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RETRACTED: Thalidomide treatment attenuates chemotherapy-induced gonadal toxicity
Thalidomide treatment attenuates chemotherapy-induced gonadal toxicity The aim of this study was to investigate gonadal-protective properties of thalidomide in a mouse model of chemotherapy-induced ovarian failure. Administration of thalidomide before alkylating chemotherapy preserved ovarian follicles and lead to the resumption of normal estrous cyclicity. (Fertil Steril 2011;95:819–22. 2011 by American Society for Reproductive Medicine.) Key Words: Thalidomide, gonadal protection, chemotherapy-induced gonadal failure, alkylating chemotherapy, busulfan, fertility, ovarian follicles Currently, there are >50,000 cancer survivors of reproductive age in the United States (1). Therefore, there is a growing interest in improving the quality of life for these patients, including preservation of their fertility. Preventing ovarian failure after cancer treatment is a major challenge among reproductive endocrinologists, and there is an ongoing search for agents with gonad-protective properties (2, 3). Thalidomide is a sedative hypnotic drug with antiemetic properties that was withdrawn from the market in the 1960s due to its teratogenic effects. It reemerged in 1998 with FDA approval for leprosy, and is currently used in a variety of oncologic, dermatologic, and inflammatory conditions. It has significant ‘‘antitumor’’ activity, both alone and as an adjunct to traditional chemotherapy (4). In reproductive-age women, thalidomide has been shown to cause reversible hypergonadotropic amenorrhea (5), with resumption of normal menses once the drug is discontinued. This amenorrhea is postulated to occur via interruption of integral angiogenic and cytokine-dependent pathways (5). In the present study, we investigated the gonad-protective properties of thalidomide in a mouse model of chemotherapy-induced ovarian failure. In addition, we studied the effects of thalidomide on ovarian funcMelanie E. Ochalski, M.D. Jennifer J. Shuttleworth, B.S. Tianjiao Chu, Ph.D. Kyle E. Orwig, Ph.D. Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh School of Medicine, Magee-Womens Research Institute, Pittsburgh, Pennsylvania Received January 18, 2010; revised September 3, 2010; accepted September 15, 2010; published online October 2, 2010. M.E.O. has nothing to disclose. J.J.S. has nothing to disclose. T.C. has nothing to disclose. K.E.O. has nothing to disclose. M.E.O. was supported by an American Medical Association seed grant and a Irene McLenahan Young Investigator Award from Magee– Womens Research Institute and Foundation (MWRI&F). K.E.O. was funded by National Institutes of Health grant nos. RR018500, AG024992, HD055475, and HD008610 as well as discretionary funds from MWRI&F. Reprint requests: Melanie E. Ochalski, M.D., Reproductive Endocrinology and Infertility, University of Pittsburgh, Magee Women’s Hospital, 300 Halket Street, Pittsburgh, PA 15213 (E-mail: ochamm@upmc. edu).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.09.020
tion, because despite this drug’s ability to ‘‘modulate fertility,’’ there is a lack of information about its effect on ovarian reserve. Forty-nine inbred C57Bl/6 mice (4 months old) were included in this study, which was approved by the University of Pittsburgh Institutional Animal Care and Use Committee (assurance no. A365401). Animals were divided into four groups. Control animals (CTL; n ¼ 13) were treated with vehicle, 1% carboxymethylcellulose (CMC), by oral gavage for 14 consecutive days. On day 7, CTL animals were treated with a single intraperitoneal (IP) dose of 50% dimethylsulfoxide (DMSO) vehicle. There were three experimental groups. The thalidomide-only group (THAL; n ¼ 12) was treated with thalidomide 100 mg/kg by oral gavage daily for 14 days and 50% DMSO vehicle on day 7. Thalidomide (Sigma Chemical Company, St. Louis. MO) was suspended in 1% CMC dissolved in deionized water. The busulfan-only group (BUS; n ¼ 12) received 1% CMC vehicle by oral gavage for 14 days and a single dose of busulfan (50 mg/ kg IP; Sigma) on day 7. This dose has been shown to produce detrimental effects on fertility (6). The THAL þ BUS group (n ¼ 12) was treated with thalidomide 100 mg/kg by oral gavage daily for 14 days. On the seventh day of thalidomide treatment, THAL þ BUS animals were treated with a single IP injection of busulfan (50 mg/kg). Estrous cyclicity was assessed by daily vaginal smears taken between 6:30 a.m. and 1:00 p.m., as previously described (7). Vaginal aspirates were read by one individual, who was blinded to treatment group. Smears were then classified into cycle stage (metestrus, diestrus, proestrus, estrus) according to the criteria of Allen (8). Animals were killed at diestrus, 90 days after the start of the study. At killing, whole blood samples as well as weights of whole body, uteri, and ovaries were obtained. Plasma FSH levels were determined by radioimmunoassay at the University of Virginia Ligand Core Laboratory (grant no. U54-HD28934). The uterus and ovaries from each animal were fixed in 4% paraformaldehyde, paraffin embedded, and serially sectioned (5 mm), followed by staining with hematoxylin and eosin. Follicles were categorized and enumerated based on the number of granulosa cell layers and the presence of the antral space, as described by Pedersen and Peters (9). Follicles were then categorized as ‘‘nongrowing’’ or ‘‘growing’’ to distinguish between the ovarian
Fertility and Sterility Vol. 95, No. 2, February 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
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FIGURE 1 Assessments of reproductive function in control (CTL), thalidomide-treated (THAL), busulfan-treated (BUS), and THAL þ BUS–treated animals. (A) Vaginal smears to assess estrous cyclicity. (B) Differential ovarian follicle counts per ovary (mean SEM). Ovarian histology of (C) CTL, (D) THAL, (E) BUS, and (F) THAL þ BUS–treated animals. Uterine histology of (G) CTL, (H) THAL, (I) BUS, and (J) THAL þ BUS–treated animals.
Ochalski. Correspondence. Fertil Steril 2011.
reserve pool and those oocytes that were recruited. Briefly, the ‘‘nongrowing’’ population consisted of an oocyte surrounded by a partial or unbroken single layer of granulosa cells, which approximates to Pedersen and Peters types 1–3b. The ‘‘growing’’ follicles
820
Ochalski et al.
Correspondence
included oocytes surrounded by multiple layers of granulosa cells (Pedersen and Peters types 4–5b) as well as the antral follicles that were characterized by a central oocyte and fluid-filled space bordered by multiple layers of granulosa cells (Pedersen and Peters
Vol. 95, No. 2, February 2011
types 6–8). Similar simplified classification systems have been described previously (10–13). For the present study, we used the classification system described by Bolon et al. (11). Only follicles with a nucleolus present were counted, to avoid overcounting a single follicle present in multiple sections. Differential follicle counts were performed blinded to treatment group. Two-sided Wilcoxon rank sum test was used to identify differences in follicle counts between treatment groups. All animals had regular cycles before initiation of treatment (data not shown). Vaginal smears indicated that all CTL animals exhibited normal estrous cyclicity throughout the experimental period. THAL-only animals exhibited a brief period of persistent diestrus before returning to normal estrous cyclicity. All BUSonly animals (100%) were acyclic, exhibiting persistent cornified or leukocytic vaginal smears. In contrast, 58% of THAL þ BUS animals returned to normal estrous cyclicity, suggesting a protective effect of thalidomide. Cyclicity data for days 74–90 after the initiation of treatment are shown in Figure 1A. Differences in vaginal cytology reflect differing hormonal milieus and give insight into the underlying mechanism of ovarian failure. The prolonged diestrus exhibited by animals treated with busulfan may indicate insufficient E2 secretion owing to reduced numbers of maturing follicles, as reported previously (14). As expected, nongrowing follicles (primordial, primary) were tenfold more numerous than growing follicles (secondary, tertiary, antral) in control animals (Fig. 1B). These data are similar to counts reported previously for age-matched control mice (13). The number of nongrowing follicles per ovary (mean SEM) was similar in CTL (297.8 24.1) and THAL-only treated animals (325.2 11.7; P¼.5). Nongrowing follicles were significantly reduced in the BUS group (5.3 0.6; P<.005). THALþ BUS animals also had fewer nongrowing follicles per ovary (60.7 11.0) than control animals (P<.01), but significantly more than the BUS-treated animals (P<.001). Similarly, growing follicle counts per ovary (Fig. 1B) were reduced in the BUS-treated group compared with CTL and THAL-treated animals (P<.01). Growing follicle counts were slightly increased in the THAL þ BUS compared with BUSonly group, but this increase did not reach the level of significance (P¼.06). However, some normal antral follicles were observed in the ovaries of THAL þ BUS–treated animals (Fig. 1F), similar to CTL (Fig. 1C) and THAL-only (Fig. 1D) animals, and these observations are consistent with the estrous cyclity data in Figure 1A. Ovaries of BUS-treated animals (Fig. 1E) revealed marked cortical fibrosis with absence of follicles. Uterine histology is also an indicator of the hormonal milieu and correlated with the presence or absence of growing follicles. CTL and THAL-treated animals exhibited endometrial glands with pseudostratified epithelium, mucous droplets, and fluid-filled
lumen (Figs. 1G and 1H). BUS-treated animals had comparatively hypotrophic endometrial glands (Fig. 1I), whereas THALþ BUS–treated animals exhibited endometrial architecture similar to control animals (Fig. 1J). Mean serum FSH levels in CTL (7.4 0.7 ng/mL) and THAL (7.4 0.5 ng/mL) animals were equivalent (P¼.98). Consistent with the decline in follicle number, FSH levels were elevated in BUS-treated animals (50 1.13 ng/mL) compared with controls (P<.05). Animals treated with THAL þ BUS also exhibited an elevated FSH (49.4 11.23 ng/mL; P<.05). Thus, although ovaries of THAL þ BUS–treated animals contained some growing follicles, which correlated with return to estrous cyclicity and preservation of uterine architecture, those follicles did not provide sufficient feedback to the brain to reduce FSH levels relative to BUS alone. The average weight of animals at the start and end of the study was not different between groups, and no adverse effects of thalidomide administration were observed. We established a model of chemotherapy-induced gonadal failure that can be used to evaluate putative gonadal-protective agents. Differential follicle counts provided a sensitive means of estimating the extent of ovarian toxicity (15), and their correlation with estrous cyclicity is an effective way of assessing reproductive function. Using these criteria, we provide evidence that thalidomide decreases the gonadal-toxic effects of an alkylating chemotherapy in female mice. Thalidomide is currently being used as a treatment for a variety of oncologic, dermatologic, and inflammatory conditions (16). However, the impact of thalidomide on ovarian function had not previously been established. We report, for the first time, normal ovarian function after thalidomide treatment of reproductive-age mice. Furthermore, thalidomide is an appealing adjunct to conventional chemotherapy regimens, because it has established anticancer properties. Thalidomide is a potent inhibitor of vascular endothelial growth factor and tumor necrosis factor a, both of which have documented effects on steroidogenesis, folliculogenesis, ovulation, leutinization, and fertility (4). By modulating these processes, thalidomide may cause a temporary senescent state in the ovary, rendering it less susceptible to chemotherapy-induced toxicity. Furthermore, thalidomide has been shown in animal models to decrease vasculature to tumors (16). If it has a similar impact on ovaries, it may effectively decrease the amount of toxic drug delivered to the gonads. Future studies will focus on elucidating the mechanisms by which thalidomide protects the ovary as well as functional assessments of preserved follicles. Acknowledgments: The authors thank Tony Battelli and Mike Bodenheimier for rodent colony maintenance and Meena Sukhwani for help with busulfan administration. FSH assays were performed by the University of Virginia Ligand Core Laboratory (grant no. U54-HD28934).
REFERENCES 1. Horner MJ RL, Krapcho M, Neyman N, Aminou R, Howlader N, Altekruse SF, et al, eds. SEER cancer statistics review, 1975–2006. Bethesda, MD: National Cancer Institute. June 2010. 2. Badawy A, Elnashar A, El-Ashry M, Shahat M. Gonadotropin-releasing hormone agonists for prevention of chemotherapy-induced ovarian damage: prospective randomized study. Fertil Steril 2009;91:694–7.
Fertility and Sterility
3. Oktay K, Sonmezer M, Oktem O, Fox K, Emons G, Bang H. Absence of conclusive evidence for the safety and efficacy of gonadotropin-releasing hormone analogue treatment in protecting against chemotherapy-induced gonadal injury. Oncologist 2007;12:1055–66. 4. Teo SK. Properties of thalidomide and its analogues: implications for anticancer therapy. AAPS J 2005;7:E14–9.
5. Dharia SP, Steinkampf MP, Cater C. Thalidomideinduced amenorrhea: case report and literature review. Fertil Steril 2004;82:460–2. 6. Generoso WM, Stout SK, Huff SW. Effects of alkylating chemicals on reproductive capacity of adult female mice. Mutat Res 1971;13:172–84. 7. Fekete E. Resources. In: Biology of the laboratory mouse, Snell GD, ed. New York: Dover Publications, 1941:89–167.
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8. Allen E. The oestrus cycle in the mouse. Am J Anat 1922;30:297–371. 9. Pedersen T, Peters H. Proposal for a classification of oocytes and follicles in the mouse ovary. J Reprod Fertil 1968;17:555–7. 10. Charleston JS, Hansen KR, Thyer AC, Charleston LB, Gougeon A, Siebert JR, et al. Estimating human ovarian nongrowing follicle number: the application of modern stereology techniques to an old problem. Hum Reprod 2007;22:2103–10. 11. Bolon B, Bucci TJ, Warbritton AR, Chen JJ, Mattison DR, Heindel JJ. Differential follicle
822
Ochalski et al.
Correspondence
counts as a screen for chemically induced ovarian toxicity in mice: results from continuous breeding bioassays. Fundam Appl Toxicol 1997;39:1–10. 12. Gougeon A, Delangle A, Arouche N, Stridsberg M, Gotteland JP, Loumaye E. Kit ligand and the somatostatin receptor antagonist, BIM-23627, stimulate in vitro resting follicle growth in the neonatal mouse ovary. Endocrinology;151:1299–1309. 13. Myers M, Britt KL, Wreford NG, Ebling FJ, Kerr JB. Methods for quantifying follicular numbers within the mouse ovary. Reproduction 2004;127:569–80.
14. Nelson JFL, Randall P, Sims C, Finch C. A longitudinal study of estrous cyclicity in aging C57/BL mice: cycle frequency, length and cytology. Biol Reprod 1982;27:327–39. 15. Mattison DR, Nightingale MS, Shiromizu K. Effects of toxic substances on female reproduction. Environ Health Perspect 1983;48: 43–52. 16. Teo SK, Stirling DI, Zeldis JB. Thalidomide as a novel therapeutic agent: new uses for an old product. Drug Discov Today 2005;10:107–14.
Vol. 95, No. 2, February 2011
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.09.020
tion, because despite this drug’s ability to ‘‘modulate fertility,’’ there is a lack of information about its effect on ovarian reserve. Forty-nine inbred C57Bl/6 mice (4 months old) were included in this study, which was approved by the University of Pittsburgh Institutional Animal Care and Use Committee (assurance no. A365401). Animals were divided into four groups. Control animals (CTL; n ¼ 13) were treated with vehicle, 1% carboxymethylcellulose (CMC), by oral gavage for 14 consecutive days. On day 7, CTL animals were treated with a single intraperitoneal (IP) dose of 50% dimethylsulfoxide (DMSO) vehicle. There were three experimental groups. The thalidomide-only group (THAL; n ¼ 12) was treated with thalidomide 100 mg/kg by oral gavage daily for 14 days and 50% DMSO vehicle on day 7. Thalidomide (Sigma Chemical Company, St. Louis. MO) was suspended in 1% CMC dissolved in deionized water. The busulfan-only group (BUS; n ¼ 12) received 1% CMC vehicle by oral gavage for 14 days and a single dose of busulfan (50 mg/ kg IP; Sigma) on day 7. This dose has been shown to produce detrimental effects on fertility (6). The THAL þ BUS group (n ¼ 12) was treated with thalidomide 100 mg/kg by oral gavage daily for 14 days. On the seventh day of thalidomide treatment, THAL þ BUS animals were treated with a single IP injection of busulfan (50 mg/kg). Estrous cyclicity was assessed by daily vaginal smears taken between 6:30 a.m. and 1:00 p.m., as previously described (7). Vaginal aspirates were read by one individual, who was blinded to treatment group. Smears were then classified into cycle stage (metestrus, diestrus, proestrus, estrus) according to the criteria of Allen (8). Animals were killed at diestrus, 90 days after the start of the study. At killing, whole blood samples as well as weights of whole body, uteri, and ovaries were obtained. Plasma FSH levels were determined by radioimmunoassay at the University of Virginia Ligand Core Laboratory (grant no. U54-HD28934). The uterus and ovaries from each animal were fixed in 4% paraformaldehyde, paraffin embedded, and serially sectioned (5 mm), followed by staining with hematoxylin and eosin. Follicles were categorized and enumerated based on the number of granulosa cell layers and the presence of the antral space, as described by Pedersen and Peters (9). Follicles were then categorized as ‘‘nongrowing’’ or ‘‘growing’’ to distinguish between the ovarian
Fertility and Sterility Vol. 95, No. 2, February 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
819
FIGURE 1 Assessments of reproductive function in control (CTL), thalidomide-treated (THAL), busulfan-treated (BUS), and THAL þ BUS–treated animals. (A) Vaginal smears to assess estrous cyclicity. (B) Differential ovarian follicle counts per ovary (mean SEM). Ovarian histology of (C) CTL, (D) THAL, (E) BUS, and (F) THAL þ BUS–treated animals. Uterine histology of (G) CTL, (H) THAL, (I) BUS, and (J) THAL þ BUS–treated animals.
Ochalski. Correspondence. Fertil Steril 2011.
reserve pool and those oocytes that were recruited. Briefly, the ‘‘nongrowing’’ population consisted of an oocyte surrounded by a partial or unbroken single layer of granulosa cells, which approximates to Pedersen and Peters types 1–3b. The ‘‘growing’’ follicles
820
Ochalski et al.
Correspondence
included oocytes surrounded by multiple layers of granulosa cells (Pedersen and Peters types 4–5b) as well as the antral follicles that were characterized by a central oocyte and fluid-filled space bordered by multiple layers of granulosa cells (Pedersen and Peters
Vol. 95, No. 2, February 2011
types 6–8). Similar simplified classification systems have been described previously (10–13). For the present study, we used the classification system described by Bolon et al. (11). Only follicles with a nucleolus present were counted, to avoid overcounting a single follicle present in multiple sections. Differential follicle counts were performed blinded to treatment group. Two-sided Wilcoxon rank sum test was used to identify differences in follicle counts between treatment groups. All animals had regular cycles before initiation of treatment (data not shown). Vaginal smears indicated that all CTL animals exhibited normal estrous cyclicity throughout the experimental period. THAL-only animals exhibited a brief period of persistent diestrus before returning to normal estrous cyclicity. All BUSonly animals (100%) were acyclic, exhibiting persistent cornified or leukocytic vaginal smears. In contrast, 58% of THAL þ BUS animals returned to normal estrous cyclicity, suggesting a protective effect of thalidomide. Cyclicity data for days 74–90 after the initiation of treatment are shown in Figure 1A. Differences in vaginal cytology reflect differing hormonal milieus and give insight into the underlying mechanism of ovarian failure. The prolonged diestrus exhibited by animals treated with busulfan may indicate insufficient E2 secretion owing to reduced numbers of maturing follicles, as reported previously (14). As expected, nongrowing follicles (primordial, primary) were tenfold more numerous than growing follicles (secondary, tertiary, antral) in control animals (Fig. 1B). These data are similar to counts reported previously for age-matched control mice (13). The number of nongrowing follicles per ovary (mean SEM) was similar in CTL (297.8 24.1) and THAL-only treated animals (325.2 11.7; P¼.5). Nongrowing follicles were significantly reduced in the BUS group (5.3 0.6; P<.005). THALþ BUS animals also had fewer nongrowing follicles per ovary (60.7 11.0) than control animals (P<.01), but significantly more than the BUS-treated animals (P<.001). Similarly, growing follicle counts per ovary (Fig. 1B) were reduced in the BUS-treated group compared with CTL and THAL-treated animals (P<.01). Growing follicle counts were slightly increased in the THAL þ BUS compared with BUSonly group, but this increase did not reach the level of significance (P¼.06). However, some normal antral follicles were observed in the ovaries of THAL þ BUS–treated animals (Fig. 1F), similar to CTL (Fig. 1C) and THAL-only (Fig. 1D) animals, and these observations are consistent with the estrous cyclity data in Figure 1A. Ovaries of BUS-treated animals (Fig. 1E) revealed marked cortical fibrosis with absence of follicles. Uterine histology is also an indicator of the hormonal milieu and correlated with the presence or absence of growing follicles. CTL and THAL-treated animals exhibited endometrial glands with pseudostratified epithelium, mucous droplets, and fluid-filled
lumen (Figs. 1G and 1H). BUS-treated animals had comparatively hypotrophic endometrial glands (Fig. 1I), whereas THALþ BUS–treated animals exhibited endometrial architecture similar to control animals (Fig. 1J). Mean serum FSH levels in CTL (7.4 0.7 ng/mL) and THAL (7.4 0.5 ng/mL) animals were equivalent (P¼.98). Consistent with the decline in follicle number, FSH levels were elevated in BUS-treated animals (50 1.13 ng/mL) compared with controls (P<.05). Animals treated with THAL þ BUS also exhibited an elevated FSH (49.4 11.23 ng/mL; P<.05). Thus, although ovaries of THAL þ BUS–treated animals contained some growing follicles, which correlated with return to estrous cyclicity and preservation of uterine architecture, those follicles did not provide sufficient feedback to the brain to reduce FSH levels relative to BUS alone. The average weight of animals at the start and end of the study was not different between groups, and no adverse effects of thalidomide administration were observed. We established a model of chemotherapy-induced gonadal failure that can be used to evaluate putative gonadal-protective agents. Differential follicle counts provided a sensitive means of estimating the extent of ovarian toxicity (15), and their correlation with estrous cyclicity is an effective way of assessing reproductive function. Using these criteria, we provide evidence that thalidomide decreases the gonadal-toxic effects of an alkylating chemotherapy in female mice. Thalidomide is currently being used as a treatment for a variety of oncologic, dermatologic, and inflammatory conditions (16). However, the impact of thalidomide on ovarian function had not previously been established. We report, for the first time, normal ovarian function after thalidomide treatment of reproductive-age mice. Furthermore, thalidomide is an appealing adjunct to conventional chemotherapy regimens, because it has established anticancer properties. Thalidomide is a potent inhibitor of vascular endothelial growth factor and tumor necrosis factor a, both of which have documented effects on steroidogenesis, folliculogenesis, ovulation, leutinization, and fertility (4). By modulating these processes, thalidomide may cause a temporary senescent state in the ovary, rendering it less susceptible to chemotherapy-induced toxicity. Furthermore, thalidomide has been shown in animal models to decrease vasculature to tumors (16). If it has a similar impact on ovaries, it may effectively decrease the amount of toxic drug delivered to the gonads. Future studies will focus on elucidating the mechanisms by which thalidomide protects the ovary as well as functional assessments of preserved follicles. Acknowledgments: The authors thank Tony Battelli and Mike Bodenheimier for rodent colony maintenance and Meena Sukhwani for help with busulfan administration. FSH assays were performed by the University of Virginia Ligand Core Laboratory (grant no. U54-HD28934).
REFERENCES 1. Horner MJ RL, Krapcho M, Neyman N, Aminou R, Howlader N, Altekruse SF, et al, eds. SEER cancer statistics review, 1975–2006. Bethesda, MD: National Cancer Institute. June 2010. 2. Badawy A, Elnashar A, El-Ashry M, Shahat M. Gonadotropin-releasing hormone agonists for prevention of chemotherapy-induced ovarian damage: prospective randomized study. Fertil Steril 2009;91:694–7.
Fertility and Sterility
3. Oktay K, Sonmezer M, Oktem O, Fox K, Emons G, Bang H. Absence of conclusive evidence for the safety and efficacy of gonadotropin-releasing hormone analogue treatment in protecting against chemotherapy-induced gonadal injury. Oncologist 2007;12:1055–66. 4. Teo SK. Properties of thalidomide and its analogues: implications for anticancer therapy. AAPS J 2005;7:E14–9.
5. Dharia SP, Steinkampf MP, Cater C. Thalidomideinduced amenorrhea: case report and literature review. Fertil Steril 2004;82:460–2. 6. Generoso WM, Stout SK, Huff SW. Effects of alkylating chemicals on reproductive capacity of adult female mice. Mutat Res 1971;13:172–84. 7. Fekete E. Resources. In: Biology of the laboratory mouse, Snell GD, ed. New York: Dover Publications, 1941:89–167.
821
8. Allen E. The oestrus cycle in the mouse. Am J Anat 1922;30:297–371. 9. Pedersen T, Peters H. Proposal for a classification of oocytes and follicles in the mouse ovary. J Reprod Fertil 1968;17:555–7. 10. Charleston JS, Hansen KR, Thyer AC, Charleston LB, Gougeon A, Siebert JR, et al. Estimating human ovarian nongrowing follicle number: the application of modern stereology techniques to an old problem. Hum Reprod 2007;22:2103–10. 11. Bolon B, Bucci TJ, Warbritton AR, Chen JJ, Mattison DR, Heindel JJ. Differential follicle
822
Ochalski et al.
Correspondence
counts as a screen for chemically induced ovarian toxicity in mice: results from continuous breeding bioassays. Fundam Appl Toxicol 1997;39:1–10. 12. Gougeon A, Delangle A, Arouche N, Stridsberg M, Gotteland JP, Loumaye E. Kit ligand and the somatostatin receptor antagonist, BIM-23627, stimulate in vitro resting follicle growth in the neonatal mouse ovary. Endocrinology;151:1299–1309. 13. Myers M, Britt KL, Wreford NG, Ebling FJ, Kerr JB. Methods for quantifying follicular numbers within the mouse ovary. Reproduction 2004;127:569–80.
14. Nelson JFL, Randall P, Sims C, Finch C. A longitudinal study of estrous cyclicity in aging C57/BL mice: cycle frequency, length and cytology. Biol Reprod 1982;27:327–39. 15. Mattison DR, Nightingale MS, Shiromizu K. Effects of toxic substances on female reproduction. Environ Health Perspect 1983;48: 43–52. 16. Teo SK, Stirling DI, Zeldis JB. Thalidomide as a novel therapeutic agent: new uses for an old product. Drug Discov Today 2005;10:107–14.
Vol. 95, No. 2, February 2011