Antiapoptotic and proliferative activity of curcumin on ovarian follicles in mice exposed to whole body ionizing radiation

Abstract

The aim of this study was to evaluate the antiapoptotic and proliferative activity of curcumin (Cur) on the ovarian follicles in mice exposed to whole body ionizing radiation (Rd). The mice were exposed to 8.3 gray whole body Rd, and Cur groups were given as a daily dose of 100 mg/kg of Cur for 10 days (10 days before Rd). The ovaries were collected 3 and 12 h after irradiation. To date, no such studies have been performed on antiapoptotic and proliferative activity of Cur on the ovarian follicles in mice exposed to whole body Rd. Analysis of mice ovary after exposure to Rd by terminal-deoxynucleotidyl-transferase-mediated dUTP nick end labeling showed that there were apoptotic cells both in the follicular wall and the antrum, and that the number of follicles showing early atresic features was high 3 h after Rd. On the other hand, analysis of mice ovary 12 h after exposure to Rd showed that the number of follicles containing apoptotic cells with advanced atresic features was significantly higher when compared to the 3-h Rd exposure group. The proliferating cell nuclear antigen -positive granulosa cells were decreased in association with follicular atresia. The groups given treatment were observed to have some benefit from Cur against the damage caused by Rd. The results of this study demonstrate that Cur prevents follicular atresia in Rd-induced apoptosis in ovarian follicles.

Keywords

Ionizing radiation ovarian follicles apoptosis TUNEL PCNA mice

Introduction

In mammals, the great majorities of ovarian follicles are degenerated and eventually disappear from the ovary through follicular atresia. The degenerating follicles showed peculiar morphological characteristics such as the increase of pyknotic nuclei in granulosa cells, hypertrophied theca layer, and ruptured or undulated basement membrane (Braw and Tsafriri, 1980; Devine et al., 2000; Hirshfield and Midgley, 1978). One of the pathologic stimuli that induces and accelerates the follicular atresia is radiation ([Rd] Kim et al., 1999). In both normal tissues and tumors, apoptosis occurs spontaneously and can be induced following irradiation (Hendry and West, 1997). According to Jarrell et al. (1986) dramatic changes occurred in the overall structure of the ovary in response to Rd exposure. Recent studies reveal that Rd induces cell apoptosis in follicles (Hendry and West, 1997), chromosomal damage of oocytes, and also impairs the ovarian functions (Chapman, 1982). Granulosa cell death in the atretic follicle involves apoptosis (Billig et al., 1993; Chun and Hsueh, 1998; Kasuya, 1995; Tilly et al., 1992; Tilly, 1996). In the irradiated mouse ovary, it was shown that the follicles were degenerated by the apoptosis of granulosa cells (Kim et al., 1999; Lee et al., 2000).

Apoptosis, a regulated form of cell death, is a physiological process essential for the normal tissue homeostasis (Kaipia and Hsueh, 1997) in the absence of immune surveillance (Kerr et al., 1994). Ovarian follicular degeneration or atresia is a hormonally controlled apoptotic process, whereby the degenerating follicles are eliminated in a coordinated fashion (Hsueh et al., 1994). It is now accepted that pyknosis of granulosa cells is an apoptotic process (Gougeon, 1996). One of the atretogenic stimuli that could accelerate the follicular atresia was γ-radiation (Kim et al., 1999). In both normal tissues and tumors, apoptosis not only occurs spontaneously but can also be induced by irradiation. Rd induced cell apoptosis (Hendry and West, 1997) and impaired the ovarian functions (Chapman, 1982). It was reported that primordial oocytes of rats and mice were more sensitive to Rd than oocytes in the growing follicles (Ataya et al., 1995).

Proliferating cell nuclear antigen (PCNA) is a 35-kDa nuclear protein that has an essential role in cell cycle regulation (Xiong et al., 1991). During S phase, PCNA pairs with cyclin D (Xiong et al., 1992), another important regulator of cell proliferation. This complex is modulated by various growth factors and other growth stimuli (Xiong et al., 1991). If appropriate stimuli are received during G1, the cells become committed to S phase, and PCNA expression increases through G1/S-phase interface, reaching a plateau during G2 (Liu et al., 1989).

Several chemical compounds and their analogues have been screened for their radioprotective ability; however, their high toxicity at optimum protective doses precluded their clinical use (Maisin, 1998). Patients might tolerate dietary agents better than other drugs because humans consume many of these dietary ingredients daily (Jagetia and Venkatesha, 2005). Turmeric, Curcuma longa (family: Zingiberaceae), is an ancient spice, a native of Southeast Asia, used from antiquity as a dye and a condiment. The use of turmeric became more popular when it was found to act as a therapeutic agent for various illnesses. The use of turmeric as an anti-inflammatory and antimicrobial agent has been recognized for more than a century. The importance of turmeric in medicine took a new twist when it was discovered that the dried rhizome of Curcuma longa is very rich in phenolics, whose structures have been identified as curcuminoids, which are chemically related to its principal ingredient, curcumin (Cur). Cur (diferuloyl methane), the natural yellow pigment in turmeric, is isolated from the rhizomes of the plant Curcuma longa. It constitutes about 3–4% of the composition of turmeric (Ammon and Wahl, 1991; Eigner and Scholz, 1999).

The aim of this study was to evaluate the antiapoptotic and proliferative activity of Cur on ovarian follicles in mice exposed to whole body Rd.

Materials and methods

Animals

Four-week-old female mice were obtained from Trakya University Animal Care and Research Unit. Animals were kept in a standard animal facility in a controlled temperature (21 ± 1°C) and photoperiod (12 L:12 D), with free access to water and commercial chow diet. Humidity ranged from 55% to 60%. All animals received human care according to the criteria outlined in the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the National Academy of Sciences and published by the National Institutes of Health.

Experimental design

A total of 40 female mice were divided into 5 experimental groups: A (control), B (3 h after Rd), C (3 h after Rd + Cur), D (12 h after Rd) and E (12 h after Rd + Cur); each group comprised 8 animals.

Drug preparation

The control group and Rd-treated groups were given serum physiologic (10 days before Rd). The Rd-treated with Cur groups were given Cur (in a dose of 100 mg/kg body weight) once a day orally using intragastric intubation for 10 days (10 days before Rd).

Irradiation and sample collection

Whole body Rd was carried out using a ⁶⁰Co source at the Department of Radiation Oncology, Faculty of Medicine, Trakya University, Edirne, Turkey. Irradiation was delivered by a cobalt-60 (⁶⁰Co) teletherapy unit (Cirus, cis-Bio Int., Gif Sur Yvette, France) at a source-surface distance of 65 cm. A single dose of 8.3 Gy Rd was given at a depth of 1.5 cm with a dose rate of 155.64 cGy/min. Each group of mice was killed by cervical dislocation at 3 and 12 h after Rd. The ovaries were collected and fixed to observe the changes in the architecture of follicles. The protocols for the study were approved by the Institutional Animal Ethical Committee of Trakya University, Edirne, Turkey.

Histopathologic evaluation

At the end of the experiment, the right ovaries were individually immersed in Bouin’s fixative, dehydrated in alcohol and embedded in paraffin. Serial sections of 5 µm were obtained, deparaffinized and stained with hematoxylin-eosin (H&E) and periodic acid-Schiff (PAS). Analysis of normal and atretic primordial, primary and preantral follicles was performed in the largest cross section of each ovary. The total number of normal and atretic follicles per ovary was calculated, and the ratio (%) of atretic follicles was calculated as (atretic follicles/total follicles) ×100 (Zhang et al., 2006).

Classification of follicles

Follicles were histologically classified into normal, early atretic and advanced atretic. Normal follicles had an intact granulosa layer with a compact and well-organized arrangement. Atretic follicles were classified based on the criteria described by Hay et al. (1976). Briefly, early atretic follicles had a minimal number of pyknotic cells, degenerated cells and/or apoptotic bodies distributed along the antral border of the granulosa layer. Advanced atretic follicles contained numerous pyknotic cells, degenerated cells and/or apoptotic bodies in the granulosa layer; while in the late atretic follicles, the granulosa layer had already disintegrated.

Immunohistochemistry for PCNA

For immunohistochemical observations, the left ovaries were fixed in Bouin’s, embedded in paraffin and sectioned at 5 µm thickness. Immunocytochemical reactions were performed according to the ABC technique described by Hsu et al. (1981). The procedure involved the following steps: (1) endogenous peroxidase activity was inhibited by 3% hydrogen peroxide (H₂O₂) in distilled water for 30 min; (2) the sections were washed in distilled water for 10 min; (3) nonspecific binding of antibodies was blocked by incubation with normal goat serum (DAKO X 0907, Carpinteria, CA, USA) with PBS, diluted 1:4; (4) the sections were incubated with specific mouse monoclonal anti-PCNA antibody (Cat. # MS-106-B, Thermo LabVision, USA) diluted 1:50 for 1 h at room temperature; (5) the sections were washed 3 times in PBS for 3 min; (6) the sections were incubated with biotinylated anti-mouse IgG (DAKO LSAB 2 Kit); (7) the sections were washed 3 times in PBS for 3 min; (8) the sections were incubated with ABC complex (DAKO LSAB 2 Kit); (9) the sections were washed 3 times in PBS for 3 min; (10) peroxidase was detected with an aminoethylcarbazole substrate kit (AEC kit; Zymed Laboratories, San Francisco, CA, USA); (11) the sections were washed in tap water for 10 min and then dehydrated; (12) the nuclei were stained with hematoxylin; and (13) the sections were mounted in DAKO paramount. All dilutions and thorough washes between steps were performed using PBS unless otherwise specified. All steps were carried out at room temperature unless otherwise specified. As a negative control, primary antibody was replaced with PBS.

Immunohistochemical staining was scored in a semiquantitative manner in order to determine the differences between the control group and the experimental groups. The numbers of the positive staining was recorded as absence (–), a few (±), few (+), medium (++), high (+++) and very high (++++). This analysis was performed in serial sections from each animal at ×400 magnification.

Ovarian apoptotic activity

Apoptosis was evaluated by the transferase-mediated dUTP nick end labeling (TUNEL) assay. The TUNEL method, which detects fragmentation of DNA in the nucleus during apoptotic cell death in situ, was employed using an apoptosis detection kit (TdT-Fragel^TM DNA Fragmentation Detection Kit, Cat. No. QIA33, Calbiochem, USA). All reagents listed below are from the kit and were prepared following the manufacturer’s instructions. Five-micrometer-thick tissue sections were deparaffinized in xylene and rehydrated through a graded ethanol series as described previously. They were then incubated with 20 mg/ml proteinase K for 20 min and rinsed in Tris-buffered saline (TBS). Endogenous peroxidase activity was inhibited by incubation with 3% H₂O₂. Sections were then incubated with equilibration buffer for 10–30 sec and then with terminal deoxynucleotidyl transferase (TdT) enzyme, in a humidified atmosphere at 37°C, for 90 min. They were subsequently treated with prewarmed working strength stop/wash buffer at room temperature for 10 min and incubated with blocking buffer for 30 min. Each step was separated by thorough washes in TBS. Labelling was revealed using diaminobenzidine (DAB), counterstaining was performed using methyl green and sections were dehydrated, cleared and mounted.

The positive staining of TUNEL cell numbers was scored in a semiquantitative manner in order to determine the differences between the control group and the experimental groups. The numbers of the positive staining was recorded as absence (–), a few (±), few (+), medium (++), high (+++) and very high (++++). This analysis was performed in serial section from each animal at ×400 magnification.

Statistical analysis

All statistical analyses were carried out using S0064 Minitab Release 13 (Lisans Number: WCP1311.00197). All data were presented in mean ± standard deviation (SD). Differences in measured parameters among the three groups were analyzed with a nonparametric test (Kruskal-Wallis). Dual comparisons between groups exhibiting significant values were evaluated with a Mann–Whitney U test. These differences were considered significant when the probability was less than 0.05.

Results

Histopathological changes

Histological changes in ovarian follicles control and 3 and 12 h after irradiation are presented in Figures 1 and 2. The control showed normal ovarian follicles (Figures 1 and 2a). In Rd-treated groups, the oocytes have a nonuniform appearance or may break up completely as in the late stages of atresia. The shape of the oocyte is often altered and in cases of severe damage, it shrinks to a fraction of its original size. Granulosa cells in atretic follicles show separation and loss of integrity. In some cases of severely damaged follicles, the pyknotic granulosa cells clump together leaving a large gap between them and the basal lamina. Cellular debris and apoptotic bodies were also increased in degenerating follicles. The connection between the zonae pellucidae and the cumulus oophoros was separated in severely degenerating follicles. Analysis of mice ovary after exposure to Rd by histopathological examination showed that the number of follicles showing early atresic features was high in 3 h after Rd. On the other hand, analysis of mice ovary 12 h after exposure to Rd showed that the number of follicles with advanced atresic features was significantly higher than the 3 h after Rd group. The degenerating follicles showed peculiar morphological characteristics such as the ruptured and undulated basement membrane and degenerated zonae pellucidae in Rd-treated groups. One frequent observation is that the zona spreads out around the oocyte. In some cases, the zona is broken and may be seen peeling off the oocyte surface (Figures 1 and 2b, d). Cur-treated animals showed an improved histological appearance in Rd-treated groups (Figures 1 and 2c, e).

The ratio of atretic follicles was increased in 3 and 12 h after irradiation. The number of atretic follicles in the Rd + Cur-treated groups was also significantly less than that in the Rd-treated groups (Figure 3).

Immunohistochemical findings

PCNA-positive cells were strongly detected in granulosa cells of the control group (Figure 4a and Table 1). However, the signal density of positive cells was significantly lower in granulosa cells of the 3 and 12 h after Rd groups. The PCNA-positive granulosa cells were decreased in association with follicular atresia. Analysis of mice ovary 12 h after exposure to Rd showed that the number of follicles containing PCNA-positive cells with advanced atresic features was significantly lower than the 3 h after Rd group (Figure 4b, d and Table 1). Cur treatment markedly raised the reactivity of PCNA in the granulosa cells (Figure 4c, e and Table 1).

Table 1.

Semiquantitative comparison of the positive staining of PCNA and TUNEL cell numbers in ovary from each group^a

	A	B	C	D	E
PCNA	++++	++±	+++	+±	++
TUNEL	+±	+++	++±	++++	+++±

Cur: curcumin, PCNA: proliferating cell nuclear antigen, Rd: radiation, TUNEL: terminal-deoxynucleotidyl-transferase-mediated dUTP nick end labelling.

^aA: control, B: 3 h after Rd, C: 3 h after Rd + Cur, D: 12 h after Rd and E: 12 h after Rd + Cur. The numbers of the positive staining was recorded as a few (±), few (+), medium (++), high (+++) and very high (++++) (n = 8 for each group).

Evaluation of apoptosis

The number of ovarian follicles with apoptotic granulosa cells was increased in irradiated mice. An increase in apoptotic granulosa cells in follicles was the primary trait of the irradiated ovaries. Analysis of mice ovary after exposure to Rd by TUNEL showed that there were apoptotic cells both in the follicular wall and in the antrum, and that the number of follicles showing early atresic features was high in 3 h after Rd group (Figure 5b and Table 1). On the other hand, analysis of mice ovary 12 h after exposure to Rd showed that the number of follicles containing apoptotic cells with advanced atresic features was significantly higher than the 3 h after Rd group (Figure 5d and Table 1). Cur treatment markedly reduced the reactivity and the number of granulosa cell apoptosis in mice ovary (Figure 5c, e and Table 1).

Discussion

In the present experiment, the antiapoptotic effects of Cur on degenerative morphological characteristics of ovarian follicles caused by whole body Rd were investigated. It was reported that Rd induced cell apoptosis (Hendry and West, 1997), chromosomal damage of oocytes and impairment of the ovarian function (Chapman, 1982).

In our present study, at the largest cross section of the ovarian slices after irradiation, the ratio of atretic follicles was calculated at each stage of oogenesis. The ratio of atretic follicles increased after irradiation. Primordial, primary, preantral and antral follicles in the irradiated group showed a remarkable degeneration. Pyknotic cellular nuclei and apoptotic body-like cell debris were observed in the antrum near the granulosa layer of the early and advanced atretic follicles. Analysis of mice ovary 12 h after exposure to Rd showed that the number of follicles containing apoptotic cells with advanced atresic features was significantly higher than the 3 h after Rd group. The atretic follicles include such morphological characteristics as gradual pyknosis of granulosa cell nuclei, reduction in granulosa cell proliferation and breakdown of the basement membrane (Braw and Tsafriri, 1980; Hirshfield and Midgley, 1978). It was reported that the follicular atresia induced by Rd was mediated by apoptosis of granulosa cell in primordial and primary (Lee et al., 2000), preantral and antral follicles (Kim and Lee, 2000).

Apoptosis is the physiological process of cell deletion and is essential for normal ovarian physiology. Follicular atresia can apparently occur at any point during follicular growth and development (Taylor et al., 1993) and follicles become atretic at any stage of their development by intrinsic or extrinsic detrimental signals. In particular, apoptosis of granulosa cells is known to contribute to the regression of the ovary (Jacquet et al., 1997). It has been reported that Rd induces cell apoptosis (Kim et al., 1999) and impairs the ovarian functions. Jacquet et al. (1997) reported that the chromosomal damage of oocytes was induced by irradiation and that the affected oocytes eventually disappeared. The detrimental effect of Rd on cell physiology gives rise to primary lesions of DNA that trigger the apoptotic response (Allan, 1992).

In ovarian follicles, apoptotic granulosa cells increased in irradiated ovaries compared to control, suggesting that follicular degeneration is accelerated by γ-Rd (Allan, 1992). Cellular debris and apoptotic bodies in degenerating follicles also increased in ovaries of mouse exposed to γ-Rd, and phagocytes were occasionally observed (Lee and Yoon, 2005). Radiotherapy is well known to cause destruction of the oocyte pool, although the precise extent of the damage is difficult to determine (Wallace et al., 2003). Rd could acutely give rise to apoptotic (Oktay et al., 1995) and inflammatory degeneration of follicular cells (Lee and Yoon, 2005). As reported by Zhang et al. (2006), it is thought that morphology in Rd-induced follicular degeneration differs according to the Rd dose.

Our study demonstrated that the frequency of PCNA-positive cells in the granulosa of healthy follicles was significantly greater than early and advanced atretic follicles in whole body Rd in mice. Analysis of mice ovary 12 h after exposure to Rd showed that the number of follicles containing PCNA-positive cells with advanced atresic features was significantly lower than 3 h after Rd group. However, pretreatment with Cur markedly raised the reactivity of PCNA in the granulosa cells in Rd-treated groups. PCNA is a sensitive marker of early events in follicular growth. Increased PCNA expression correlates with the earliest signs of granulosa cell growth. In addition, oocytes begin to express PCNA early in follicular growth, before beginning to enlarge. PCNA immunoreactivity remains prevalent in granulosa and theca cells of follicles in subsequent stages of follicle growth and progressively diminishes with advancing atresia (Oktay et al., 1995). There is ample evidence that the PCNA is a reliable indicator of cell proliferation (Chang et al., 1990; Hall et al., 1990; Liu et al., 1989). Increased PCNA expression in the oocyte nucleus could also reflect increased expression of growth factors in the oocyte since PCNA expression can be stimulated by various growth factors even in quiescent cells (Jaskulski et al., 1988). As reported by Oktay et al. (1995), the expression of PCNA, an essential regulator of the cell cycle, appears to mark the initiation of follicle growth, coinciding with and in some cases preceding the first sign of granulosa cell enlargement and preceding oocyte enlargement. In addition, PCNA staining was accomplished as a measure of proliferation in the whole body-irradiated ovarian follicles. Proliferation of granulosa cells reduced and the incidence of follicular atresia increased in the ovarian follicles of the irradiated mice compared to those of the control and Cur-pretreated group.

In our present study, pretreatment with Cur may lead to a decrease in the irradiation-caused degeneration of the ovarian follicles. An interesting aspect of Cur’s activity is the ability to exert radioprotective effects in normal cells. It has been suggested that Cur’s ability to reduce oxidative stress and inhibit transcription of genes related to oxidative stress and inflammatory responses may afford protection against the harmful effects of Rd (Jagetia, 2007).

It is a well-established fact that Rd induces reactive oxygen species that follows a cascade of events leading to DNA damage that includes single- or double-strand breaks, base damage, and DNA–DNA or DNA–protein cross-links, and these lesions cluster as complex, local, multiple damaged sites. The DNA double-strand breaks are considered the most lethal events after ionizing Rd and have been found to be the main target of cell killing by Rd. The radioprotective activity of Cur might not be due to a single mechanism but due to several mechanisms. The scavenging of Rd-induced free radicals and the elevation in cellular antioxidants by Cur in irradiated systems could be one of the main leading mechanisms. Upregulation of enzymes like catalase, glutathione transferase, glutathione peroxidase, superoxide dismutase and their mRNAs might be another mechanism of radioprotection by Cur. Reduction in lipid peroxidation and elevation in glutathione and increase in sulfydryl groups might also contribute to some extent for its radioprotective activity (Jagetia, 2007).

In conclusion, these findings suggest that Cur pretreatment has the antiapoptotic and proliferative activity on ovarian follicular atresia induced by whole body Rd in mice.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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