‐Px (glutathione peroxidase) activity was detected by UV colorimetric assay, and MDA (malondialdehyde) content was determined by thiobarbituric acid assay. Apoptosis induced by melamine was determined by flow cytometry. The IC50 increased when NRK‐52e cells were treated with both melamine and trolox compared with melamine only. SOD and GSH‐Px activities were decreased, but MDA content was increased by melamine in a dose‐dependent manner. Trolox significantly enhanced SOD and GSH‐Px activity in melamine‐treated NRK‐52e cells, but it decreased their MDA content. LDH (lactate dehydrogenase) activity and the level of ROS (reactive oxygen species) of the NRK‐52e cells were enhanced by melamine compared with the control. Furthermore, the apoptosis rate increased in NRK‐52e cells treated with melamine, whereas trolox was protective. These results show that melamine has an obvious adverse effect on proliferation of NRK‐52e cells, causing oxidative damage and apoptosis, thus providing a novel insight into renal cytotoxicology of melamine. Trolox ameliorates the effect on melamine toxicity.