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Pigment epithelium-derived factor exerts antioxidative effects in granulosa cells.

Significance Statement

PEDF protects the ovaries from oxidative stress and chemotherapy

About  10% of cancers in women occur under the age of 45. Fortunately, cancers in more than 90% of girls and young women are curable by current treatments. However, these treatments can cause premature ovarian failure, which stresses the importance of fertility preservation for improving the quality of life of cancer survivors. Unfortunately, current solutions are very limited. Doxorubicin (DXR), a cornerstone of many chemotherapeutic protocols, which is used for treating a wide spectrum of malignancies, induces ovarian toxicity, manifested by reduced ovulation rate, and interferes with mitochondrial function, leading to oxidative-stress (OS). OS is a state characterized by an imbalance between pro-oxidants and defensive antioxidants. It plays a key role in the pathogenesis of female infertility by damaging lipids, proteins and DNA in the various ovarian components, including granulosa cells. Pigment epithelium-derived factor (PEDF) is a multifunctional protein that was recently found by us to be expressed in and secreted from granulosa cells of human and rodents origin. We showed that PEDF has an anti-oxidative role in the ovary. We found that granulosa cells apoptosis is correlated with the reduction in PEDF mRNA level, when exposed to H2O2. The rate of H2O2-induced apoptosis was significantly attenuated in granulosa cells treated with recombinant PEDF (rPEDF). Granulosa cells, of human and rodents origin, express the pro-survival PEDF receptor, PNPLA2. Stimulation of granulosa cells by rPEDF induced phosphorylation of AKT. Interestingly, we showed that PEDF exerts its anti-oxidative activity through the AKT signaling pathway. Thus, we hypothesize that PEDF can protect ovaries under OS conditions as chemotherapy. After establishing PEDF as an important ovarian regulator, we evaluated whether DXR can affect PEDF mRNA. ICR female mice were super-ovulated (to synchronize the mice population), DXR was administered 24h after injection of PMSG. The level of PEDF mRNA in the ovaries was evaluated 24h after hCG administration (Fig.1A). We found that DXR significantly reduced the level of PEDF mRNA (Fig.1B). DXR was found to be detrimental to mice oocytes at the MII stage; thus, our aim was to evaluate whether rPEDF can protect the MII oocytes from their DXR-induced apoptosis. We treated superovulated mice that were pre-exposed to DXR with rPEDF (Fig.1C) and followed the number of ovulated oocyte (Fig.1D). We found that administration of rPEDF after DXR treatment significantly increased the number of ovulated oocytes. We suggest that maintaining the physiological ovarian PEDF level may protect the follicular pool.

Figure Legend: Doxorubicin (DXR) reduces PEDF mRNA levels in mice ovaries. A. Illustration of the experimental setting: seven weeks old ICR female mice (n = 5 / group) were superovulated according to the standard pregnant mares’ serum/ human chorionic gonadotropin (PMSG/hCG) protocol. Twenty four hours post PMSG mice were administered with DXR or saline (control). Ovaries were excised twenty-four hours post hCG. B. PEDF mRNA level in ovaries of control and DXR-treated superovulated mice (according to protocol A) Total RNA was extracted from the ovaries and changes in PEDF mRNA level (relative quantification; RQ) were measured by qPCR using specific primers for PEDF; calibrated with HPRT1. Bars are Mean ± SEM (*P < 0.05) – significantly different from control value. rPEDF improves ovulation under DXR treatment. C. Illustration of the experimental setting: seven weeks old ICR female mice (n = 4 / group) were treated with rPEDF or TRIS (control), a day after DXR or saline (control) administration. On the fourth day of the experiment mice were superovulated (PMSG + hCG). Mice were sacrificed 16-17 hours after hCG administration and oocytes were collected. D. Number of ovulated oocytes in control, DXR-treated and DXR+rPEDF-treated superovulated mice (according to protocol C). Bars are Mean ± SEM, (*P < 0.05) – significantly different from DXR value.

Pigment epithelium-derived factor exerts antioxidative effects in granulosa cells.. Global Medical Discovery

 

 

 

 

 

 

 

 

 

 

Journal Reference

Bar-Joseph H1, Ben-Ami I2, Ron-El R2, Shalgi R1, Chuderland D3. Fertil Steril. 2014 Sep;102(3):891-898.e3.

1Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv, Tel Aviv, Israel.

2IVF and Infertility Unit, Department of Obstetrics and Gynecology, Assaf Harofeh Medical Center, Zerifin Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

3Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv, Tel Aviv, Israel.

Electronic address: [email protected]

 

Abstract

OBJECTIVE:

To determine whether supplementing granulosa cells cultures with pigment epithelium-derived factor (PEDF) can protect them from oxidative stress.

DESIGN:

We used either granulosa cell line or human primary granulosa cell culture from  women undergoing in vitro fertilization (IVF) treatments.

SETTING:

University research facilities.

ANIMAL(S):

Imprinting control region female mice.

INTERVENTION(S):

Recombinant PEDF (rPEDF) was added to cultures of either primary  granulosa cell culture or granulosa cell line in the present or absence of H2O2 triggering.

MAIN OUTCOME MEASURE(S):

We followed cell viability with the use of methylthiazolyl tetrazolium assay and tracked PEDF mechanism of action with the use of Western blot analysis, measuring the level of SOD-1 and GPX-1 mRNA, protein level of BAX, and phosphorylation of AKT.

RESULT(S):

We found that granulosa cell viability and the level of PEDF mRNA were both significantly reduced, in a dose-dependent manner, after exposure to H2O2. The rate of H2O2-induced apoptosis was significantly attenuated in granulosa cells treated with rPEDF. We showed that granulosa cells, of both humans and rodents, express the PEDF receptor, PNPLA2; once stimulated by rPEDF, the cells exhibited phosphorylation of AKT. Finally, we showed that PEDF exerts its antioxidative activity through the AKT signaling pathway.

CONCLUSION(S):

This study demonstrates that PEDF represents a novel intrinsic antioxidant of granulosa cells.

Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

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