Fenglei Chen

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.

Construction and expression of lentiviral vectors encoding recombinant mouse CREBZF in NIH 3T3 cells.

CREBZF, also known as Zhangfei or SMILE, is a member of the CREB/ATF protein family. CREBZF has mainly been considered as a basic region-leucine zipper transcription factor that functions in coordination with other transcription factors and plays a role in latent HSV-1 infection, apoptosis and the mammalian endoplasmic reticulum stress and unfolded protein response. In this study, we constructed recombinant lentiviral vectors for CREBZF short hairpin RNA (shRNA) expression and over-expression to improve understanding of the mechanisms regulating CREBZF. The CREBZF ORF sequence was cloned into the lentiviral shuttle plasmid pCD513B-1, and various shRNA oligonucleotides and one negative control (shN) were cloned into the pCD513B-U6 expression vector. The recombinant lentivirus was packaged and transduced into NIH 3T3 cells. CREBZF mRNA and protein expression were examined using real-time reverse transcription-polymerase chain reaction (RT-qPCR) and western blotting, respectively. The over-expression vector and the most effective shRNA vector significantly affected the expression of CREBZF mRNA and protein. Both of the CREBZF recombinant lentiviral vectors were successfully constructed. The over-expression vector significantly increased the expression of exogenous CREBZF and inhibited the growth of NIH 3T3 cells compared to controls. The most effective shRNA lentiviral vector, pCD513B-U6-CREBZF-shRNA-3, was transformed, leading to significant knockdown of the CREBZF gene. We conclude that CREBZF the recombinant lentiviral vectors are promising tools for regulating the expression of CREBZF in NIH 3T3 cells.

GRP78 expression and regulation in the mouse uterus during embryo implantation

The aim of this study was to investigate the spatiotemporal expression and regulation of GRP78 in the mouse uterus during the peri-implantation period. The GRP78 protein was mainly detected in the luminal and glandular epithelia on days 1–4 of pregnancy. On day 5 of pregnancy, the GRP78 protein was more highly observed around the implanted embryo at the implantation site. There was no detectable GRP78 protein signal on day 5 of pseudopregnancy. GRP78 mRNA and protein levels gradually increased on days 6–8 of pregnancy, and the expression pattern was also expanded, coinciding with the development of decidua. Similarly, GRP78 expression was also strongly expressed in decidualised cells following artificial decidualisation. Compared with the results obtained with the delayed uterus, a high level of GRP78 expression was detected in the implantation-activated uterus. In the uteri of ovariectomised mice, GRP78 expression increased and reached its highest level after injection of oestrogen, and progesterone seemed to have an antagonistic effect on oestrogen up-regulation of GRP78 expression. Our data indicate that GRP78 might play an important role during the process of mouse embryo implantation, and GRP78 expression was mainly regulated by active blastocysts and maternal oestrogen.

Herp depletion inhibits zearalenone-induced cell death in RAW 264.7 macrophages

Herp is an endoplasmic reticulum (ER) membrane protein and strongly induced by the ER stress that not only participates in the unfolded protein response (UPR) under the ER stress, but also in cell autophagy under glucose starvation (GS). However, we do not know whether Herp plays any roles in other responses, such as zearalenone (ZEA). In this study, we constructed recombinant lentiviral vectors for Herp shRNA expression and generated stable Herp knockdown RAW 264.7 macrophages. Flow cytometry analysis showed Herp depletion could inhibit cell death induced by ZEA. Western blot analysis revealed that Herp depletion could up-regulate autophagy-related protein LC3-I conversion into LC3-II and the expression of ER stress-related protein CHOP. These results suggest that Herp depletion inhibits cell death by up-regulating autophagy.

GRP78 expression and immunohistochemical localization in the female reproductive tract of mice

The 78-kDa glucose-regulated protein (GRP78) is an endoplasmic reticulum chaperone, with multiple functional roles in protein processing and provision of cellular protection. However, the physiological role of GRP78 in embryo development is not clear. Localization of GRP78 and expression of its mRNA in the reproductive organs throughout the estrous cycle in mice were investigated by immunohistochemistry and real-time polymerase chain reaction, respectively. Whereas there was intense staining for GRP78 in the oviduct at estrus, the ciliated cells of isthmus had better staining than those of infundibulum and ampulla at all phases of the cycle. Furthermore, GRP78 was located in the uterine luminal and glandular epithelial cells throughout the estrous cycle, particularly during the estrus phase. However, levels of GRP78 mRNA in the oviduct and uterus varied during the cycle, with peaks at estrus. In conclusion, GRP78 expression varied with the phase of the murine estrous cycle; this might be related to gamete transport, fertilization and early development of the zygote/embryo.