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Natural and radiation-induced degeneration of primordial and primary follicles in mouse ovary
Animal Reproduction Science 59 Ž2000. 109–117 www.elsevier.comrlocateranireprosci
Natural and radiation-induced degeneration of primordial and primary follicles in mouse ovary Chang Joo Lee a,b, Ho Hyun Park b, Byung Rok Do b, Yong-Dal Yoon b, Jin Kyu Kim a,) b
a Korea Atomic Energy Research Institute, Yusong P.O. Box 105, Taejon 305-600, South Korea Department of Biology, College of Natural Sciences, Hanyang UniÕersity, Seoul 133-791, South Korea
Received 7 July 1999; received in revised form 30 November 1999; accepted 13 December 1999
Abstract The present study deals with the morphological changes of the degenerating primordial and primary follicles induced by g-radiation. Prepubertal female mice of 3 weeks old ICR strain were g-irradiated with the dose of LD 80Ž30. Ž8.3 Gy.. The ovaries were collected at 3, 6 and 12 h after irradiation. The largest cross-sections were prepared by histological semithin sections for microscopical observations. The ratio Ž%. of normal to atretic follicles decreased with time after the irradiation in primordial follicles and in primary follicles as well. At 6 h after irradiation, the number of degenerated primordial follicles increased. Germinal vesicles disappeared and lipid droplets increased in number. Granulosa cells became round in shape and apoptotic cells started to appear. The ooplasmic membrane was not recognizable. The ratio of normal to atretic primordial follicles in the control group was 62.5. Then it became lower with time after the irradiation. It went down to 51.6, 49.0, 11.1 and 7.1 at 0, 3, 6 and 12 h, respectively. The ratio of normal to atretic primary follicles in the control mouse ovary was 81.3. It was 80.0, 75.0, 45.5 and 33.3 at 0, 3, 6 and 12 h after irradiation, respectively. It is concluded that the ionizing radiation acutely induces the degeneration of primordial and primary follicles. The pattern of degeneration is one of the following: Ž1. apoptosis of one or more granulosa cells with a relatively intact oocyte, Ž2. apoptosis of an oocyte with intact follicle cells, or Ž3. apoptotic degenerations of both kinds of cells. These results can provide morphological clues for the identification of the degenerating
)
Corresponding author. Tel.: q82-42-868-2057; fax: q82-42-868-2091. E-mail address: [email protected] ŽJ.K. Kim..
0378-4320r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 0 0 . 0 0 0 7 2 - 5
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C.J. Lee et al.r Animal Reproduction Science 59 (2000) 109–117
primordial and primary follicles in normal and irradiated mouse ovaries. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Mouse; Ovary; Primary follicle; Primordial follicle; Radiation
1. Introduction A primordial follicle is the smallest follicle within the ovary and contains an oocyte surrounded by a single layer of flattened somatic cells ŽMeredith and Doolin, 1997.. The formation of the primordial follicles occurs around the time of birth in rodents ŽHirshfield, 1991.. By unproved mechanisms, the primordial follicles initiate growth into primary, and consequently, secondary stages before acquiring an antral cavity. Except for a few primordial follicles, most of them remain as resting, quiescent, and nongrowing pool of follicles that will be progressively depleted throughout the reproductive life span of the female. Most of the ovarian follicles undergo a degenerative process called atresia during the reproductive life in mammals ŽByskov, 1978.. However, the precise mechanism of follicular atresia has not been elucidated yet. 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 ŽHirshfield and Midgley, 1978; Braw and Tsafriri, 1980.. However, most of the research works regarding the follicular atresia have been focused on the growing follicles. In the mammalian ovary, two major stages of cell degeneration can be distinguished: the degeneration of germ cells, defined as attrition, which accounts for the main loss of oocytes ŽBeaumont and Mandl, 1962. and occurs prenatally; and the follicular degeneration, defined as atresia, which occurs during postnatal reproductive life ŽKaipia and Hsueh, 1997.. Apoptosis, a regulated form of cell death, is a physiological process essential for the normal tissue homeostasis ŽKaipia and Hsueh, 1997. in the absence of immune surveillance ŽKerr et al., 1994.. Ovarian follicular degeneration or atresia is a hormonally controlled apoptotic process, whereby the degenerating follicles are eliminated in a coordinated fashion ŽHsueh et al., 1994.. It is now accepted that pyknosis of granulosa cells is an apoptotic process ŽHughes and Gorospe, 1991; Billig et al., 1993; Hurwitz and Adashi, 1993; Gougeon, 1996.. One of the atretogenic stimuli that could accelerate the follicular atresia was g-radiation ŽKim et al., 1999.. In both normal tissues and tumors, apoptosis not only occurs spontaneously but can also be induced by irradiation ŽHendry and West, 1997.. Radiation induced cell apoptosis ŽHendry and West, 1997. and impaired the ovarian functions ŽChapman, 1982.. It was reported that primordial oocytes of rats and mice were more sensitive to radiation than oocytes in the growing follicles ŽAtaya et al., 1995.. However, the radiation sensitivities between the resting and nongrowing primordial and the activated primary follicles were not morphologically demonstrated. Moreover, the morphological changes of the primordial and primary follicles occurred at natural and artificial degeneration processes were also not depicted yet. Therefore, the present experiments have been performed to analyze the morphological features of the normal and the degenerating primordial and primary follicles in the natural and the irradiated mouse ovary.
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2. Materials and methods 2.1. Experimental animals Prepubertal female mice ŽICR strain. aged 3 weeks were used to avoid the intervening results, which could come from the reproductive cycles. Female mice were obtained from Toxicology Research Center, Korea Research Institute of Chemical Technology, Taejon, Republic of Korea. The mice were maintained in a 238C-controlled animal care room with lightrdark Ž12r12 h.. The animals had free access to tap water and commercial chow during the experiments. 2.2. Irradiation Five mice per group were whole-body irradiated with g-radiation from 60 Co isotopic source Ždose rate: 6.94 cGyrmin, source strength: approximately 150 TBq, Panoramic Irradiator, Atomic Energy of Canada, Limited. at Korea Atomic Energy Research Institute ŽKAERI. as previously reported by Kim et al. Ž1999.. The radiation dose was 8.3 Gy, which was LD 80Ž30. for the mice. The irradiation was done for 2 h. As a control, the sham-irradiation was carried out by placing the mice in the preparation room during the irradiation period. Groups of mice were killed by cervical dislocation before irradiation, and at 0, 3, 6 and 12 h after the end of the 2-h irradiation. 2.3. Histological preparation After irradiation, mice of each group were kept in a 238C-controlled animal care room at KAERI. Each group of mice was killed by cervical dislocation at 0, 3, 6 and 12 h after irradiation. The ovaries were collected and fixed to observe the changes in the architecture of primordial and primary follicles. Postfixation using 1% of osmium tetraoxide ŽSigma, MO. was conducted for 2 h at 48C after prefixation with 2.5% glutaraldehyder0.1 M phosphate buffer ŽpH 7.3.. Embedding of specimens after an alcoholic dehydration and displacement by propylene oxide was carried out in epon mixture wPolyrBed 812 resin ŽEpon 812.:dodecenylsuccinic anhydride:nadic methyl anhydride:2,4,6-tri Ždimethylaminomethyl. phenol ŽDMP30. s 19.3:12.3:9.4:0.6 ml, Polysciencesx. Using ultramicrotome ŽLeica., semithin sections were prepared by 1 mm in thickness and stained with 1% toluidine blue O in 1% borax solution. The largest cross-sections were used in this study. Observation of morphological changes was done under a light microscope ŽOlympus BX50.. 2.4. Identification of oÕarian status Normal primordial follicles were identified by the presence of normal granulosa cells surrounding the healthy oocyte. Follicles with shrunk oocytes, with one or more
112
Primordial follicle
Granulosa cells Germinal vesicle Ooplasmic membrane Ooplasm Basement membrane
Primary follicle
Normal
Atretic
Normal
Atretic
Flattened or round, no pyknotic Round and clear Clear, regular Even, clear Clear, thin, regular
Irregular or amorphous, apoptotic Irregular, absent Unclear, irregular Uneven, unclear, dark Unclear, relatively thick, irregular
Round, no pyknotic, some cells mitotic Round and clear Clear, regular Even, clear Clear, regular
Pyknosis of one or more cells Irregular, absent Unclear, irregular Uneven, unclear, dark Unclear, irregular
C.J. Lee et al.r Animal Reproduction Science 59 (2000) 109–117
Table 1 Criteria for the identification of normal and atretic primordial follicles in the present experiment
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pyknotic granulosa cells, andror with apoptotic oocyte or without oocyte were classified into atretic ones based on the criteria listed in Table 1. The criteria for classification of the primary follicles were also described in Table 1. Under a microscope, the number of normal and atretic follicles was counted and their ratio was calculated. Data were expressed as mean " SEM and the statistical differences between the experimental groups were considered when p value was smaller than 0.05.
Fig. 1. Microphotographs of the primordial and the primary follicles in normal and irradiated mouse ovaries. The identification of normal and atretic primordial and primary follicles based on the criteria depicted in Table 1. Panel A shows morphologically the normal primordial follicles in the sham-irradiated mouse ovary. Panel B shows the atretic primordial follicles. Four degenerating primordial follicles were shown. In the right-upper primordial follicle, the apoptotic nucleus of oocyte was identified. In the left-upper and middle primordial follicles, the remnants of degenerating oocyte nuclei were shown. In the left-lower primordial follicle, the oocyte was no longer observable, and the granulosa cells, instead of the oocyte, were filled in a basement membrane. These primordial follicular changes were observed in the irradiated mouse ovaries at 6 h after irradiation. In panel C, in a basement membrane, some of the granulosa cells were recognized pyknotic. The peculiar changes in the primordial follicular shapes were shown at 12 h after irradiation, and also shown in the sham-irradiated normal mouse ovaries. In panel D, a normal primary follicle with a mitotic granulosa cell was shown. The upper primary follicle in panel E was shown with a pyknotic or apoptotic nucleus, and the lower primary follicle had a degenerated remnant of oocyte nucleus. These follicles were shown 6 h after irradiation. In panel F, the primary follicle with apoptotic granulosa cells and without oocyte was shown at 12 h after irradiation. Bars: 10 mm. Thin arrows, basement membranes; thick arrows, oocytes; arrow heads, granulosa cells; white thick arrow, follicles with pyknotic or apoptotic granulosa cells; white thin arrow, mitotic granulosa cell.
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3. Results The ratio of normal to atretic follicles was significantly reduced at 6 h after irradiation. In the sham-irradiated control ovaries, the primordial follicles might be degenerated by the gradual, somewhat long-lasting, procedure. In the irradiated ones, however, it happened acutely. Since the degenerating patterns of ovarian primordial, follicles were diverse in the control mouse, various types of primordial follicles were observed. The normal primordial follicles with two or three flattened granulosa cells possessed round ooplasmic membrane, clear germinal vesicle, and one or more nucleoli as shown in Fig. 1. Mitochondria were randomly distributed throughout the ooplasm ŽFig. 1.. At 6 h after irradiation, the degenerated primordial follicles were considerably increased. Germinal vesicles disappeared, and lipid droplets increased. No more ooplasmic membrane was observable. Granulosa cells became round in their shape, and apoptotic cells started to appear. Dark-stained germinal vesicles and apoptotic granulosa cells were also seen. The ratio of normal to atretic follicles in control group was 62.5. This ratio decreased with time after the irradiation. The ratio was 51.6, 49.0, 11.1 and 7.1 at 0, 3, 6 and 12 h, respectively ŽFig. 2.. The degenerating primordial follicles had one of the following characteristics: Ž1. relatively normal oocyte with apoptotic granulosa cells, Ž2. degenerating oocyte with intact granulosa cells, and Ž3. apoptotic or pyknotic oocyte and granulosa cells. The primary follicle with a degenerating oocyte was observed. Follicles with apoptotic or pyknotic granulosa cells and with a relatively intact oocyte increased, similarly with those in primordial follicles ŽFig. 2.. The ratio of normal to atretic follicles in the
Fig. 2. Changes in the ratio of normal to atretic primordial and primary follicles in the control and irradiated mouse ovaries. The irradiation time in the present experiment was 2 h. Time 0 means the end of the irradiation. The number of mice in each experimental group was five. The ratio decreased steeply at 6 h after irradiation, and decreased further at 12 h after irradiation. Data were expressed as mean"SEM. a: significantly different from that of group ‘y2 h’ Ž p- 0.01..
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control mouse ovary was 81.3. This ratio decreased down to 80.0, 75.0, 45.5 and 33.3 at 0, 3, 6 and 12 h after irradiation, respectively.
4. Discussion Even though it has been widely accepted that radiation had a detrimental effect on the ovarian follicles in mammals, the reports concerning the changes of morphological characteristics of primordial and primary follicles induced by radiation were quite limited. In the present experiment, the changes of morphological characteristics of primordial and primary follicles caused by radiation were investigated. Erickson Ž1966. reported that primordial follicles were depleted as a consequence of either oocyte attrition or initiation of growth. The result of the present experiment showed that g-radiation had effects of oocyte attrition, as well as the induction of apoptosis of granulosa cells in the primordial and primary follicles. There were three kinds of degenerating patterns of primordial and primary follicles. First, follicles, which were recognized by the presence of the apoptotic oocytes had intact granulosa cells. Second, follicles had one or more apoptotic granulosa cells with relatively intact oocyte membrane. And, finally, follicles with both of the above characteristic changes were observed. Apoptotic granulosa cells and defected oocytes were easily recognized by the dark-stained nuclei. The most characteristic morphology of the degenerated primordial follicles was the lack of oocytes with two or more malformed granulosa cell-like cells. These malformed cells were placed in the basement membrane of the degenerated primordial follicles. The fact that the morphological changes were induced in a short time period after irradiation indicates that the primordial follicles have considerably greater susceptibility to ionizing radiation than primary follicles. In this regard, there is a great possibility that primary follicles, which are not degenerated, grow to secondary follicles. Also, it is thought that granulosa cells in a follicle are categorized into two groups. One type is radioresistant, and the other, radiosensitive. It can be thought that the radiation sensitivities between the resting and nongrowing primordial and the activated primary follicles were different. Ratts et al. Ž1995. reported that there were numerous primordial follicle-like structures in bcl-2 deficient mouse ovaries. In the present study, the irradiated mouse ovary contained numerous oocyte-deficient primordial follicles at 12 h after irradiation. In comparison, apoptotic granulosa cells and oocytes of the primordial follicles were shown until 6 h after irradiation. The degeneration of primordial follicles caused by g-radiation showed an acute progression. Nearly all the primordial follicles were degenerated within 12 h after irradiation. It was reported that there were no adequate morphological markers of degeneration in primordial follicles ŽEdwards et al., 1977.. However, in the present study, the primordial follicular degeneration was induced by g-irradiation and the morphological changes of primordial follicles with time lapse were marked and easily recognized by the presence of the apoptotic granulosa cells or degenerating oocytes, or both. Radiation could acutely give rise to the apoptotic degeneration of follicular cells in the present experimental scheme. The possible explanation of such an acute follicular degeneration
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lies on the high radiation dose used in this study, at which 80% of the irradiated animals will die within 30 days. The present study provides morphological clues for the microscopical identification of the degenerating primordial and primary follicles. The result indicates that the degeneration of primordial follicles goes much faster than that of primary follicles after irradiation and that the primordial follicles have higher sensitivity to the ionizing radiation than the primary follicles. In any case, the pattern of degeneration is one of the following: Ž1. apoptosis of one or more granulosa cells with relatively intact oocyte, Ž2. apoptosis of oocyte with intact follicle cells, or Ž3. apoptotic degenerations of both cells.
5. Conclusion The biological meaning of the present study is the identification of morphological changes of the degenerating primordial and primary follicles in the g-irradiated mouse ovary. The degeneration of primordial follicles is much faster than that of the primary follicles. The degenerations are mediated by apoptosis of oocytes and granulosa cells in both follicles. The present study provides morphological clues for the visual identification of the degenerating primordial and primary follicles. The result can give light on efforts to elucidate the follicular atresia mechanism.
Acknowledgements This study has been carried out under the Nuclear R & D Program by the Ministry of Science and Technology ŽMOST. of Korea.
References Ataya, K., Pydyn, E., Ramahi-Ataya, Orton, C.G., 1995. Is radiation-induced ovarian failure in rhesus monkeys preventable by luteining hormone-releasing hormone agonists? preliminary observations. J. Clin. Endocrinol. Metab. 80, 790–795. Beaumont, H.M., Mandl, A.M., 1962. A quantitative and cytological study of oogonia and oocytes in the fetal and neonatal rat. Proc. R. Soc. London, Ser. B 155, 557–579. Billig, H., Furuta, I., Hsueh, A.J., 1993. Estrogens inhibit and androgens enhance ovarian granulosa cell apoptosis. Endocrinology 133, 2204–2212. Braw, R.H., Tsafriri, A., 1980. Follicles explants from pentobarbitone-treated rats provide a model for atresia. J. Reprod. Fertil. 59, 259–265. Byskov, A.G., 1978. Follicular atresia. In: Jones, R. ŽEd.., The Vertebrate Ovary. Plenum, New York, pp. 533–562. Chapman, R.M., 1982. Effect of cytotoxic therapy on sexuality and gonadal function. Semin. Oncol. 9, 84–94. Edwards, R.G., Fowler, R.E., Gore-Langton, R.E., Gosden, R.G., Jones, E.C., Readhead, C., Steptoe, P.C., 1977. Normal and abnormal follicular growth in mouse, rat, and human ovaries. J. Reprod. Fertil. 51, 237–263. Erickson, B.H., 1966. Development and radio-response of the prenatal bovine ovary. J. Reprod. Fertil. 11, 97–105.
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Gougeon, A., 1996. Regulation of ovarian follicular development in primates: facts and hypotheses. Endocr. Rev. 17, 121–155. Hendry, J.H., West, C.M., 1997. Apoptosis and mitotic cell death: their relative contributions to normal-tissue and tumour radiation response. Int. J. Radiat. Biol. 71, 709–719. Hirshfield, A.N., 1991. Development of follicles in the mammalian ovary. Int. Rev. Cytol. 124, 43–101. Hirshfield, A.N., Midgley, A.R., 1978. Morphometric analysis of follicular development in the rat. Biol. Reprod. 19, 606–611. Hsueh, A.J., Billig, H., Tsafriri, A., 1994. Ovarian follicle atresia: a hormonally controlled apoptotic process. Endocr. Rev. 15, 707–724. Hughes, F.M. Jr., Gorospe, W.C., 1991. Biochemical identification of apoptosis Žprogrammed cell death. in granulosa cells: evidence for a potential mechanism underlying follicular atresia. Endocrinology 129, 2415–2422. Hurwitz, A., Adashi, E.Y., 1993. Ovarian follicular atresia as an apoptotic process. In: Adashi, E.Y., Leung, P.C.K. ŽEds.., The Ovary. Raven Press, New York, pp. 473–485. Kaipia, A., Hsueh, A.J., 1997. Regulation of ovarian follicle atresia. Annu. Rev. Physiol. 59, 349–363. Kerr, J.F., Winterford, C.M., Harmon, B.V., 1994. Apoptosis: its significance in cancer and cancer therapy. Cancer 73, 2013–2026. Kim, J.K., Lee, C.J., Song, K.W., Do, B.R., Yoon, Y.D., 1999. g-Radiation accelerates ovarian follicular atresia in immature mice. In Vivo 13, 21–24. Meredith, S., Doolin, D., 1997. Timing of activation of primordial follicles in mature rats is only slightly affected by fetal stage at meiotic arrest. Biol. Reprod. 57, 63–67. Ratts, V.S., Flaws, J.A., Kolp, R., Sorenson, C.M., Tilly, J.L., 1995. Ablation of bcl-2 gene expression decreases the numbers of oocytes and primordial follicles established in the after-natal female mouse gonad. Endocrinology 136, 3665–3668.
Natural and radiation-induced degeneration of primordial and primary follicles in mouse ovary Chang Joo Lee a,b, Ho Hyun Park b, Byung Rok Do b, Yong-Dal Yoon b, Jin Kyu Kim a,) b
a Korea Atomic Energy Research Institute, Yusong P.O. Box 105, Taejon 305-600, South Korea Department of Biology, College of Natural Sciences, Hanyang UniÕersity, Seoul 133-791, South Korea
Received 7 July 1999; received in revised form 30 November 1999; accepted 13 December 1999
Abstract The present study deals with the morphological changes of the degenerating primordial and primary follicles induced by g-radiation. Prepubertal female mice of 3 weeks old ICR strain were g-irradiated with the dose of LD 80Ž30. Ž8.3 Gy.. The ovaries were collected at 3, 6 and 12 h after irradiation. The largest cross-sections were prepared by histological semithin sections for microscopical observations. The ratio Ž%. of normal to atretic follicles decreased with time after the irradiation in primordial follicles and in primary follicles as well. At 6 h after irradiation, the number of degenerated primordial follicles increased. Germinal vesicles disappeared and lipid droplets increased in number. Granulosa cells became round in shape and apoptotic cells started to appear. The ooplasmic membrane was not recognizable. The ratio of normal to atretic primordial follicles in the control group was 62.5. Then it became lower with time after the irradiation. It went down to 51.6, 49.0, 11.1 and 7.1 at 0, 3, 6 and 12 h, respectively. The ratio of normal to atretic primary follicles in the control mouse ovary was 81.3. It was 80.0, 75.0, 45.5 and 33.3 at 0, 3, 6 and 12 h after irradiation, respectively. It is concluded that the ionizing radiation acutely induces the degeneration of primordial and primary follicles. The pattern of degeneration is one of the following: Ž1. apoptosis of one or more granulosa cells with a relatively intact oocyte, Ž2. apoptosis of an oocyte with intact follicle cells, or Ž3. apoptotic degenerations of both kinds of cells. These results can provide morphological clues for the identification of the degenerating
)
Corresponding author. Tel.: q82-42-868-2057; fax: q82-42-868-2091. E-mail address: [email protected] ŽJ.K. Kim..
0378-4320r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 0 0 . 0 0 0 7 2 - 5
110
C.J. Lee et al.r Animal Reproduction Science 59 (2000) 109–117
primordial and primary follicles in normal and irradiated mouse ovaries. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Mouse; Ovary; Primary follicle; Primordial follicle; Radiation
1. Introduction A primordial follicle is the smallest follicle within the ovary and contains an oocyte surrounded by a single layer of flattened somatic cells ŽMeredith and Doolin, 1997.. The formation of the primordial follicles occurs around the time of birth in rodents ŽHirshfield, 1991.. By unproved mechanisms, the primordial follicles initiate growth into primary, and consequently, secondary stages before acquiring an antral cavity. Except for a few primordial follicles, most of them remain as resting, quiescent, and nongrowing pool of follicles that will be progressively depleted throughout the reproductive life span of the female. Most of the ovarian follicles undergo a degenerative process called atresia during the reproductive life in mammals ŽByskov, 1978.. However, the precise mechanism of follicular atresia has not been elucidated yet. 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 ŽHirshfield and Midgley, 1978; Braw and Tsafriri, 1980.. However, most of the research works regarding the follicular atresia have been focused on the growing follicles. In the mammalian ovary, two major stages of cell degeneration can be distinguished: the degeneration of germ cells, defined as attrition, which accounts for the main loss of oocytes ŽBeaumont and Mandl, 1962. and occurs prenatally; and the follicular degeneration, defined as atresia, which occurs during postnatal reproductive life ŽKaipia and Hsueh, 1997.. Apoptosis, a regulated form of cell death, is a physiological process essential for the normal tissue homeostasis ŽKaipia and Hsueh, 1997. in the absence of immune surveillance ŽKerr et al., 1994.. Ovarian follicular degeneration or atresia is a hormonally controlled apoptotic process, whereby the degenerating follicles are eliminated in a coordinated fashion ŽHsueh et al., 1994.. It is now accepted that pyknosis of granulosa cells is an apoptotic process ŽHughes and Gorospe, 1991; Billig et al., 1993; Hurwitz and Adashi, 1993; Gougeon, 1996.. One of the atretogenic stimuli that could accelerate the follicular atresia was g-radiation ŽKim et al., 1999.. In both normal tissues and tumors, apoptosis not only occurs spontaneously but can also be induced by irradiation ŽHendry and West, 1997.. Radiation induced cell apoptosis ŽHendry and West, 1997. and impaired the ovarian functions ŽChapman, 1982.. It was reported that primordial oocytes of rats and mice were more sensitive to radiation than oocytes in the growing follicles ŽAtaya et al., 1995.. However, the radiation sensitivities between the resting and nongrowing primordial and the activated primary follicles were not morphologically demonstrated. Moreover, the morphological changes of the primordial and primary follicles occurred at natural and artificial degeneration processes were also not depicted yet. Therefore, the present experiments have been performed to analyze the morphological features of the normal and the degenerating primordial and primary follicles in the natural and the irradiated mouse ovary.
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2. Materials and methods 2.1. Experimental animals Prepubertal female mice ŽICR strain. aged 3 weeks were used to avoid the intervening results, which could come from the reproductive cycles. Female mice were obtained from Toxicology Research Center, Korea Research Institute of Chemical Technology, Taejon, Republic of Korea. The mice were maintained in a 238C-controlled animal care room with lightrdark Ž12r12 h.. The animals had free access to tap water and commercial chow during the experiments. 2.2. Irradiation Five mice per group were whole-body irradiated with g-radiation from 60 Co isotopic source Ždose rate: 6.94 cGyrmin, source strength: approximately 150 TBq, Panoramic Irradiator, Atomic Energy of Canada, Limited. at Korea Atomic Energy Research Institute ŽKAERI. as previously reported by Kim et al. Ž1999.. The radiation dose was 8.3 Gy, which was LD 80Ž30. for the mice. The irradiation was done for 2 h. As a control, the sham-irradiation was carried out by placing the mice in the preparation room during the irradiation period. Groups of mice were killed by cervical dislocation before irradiation, and at 0, 3, 6 and 12 h after the end of the 2-h irradiation. 2.3. Histological preparation After irradiation, mice of each group were kept in a 238C-controlled animal care room at KAERI. Each group of mice was killed by cervical dislocation at 0, 3, 6 and 12 h after irradiation. The ovaries were collected and fixed to observe the changes in the architecture of primordial and primary follicles. Postfixation using 1% of osmium tetraoxide ŽSigma, MO. was conducted for 2 h at 48C after prefixation with 2.5% glutaraldehyder0.1 M phosphate buffer ŽpH 7.3.. Embedding of specimens after an alcoholic dehydration and displacement by propylene oxide was carried out in epon mixture wPolyrBed 812 resin ŽEpon 812.:dodecenylsuccinic anhydride:nadic methyl anhydride:2,4,6-tri Ždimethylaminomethyl. phenol ŽDMP30. s 19.3:12.3:9.4:0.6 ml, Polysciencesx. Using ultramicrotome ŽLeica., semithin sections were prepared by 1 mm in thickness and stained with 1% toluidine blue O in 1% borax solution. The largest cross-sections were used in this study. Observation of morphological changes was done under a light microscope ŽOlympus BX50.. 2.4. Identification of oÕarian status Normal primordial follicles were identified by the presence of normal granulosa cells surrounding the healthy oocyte. Follicles with shrunk oocytes, with one or more
112
Primordial follicle
Granulosa cells Germinal vesicle Ooplasmic membrane Ooplasm Basement membrane
Primary follicle
Normal
Atretic
Normal
Atretic
Flattened or round, no pyknotic Round and clear Clear, regular Even, clear Clear, thin, regular
Irregular or amorphous, apoptotic Irregular, absent Unclear, irregular Uneven, unclear, dark Unclear, relatively thick, irregular
Round, no pyknotic, some cells mitotic Round and clear Clear, regular Even, clear Clear, regular
Pyknosis of one or more cells Irregular, absent Unclear, irregular Uneven, unclear, dark Unclear, irregular
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Table 1 Criteria for the identification of normal and atretic primordial follicles in the present experiment
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pyknotic granulosa cells, andror with apoptotic oocyte or without oocyte were classified into atretic ones based on the criteria listed in Table 1. The criteria for classification of the primary follicles were also described in Table 1. Under a microscope, the number of normal and atretic follicles was counted and their ratio was calculated. Data were expressed as mean " SEM and the statistical differences between the experimental groups were considered when p value was smaller than 0.05.
Fig. 1. Microphotographs of the primordial and the primary follicles in normal and irradiated mouse ovaries. The identification of normal and atretic primordial and primary follicles based on the criteria depicted in Table 1. Panel A shows morphologically the normal primordial follicles in the sham-irradiated mouse ovary. Panel B shows the atretic primordial follicles. Four degenerating primordial follicles were shown. In the right-upper primordial follicle, the apoptotic nucleus of oocyte was identified. In the left-upper and middle primordial follicles, the remnants of degenerating oocyte nuclei were shown. In the left-lower primordial follicle, the oocyte was no longer observable, and the granulosa cells, instead of the oocyte, were filled in a basement membrane. These primordial follicular changes were observed in the irradiated mouse ovaries at 6 h after irradiation. In panel C, in a basement membrane, some of the granulosa cells were recognized pyknotic. The peculiar changes in the primordial follicular shapes were shown at 12 h after irradiation, and also shown in the sham-irradiated normal mouse ovaries. In panel D, a normal primary follicle with a mitotic granulosa cell was shown. The upper primary follicle in panel E was shown with a pyknotic or apoptotic nucleus, and the lower primary follicle had a degenerated remnant of oocyte nucleus. These follicles were shown 6 h after irradiation. In panel F, the primary follicle with apoptotic granulosa cells and without oocyte was shown at 12 h after irradiation. Bars: 10 mm. Thin arrows, basement membranes; thick arrows, oocytes; arrow heads, granulosa cells; white thick arrow, follicles with pyknotic or apoptotic granulosa cells; white thin arrow, mitotic granulosa cell.
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3. Results The ratio of normal to atretic follicles was significantly reduced at 6 h after irradiation. In the sham-irradiated control ovaries, the primordial follicles might be degenerated by the gradual, somewhat long-lasting, procedure. In the irradiated ones, however, it happened acutely. Since the degenerating patterns of ovarian primordial, follicles were diverse in the control mouse, various types of primordial follicles were observed. The normal primordial follicles with two or three flattened granulosa cells possessed round ooplasmic membrane, clear germinal vesicle, and one or more nucleoli as shown in Fig. 1. Mitochondria were randomly distributed throughout the ooplasm ŽFig. 1.. At 6 h after irradiation, the degenerated primordial follicles were considerably increased. Germinal vesicles disappeared, and lipid droplets increased. No more ooplasmic membrane was observable. Granulosa cells became round in their shape, and apoptotic cells started to appear. Dark-stained germinal vesicles and apoptotic granulosa cells were also seen. The ratio of normal to atretic follicles in control group was 62.5. This ratio decreased with time after the irradiation. The ratio was 51.6, 49.0, 11.1 and 7.1 at 0, 3, 6 and 12 h, respectively ŽFig. 2.. The degenerating primordial follicles had one of the following characteristics: Ž1. relatively normal oocyte with apoptotic granulosa cells, Ž2. degenerating oocyte with intact granulosa cells, and Ž3. apoptotic or pyknotic oocyte and granulosa cells. The primary follicle with a degenerating oocyte was observed. Follicles with apoptotic or pyknotic granulosa cells and with a relatively intact oocyte increased, similarly with those in primordial follicles ŽFig. 2.. The ratio of normal to atretic follicles in the
Fig. 2. Changes in the ratio of normal to atretic primordial and primary follicles in the control and irradiated mouse ovaries. The irradiation time in the present experiment was 2 h. Time 0 means the end of the irradiation. The number of mice in each experimental group was five. The ratio decreased steeply at 6 h after irradiation, and decreased further at 12 h after irradiation. Data were expressed as mean"SEM. a: significantly different from that of group ‘y2 h’ Ž p- 0.01..
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control mouse ovary was 81.3. This ratio decreased down to 80.0, 75.0, 45.5 and 33.3 at 0, 3, 6 and 12 h after irradiation, respectively.
4. Discussion Even though it has been widely accepted that radiation had a detrimental effect on the ovarian follicles in mammals, the reports concerning the changes of morphological characteristics of primordial and primary follicles induced by radiation were quite limited. In the present experiment, the changes of morphological characteristics of primordial and primary follicles caused by radiation were investigated. Erickson Ž1966. reported that primordial follicles were depleted as a consequence of either oocyte attrition or initiation of growth. The result of the present experiment showed that g-radiation had effects of oocyte attrition, as well as the induction of apoptosis of granulosa cells in the primordial and primary follicles. There were three kinds of degenerating patterns of primordial and primary follicles. First, follicles, which were recognized by the presence of the apoptotic oocytes had intact granulosa cells. Second, follicles had one or more apoptotic granulosa cells with relatively intact oocyte membrane. And, finally, follicles with both of the above characteristic changes were observed. Apoptotic granulosa cells and defected oocytes were easily recognized by the dark-stained nuclei. The most characteristic morphology of the degenerated primordial follicles was the lack of oocytes with two or more malformed granulosa cell-like cells. These malformed cells were placed in the basement membrane of the degenerated primordial follicles. The fact that the morphological changes were induced in a short time period after irradiation indicates that the primordial follicles have considerably greater susceptibility to ionizing radiation than primary follicles. In this regard, there is a great possibility that primary follicles, which are not degenerated, grow to secondary follicles. Also, it is thought that granulosa cells in a follicle are categorized into two groups. One type is radioresistant, and the other, radiosensitive. It can be thought that the radiation sensitivities between the resting and nongrowing primordial and the activated primary follicles were different. Ratts et al. Ž1995. reported that there were numerous primordial follicle-like structures in bcl-2 deficient mouse ovaries. In the present study, the irradiated mouse ovary contained numerous oocyte-deficient primordial follicles at 12 h after irradiation. In comparison, apoptotic granulosa cells and oocytes of the primordial follicles were shown until 6 h after irradiation. The degeneration of primordial follicles caused by g-radiation showed an acute progression. Nearly all the primordial follicles were degenerated within 12 h after irradiation. It was reported that there were no adequate morphological markers of degeneration in primordial follicles ŽEdwards et al., 1977.. However, in the present study, the primordial follicular degeneration was induced by g-irradiation and the morphological changes of primordial follicles with time lapse were marked and easily recognized by the presence of the apoptotic granulosa cells or degenerating oocytes, or both. Radiation could acutely give rise to the apoptotic degeneration of follicular cells in the present experimental scheme. The possible explanation of such an acute follicular degeneration
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lies on the high radiation dose used in this study, at which 80% of the irradiated animals will die within 30 days. The present study provides morphological clues for the microscopical identification of the degenerating primordial and primary follicles. The result indicates that the degeneration of primordial follicles goes much faster than that of primary follicles after irradiation and that the primordial follicles have higher sensitivity to the ionizing radiation than the primary follicles. In any case, the pattern of degeneration is one of the following: Ž1. apoptosis of one or more granulosa cells with relatively intact oocyte, Ž2. apoptosis of oocyte with intact follicle cells, or Ž3. apoptotic degenerations of both cells.
5. Conclusion The biological meaning of the present study is the identification of morphological changes of the degenerating primordial and primary follicles in the g-irradiated mouse ovary. The degeneration of primordial follicles is much faster than that of the primary follicles. The degenerations are mediated by apoptosis of oocytes and granulosa cells in both follicles. The present study provides morphological clues for the visual identification of the degenerating primordial and primary follicles. The result can give light on efforts to elucidate the follicular atresia mechanism.
Acknowledgements This study has been carried out under the Nuclear R & D Program by the Ministry of Science and Technology ŽMOST. of Korea.
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