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Abnormal accumulation of corpora lutea in ovaries of the senescence accelerated mouse prone (SAMP1)
International Congress Series 1260 (2004) 179 – 185
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Abnormal accumulation of corpora lutea in ovaries of the senescence accelerated mouse prone (SAMP1) Noboru Manabe *, Minako Kiso, Munetake Shimabe, Nami Nakai-Sugimoto, Hajime Miyamoto Unit of Anatomy and Cell Biology, Department of Animal Sciences, Kyoto University, Kyoto 606-8502, Japan Received 18 June 2003; received in revised form 18 June 2003; accepted 3 September 2003
Abstract. Senescence accelerated mouse prone (SAMP) mice with a shortened life span show accelerated changes in many of the signs of aging and have a shorter reproductive life span than SAM resistant (SAMR) controls. The reproductive senescence of SAMP is more accelerated than that of SAMR. In the present study, we found abnormal accumulation of luteal bodies (LBs) only in the ovaries of SAMP1 mice and examined the mechanism of abnormality in luteal cell regression in the ovaries of SAMP1 mice. No significant changes were detected in serum progesterone or 17h-estradiol levels during the estrus cycle between SAMP1 and SAMR1 mice. In abnormally accumulated LBs of SAMP1 mice, extremely high levels of 20a-hydroxysteroid dehydrogenase, which catalyzes the conversion of progesterone to the inactive form, 20a-hydroxyprogesterone, were demonstrated histochemically. However, no marked differences in the activity of 17h-hydroxysteroid dehydrogenase activity were detected histochemically. Moreover, no apoptotic cells were detected in the abnormally accumulated LBs of SAMP1 mice. The SAMP1 mouse may be a useful model in which to examine the mechanism of regression of LBs in mammalian ovaries. D 2003 Elsevier B.V. All rights reserved. Keywords: Corpus luteum; Abnormal accumulation; Apoptosis; Ovaries; Senescence accelerated mouse prone
1. Introduction The senescence accelerated mouse (SAMP1) is a useful animal model of accelerated senescence and consists of two related strains: SAM resistant and prone (SAMR and SAMP, respectively) [1]. Previously, we showed that age-dependent changes in reproductive functions are more severe in SAMP1 mice than in other murine models [2 –6]. We compared the morphometrical parameters of spermatogenic cells of male SAMP1 mice with those of SAMR1 mice after birth until 40 weeks old (approximately the mean life span of the SAMP1 strain) [2,3]. These results indicated that testicular maturation * Corresponding author. Tel.: +81-75-753-6326; fax: +81-75-753-6345. E-mail address: [email protected] (N. Manabe). 0531-5131/ D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0531-5131(03)01425-0
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begins at an earlier age in SAMP1 mice than in SAMR1 mice, and that signs of testicular deterioration are evident only in SAMP1 mice at 40-week-old. Moreover, we examined the female reproductive properties and early embryonic development of SAMP1 and SAMR1 mice [4,5]. The reproductive senescence of SAMP1 mice is more accelerated than that of SAMR1 mice. The reproductive life span of SAMP1 mice was shorter than that of SAMR1 mice, and the total number of SAMP1 pups was 41.7% fewer than in SAMR1. Cell cleavage was delayed in embryos of SAMP1 mice compared to SAMR1 mice. Even in young SAMP1 mice (15-week-old), a reduced litter size and lower incidence of regular estrus cycles were noted compared with normal SAMR1 mice. During these investigations, we found the abnormal accumulation of luteal bodies (LBs) in the ovaries of SAMP1 mice with regular estrus cycles. However, the mechanism behind the abnormal accumulation is not understood. The aim of the present study, therefore, was to determine the mechanism of the abnormalities in luteal cell regression in the ovaries of SAMP1 mice. We examined the changes in serum progesterone and 17h-estradiol levels in SAM mice during the estrus cycle, enzymehistochemically demonstrated the activities of 17h-hydroxysteroid dehydrogenase (17hHSD) and 20a-hydroxysteroid dehydrogenase (20a-HSD) to determine steroid hormone production in the ovaries, and histochemically detected apoptotic cells by terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated deoxyuridine triphosphate nick end-labeling (TUNEL) in the LBs of SAMR1 and SAMP1 mice. 2. Peripheral blood 17B-estradiol and progesterone levels and enzyme-histochemistry for 17B-HSD and 20A-HSD Female SAMP1 (30-week-old) mice and age-matched SAMR1 mice were checked daily, and only those without severe changes, such as severe loss of activity, hair loss and lack of hair glossiness, skin coarseness, periophthalmic lesions, and increased lordokyphosis of the spine, were used. They received humane care as outlined in the Guide for the Care and Use of Laboratory Animals (Kyoto University Animal Care Committee according to NIH no. 8623; revised 1999). Estrus cycle stages were determined by daily examination of vaginal cytology, and animals showing more than two consecutive 4-day estrus cycles were used. They were bled at different stages in the estrus cycle (proestrus, 13:00 and 23:00; estrus, 13:00; metestrus, 13:00 and 23:00; diestrus, 13:00). Serum 17h-estradiol and progesterone were measured in each sample by 125I radioimmunoassay. No significant differences were noted in serum 17h-estradiol or progesterone levels throughout the regular 4-day estrus cycles between the SAMR and SAMP mice. Animals were sacrificed under ether anesthesia in the afternoon (approximately 14:00) at the proestrus, estrus, metestrus and diestrus stages. Ovaries were rapidly removed, weighed, put on filter paper, mounted in OCT compound (Miles, Elkhart, IN, USA), and frozen in a dry ice-isopentane mixture (Wako, Osaka, Japan), and then serial frozen sections (5-Am thick) were cut on a cryostat (Jung CM1500; Leica, Heidelberg, Germany) and mounted on glass slides precoated with 3-aminopropyltriethoxysilane (Sigma Aldrich Chemicals, St. Louis, MO, USA). Frozen sections were dipped in incubation media for 17h-HSD activity [10 ml of 0.1 M phosphate-buffered saline (PBS; pH 7.4) containing 1.8 mg of 17h-estradiol (Sigma) in 0.5 ml of acetone, 4 mg of h-nicotinamide adenine dinucleotide (Sigma) and 2 mg of nitro blue tetrazolium salt
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(NBT; Sigma)] and 20a-HSD activity [14 ml of 200 mM Tris – HCl buffer, pH 8.0, and 14 ml of 50% polyvinyl-pyrolidone containing 35 mg of NBT, 35 mg of h-nicotinamide adenine dinucleotide phosphate (Sigma), 70 mg of ethylenediaminetetraacetic acid disodium salt (Wako), and 35 mg of 20a-hydroxypregn-4-en-3-on (Sigma) in 7 ml of N,N-dimethylformamide (Wako)] at 36 jC for 1 h [6]. The slides were washed with PBS, mounted with glycerol, and examined with a microscope (BX50, Olympus, Tokyo, Japan). Adjoining sections from each specimen were stained with hematoxylin and eosin for conventional histopathological evaluation. A significant increase in the ovary weight of SAMP mice (130.5 F 42.1 mg) was noted in comparison with that of SAMR mice (17.6 F 2.3 mg) (P < 0.01). Many LBs (more than 50 LBs/ovary) were accumulated in the ovaries of SAMP mice (Fig. 1B), but they were much fewer in the ovaries of SAMR1 mice (less than 20 LBs/ovary; Fig. 1A). No marked differences in the number of LBs were seen between the ovaries of SAMR1 mice and age-matched ICR mice, used as normal healthy controls. Most of the LBs accumulated in SAMP1 ovaries were considered to be abnormally accumulated. Thus, the more than sevenfold increase in ovary weight of SAMP1 mice was caused by the abnormal accumulation of LBs. No marked differences were seen in intensity of 17h-HSD staining between the LBs of SAMR1 mice and those of SAMP1 mice (data not shown). Extremely strong 20a-HSD activity, however, was demonstrated in the abnormally accumulated LBs of SAMP mice (Fig. 1D) compared with LBs of SAMR1 mice (Fig. 1C). During the estrus cycle, no differences in the kinetics of 17h-estradiol and progesterone in peripheral blood were observed between SAMP1 and SAMR1 mice, indicating that there are no differences in female hormonal regulation between the two strains. However, high levels of 20a-HSD activity were histochemically demonstrated in luteal cells of abnormally accumulated LBs of SAMP1 mice. Moreover, high levels of 17h-HSD activity were also demonstrated in these cells by an enzyme histochemical method. Steroid hormones synthesized and secreted from the ovaries coordinate the function of the entire female reproductive system, and increased 17h-estradiol secretion characterizes the follicular phase of the estrus cycle, reflecting the specialized endocrine function of the preovulatory follicle, and the steroid hormone balance in peripheral blood reflects folliculogenesis and ovulation [5]. In luteal cells of rodents, progesterone is catabolized to 20a-hydroxyprogesterone, the physiologically inactive form, by 20aHSD. Thus, the present results indicated that progesterone is synthesized and then rapidly catabolized into an inactive steroid in the luteal cells of the abnormally accumulated LBs in SAMP1 ovaries, that such catabolism does not affect the normal progesterone level in peripheral blood of SAMP1 mice, and that SAMP1 mice show normal consecutive 4-day estrus cycles. In our preliminary study, extremely low concentrations of serum 17h-estradiol were detected in SAMP1 mice with irregular estrus cycles, and such an irregular secretion of 17h-estradiol is considered to reflect the age-related deterioration of reproductive functions in the ovaries of SAMP1 mice [4,5]. The abnormal accumulation of LBs is a unique phenomenon seen only in SAMP1 mice. Our preliminary study showed that a low level of 20a-HSD activity was histochemically demonstrated in LBs of aging ICR mice after functional regression (data not shown). These findings indicated that functional regression, i.e. a decrease in progesterone production, occurs in the luteal cells of abnormally accumulated LBs in SAMP1 mice.
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Fig. 1. Representative frozen sections of ovaries of SAMR1 (A and C) and SAMP1 (B and D) mice were stained with hematoxylin and eosin (A and B) and histochemically demonstrated 20a-HSD activity (C and D). Many luteal bodies (asterisks) were accumulated only in SAMP1 ovaries (B), not in SAMR1 ovaries (A). Extremely strong staining of 20a-HSD activity was demonstrated in abnormally accumulated luteal bodies (asterisks) of SAMP1 ovaries (D), and weak/moderate activity was seen in the luteal bodies (asterisk) of SAMR1 ovaries (C). Bars indicate 100 Am.
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3. In situ detection of apoptotic cells and DNA electrophoresis Ovarian tissue sections were stained by the TUNEL method using a commercial kit (Apop Tag; Oncor Gaithersburg, MD, USA) to allow visualization of the apoptotic cells [7]. Briefly, the sections were fixed with 10% buffered formalin, washed with PBS, incubated with 20 Ag/ml of proteinase K (Sigma) in PBS for 15 min at room temperature (RT: 22– 25 jC), and then immersed in 2% H2O2 in methanol for 5 min to inhibit endogenous peroxidase activity. After preincubation with equilibration buffer for 10 min at 37 jC, sections were incubated with TdT solution containing 45 AM ddATP and 5 AM digoxigenin (DIG)-ddUTP for 1 h at 37 jC, and then immersed in double-strength salt sodium citrate buffer to stop the labeling reaction. After washing, the sections were incubated with peroxidase-labeled anti-DIG antibody solution for 30 min at RT and then reacted with 0.05% 3,3-diaminobenzidine and 0.002% H2O2 in 50 mM Tris – HCl, pH 7.2, for 1 min at RT. After washing with distilled water, the sections dehydrated through a graded ethanol series were mounted with Eukitt (Kindler, Freiburg, Germany). During the regression of LBs, many apoptotic/TUNEL-positive cells were demonstrated among luteal cells in the ovaries of SAMR1 mice (Fig. 2A). In abnormally accumulated LBs of SAMP1 mice, however, no apoptotic cells were detected (Fig. 2B). To assess the DNA fragmentation in luteal cells, LBs were isolated under a surgical dissecting microscope (SZ11, Olympus), and then LBs were homogenized. DNA
Fig. 2. Representative sections of luteal bodies of SAMR1 (A) and SAMP1 (B) ovaries were histochemically stained to detect the apoptotic cells by TUNEL method. During regression of the luteal bodies, many TUNELpositive cells (arrows) were detected among the luteal cells in the ovaries of SAMR1 mice (A). In abnormally accumulated LBs in the ovaries of SAMP1 mice, no TUNEL-positive apoptotic cells were seen (B).
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fractions were separated by centrifugation at 9000 g for 20 min at 4 jC, and then DNA contents were determined. DNA samples were electrophoresed in 2% agarose gels with 40 mM Tris –acetate, pH 8.1, containing 2 mM EDTA, 18 mM NaCl and 10 Ag/ml ethidium bromide at 60 V for 90 min. Gels were photographed on an ultraviolet transilluminator. DNA samples from LBs of SAMR1 ovaries displayed a ladder pattern, a biochemical hallmark of apoptosis (Fig. 3; lanes 5 –9). No such ladder pattern was seen in the DNA samples prepared from LBs of SAMP1 ovaries (Fig. 3; lanes 1– 4), indicating that no apoptosis occurred in luteal cells of abnormally accumulated LBs in SAMP1 ovaries. These histochemical and biochemical findings confirmed that the arrest of morphological regression, i.e. deficiency of apoptosis, occurs in the luteal cells of abnormally accumulated LBs in SAMP1 mice. In other words, functional regression occurs but structural regression is inhibited by blockage of apoptosis in the luteal cells of abnormally accumulated LBs in SAMP mice. The SAMP mouse is considered to be a useful animal model in which to study the local regulatory mechanism for the regression of LBs in murine ovaries [8,9].
Fig. 3. Electrophoretic analysis of DNA fragments in luteal bodies (LBs) of SAMR1 (lanes 5 – 10) and SAMP1 (lanes 1 – 4) ovaries. DNA samples from LBs of SAMR1 ovaries displayed a ladder pattern (lanes 5 – 10), but no such ladder pattern was seen in the DNA samples prepared from LBs of SAMP1 ovaries (lanes 1 – 4), indicating that no apoptosis occurred in the luteal cells of abnormally accumulated LBs in SAMP1 ovaries. M: molecular weight marker.
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Acknowledgements This work was supported by a Grant-in-Aid for Creative Scientific Research (13GS0008) and for Scientific Research (A) (13027241) to N.M. from the Ministry of Education, Culture, Sports, Science and Technology in Japan. References [1] T. Takeda, M. Hosokawa, S. Takeshita, M. Irino, K. Higuchi, T. Matsushita, Y. Tomita, K. Yasuhira, H. Hamamoto, K. Shimizu, M. Ishii, T. Yamamuro, A new model of accelerated senescence, Mech. Ageing Dev. 17 (1981) 183 – 194. [2] H. Miyamoto, N. Manabe, Y. Akiyama, Y. Mitani, M. Sugimoto, E. Sato, Quantitative studies on spermatogenesis in the senescence accelerated mouse, in: T. Takeda, M. Hosokawa (Eds.), The SAM Model, Elsevier, Amsterdam, 1994, pp. 275 – 278. [3] H. Miyamoto, N. Manabe, Y. Akiyama, T. Watanabe, M. Sugimoto, E. Sato, A morphometric study of spermatogenesis in the testes of mice of a senescence accelerated strain, Experientia 50 (1994) 808 – 811. [4] H. Miyamoto, N. Manabe, T. Watanabe, C. Aruga, Y. Mitani, N. Sugimoto, M. Sugimoto, E. Sato, Female reproductive characteristics of the senescence accelerated mouse, in: T. Takeda, M. Hosokawa (Eds.), The SAM model, Elsevier, Amsterdam, 1994, pp. 279 – 282. [5] H. Miyamoto, N. Manabe, Y. Mitani, N. Sugimoto, T. Watanabe, C. Aruga, E. Sato, Female reproductive properties and prenatal development of a senescence accelerated mouse strain, J. Exp. Zool. 272 (1995) 116 – 122. [6] M. Kiso, N. Manabe, K. Komatsu, N. Nisioka, N. Nakai-Sugimoto, H. Miyamoto, Abnormal accumulation of luteal bodies in ovaries of the senescence accelerated mouse (SAM), J. Reprod. Dev. 47 (2001) 153 – 164. [7] N. Manabe, Y. Imai, H. Ohno, Y. Takahagi, M. Sugimoto, H. Miyamoto, Apoptosis occurs in granulosa cells but not cumulus cells in the atretic antral follicles in the pig ovaries, Experientia 52 (1996) 647 – 651. [8] K. Komatsu, N. Manabe, M. Kiso, M. Shimabe, H. Miyamoto, Changes in localization of immune cells and cytokines in corpora lutea during luteolysis in murine ovaries, J. Exp. Zool. 296 (2003) 152 – 159. [9] K. Komatsu, N. Manabe, M. Kiso, M. Shimabe, H. Miyamoto, Soluble Fas (FasB) regulates luteal cell apoptosis during luteolysis in murine ovaries, Mol. Reprod. Dev. 65 (2003) 196 – 205.
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Abnormal accumulation of corpora lutea in ovaries of the senescence accelerated mouse prone (SAMP1) Noboru Manabe *, Minako Kiso, Munetake Shimabe, Nami Nakai-Sugimoto, Hajime Miyamoto Unit of Anatomy and Cell Biology, Department of Animal Sciences, Kyoto University, Kyoto 606-8502, Japan Received 18 June 2003; received in revised form 18 June 2003; accepted 3 September 2003
Abstract. Senescence accelerated mouse prone (SAMP) mice with a shortened life span show accelerated changes in many of the signs of aging and have a shorter reproductive life span than SAM resistant (SAMR) controls. The reproductive senescence of SAMP is more accelerated than that of SAMR. In the present study, we found abnormal accumulation of luteal bodies (LBs) only in the ovaries of SAMP1 mice and examined the mechanism of abnormality in luteal cell regression in the ovaries of SAMP1 mice. No significant changes were detected in serum progesterone or 17h-estradiol levels during the estrus cycle between SAMP1 and SAMR1 mice. In abnormally accumulated LBs of SAMP1 mice, extremely high levels of 20a-hydroxysteroid dehydrogenase, which catalyzes the conversion of progesterone to the inactive form, 20a-hydroxyprogesterone, were demonstrated histochemically. However, no marked differences in the activity of 17h-hydroxysteroid dehydrogenase activity were detected histochemically. Moreover, no apoptotic cells were detected in the abnormally accumulated LBs of SAMP1 mice. The SAMP1 mouse may be a useful model in which to examine the mechanism of regression of LBs in mammalian ovaries. D 2003 Elsevier B.V. All rights reserved. Keywords: Corpus luteum; Abnormal accumulation; Apoptosis; Ovaries; Senescence accelerated mouse prone
1. Introduction The senescence accelerated mouse (SAMP1) is a useful animal model of accelerated senescence and consists of two related strains: SAM resistant and prone (SAMR and SAMP, respectively) [1]. Previously, we showed that age-dependent changes in reproductive functions are more severe in SAMP1 mice than in other murine models [2 –6]. We compared the morphometrical parameters of spermatogenic cells of male SAMP1 mice with those of SAMR1 mice after birth until 40 weeks old (approximately the mean life span of the SAMP1 strain) [2,3]. These results indicated that testicular maturation * Corresponding author. Tel.: +81-75-753-6326; fax: +81-75-753-6345. E-mail address: [email protected] (N. Manabe). 0531-5131/ D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0531-5131(03)01425-0
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begins at an earlier age in SAMP1 mice than in SAMR1 mice, and that signs of testicular deterioration are evident only in SAMP1 mice at 40-week-old. Moreover, we examined the female reproductive properties and early embryonic development of SAMP1 and SAMR1 mice [4,5]. The reproductive senescence of SAMP1 mice is more accelerated than that of SAMR1 mice. The reproductive life span of SAMP1 mice was shorter than that of SAMR1 mice, and the total number of SAMP1 pups was 41.7% fewer than in SAMR1. Cell cleavage was delayed in embryos of SAMP1 mice compared to SAMR1 mice. Even in young SAMP1 mice (15-week-old), a reduced litter size and lower incidence of regular estrus cycles were noted compared with normal SAMR1 mice. During these investigations, we found the abnormal accumulation of luteal bodies (LBs) in the ovaries of SAMP1 mice with regular estrus cycles. However, the mechanism behind the abnormal accumulation is not understood. The aim of the present study, therefore, was to determine the mechanism of the abnormalities in luteal cell regression in the ovaries of SAMP1 mice. We examined the changes in serum progesterone and 17h-estradiol levels in SAM mice during the estrus cycle, enzymehistochemically demonstrated the activities of 17h-hydroxysteroid dehydrogenase (17hHSD) and 20a-hydroxysteroid dehydrogenase (20a-HSD) to determine steroid hormone production in the ovaries, and histochemically detected apoptotic cells by terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated deoxyuridine triphosphate nick end-labeling (TUNEL) in the LBs of SAMR1 and SAMP1 mice. 2. Peripheral blood 17B-estradiol and progesterone levels and enzyme-histochemistry for 17B-HSD and 20A-HSD Female SAMP1 (30-week-old) mice and age-matched SAMR1 mice were checked daily, and only those without severe changes, such as severe loss of activity, hair loss and lack of hair glossiness, skin coarseness, periophthalmic lesions, and increased lordokyphosis of the spine, were used. They received humane care as outlined in the Guide for the Care and Use of Laboratory Animals (Kyoto University Animal Care Committee according to NIH no. 8623; revised 1999). Estrus cycle stages were determined by daily examination of vaginal cytology, and animals showing more than two consecutive 4-day estrus cycles were used. They were bled at different stages in the estrus cycle (proestrus, 13:00 and 23:00; estrus, 13:00; metestrus, 13:00 and 23:00; diestrus, 13:00). Serum 17h-estradiol and progesterone were measured in each sample by 125I radioimmunoassay. No significant differences were noted in serum 17h-estradiol or progesterone levels throughout the regular 4-day estrus cycles between the SAMR and SAMP mice. Animals were sacrificed under ether anesthesia in the afternoon (approximately 14:00) at the proestrus, estrus, metestrus and diestrus stages. Ovaries were rapidly removed, weighed, put on filter paper, mounted in OCT compound (Miles, Elkhart, IN, USA), and frozen in a dry ice-isopentane mixture (Wako, Osaka, Japan), and then serial frozen sections (5-Am thick) were cut on a cryostat (Jung CM1500; Leica, Heidelberg, Germany) and mounted on glass slides precoated with 3-aminopropyltriethoxysilane (Sigma Aldrich Chemicals, St. Louis, MO, USA). Frozen sections were dipped in incubation media for 17h-HSD activity [10 ml of 0.1 M phosphate-buffered saline (PBS; pH 7.4) containing 1.8 mg of 17h-estradiol (Sigma) in 0.5 ml of acetone, 4 mg of h-nicotinamide adenine dinucleotide (Sigma) and 2 mg of nitro blue tetrazolium salt
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(NBT; Sigma)] and 20a-HSD activity [14 ml of 200 mM Tris – HCl buffer, pH 8.0, and 14 ml of 50% polyvinyl-pyrolidone containing 35 mg of NBT, 35 mg of h-nicotinamide adenine dinucleotide phosphate (Sigma), 70 mg of ethylenediaminetetraacetic acid disodium salt (Wako), and 35 mg of 20a-hydroxypregn-4-en-3-on (Sigma) in 7 ml of N,N-dimethylformamide (Wako)] at 36 jC for 1 h [6]. The slides were washed with PBS, mounted with glycerol, and examined with a microscope (BX50, Olympus, Tokyo, Japan). Adjoining sections from each specimen were stained with hematoxylin and eosin for conventional histopathological evaluation. A significant increase in the ovary weight of SAMP mice (130.5 F 42.1 mg) was noted in comparison with that of SAMR mice (17.6 F 2.3 mg) (P < 0.01). Many LBs (more than 50 LBs/ovary) were accumulated in the ovaries of SAMP mice (Fig. 1B), but they were much fewer in the ovaries of SAMR1 mice (less than 20 LBs/ovary; Fig. 1A). No marked differences in the number of LBs were seen between the ovaries of SAMR1 mice and age-matched ICR mice, used as normal healthy controls. Most of the LBs accumulated in SAMP1 ovaries were considered to be abnormally accumulated. Thus, the more than sevenfold increase in ovary weight of SAMP1 mice was caused by the abnormal accumulation of LBs. No marked differences were seen in intensity of 17h-HSD staining between the LBs of SAMR1 mice and those of SAMP1 mice (data not shown). Extremely strong 20a-HSD activity, however, was demonstrated in the abnormally accumulated LBs of SAMP mice (Fig. 1D) compared with LBs of SAMR1 mice (Fig. 1C). During the estrus cycle, no differences in the kinetics of 17h-estradiol and progesterone in peripheral blood were observed between SAMP1 and SAMR1 mice, indicating that there are no differences in female hormonal regulation between the two strains. However, high levels of 20a-HSD activity were histochemically demonstrated in luteal cells of abnormally accumulated LBs of SAMP1 mice. Moreover, high levels of 17h-HSD activity were also demonstrated in these cells by an enzyme histochemical method. Steroid hormones synthesized and secreted from the ovaries coordinate the function of the entire female reproductive system, and increased 17h-estradiol secretion characterizes the follicular phase of the estrus cycle, reflecting the specialized endocrine function of the preovulatory follicle, and the steroid hormone balance in peripheral blood reflects folliculogenesis and ovulation [5]. In luteal cells of rodents, progesterone is catabolized to 20a-hydroxyprogesterone, the physiologically inactive form, by 20aHSD. Thus, the present results indicated that progesterone is synthesized and then rapidly catabolized into an inactive steroid in the luteal cells of the abnormally accumulated LBs in SAMP1 ovaries, that such catabolism does not affect the normal progesterone level in peripheral blood of SAMP1 mice, and that SAMP1 mice show normal consecutive 4-day estrus cycles. In our preliminary study, extremely low concentrations of serum 17h-estradiol were detected in SAMP1 mice with irregular estrus cycles, and such an irregular secretion of 17h-estradiol is considered to reflect the age-related deterioration of reproductive functions in the ovaries of SAMP1 mice [4,5]. The abnormal accumulation of LBs is a unique phenomenon seen only in SAMP1 mice. Our preliminary study showed that a low level of 20a-HSD activity was histochemically demonstrated in LBs of aging ICR mice after functional regression (data not shown). These findings indicated that functional regression, i.e. a decrease in progesterone production, occurs in the luteal cells of abnormally accumulated LBs in SAMP1 mice.
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Fig. 1. Representative frozen sections of ovaries of SAMR1 (A and C) and SAMP1 (B and D) mice were stained with hematoxylin and eosin (A and B) and histochemically demonstrated 20a-HSD activity (C and D). Many luteal bodies (asterisks) were accumulated only in SAMP1 ovaries (B), not in SAMR1 ovaries (A). Extremely strong staining of 20a-HSD activity was demonstrated in abnormally accumulated luteal bodies (asterisks) of SAMP1 ovaries (D), and weak/moderate activity was seen in the luteal bodies (asterisk) of SAMR1 ovaries (C). Bars indicate 100 Am.
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3. In situ detection of apoptotic cells and DNA electrophoresis Ovarian tissue sections were stained by the TUNEL method using a commercial kit (Apop Tag; Oncor Gaithersburg, MD, USA) to allow visualization of the apoptotic cells [7]. Briefly, the sections were fixed with 10% buffered formalin, washed with PBS, incubated with 20 Ag/ml of proteinase K (Sigma) in PBS for 15 min at room temperature (RT: 22– 25 jC), and then immersed in 2% H2O2 in methanol for 5 min to inhibit endogenous peroxidase activity. After preincubation with equilibration buffer for 10 min at 37 jC, sections were incubated with TdT solution containing 45 AM ddATP and 5 AM digoxigenin (DIG)-ddUTP for 1 h at 37 jC, and then immersed in double-strength salt sodium citrate buffer to stop the labeling reaction. After washing, the sections were incubated with peroxidase-labeled anti-DIG antibody solution for 30 min at RT and then reacted with 0.05% 3,3-diaminobenzidine and 0.002% H2O2 in 50 mM Tris – HCl, pH 7.2, for 1 min at RT. After washing with distilled water, the sections dehydrated through a graded ethanol series were mounted with Eukitt (Kindler, Freiburg, Germany). During the regression of LBs, many apoptotic/TUNEL-positive cells were demonstrated among luteal cells in the ovaries of SAMR1 mice (Fig. 2A). In abnormally accumulated LBs of SAMP1 mice, however, no apoptotic cells were detected (Fig. 2B). To assess the DNA fragmentation in luteal cells, LBs were isolated under a surgical dissecting microscope (SZ11, Olympus), and then LBs were homogenized. DNA
Fig. 2. Representative sections of luteal bodies of SAMR1 (A) and SAMP1 (B) ovaries were histochemically stained to detect the apoptotic cells by TUNEL method. During regression of the luteal bodies, many TUNELpositive cells (arrows) were detected among the luteal cells in the ovaries of SAMR1 mice (A). In abnormally accumulated LBs in the ovaries of SAMP1 mice, no TUNEL-positive apoptotic cells were seen (B).
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fractions were separated by centrifugation at 9000 g for 20 min at 4 jC, and then DNA contents were determined. DNA samples were electrophoresed in 2% agarose gels with 40 mM Tris –acetate, pH 8.1, containing 2 mM EDTA, 18 mM NaCl and 10 Ag/ml ethidium bromide at 60 V for 90 min. Gels were photographed on an ultraviolet transilluminator. DNA samples from LBs of SAMR1 ovaries displayed a ladder pattern, a biochemical hallmark of apoptosis (Fig. 3; lanes 5 –9). No such ladder pattern was seen in the DNA samples prepared from LBs of SAMP1 ovaries (Fig. 3; lanes 1– 4), indicating that no apoptosis occurred in luteal cells of abnormally accumulated LBs in SAMP1 ovaries. These histochemical and biochemical findings confirmed that the arrest of morphological regression, i.e. deficiency of apoptosis, occurs in the luteal cells of abnormally accumulated LBs in SAMP1 mice. In other words, functional regression occurs but structural regression is inhibited by blockage of apoptosis in the luteal cells of abnormally accumulated LBs in SAMP mice. The SAMP mouse is considered to be a useful animal model in which to study the local regulatory mechanism for the regression of LBs in murine ovaries [8,9].
Fig. 3. Electrophoretic analysis of DNA fragments in luteal bodies (LBs) of SAMR1 (lanes 5 – 10) and SAMP1 (lanes 1 – 4) ovaries. DNA samples from LBs of SAMR1 ovaries displayed a ladder pattern (lanes 5 – 10), but no such ladder pattern was seen in the DNA samples prepared from LBs of SAMP1 ovaries (lanes 1 – 4), indicating that no apoptosis occurred in the luteal cells of abnormally accumulated LBs in SAMP1 ovaries. M: molecular weight marker.
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