Pengfei Lin

Mycotoxin zearalenone induces apoptosis in mouse Leydig cells via an endoplasmic reticulum stress-dependent signalling pathway

Zearalenone (ZEN) is a Fusarium mycotoxin that causes several reproductive disorders and genotoxic effects. This study demonstrated the involvement of endoplasmic reticulum (ER) stress in ZEN-induced mouse Leydig cell death. Our study showed that ZEN reduced cell proliferation in a murine Leydig tumour cell line in a dose-dependent manner. The involvement of apoptosis as a major cause of ZEN-induced cell death was further confirmed by the results of a caspase-3 activity assay, which showed a ZEN dose-dependent increase in cell death. Treatment of MLTC-1 and primary mouse Leydig cells with ZEN upregulated the expression of the ER stress-typical markers GRP78, CHOP and caspase-12 protein. Further, pre-treating the cells with 4-phenylbutyrate or knocking down GRP78 using lentivirus-encoded shRNA significantly diminished ZEN-induced apoptosis and inhibited the expression of CHOP and caspase-12. In summary, these results suggest that the activation of an ER stress pathway plays a key role in ZEN-induced apoptosis in the mouse Leydig cells.

Endoplasmic reticulum stress is involved in granulosa cell apoptosis during follicular atresia in goat ovaries

Follicular atresia is primarily induced by granulosa cell apoptosis, but description of the apoptotic pathway in granulosa cells is incomplete. In this study, we explored the possibility that endoplasmic reticulum (ER) stress could be involved in granulosa cell apoptosis during goat follicular atresia. Immunohistochemical analysis revealed that DNA damage‐inducible transcript 3 (DDIT3) and glucose‐regulated protein 78 (Grp78) were observed in scattered apoptotic granulosa cells of atretic follicles. Grp78 and DDIT3 mRNA and protein were upregulated in granulosa cells during follicular atresia, although DDIT3 was not significantly different between early atretic and progressed atretic follicles. Spontaneous apoptosis was also observed in vitro in granulosa cells induced by serum deprivation or by the ER stress agent tunicamycin, both inducing similar increases in DDIT3 mRNA. Activating transcription factor‐6 (ATF6) and ATF4 mRNAs were significantly increased during granulosa cell apoptosis in vivo; in contrast to ATF6, ATF4 mRNA was attenuated after 16 hr of culture despite the persistence of ER stress. Taken together, ER stress‐dependent DDIT3 pathways may play an important role in the regulation of selective granulosa cell apoptosis in goat ovaries during early follicular atresia. Serum deprivation could also increase apoptosis of cultured granulosa cells through the ER stress pathway as both ATF6 and PERK/eIF2α/ATF4 signaling have been implicated in the granulosa cell apoptosis of atretic follicles. Mol. Reprod. Dev. 79:423–432, 2012.

Reference gene selection for real-time quantitative PCR analysis of the mouse uterus in the peri-implantation period.

The study of uterine gene expression patterns is valuable for understanding the biological and molecular mechanisms that occur during embryo implantation. Real-time quantitative RT-PCR (qRT-PCR) is an extremely sensitive technique that allows for the precise quantification of mRNA abundance; however, selecting stable reference genes suitable for the normalization of qRT-PCR data is required to avoid the misinterpretation of experimental results and erroneous analyses. This study employs several mouse models, including an early pregnancy, a pseudopregnancy, a delayed implantation and activation, an artificial decidualization and a hormonal treatment model; ten candidate reference genes (PPIA, RPLP0, HPRT1, GAPDH, ACTB, TBP, B2M, 18S, UBC and TUBA) that are found in uterine tissues were assessed for their suitability as internal controls for relative qRT-PCR quantification. GeNormPLUS, NormFinder, and BestKeeper were used to evaluate these candidate reference genes, and all of these methods identified RPLP0 and GAPDH as the most stable candidates and B2M and 18S as the least stable candidates. However, when the different models were analyzed separately, the reference genes exhibited some variation in their expression levels.

Luman recruiting factor regulates endoplasmic reticulum stress in mouse ovarian granulosa cell apoptosis

Follicular atresia is primarily induced by granulosa cell apoptosis; however, the molecular mechanisms that control apoptotic cell death in granulosa cells remain poorly understood. The present studies were undertaken to investigate the role of a novel endoplasmic reticulum stress-regulated gene Luman recruiting factor (LRF) in granulosa cell apoptosis during mouse follicular atresia. Based on immunohistochemistry and confocal laser scanning microscope analysis, LRF protein was localized in the cytoplasm of apoptotic granulosa cells, similar to localization of the LRF, Luman, CCAAT/enhancer-binding protein homologous protein and caspase-12 proteins were localized in apoptotic granulosa cells. However, glucose-regulated protein 78 protein was only present in healthy cells of the mural granulosa cell layers. A spontaneous onset of apoptotic cell death of granulosa cells was induced by thapsigargin or tunicamycin treatment in vitro, which was closely related to the increase of LRF, Luman, CCAAT/enhancer-binding protein homologous protein, and caspase-12 mRNA. Taken together, LRF might be involved in inducing apoptosis of granulosa cells through the endoplasmic reticulum stress pathway and might have a key role in mouse follicular selection.

Endoplasmic Reticulum Stress Cooperates in Zearalenone-Induced Cell Death of RAW 264.7 Macrophages

Zearalenone (ZEA) is a fungal mycotoxin that causes cell apoptosis and necrosis. However, little is known about the molecular mechanisms of ZEA toxicity. The objective of this study was to explore the effects of ZEA on the proliferation and apoptosis of RAW 264.7 macrophages and to uncover the signaling pathway underlying the cytotoxicity of ZEA in RAW 264.7 macrophages. This study demonstrates that the endoplasmic reticulum (ER) stress pathway cooperated in ZEA-induced cell death of the RAW 264.7 macrophages. Our results show that ZEA treatment reduced the viability of RAW 264.7 macrophages in a dose- and time-dependent manner as shown by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT) and flow cytometry assay. Western blots analysis revealed that ZEA increased the expression of glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP), two ER stress-related marker genes. Furthermore, treating the cells with the ER stress inhibitors 4-phenylbutyrate (4-PBA) or knocking down CHOP, using lentivirus encoded short hairpin interfering RNAs (shRNAs), significantly diminished the ZEA-induced increases in GRP78 and CHOP, and cell death. In summary, our results suggest that ZEA induces the apoptosis and necrosis of RAW 264.7 macrophages in a dose- and time-dependent manner via the ER stress pathway in which the activation of CHOP plays a critical role.

Expression Pattern Implicates a Potential Role for Luman Recruitment Factor in the Process of Implantation in Uteri and Development of Preimplantation Embryos in Mice

Abstract Luman/CREB3 recruitment factor (LRF or CREBRF) was identified as a regulator of Luman (or CREB3) that is involved in the unfolded protein response during endoplasmic reticulum stress. Luman is implicated in a multitude of functions ranging from viral infection and immunity to cancer. The biological function of LRF, however, is unknown. In this paper, we report that uteri of pregnant mice and embryos displayed enhanced LRF expression at all stages, and the expressed LRF was found to be localized specifically at implantation sites. On the other hand, uteri of mice induced for delayed implantation or pseudopregnant mice showed low levels of LRF expression, suggesting that LRF mediates uterine receptivity during implantation. Further, expression of LRF was found to be modulated by steroid hormones such as progesterone and estradiol. This study thereby identifies a potential role for LRF in the process of implantation in uteri and development of preimplantation embryos in mice.

Apoptosis inducing factor gene depletion inhibits zearalenone-induced cell death in a goat Leydig cell line

Zearalenone (ZEA) is a contaminant of human food and animal feedstuffs that causes health hazards. However, the signal pathways underlying ZEA toxicity remain elusive. The aims of this study were to determine which pathways are involved in ZEA-induced cell death and investigate the effect of apoptosis inducing factor (AIF) on cell death during ZEA treatment in the immortalized goat Leydig cell line hTERT-GLC. This study showed that ZEA-induced cell death in hTERT-GLCs works via endoplasmic reticulum (ER) stress, the caspase-dependent pathway, the caspase-independent pathway and autophagy. Recombinant lentiviral vectors were constructed to silence AIF expression in hTERT-GLCs. Flow cytometry results showed that knockdown of AIF diminished ZEA-induced cell apoptosis in hTERT-GLCs. Furthermore, we found AIF depletion down-regulated phosphoIRE1α, GRP78, CHOP and promoted the switch of LC3-I to LC3-II. Therefore, ZEA induces cytotoxicity in hTERT-GLCs via different pathways, while AIF-mediated signaling plays a critical role in ZEA-induced cell death in hTERT-GLCs.

Brucella suis Vaccine Strain 2 Induces Endoplasmic Reticulum Stress that Affects Intracellular Replication in Goat Trophoblast Cells In vitro

Brucella has been reported to impair placental trophoblasts, a cellular target where Brucella efficiently replicates in association with the endoplasmic reticulum (ER), and ultimately trigger abortion in pregnant animals. However, the precise effects of Brucella on trophoblast cells remain unclear. Here, we describe the infection and replication of Brucella suis vaccine strain 2 (B.suis.S2) in goat trophoblast cells (GTCs) and the cellular and molecular responses induced in vitro. Our studies demonstrated that B.suis.S2 was able to infect and proliferate to high titers, hamper the proliferation of GTCs and induce apoptosis due to ER stress. Tunicamycin (Tm), a pharmacological chaperone that strongly mounts ER stress-induced apoptosis, inhibited B.suis.S2 replication in GTCs. In addition, 4 phenyl butyric acid (4-PBA), a pharmacological chaperone that alleviates ER stress-induced apoptosis, significantly enhanced B.suis.S2 replication in GTCs. The Unfolded Protein Response (UPR) chaperone molecule GRP78 also promoted B.suis.S2 proliferation in GTCs by inhibiting ER stress-induced apoptosis. We also discovered that the IRE1 pathway, but not the PERK or ATF6 pathway, was activated in the process. However, decreasing the expression of phosphoIRE1α and IRE1α proteins with Irestatin 9389 (IRE1 antagonist) in GTCs did not affect the proliferation of B.suis.S2. Although GTC implantation was not affected upon B.suis.S2 infection, progesterone secretion was suppressed, and prolactin and estrogen secretion increased; these effects were accompanied by changes in the expression of genes encoding key steroidogenic enzymes. This study systematically explored the mechanisms of abortion in Brucella infection from the viewpoint of pathogen invasion, ER stress and reproductive endocrinology. Our findings may provide new insight for understanding the mechanisms involved in goat abortions caused by Brucella infection.

Brucella suis vaccine strain S2-infected immortalized caprine endometrial epithelial cell lines induce non-apoptotic ER-stress

Brucella, which is regarded as an intracellular pathogen responsible for a zoonotic disease called brucellosis, survives and proliferates within several types of phagocytic and non-phagocytic cells. Brucella infects not only their preferred hosts but also other domestic and wild animal species, inducing abortion and infertility. Therefore, the interaction between uterine cells and Brucella is important for understanding the pathogenesis of this disease. In this study, we describe the Brucella suis vaccine strain S2 (B.suis.S2) infection and replication in the immortalized caprine endometrial epithelial cell line hTERT-EECs and the induced cellular and molecular response modulation in vitro. We found that B.suis S2 was able to infect and replicate to high titers and inhibit the proliferation of EECs and induce non-apoptotic pathways, as determined by B.suis.S2 detection using MTT and acridine orange/ethidium bromide (AO/EB) staining and flow cytometry. We explored the evidence of non-apoptotic pathways using real-time quantitative RT-PCR and by western blot analysis. Finally, we discovered the over-expression of GRP78, ATF4, ATF6, PERK, eIF2α, CHOP, and cytochrome c (Cyt-c) but not IRE1, xbp-1, and caspase-3 in B.suis.S2 (HK)-attacked and B.suis.S2-infected cells, suggesting that the molecular mechanism of ER stress sensor activation by B.suis.S2 is basically concomitant with that by B.suis.S2 (HK) and that ER stress, especially the PERK pathway, plays an important role in the process of B.suis.S2 infecting EEC, which may, in part, explain the role of the uterus in the pathogenesis of B.suis.S2.

THE EXPRESSION AND LOCALIZATION OF LRF IN THE FEMALE REPRODUCTIVE TRACT OF CYCLING MICE THROUGHOUT THE ESTROUS CYCLE

In this article, the expression patterns of LRF in the mouse oviduct, uterus, and ovary were checked during estrous cycle. The expression of LRF mRNA and protein were remarkably changed in the ovary, oviduct, and uterus at four phases. LRF immunostaining was detected in the follicle from primary to antral follicle, luminal and glandular epithelial cells of the uterus, and cilia of the ciliated cells of the oviduct at all phase. Our findings suggested that LRF may be related to the processes of development and maturation of oocyte, gamete transport, and the development of early embryo.