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. 2018 Sep 3;16(1):85.
doi: 10.1186/s12958-018-0404-4.

Alleviation of endoplasmic reticulum stress protects against cisplatin-induced ovarian damage

Yuping Wu  1 , Congshun Ma  2 , Huihui Zhao  1 , Yuxia Zhou  1 , Zhenguo Chen  3 , Liping Wang  4
Affiliations

Alleviation of endoplasmic reticulum stress protects against cisplatin-induced ovarian damage

Yuping Wu et al. Reprod Biol Endocrinol. .

Abstract

Background: Cisplatin (CDDP), a widely used chemotherapeutic agent, can induce excessive granulosa cell apoptosis, follicle loss and even premature ovarian insufficiency (POI). However, the mechanism remains elusive, although some studies have indicated the involvement of endoplasmic reticulum stress (ERS). The aim of our study was to investigate the possible mechanism ERS in CDDP-induced granulosa cell apoptosis and follicle loss.

Methods: A POI mouse model was generated by CDDP. The ovaries samples were collected and processed for isobaric tags for relative and absolute quantification analysis (iTRAQ) to screen out our interested proteins of HSPA5 and HSP90AB1, and the decline in their expression were verified by a real-time quantitative PCR and a western blotting assay. In vitro, human granulosa cells, KGN and COV434 cells were transfected with siRNA targeting HSPA5 and HSP90AB1 and then treated with CDDP, or treated with CDDP with/without CDDP+ 4-phenylbutyric acid (4-PBA) and 3-methyladenine (3-MA). The levels of ERS, autophagy and apoptosis were evaluated by western blotting, DALGreen staining and flow cytometry. In vivo, ovaries from mice that received intraperitoneal injections of saline, CDDP, CDDP+ 4-PBA and CDDP+ 3-MA were assayed by immunofluorescence, hematoxylin and eosin (H&E) staining for follicle counting, and terminal-deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining for cell apoptosis assay. The plasma hormone levels were measured by an enzyme-linked immunosorbent assay (ELISA) kit.

Results: We have clarified the relationships between ERS, autophagy, and apoptosis in CDDP-induced granulosa cell apoptosis, both in vitro and in vivo. Alleviating ERS by inhibiting HSPA5 and HSP90AB1 attenuated CDDP-induced autophagy and apoptosis. 4-PBA treatment significantly attenuated CDDP-induced cell autophagy and apoptosis in cultured KGN and COV434 cells. However, inhibiting cell autophagy with 3-MA negligibly restored the CDDP-induced changes in ERS and apoptosis. In vivo experiments also demonstrated that treatment with 4-PBA, but not 3-MA, prevented CDDP-induced ovarian damage and hormone dysregulation.

Conclusions: CDDP-induced ERS could promote autophagy and apoptosis in granulosa cells, causing excessive follicle loss and endocrine disorders. Alleviation of ERS with 4-PBA, but not of autophagy with 3-MA, protect against CDDP-induced granulosa cell apoptosis and ovarian damage. Thus, 4-PBA can be used to protect the ovary during chemotherapy in women.

Keywords: 4-PBA; Cisplatin; Endoplasmic reticulum stress; Granulosa cell apoptosis; Ovarian damage.

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Conflict of interest statement

Ethics approval

All animal experiments were approved by the Southern Medical University Committee on the Use and Care of Animals and were performed in accordance with the Committee’s guidelines and regulations.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
CDDP decreases the expressions of HSPA5 and HSP90AB1 in ovaries. a qPCR showed that both mRNA levels of Hspa5 and Hsp90ab1 were decreased in CDDP-treated ovaries. b western blotting showed that both protein levels of HSPA5 and HSP90AB1 were decreased in CDDP-treated ovaries. c Quantification of the results in b. Protein levels were normalized to that of Tubulin. Data are presented as mean ± SD. n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001
Fig. 2
Fig. 2
Inhibition of ERS depresses CDDP-induced ERS, autophagy and apoptosis in granulosa cells. a Immunofluorescence of HSP90AB1 and HSPA5 in the saline and CDDP-treated ovaries. Nuclei were stained with DAPI. White arrow indicates primordial follicle; red, primary follicle; yellow, secondary follicle; green, antral follicle. Red scale bars = 80 μm, White scale bars = 10 μm (b) Immunoblotting of HSP90AB1 and HSPA5 in the KGN cells treated with CDDP at indicated concentrations (0, 2, 5, 10, 25 and 50 μM) for 24 h. c Immunoblotting of HSP90AB1 and HSPA5 in the KGN cells treated with CDDP at 50 μM for indicated times (0, 4, 8, 16, 24 h). d KGN and COV434 cells were transfected with HSPA5 and HSP90AB1-specific siRNA and NC-siRNA respectively for 48 h, and then treated with 50 μM CDDP for indicated times. Immunoblotting was carried out to detect the protein levels of ERS-, autophagy- and apoptosis-related genes. e KGN and COV434 cells were treated with 50 μM CDDP with/without 5 mM 4-PBA for indicated times. ERS, autophagy and apoptosis levels were detected by western blot. f KGN cells were treated with 50 μM CDDP with/without 5 mM 4-PBA for 24 h and then stained with Annexin V-FITC and PI. Both cells at early (annexin Vpos; PIneg) and late apoptotic stages (annexin Vpos; PIpos) were counted. g Quantification of the results in f. * P < 0.05, ** P < 0.01
Fig. 3
Fig. 3
Suppressing autophagy hardly alleviates CDDP-induced cell apoptosis or ERS. a Autophagy levels in KGN cells after indicated treatments by DALGreen staining. Positive autophagosomes were observed by fluorescence microscopy. White arrow indicates autophagosome-negative cell and the red indicates autophagsome-positive cell. Scale bars = 80 μm. b Quantification of the positive phagosomes in a. c KGN and COV434 cells were treated with 50 μM CDDP with/without 5 mM 3-MA for indicated times. ERS, autophagy and apoptosis levels were detected by western blotting. d KGN cells were treated with 50 μM CDDP with/without 5 mM 3-MA for 24 h and then stained with Annexin V-FITC and PI. e Quantification of the results in d. * P < 0.05
Fig. 4
Fig. 4
Effects of 4-PBA and 3-MA on ovarian histology in CDDP-treated mice. Mice were treated with saline, CDDP, and CDDP with 4-PBA or 3-MA for 1 day, 3 days and 7 days, respectively. a H&E staining showed the ovarian histology in each group. Five sections (taken 100 μm apart) from an ovary were photographed for follicle assessment. Scale bars = 40 μm (upper panels) and 4 μm (lower panels). b Comparison of the total number of healthy and atresia follicles among groups. Healthy follicles include healthy primordial, primary, secondary and antral follicles. c Comparison of the number of healthy primordial, primary, secondary and antral follicles among groups. n = 5, * P < 0.05, ** P < 0.01
Fig. 5
Fig. 5
Effects of 4-PBA and 3-MA on ovarian function in CDDP-treated mice. a Granulosa cell apoptosis in ovarian sections from each group was measured by fluorescent TUNEL staining. Green fluorescences indicate TUNEL-positive apoptotic cells (red arrow). The level of apoptosis is present as the total number of apoptotic granulosa cell on five sections (taken 100 μm apart) from an ovary. Scale bars =40 μm. b Quantification of TUNEL-positive apoptotic cells in each group. c Plasma E2 and FSH levels by ELISA in mice with indicated treatment for 7 days. d Immunoblotting of the protein levels of ERS-, autophagy- and apoptosis-related proteins in each group. Protein extractions were from mice with indicated treatment for 7 days. n = 5, * P < 0.05, ** P < 0.01
Fig. 6
Fig. 6
A schematic mechanism of ERS in CDDP-induced ovarian damage. Exogenous CDDP increases the accumulation misfolded proteins in ER in granulosa cells, which subsequently enhance HSPA5 and HSP90AB1 expressions, leading to activation of ERS. ERS promotes cell autophagy and apoptosis. Excessive granulosa cell apoptosis induces follicular atresia contributing to ovarian dysfunction. 4-PBA can alleviate ERS, suppress cell autophagy and apoptosis, preserve follicles, and thus prevent against CDDP-induced ovarian damage
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References

    1. Goswami D, Conway GS. Premature ovarian failure. Hum Reprod Update. 2005;11:391–410. doi: 10.1093/humupd/dmi012. - DOI - PubMed
    1. European Society for Human R, embryology guideline group on POI. Webber L, Davies M, Anderson R, Bartlett J, Braat D, cartwright B, Cifkova R, de Muinck Keizer-Schrama S, et al. ESHRE guideline: management of women with premature ovarian insufficiency. Hum Reprod. 2016;31:926–937. doi: 10.1093/humrep/dew027. - DOI - PubMed
    1. Tatone C, Amicarelli F. The aging ovary--the poor granulosa cells. Fertil Steril. 2013;99:12–17. doi: 10.1016/j.fertnstert.2012.11.029. - DOI - PubMed
    1. Uyar A, Torrealday S, Seli E. Cumulus and granulosa cell markers of oocyte and embryo quality. Fertil Steril. 2013;99:979–997. doi: 10.1016/j.fertnstert.2013.01.129. - DOI - PMC - PubMed
    1. Adhikari D, Liu K. Molecular mechanisms underlying the activation of mammalian primordial follicles. Endocr Rev. 2009;30:438–464. doi: 10.1210/er.2008-0048. - DOI - PubMed

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