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γ-Radiation-induced follicular degeneration in the prepubertal mouse ovary
Mutation Research 578 (2005) 247–255
␥-Radiation-induced follicular degeneration in the prepubertal mouse ovary Chang Joo Lee, Yong-Dal Yoon ∗ Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791, Republic of Korea Received 3 February 2005; received in revised form 22 May 2005; accepted 24 May 2005 Available online 30 August 2005
Abstract Prepubertal mice were whole-body irradiated with a mean lethal dose (LD50 ) of ␥-radiation using a 60 Co source with a total dose of 7.2 Gy and a dose rate of 12.0 cGy/min. At day 0 before the irradiation and at day 1, 2, and 3 after the irradiation, the ovaries were collected and the morphological changes were assessed. The ratios (%) of atretic or polymorphonuclear leukocytes (neutrophil)-infiltrated follicles in the largest cross sections were calculated. In the early atretic follicle of the control mouse ovary, both apoptotic and mitotic cells were observed and occasionally neutrophils were infiltrated into the follicle cavity. However, in the atretic follicles 2 days post-irradiation, numerous cell fragments, apoptotic cells and bodies, and especially, a number of neutrophils were observed. In the non-irradiated control, the ratios of atretic follicles were 58.0 ± 8.6 and 27.3 ± 11.2 (mean ± S.E.M.) in antral and preantral follicles, respectively. The ratios of the number of antral and preantral follicles with one or more neutrophils to the total number of atretic follicles were 29.3 ± 12.0. At 2 days post-irradiation, the ratios of atretic follicles were increased to 94.0 ± 3.4 and 86.9 ± 7.6 in antral and preantral follicles, respectively. The ratios of neutrophil-containing follicles among the atretic one were increased to 65.9 ± 11.5 and 57.8 ± 15.4 at 2 and 3 days after the irradiation, respectively. Taken together, the present results show that ␥-radiation induces apoptotic and inflammatory degeneration of mouse ovarian follicles. Besides, neutrophils may be involved in the acute atretic degeneration in ␥-irradiated mouse ovarian follicles. © 2005 Elsevier B.V. All rights reserved. Keywords: Apoptosis; Inflammation; Neutrophil; Atresia; Radiation; Ovarian follicle; Mouse
1. Introduction In mammals, the great majorities of ovarian follicles are degenerated and eventually disappear from the ∗ Corresponding author. Tel.: +82 2 2220 0955; fax: +82 2 2294 0955. E-mail address: [email protected] (Y.-D. Yoon).
0027-5107/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2005.05.019
ovary through follicle 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 [1–3]. One of the pathologic stimuli that induces and accelerates the follicle atresia is radiation [4]. In both normal tissues and tumors apoptosis occurs spontaneously and can be
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induced following irradiation [5]. According to Jarrell et al. [6], dramatic changes broke out in the overall structure of the ovary in response to radiation. Recent studies reveal that radiation induces cell apoptosis in follicles [5], chromosomal damage of oocytes, and also impairs the ovarian functions [7]. Granulosa cell death in the atretic follicle involves apoptosis [8–12]. In the irradiated mouse ovary, it was shown that the follicles degenerated by the apoptosis of granulosa cells [4,13]. Morphologically, the degenerating granulosa cells in the follicles were phagocytosed by the neighboring intact granulosa cells or the macrophages [14–16]. Evidently, macrophages participated in the elimination of apoptotic and inflammatory cellular debris. However, in human antral follicles, relatively large numbers of polymorphonuclear leukocytes (neutrophils) were contained within the theca vasculature and the density of neutrophil was greater in atretic than in healthy follicles [17]. It implicates that the follicular immune cells such as neutrophils and macrophages are participated in the ovarian physiology, including follicle atresia. The mice were whole-body irradiated for the induction of artificial atresia of the ovarian follicles, and the morphological assessment was carried out to obtain the evidences that the immune cells participated in the follicular degenerating process induced by ␥-irradiation in the mouse ovary. 2. Materials and methods 2.1. Animals Three weeks old female mice (ICR strain) were obtained from Toxicology Research Center, Korea Research Institute of Chemical Technology, Taejon, Korea. The mice were raised in a 22 ◦ C controlled animal care room with the 12-h light:12-h dark cycle. The animals had free access to tap water and commercial chow during the experiments. 2.2. γ-Irradiation The whole-body ␥-irradiation was carried out with a isotopic source (dose rate: 12.0 cGy/min; source strength: approximately 150 TBq, Panoramic Irradiator, Atomic Energy of Canada Ltd.) at Korea Atomic Energy Research Institute as previously reported by Kim et al. [4] and Kim and Lee [18]. The radiation dose
was LD50(30) (7.2 Gy) and the irradiation time was 1 h. The number of mice used in the present experiment was five per group of experiment. 2.3. Histology Before the irradiation (day 0) and 1, 2, and 3 days after the irradiation, the left ovaries were dissected out and immediately fixed in 2.5% glutaraldehyde/0.1 M phosphate buffer (pH 7.3). Post fixation was followed using 1% of osmium tetroxide (Sigma Chem. Co., MO) for 2 h at 4 ◦ C. Embedding of specimens after alcoholic dehydration and displacement by propylene oxide was carried out in epon mixture [Poly/Bed 812 resin (Epon 812):dodecenylsuccinic anhydride:nadic methyl anhydride:2,4,6-tri(dimethylaminomethyl) phenol (DMP30) = 19.3:12.3:9.4:0.6 ml, Polysciences Inc.]. Using ultramicrotome (Leica), semi-thin sections of 1 m thickness were prepared and stained with 1% toluidine blue O in 1% borax solution. The right ovaries were fixed in 10% neutral buffered formalin for 12 h. Following the routine histologic preparation, the samples were embedded in paraffin, and sectioned into 4 m in thickness. Serial sections were stained with hematoxylin and eosin for the evaluation of the follicle status. Periodic acid Schiffs’ (PAS) staining was carried out according to Bancroft and Stevens [19]. The tissues were oxidized in 0.5% periodic acid solution for 15 min. After washing in tap water, the sections were incubated in Schiffs’ solution (basic fuchsin, 1 g; 1 N HCl, 20 ml; anhydrous sodium metabisulfate, 2 g) for 30 min. The counterstain with Harris hematoxylin was followed for 3 min. To identify neutrophil in a follicle cavity further, 7/4 rat monoclonal mouse antibody (Serotec) was used according to manufacturer’s guidelines. The polymorphic myelomonocytic antigen 7/4 is expressed at high levels on polymorphonuclear leukocytes [20], but is absent on resident tissue macrophages [21]. After the incubation with 7/4 antibody, biotin-labeled goat anti-mouse IgG (ImmunoTag) was applied. It was stained using 3,3 -diaminobenzidine tetrahydrochloride and counterstained with 1% of light green.
60 Co
2.4. Light microscopy The largest cross sections were observed in this study. Observation of morphological changes was done
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under a light microscope (Olympus BX5O) with the magnifications of ×400 or ×1000. Preantral and antral follicles in the largest cross sections of the whole ovaries were observed. The identification criteria were largely based on Pedersen and Peters [22] and Kim and Lee [18]. Follicles with one or more mitotic granulosa cells and linear membrana granulosa, and without pyknotic granulosa cells as well as with linear and peculiar zona pellucida were classified into normal [18]. Follicles undefined by above criteria were regarded as atretic. The preantral and antral follicles were identified by the comparison of the serial sections. Neutrophils were identified by the PAS staining and re-identified by neutrophil immunohistochemistry. Under a light microscope, the number of normal and atretic follicles was counted. Among the atretic follicles, the number of follicles containing one or more neutrophils was obtained and calculated the ratio by the equation [(number of follicle containing neutrophil/total atretic antral and preantral follicle) × 100]. To compare the follicle status in the ovary, the ratio (%) of normal follicles was calculated by the equation [(normal follicles/total follicles) × 100]. 2.5. Electron microscopy Ultrathin sections stained with lead citrate and uranyl acetate were examined and photographed under a transmission electron microscope (Jeol, JEM-1200 EX II). 2.6. Statistical analysis Numerical values were expressed as the mean ± S.E.M. Statistical analysis was carried out by ANOVA followed by the Student–Newman–Keuls method for multiple comparisons among means. Significance was considered at the 0.05 level.
3. Results In the present study, using the immature mouse ovary, we evaluated morphological degeneration of preantral and antral follicles induced by ␥-radiation. Normal follicles contained mitotic granulosa and no apoptotic cells in the membrana granulosa. The oocyte and zona pellucida had healthy appearances (Fig. 1A).
Fig. 1. Microphotographs of normal and atretic follicles in the control and ␥-irradiated mouse ovary. (A) In the normal early antral follicle of the control mice ovary, mitotic cell (long arrow) was present, and no apoptotic or pyknotic cells were found. Bar, 20 m. (B) In the atretic early antral follicle of the control mouse ovary, mitotic (long arrow) and apoptotic cells (short thin arrow) and bodies (open arrow) were observed in the follicle cavity. Note the neutrophil infiltration into the cavity (short thick arrow). Bar, 20 m. (C) In a severely atretic follicle of the ␥-irradiated mouse ovary, numerous neutrophils (short thick arrows) were found around the basement membrane. Theca layer was hypertrophied. Macrophage (arrow head) and numerous cell fragments (bold arrow) were found in the follicle cavity. Bar, 50 m. Abbreviations: ZP, zona pellucida; BM, basement membrane; TC, theca layer; O, oocyte.
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In the early atretic follicle of the control mouse ovary obtained at day 0, both apoptotic and mitotic cells were observed. Occasionally, neutrophils infiltrated into the follicle cavity. Mostly, neutrophils were observed in the theca layer. In the follicle cavity, the number of cell fragments was relatively few (Fig. 1B). However, in the severely atretic follicles at 2 days post-irradiation, numerous cell fragments, apoptotic cells and bodies, and, especially, neutrophils were observed in the antral cavity. Macrophages engulfing the apoptotic bodies were also found. Neutrophils infiltrated into the follicle cavity presumably across the basement membrane (Fig. 1C). Such follicles had apoptotic and mitotic cells as well. Some granulosa cells were ruptured and in the follicle cav-
ity, cell fragments were abnormally increased in number. In the control mouse ovary, it was observed that the early atretic antral follicles contained both apoptotic and mitotic granulosa cells in the membrana granulosa. No neutrophils were found around the apoptotic bodies. The antrum of the atretic antral follicles with shrunken oocytes contained relatively few cell fragments. In this kind of follicles, macrophage or macrophage-like phagocytic cells engulfing the apoptotic bodies were observed. Apoptosis of granulosa cell was easily identified by the presence of its condensed or marginated nucleus. In the atretic follicle of the control mouse ovary, it was observed that granulosa cells engulfed apoptotic bodies.
Fig. 2. Electron microphotographs of the atretic follicles in ␥-irradiated mouse ovary. (A) In an early atretic follicle, neutrophil (short thick arrow) was observed between the basement membrane and theca layer. Apoptotic cell (thin arrow) and a few cell fragments were found in granulosa layer. Bar, 5 m. Original magnification: ×1500. (B) Neutrophils were observed in the theca-granulosa region. Left upper neutrophil was located in theca and right two neutrophils were in granulosa layer. The basement membrane was indicated by small arrow heads according to the line. Bar, 2 m. Original magnification: ×4000. (C) In a severely atretic follicle, numerous neutrophils (small thick arrows) and macrophages (short-tailed arrows) were found. One neutrophil was phagocytosed by a macrophage in this panel. Bar, 2 m. Original magnification: ×2000. (D) Numerous neutrophils (short thick arrow) were found in the severely atretic follicles. Apoptotic cells (long thin arrow) were also found in the acute inflammatory region of the follicle. The cell membrane was not observed and the cellular components of granulosa cells were leaked out. Bar, 5 m. Original magnification: ×1500.
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At 2 and 3 days post-irradiation, neutrophils appeared near the basement membrane of the atretic follicles. These follicles had both apoptotic and normal granulosa cells (Fig. 2A). A number of neutrophils were recruited around the basement membrane (Fig. 2B). In the follicle cavity, occasionally, it was observed that the infiltrated neutrophils were engulfed by the macrophage (Fig. 2C). In the atretic follicular cavity, numerous neutrophils were observed. The granulosa cells were ruptured and the cellular components were leaked out as an inflammation (Fig. 2D). By comparisons with hematoxylin and eosin stain (Fig. 3A), neutrophils in the ovarian sections were further iden-
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tified by PAS, and by immunohistochemical staining with 7/4 monoclonal antibody (Fig. 3B and C). Morphologically, macrophage and macrophage-like phagocytic cells were observed in the follicular cavities as shown in Fig. 4. Obviously, two kinds of morphologically different phagocytic cells were present in the degenerating follicles. One was macrophage or macrophage-like giant cells, and the other was the phagocytic granulosa cells. These cells engulfed the apoptotic bodies and cell debris present in the follicle cavity. The frequency of atretic antral (Fig. 5) and preantral follicles induced by ␥-radiation was increased at 2 and
Fig. 3. Microphotographs of the ovarian follicles in mice. The ovarian section was stained with hematoxylin and eosin (A); periodic acid Schiffs’ (B); immunohistochemical localization for neutrophil with 7/4 monoclonal antibody was applied (C). (D) and (E) are high power magnification of (B) and (C), respectively. Open arrows, atretic follicles; thin arrows, atretic bodies; thick arrows, neutrophils. Original magnification: (A–C)—×1500; (D and E)—×400. Bars: (A–C)—100 m; (D and E)—50 m.
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Fig. 5. Ratio of the number of follicles containing neutrophils to that of total preantral and antral atretic follicles in ␥-irradiated mouse ovary. The ratio (%) of the numbers of atretic and neutrophilcontaining follicles in the largest cross section were counted and presented as mean ± S.E.M. Number of mice in a group was 5. *P < 0.05 significantly different when compared to day 0.
The ratios of the number of antral and preantral follicles with one or more neutrophils to the entire number of follicles in the largest cross sections were 29.3 ± 12.0 in the non-irradiated control mouse
Fig. 4. Macrophages and macrophage-like granulosa cells in the mouse ovarian follicles. Macrophages and macrophage-like granulosa cells engulfing cell debris and/or apoptotic bodies were observed in the relatively normal (A) and atretic follicles with hypertrophied theca layer (B) in mouse ovaries. These types of cell clearance were identified in the control as well as ␥-irradiated mouse ovarian follicles. (A) Bar, 50 m. Original magnification, ×200. Insert: bar, 10 m. Original magnification, ×1000; (B) Bar, 20 m. Original magnification, ×400. Insert: bar, 10 m. Original magnification, ×1000. O, oocyte; GC, granulosa cells; ZP, zona pellucida. Thin arrows, apoptotic bodies; thick arrows, macrophage or macrophagelike cells; white arrow, mitotic cell.
3 days after irradiation (P < 0.05). In the control group before the irradiation, the atretic ratios were 58.0 ± 8.6 and 27.3 ± 11.2 (mean ± S.E.M., n = 5) in antral and preantral follicles, respectively. At 2 days after irradiation, the ratios were increased to 94.0 ± 3.4 and 86.9 ± 7.6 in antral and preantral follicles, respectively (P < 0.05).
Fig. 6. Change of atretic ratio of preantral and antral follicles in ␥irradiated mouse ovary. The immature mice were ␥-irradiated at a dose of 7.2 Gy (LD50 ) for 1 h. Before the irradiation and 1, 2, and 3 days after irradiation, histological changes of left ovaries were observed. In the largest cross sections, the atretic follicles were counted under a microscope. Follicles were identified by comparison of the serial sections. Follicles with one or more apoptotic granulosa cells and bodies were classified as atretic. The atretic ratio (%) was obtained from the division of the number of atretic follicle by that of the total follicles and presented as mean ± S.E.M. The number of mice in a group was 5. *P < 0.05 significantly different when compared to day 0. White bars, preantral follicles; dashed bars, antral follicles.
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(Fig. 6). The ratios were increased to 65.9 ± 11.5 and 57.8 ± 15.4 at 2 and 3 days after the irradiation, respectively (P < 0.05).
4. Discussion Ovarian follicular growth and development is an integrated process encompassing both extraovarian signals and intraovarian factors [23]. The predominant event in the ovary is the loss of follicles through atresia during the reproductive period in mammals. However, the precise mechanism of follicle atresia is not proven yet. It has been suggested that macrophage has the potential relevance as a major cellular component of the immune system within the ovary [24,25]. Besides having a role in secretion of cytokines, macrophages in the ovary participated in the elimination of cellular debris originating from the apoptotic processes. During follicle development, gonadotropins, together with local ovarian growth factors and cytokines, as well as estrogens, activate different intracellular pathways to rescue follicles from apoptotic demise. In contrast, some cytokines such as tumor necrosis factor-␣, Fas ligand, presumably acting through receptors with a death domain, and androgens are atretogenic factors [26]. Azuma et al. [27] reported that macrophages modulate steroid production by granulosa–luteal cells. It was reported that macrophages were abundant in human atretic follicles [28] and resident macrophages have been detected in the human ovary [29]. Previously, it was reported that a high dose of radiation induced acute degeneration of primordial and primary [13], and preantral and antral follicles [18]. In the present experiment, the ratios (%) of atretic antral and preantral follicles in the control group were 58 and 27%, respectively. At 2 and 3 days post-irradiation, the ratios were increased to 94 and 87% in antral and preantral follicles, respectively. We observed that macrophages and macrophage-like phagocytes participated in the degenerating processes of ␥-irradiated mouse ovarian follicles. In our results, it was identified that the follicle degeneration after a mean lethal dose of ␥-irradiation was taking place on granulosa cells through both the apoptosis and inflammation. The apoptotic cell fragments and even the neutrophils were eliminated by
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the phagocytic macrophages or macrophage-like granulosa cells. It was reported that, in the initial stage of atretic follicle of the rabbit [11] and guinea pig ovary [30], two kinds of phagocytic cells were identified: one was intact granulosa cells ingesting neighboring dead granulosa cells, and the other was large round cells identified as macrophages. The present results showed that neutrophils in the ␥-radiation-induced atretic follicles were recruited into the follicle cavity. It suggests that neutorphils, with the macrophages and the normal granulosa cells were actively involved in the atretic degeneration of the ovarian follicles. Morphologically, apoptosis involves rapid condensation and budding of the cell, with the formation of membrane-enclosed apoptotic bodies containing wellpreserved organelles, which are phagocytosed and digested by nearby resident cells without inflammatory immune response [31]. In the present study, the phagocytosis of neutrophils by macrophage and the presence of apoptotic cells and bodies meant that the follicles after receiving the detrimental radiation degenerated not only by apoptosis but also by the inflammatory immune response. By a morphometric analysis of human ovarian follicles, Chang et al. [17] reported that the number of neutrophils were two-fold greater in atretic than in healthy follicles. However, they observed that the neutrophils were contained within the theca vasculature. About 29% of the atretic antral and preantral follicles contained neutrophils in the follicle cavity of the control mouse ovary. The ratios were increased at 2 and 3 days post-irradiation to 66 and 58%, respectively. Especially, follicles in the advanced stage of atresia contained more neutrophils than the early atretic ones. As reported by Kim et al. [4], it can be thought that the radiation induced the acute and accelerated follicle degeneration, so the apoptotic fragments were not sufficiently eliminated by phagocytes. Though we did not obtain the biochemical evidences about the triggering mechanism of the neutrophil recruitment into the follicle, it was found that the immune cells infiltrated into the follicle antrum, definitely across the basement membrane. The presence of neutrophils in the theca of developing antral follicles was associated with expression of interleukin8 (IL-8) in the follicle wall [17]. It can be thought that the recruitment of neutrophils might be mediated by the chemoattractant such as IL-8 secreted from the granulosa cells or resident macrophages of the degenerating
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follicles. The infiltration of neutrophils into the follicle cavity seems to be a consequential rather than a primary cause of follicle degeneration. It might be discussed that the acute inflammatory immune response occurred in the follicles containing the excess cell debris. This suggests that the clearance mechanism of the damaged follicles may differ according to the atretic stimuli and to the status of follicles. In summary, the present results show that a mean lethal dose of ␥-radiation induces the apoptotic and inflammatory degeneration of mouse ovarian follicles. Besides, the immune cells such as macrophages, macrophage-like granulosa cells and even neutrophils may be involved in the acute atretic degeneration in ␥-irradiated mouse ovarian follicles.
[10] [11]
[12] [13]
[14]
[15]
Acknowledgement [16]
This study was supported by Grants from Korea Science and Engineering Foundation (ABRL; R14-2003036-010020) to YD Yoon.
[17]
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␥-Radiation-induced follicular degeneration in the prepubertal mouse ovary Chang Joo Lee, Yong-Dal Yoon ∗ Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791, Republic of Korea Received 3 February 2005; received in revised form 22 May 2005; accepted 24 May 2005 Available online 30 August 2005
Abstract Prepubertal mice were whole-body irradiated with a mean lethal dose (LD50 ) of ␥-radiation using a 60 Co source with a total dose of 7.2 Gy and a dose rate of 12.0 cGy/min. At day 0 before the irradiation and at day 1, 2, and 3 after the irradiation, the ovaries were collected and the morphological changes were assessed. The ratios (%) of atretic or polymorphonuclear leukocytes (neutrophil)-infiltrated follicles in the largest cross sections were calculated. In the early atretic follicle of the control mouse ovary, both apoptotic and mitotic cells were observed and occasionally neutrophils were infiltrated into the follicle cavity. However, in the atretic follicles 2 days post-irradiation, numerous cell fragments, apoptotic cells and bodies, and especially, a number of neutrophils were observed. In the non-irradiated control, the ratios of atretic follicles were 58.0 ± 8.6 and 27.3 ± 11.2 (mean ± S.E.M.) in antral and preantral follicles, respectively. The ratios of the number of antral and preantral follicles with one or more neutrophils to the total number of atretic follicles were 29.3 ± 12.0. At 2 days post-irradiation, the ratios of atretic follicles were increased to 94.0 ± 3.4 and 86.9 ± 7.6 in antral and preantral follicles, respectively. The ratios of neutrophil-containing follicles among the atretic one were increased to 65.9 ± 11.5 and 57.8 ± 15.4 at 2 and 3 days after the irradiation, respectively. Taken together, the present results show that ␥-radiation induces apoptotic and inflammatory degeneration of mouse ovarian follicles. Besides, neutrophils may be involved in the acute atretic degeneration in ␥-irradiated mouse ovarian follicles. © 2005 Elsevier B.V. All rights reserved. Keywords: Apoptosis; Inflammation; Neutrophil; Atresia; Radiation; Ovarian follicle; Mouse
1. Introduction In mammals, the great majorities of ovarian follicles are degenerated and eventually disappear from the ∗ Corresponding author. Tel.: +82 2 2220 0955; fax: +82 2 2294 0955. E-mail address: [email protected] (Y.-D. Yoon).
0027-5107/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2005.05.019
ovary through follicle 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 [1–3]. One of the pathologic stimuli that induces and accelerates the follicle atresia is radiation [4]. In both normal tissues and tumors apoptosis occurs spontaneously and can be
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induced following irradiation [5]. According to Jarrell et al. [6], dramatic changes broke out in the overall structure of the ovary in response to radiation. Recent studies reveal that radiation induces cell apoptosis in follicles [5], chromosomal damage of oocytes, and also impairs the ovarian functions [7]. Granulosa cell death in the atretic follicle involves apoptosis [8–12]. In the irradiated mouse ovary, it was shown that the follicles degenerated by the apoptosis of granulosa cells [4,13]. Morphologically, the degenerating granulosa cells in the follicles were phagocytosed by the neighboring intact granulosa cells or the macrophages [14–16]. Evidently, macrophages participated in the elimination of apoptotic and inflammatory cellular debris. However, in human antral follicles, relatively large numbers of polymorphonuclear leukocytes (neutrophils) were contained within the theca vasculature and the density of neutrophil was greater in atretic than in healthy follicles [17]. It implicates that the follicular immune cells such as neutrophils and macrophages are participated in the ovarian physiology, including follicle atresia. The mice were whole-body irradiated for the induction of artificial atresia of the ovarian follicles, and the morphological assessment was carried out to obtain the evidences that the immune cells participated in the follicular degenerating process induced by ␥-irradiation in the mouse ovary. 2. Materials and methods 2.1. Animals Three weeks old female mice (ICR strain) were obtained from Toxicology Research Center, Korea Research Institute of Chemical Technology, Taejon, Korea. The mice were raised in a 22 ◦ C controlled animal care room with the 12-h light:12-h dark cycle. The animals had free access to tap water and commercial chow during the experiments. 2.2. γ-Irradiation The whole-body ␥-irradiation was carried out with a isotopic source (dose rate: 12.0 cGy/min; source strength: approximately 150 TBq, Panoramic Irradiator, Atomic Energy of Canada Ltd.) at Korea Atomic Energy Research Institute as previously reported by Kim et al. [4] and Kim and Lee [18]. The radiation dose
was LD50(30) (7.2 Gy) and the irradiation time was 1 h. The number of mice used in the present experiment was five per group of experiment. 2.3. Histology Before the irradiation (day 0) and 1, 2, and 3 days after the irradiation, the left ovaries were dissected out and immediately fixed in 2.5% glutaraldehyde/0.1 M phosphate buffer (pH 7.3). Post fixation was followed using 1% of osmium tetroxide (Sigma Chem. Co., MO) for 2 h at 4 ◦ C. Embedding of specimens after alcoholic dehydration and displacement by propylene oxide was carried out in epon mixture [Poly/Bed 812 resin (Epon 812):dodecenylsuccinic anhydride:nadic methyl anhydride:2,4,6-tri(dimethylaminomethyl) phenol (DMP30) = 19.3:12.3:9.4:0.6 ml, Polysciences Inc.]. Using ultramicrotome (Leica), semi-thin sections of 1 m thickness were prepared and stained with 1% toluidine blue O in 1% borax solution. The right ovaries were fixed in 10% neutral buffered formalin for 12 h. Following the routine histologic preparation, the samples were embedded in paraffin, and sectioned into 4 m in thickness. Serial sections were stained with hematoxylin and eosin for the evaluation of the follicle status. Periodic acid Schiffs’ (PAS) staining was carried out according to Bancroft and Stevens [19]. The tissues were oxidized in 0.5% periodic acid solution for 15 min. After washing in tap water, the sections were incubated in Schiffs’ solution (basic fuchsin, 1 g; 1 N HCl, 20 ml; anhydrous sodium metabisulfate, 2 g) for 30 min. The counterstain with Harris hematoxylin was followed for 3 min. To identify neutrophil in a follicle cavity further, 7/4 rat monoclonal mouse antibody (Serotec) was used according to manufacturer’s guidelines. The polymorphic myelomonocytic antigen 7/4 is expressed at high levels on polymorphonuclear leukocytes [20], but is absent on resident tissue macrophages [21]. After the incubation with 7/4 antibody, biotin-labeled goat anti-mouse IgG (ImmunoTag) was applied. It was stained using 3,3 -diaminobenzidine tetrahydrochloride and counterstained with 1% of light green.
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2.4. Light microscopy The largest cross sections were observed in this study. Observation of morphological changes was done
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under a light microscope (Olympus BX5O) with the magnifications of ×400 or ×1000. Preantral and antral follicles in the largest cross sections of the whole ovaries were observed. The identification criteria were largely based on Pedersen and Peters [22] and Kim and Lee [18]. Follicles with one or more mitotic granulosa cells and linear membrana granulosa, and without pyknotic granulosa cells as well as with linear and peculiar zona pellucida were classified into normal [18]. Follicles undefined by above criteria were regarded as atretic. The preantral and antral follicles were identified by the comparison of the serial sections. Neutrophils were identified by the PAS staining and re-identified by neutrophil immunohistochemistry. Under a light microscope, the number of normal and atretic follicles was counted. Among the atretic follicles, the number of follicles containing one or more neutrophils was obtained and calculated the ratio by the equation [(number of follicle containing neutrophil/total atretic antral and preantral follicle) × 100]. To compare the follicle status in the ovary, the ratio (%) of normal follicles was calculated by the equation [(normal follicles/total follicles) × 100]. 2.5. Electron microscopy Ultrathin sections stained with lead citrate and uranyl acetate were examined and photographed under a transmission electron microscope (Jeol, JEM-1200 EX II). 2.6. Statistical analysis Numerical values were expressed as the mean ± S.E.M. Statistical analysis was carried out by ANOVA followed by the Student–Newman–Keuls method for multiple comparisons among means. Significance was considered at the 0.05 level.
3. Results In the present study, using the immature mouse ovary, we evaluated morphological degeneration of preantral and antral follicles induced by ␥-radiation. Normal follicles contained mitotic granulosa and no apoptotic cells in the membrana granulosa. The oocyte and zona pellucida had healthy appearances (Fig. 1A).
Fig. 1. Microphotographs of normal and atretic follicles in the control and ␥-irradiated mouse ovary. (A) In the normal early antral follicle of the control mice ovary, mitotic cell (long arrow) was present, and no apoptotic or pyknotic cells were found. Bar, 20 m. (B) In the atretic early antral follicle of the control mouse ovary, mitotic (long arrow) and apoptotic cells (short thin arrow) and bodies (open arrow) were observed in the follicle cavity. Note the neutrophil infiltration into the cavity (short thick arrow). Bar, 20 m. (C) In a severely atretic follicle of the ␥-irradiated mouse ovary, numerous neutrophils (short thick arrows) were found around the basement membrane. Theca layer was hypertrophied. Macrophage (arrow head) and numerous cell fragments (bold arrow) were found in the follicle cavity. Bar, 50 m. Abbreviations: ZP, zona pellucida; BM, basement membrane; TC, theca layer; O, oocyte.
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In the early atretic follicle of the control mouse ovary obtained at day 0, both apoptotic and mitotic cells were observed. Occasionally, neutrophils infiltrated into the follicle cavity. Mostly, neutrophils were observed in the theca layer. In the follicle cavity, the number of cell fragments was relatively few (Fig. 1B). However, in the severely atretic follicles at 2 days post-irradiation, numerous cell fragments, apoptotic cells and bodies, and, especially, neutrophils were observed in the antral cavity. Macrophages engulfing the apoptotic bodies were also found. Neutrophils infiltrated into the follicle cavity presumably across the basement membrane (Fig. 1C). Such follicles had apoptotic and mitotic cells as well. Some granulosa cells were ruptured and in the follicle cav-
ity, cell fragments were abnormally increased in number. In the control mouse ovary, it was observed that the early atretic antral follicles contained both apoptotic and mitotic granulosa cells in the membrana granulosa. No neutrophils were found around the apoptotic bodies. The antrum of the atretic antral follicles with shrunken oocytes contained relatively few cell fragments. In this kind of follicles, macrophage or macrophage-like phagocytic cells engulfing the apoptotic bodies were observed. Apoptosis of granulosa cell was easily identified by the presence of its condensed or marginated nucleus. In the atretic follicle of the control mouse ovary, it was observed that granulosa cells engulfed apoptotic bodies.
Fig. 2. Electron microphotographs of the atretic follicles in ␥-irradiated mouse ovary. (A) In an early atretic follicle, neutrophil (short thick arrow) was observed between the basement membrane and theca layer. Apoptotic cell (thin arrow) and a few cell fragments were found in granulosa layer. Bar, 5 m. Original magnification: ×1500. (B) Neutrophils were observed in the theca-granulosa region. Left upper neutrophil was located in theca and right two neutrophils were in granulosa layer. The basement membrane was indicated by small arrow heads according to the line. Bar, 2 m. Original magnification: ×4000. (C) In a severely atretic follicle, numerous neutrophils (small thick arrows) and macrophages (short-tailed arrows) were found. One neutrophil was phagocytosed by a macrophage in this panel. Bar, 2 m. Original magnification: ×2000. (D) Numerous neutrophils (short thick arrow) were found in the severely atretic follicles. Apoptotic cells (long thin arrow) were also found in the acute inflammatory region of the follicle. The cell membrane was not observed and the cellular components of granulosa cells were leaked out. Bar, 5 m. Original magnification: ×1500.
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At 2 and 3 days post-irradiation, neutrophils appeared near the basement membrane of the atretic follicles. These follicles had both apoptotic and normal granulosa cells (Fig. 2A). A number of neutrophils were recruited around the basement membrane (Fig. 2B). In the follicle cavity, occasionally, it was observed that the infiltrated neutrophils were engulfed by the macrophage (Fig. 2C). In the atretic follicular cavity, numerous neutrophils were observed. The granulosa cells were ruptured and the cellular components were leaked out as an inflammation (Fig. 2D). By comparisons with hematoxylin and eosin stain (Fig. 3A), neutrophils in the ovarian sections were further iden-
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tified by PAS, and by immunohistochemical staining with 7/4 monoclonal antibody (Fig. 3B and C). Morphologically, macrophage and macrophage-like phagocytic cells were observed in the follicular cavities as shown in Fig. 4. Obviously, two kinds of morphologically different phagocytic cells were present in the degenerating follicles. One was macrophage or macrophage-like giant cells, and the other was the phagocytic granulosa cells. These cells engulfed the apoptotic bodies and cell debris present in the follicle cavity. The frequency of atretic antral (Fig. 5) and preantral follicles induced by ␥-radiation was increased at 2 and
Fig. 3. Microphotographs of the ovarian follicles in mice. The ovarian section was stained with hematoxylin and eosin (A); periodic acid Schiffs’ (B); immunohistochemical localization for neutrophil with 7/4 monoclonal antibody was applied (C). (D) and (E) are high power magnification of (B) and (C), respectively. Open arrows, atretic follicles; thin arrows, atretic bodies; thick arrows, neutrophils. Original magnification: (A–C)—×1500; (D and E)—×400. Bars: (A–C)—100 m; (D and E)—50 m.
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Fig. 5. Ratio of the number of follicles containing neutrophils to that of total preantral and antral atretic follicles in ␥-irradiated mouse ovary. The ratio (%) of the numbers of atretic and neutrophilcontaining follicles in the largest cross section were counted and presented as mean ± S.E.M. Number of mice in a group was 5. *P < 0.05 significantly different when compared to day 0.
The ratios of the number of antral and preantral follicles with one or more neutrophils to the entire number of follicles in the largest cross sections were 29.3 ± 12.0 in the non-irradiated control mouse
Fig. 4. Macrophages and macrophage-like granulosa cells in the mouse ovarian follicles. Macrophages and macrophage-like granulosa cells engulfing cell debris and/or apoptotic bodies were observed in the relatively normal (A) and atretic follicles with hypertrophied theca layer (B) in mouse ovaries. These types of cell clearance were identified in the control as well as ␥-irradiated mouse ovarian follicles. (A) Bar, 50 m. Original magnification, ×200. Insert: bar, 10 m. Original magnification, ×1000; (B) Bar, 20 m. Original magnification, ×400. Insert: bar, 10 m. Original magnification, ×1000. O, oocyte; GC, granulosa cells; ZP, zona pellucida. Thin arrows, apoptotic bodies; thick arrows, macrophage or macrophagelike cells; white arrow, mitotic cell.
3 days after irradiation (P < 0.05). In the control group before the irradiation, the atretic ratios were 58.0 ± 8.6 and 27.3 ± 11.2 (mean ± S.E.M., n = 5) in antral and preantral follicles, respectively. At 2 days after irradiation, the ratios were increased to 94.0 ± 3.4 and 86.9 ± 7.6 in antral and preantral follicles, respectively (P < 0.05).
Fig. 6. Change of atretic ratio of preantral and antral follicles in ␥irradiated mouse ovary. The immature mice were ␥-irradiated at a dose of 7.2 Gy (LD50 ) for 1 h. Before the irradiation and 1, 2, and 3 days after irradiation, histological changes of left ovaries were observed. In the largest cross sections, the atretic follicles were counted under a microscope. Follicles were identified by comparison of the serial sections. Follicles with one or more apoptotic granulosa cells and bodies were classified as atretic. The atretic ratio (%) was obtained from the division of the number of atretic follicle by that of the total follicles and presented as mean ± S.E.M. The number of mice in a group was 5. *P < 0.05 significantly different when compared to day 0. White bars, preantral follicles; dashed bars, antral follicles.
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(Fig. 6). The ratios were increased to 65.9 ± 11.5 and 57.8 ± 15.4 at 2 and 3 days after the irradiation, respectively (P < 0.05).
4. Discussion Ovarian follicular growth and development is an integrated process encompassing both extraovarian signals and intraovarian factors [23]. The predominant event in the ovary is the loss of follicles through atresia during the reproductive period in mammals. However, the precise mechanism of follicle atresia is not proven yet. It has been suggested that macrophage has the potential relevance as a major cellular component of the immune system within the ovary [24,25]. Besides having a role in secretion of cytokines, macrophages in the ovary participated in the elimination of cellular debris originating from the apoptotic processes. During follicle development, gonadotropins, together with local ovarian growth factors and cytokines, as well as estrogens, activate different intracellular pathways to rescue follicles from apoptotic demise. In contrast, some cytokines such as tumor necrosis factor-␣, Fas ligand, presumably acting through receptors with a death domain, and androgens are atretogenic factors [26]. Azuma et al. [27] reported that macrophages modulate steroid production by granulosa–luteal cells. It was reported that macrophages were abundant in human atretic follicles [28] and resident macrophages have been detected in the human ovary [29]. Previously, it was reported that a high dose of radiation induced acute degeneration of primordial and primary [13], and preantral and antral follicles [18]. In the present experiment, the ratios (%) of atretic antral and preantral follicles in the control group were 58 and 27%, respectively. At 2 and 3 days post-irradiation, the ratios were increased to 94 and 87% in antral and preantral follicles, respectively. We observed that macrophages and macrophage-like phagocytes participated in the degenerating processes of ␥-irradiated mouse ovarian follicles. In our results, it was identified that the follicle degeneration after a mean lethal dose of ␥-irradiation was taking place on granulosa cells through both the apoptosis and inflammation. The apoptotic cell fragments and even the neutrophils were eliminated by
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the phagocytic macrophages or macrophage-like granulosa cells. It was reported that, in the initial stage of atretic follicle of the rabbit [11] and guinea pig ovary [30], two kinds of phagocytic cells were identified: one was intact granulosa cells ingesting neighboring dead granulosa cells, and the other was large round cells identified as macrophages. The present results showed that neutrophils in the ␥-radiation-induced atretic follicles were recruited into the follicle cavity. It suggests that neutorphils, with the macrophages and the normal granulosa cells were actively involved in the atretic degeneration of the ovarian follicles. Morphologically, apoptosis involves rapid condensation and budding of the cell, with the formation of membrane-enclosed apoptotic bodies containing wellpreserved organelles, which are phagocytosed and digested by nearby resident cells without inflammatory immune response [31]. In the present study, the phagocytosis of neutrophils by macrophage and the presence of apoptotic cells and bodies meant that the follicles after receiving the detrimental radiation degenerated not only by apoptosis but also by the inflammatory immune response. By a morphometric analysis of human ovarian follicles, Chang et al. [17] reported that the number of neutrophils were two-fold greater in atretic than in healthy follicles. However, they observed that the neutrophils were contained within the theca vasculature. About 29% of the atretic antral and preantral follicles contained neutrophils in the follicle cavity of the control mouse ovary. The ratios were increased at 2 and 3 days post-irradiation to 66 and 58%, respectively. Especially, follicles in the advanced stage of atresia contained more neutrophils than the early atretic ones. As reported by Kim et al. [4], it can be thought that the radiation induced the acute and accelerated follicle degeneration, so the apoptotic fragments were not sufficiently eliminated by phagocytes. Though we did not obtain the biochemical evidences about the triggering mechanism of the neutrophil recruitment into the follicle, it was found that the immune cells infiltrated into the follicle antrum, definitely across the basement membrane. The presence of neutrophils in the theca of developing antral follicles was associated with expression of interleukin8 (IL-8) in the follicle wall [17]. It can be thought that the recruitment of neutrophils might be mediated by the chemoattractant such as IL-8 secreted from the granulosa cells or resident macrophages of the degenerating
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follicles. The infiltration of neutrophils into the follicle cavity seems to be a consequential rather than a primary cause of follicle degeneration. It might be discussed that the acute inflammatory immune response occurred in the follicles containing the excess cell debris. This suggests that the clearance mechanism of the damaged follicles may differ according to the atretic stimuli and to the status of follicles. In summary, the present results show that a mean lethal dose of ␥-radiation induces the apoptotic and inflammatory degeneration of mouse ovarian follicles. Besides, the immune cells such as macrophages, macrophage-like granulosa cells and even neutrophils may be involved in the acute atretic degeneration in ␥-irradiated mouse ovarian follicles.
[10] [11]
[12] [13]
[14]
[15]
Acknowledgement [16]
This study was supported by Grants from Korea Science and Engineering Foundation (ABRL; R14-2003036-010020) to YD Yoon.
[17]
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