The National Institute of Health Sciences (NIHS) and 18 pharmaceutical companies of the Japan Pharmaceutical Manufacturers Association (JPMA) have conducted a validation study intended to evaluate whether a 2-week repeated general toxicity period with histopathological examination is sufficient to detect ovarian toxicity or not. The current repeated dose general toxicity study is considered to be insufficient in terms of evaluating female reproductive function due to a lack of evidence indicating that it is adequate. Evaluation of ovarian toxicity by comprehensive histopathological examination of the female reproductive organs based on the underlying morphology of a normal cycle of the reproductive tract including the ovary and additional immunohistochemical staining with proliferative cell nuclear antigen (PCNA) to identify small follicles may be a good tool to assess female reproductive function. In the collaborative study, 2- or 4-week repeated dose toxicity studies with ovarian histopathological examinations were conducted. A female fertility study was also conducted to compare the results with those of the ovarian histopathological findings. A total of 17 test substances were evaluated and categorized into hormone analogues, primordial follicle damaging agents, metabolite imbalance inducers, and endocrine imbalance inducers. Based on the results, ovarian toxicity could be detected by a careful histopatholgical examination. A 2-week dosing period may be sufficient for the evaluation of ovarian toxicity, except for cytotoxic compounds such as alkylating agents. The pathological findings of ovarian toxicity (decreases in follicles, increases in atretic follicles, increases in currently formed corpora lutea, etc) reflected the female fertility parameters (irregular estrous cycle, pre-implantation loss).

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As a part of the collaborative study to evaluate the relationship between histopathological changes of the ovary and functional changes in female fertility, 2- and 4-week repeated-dose toxicity studies and a female fertility study were conducted using female Crl:CD(SD) rats using a synthetic progestagen of medroxyprogesterone acetate (MPA) as a test compound. MPA was administered to female rats by gavage at 0, 0.4 , 2.0 and 10 mg/kg/day for 2 and 4 weeks to assess the histopathological changes of ovaries and to pregnant rats at the same doses from 2 weeks prior to mating until day 7 of gestation to examine female fertility. The number of non-pregnant female rats with irregular estrous cycle increased in number and there was a decrease in weight of ovaries was observed at doses ≥ 2.0 mg/kg in the 2- and 4-week-treatment groups. The histopathological examination revealed an increased number of large atretic follicles and decreases in currently formed corpora lutea and previously-formed large or small ones were observed at the same doses in the 2- and 4-week treatment groups. In female fertility study, the number of animals with an irregular estrous cycle and elongation of mean estrous cycle increased at 0.4 mg/kg, with no changes in fertility. A decreased number of copulating animals and a decreased gestation rate with low preimplantation loss were observed in the 2.0 mg/kg-treatment group and no copulation was observed in the group treated with 10 mg/kg. Based on these results, changes in fertility induced by MPA correlated well with histopathological changes of ovaries after 2 and 4 weeks of treatment, which suggests that a 2 weeks administration period is sufficient to detect ovarian toxicity of MPA with repeated dosing.

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In order to assess ovarian pathological changes and their relationship to changes in female fertility parameters, mifepristone, a progesterone receptor antagonist, was selected as the test article and was administered orally to female rats at dose levels of 0, 0.8, 4, 20 and 100 mg/kg for 2 or 4 weeks in repeated dose-toxicity studies and in a female fertility study at dose levels of 0, 0.8, 4 and 20 mg/kg from > 2 weeks before copulation to postcoital day 7. In the repeated dose toxicity studies, persistent estrus was seen in the vaginal smears, and multiple cysts in the ovaries at necropsy, increases in luteinized cysts and hypertrophy of previously formed corpora lutea were observed in the histopathological examination of ovaries in rats receiving 20 mg/kg or more for 2 or 4 weeks. In female fertility studies, persistent vaginal cornification was also observed at 20 mg/kg and the precoital interval was significantly shortened. All of the animals were completely infertile when dosed with 20 mg/kg during the post-coital period. An increase in pre-implantation losses was observed in the animals treated with 20 mg/kg during the pre-coital phase, while treatment with 4 mg/kg mifepristone during the post-coital phase induced an increase in post-implantation losses. These results suggested that a 2-week administration period would be sufficient to detect the ovarian toxicity of mifepristone in repeated dose toxicity study and the pathological findings in the ovaries would reflect the alterations in female reproductive endpoints in the female fertility study.

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To assess whether ovarian histopathological examination in repeated-dose rodent toxicity study could reliably anticipate toxic effects on female reproductive function and to assess whether ovarian change could be detected in a 2-week repeated-dose toxicity study, tamoxifen was administrated orally to female rats at 0.005, 0.03, or 0.2 mg/kg/day for 2 and 4 weeks in the repeated-dose toxicity studies, and for 2 weeks prior to cohabitation, during cohabitation, and through Gestation Day 7 in a female fertility study. The relationship between ovarian histopathological findings and fertility results was investigated. Findings at 0.03 and 0.2 mg/kg/day included decreases in body weight gains associated with decreases in food consumption, in 2- and 4-week repeated-dose toxicity studies and fertility study. The ovarian histopathological findings included increases in large atretic follicles, increases in interstitium cells and absence of newly-formed corpus lutea at 0.2 mg/kg/day in the 2-week study and at 0.03 and 0.2 mg/kg/day in the 4-week study. The treatment induced estrogenic and antiestrogenic reactions in the uterus, while mucinous degeneration was detected in the vagina. Effects on female fertility consisted primarily of disturbance of estrus cycle and decreases in numbers of pregnant rats which were considered to be related to ovarian histopathological changes. Based on these findings, ovarian histopathological evaluation in the repeated-dose toxicity study could anticipate the effects of tamoxifen on female fertility via ovarian dysfunction at slightly toxic doses, and 2-week treatment of tamoxifen at appropriate dose could be sufficient to detect ovarian toxicity by microscopic examination.

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To determine the optimal administration period for evaluation of ovarian toxicity of 4-vinylcyclohexene diepoxide (VCD), VCD was intraperitoneally administered to female Sprague-Dawley rats at 0 (Control), 5, 20 and 80 mg/kg once a day for 2 or 4 weeks (2- or 4-week study). To identify small follicles, serial sections of the ovaries were stained with routine hematoxylin and eosin (HE) and proliferating cell nuclear antigen (PCNA) immunohistochemistry. In the 4-week study, decrease in small follicles was observed in the ovaries at 20 and 80 mg/kg. In the 2-week study, the same change was also observed at 80 mg/kg. Identification of small follicles using PCNA-stained slides was easier than that using HE-stained slides. In conclusion, histopathological findings in the ovaries are important for evaluation of female reproductive toxicity of VCD, and ovarian toxicity of VCD can be detected by administration for 2 weeks at an appropriate dose level. Furthermore, PCNA immunohistochemistry is effective for evaluation of small follicle destruction in chemical-induced ovarian toxicity.

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As part of a collaborative project, 4-vinylcyclohexene diepoxide (VCD), an ovarian toxicant, was intraperitoneally administered to female Sprague-Dawley rats at 0, 5, 20 or 80 mg/kg from 2 weeks prior to mating to Day 7 of gestation. At necropsy, the number of implanted embryos, rate of implantation decreased and the rate of preimplantation loss showed an increasing tendency in the 80 mg/kg group. As for organ weight, decreases in absolute and relative ovary weight were observed in the 80 mg/kg group. Histopathologically, the ovaries showed a decrease in number of small follicles at 80 mg/kg.

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Busulfan, an antineoplastic agent that targets small follicles (primordial and primary follicles), was given orally to female Sprague-Dawley rats (0, 0.1, 0.5, or 1.5 mg/kg/day; n = 10 in each group) for 2 or 4 weeks to assess the optimal administration period for detection of the toxic effects on ovarian morphology. Isolated ovaries were used for histopathological analysis and follicle counts. In addition, a female fertility study was conducted by giving the same dose levels of busulfan from 2 weeks before mating to day 7 of pregnancy to determine the non-observed-adverse-effect-level (NOAEL) for female reproduction. In the 2-week study, all rats treated with busulfan showed normal estrous cyclicity and no toxicological changes in weight or histopathology of the ovaries. In the 4-week study, a decrease in small follicles was found histopathologically in 1 rat, even at 0.5 mg/kg, and in 4 rats at 1.5 mg/kg. Proliferating cell nuclear antigen immunohistochemistry of the follicles confirmed the above decrease in number of small follicles at 1.5 mg/kg. In the female fertility study, increases in dead embryos and post-implantation loss were found in rats at 1.5 mg/kg. Taken together, the NOAELs were 1.5 mg/kg for reproductive performance and 0.5 mg/kg for early embryonic development. In conclusion, the present study indicates that a 4-week administration period and appropriate assessment, including careful histopathological analysis of stage-based follicles are needed to detect small follicle depletion in a general toxicity study used as a first-titer screen.

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The main aim of the present study is to determine the optimal administration period of cisplatin with regards to its toxic effects on ovarian morphology in the repeated-dose toxicity study. Cisplatin was administered to female SD rats intraperitoneally once daily at dose levels of 0.25, 0.5, 1.0 and 2.0 mg/kg for 2 weeks, or at dose levels of 0.125, 0.25 and 0.5 mg/kg for 4 weeks in the repeated-dose toxicity study. In the female fertility study, 0.25, 0.5 and 1.0 mg/kg of cisplatin were administered in the same manner from 14 days prior to mating to Day 7 of gestation. In the repeated-dose toxicity study, a decrease in large follicle, an increase in atresia of medium and large follicles, and/or a decrease in currently formed corpus luteum were observed in animals receiving 1.0 and 2.0 mg/kg for 2 weeks, and decreases in small and/or large follicles and an increase in atresia of large follicle were observed in animals receiving 0.25 and 0.5 mg/kg for 4 weeks on the histopathological examination of the ovaries. In the female fertility study, the copulation and fertility indices in the animals receiving 1.0 mg/kg tended to be lower than those in the control animals. In conclusion, histopathological changes in the ovary that were attributable to cisplatin dosing were detected by detailed observation of the ovary in the 2-week study; and therefore, a 2-week administration period is sufficient to evaluate the ovarian toxicity of cisplatin.

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The main focus of this study was to determine the optimal administration period concerning the toxic effects on ovarian morphological changes in the repeated dose toxicity study. In order to assess the morphological and functional changes induced in the ovary by cyclophosphamide (CP), the compound was administrated to female rats at dose levels of 0, 5, 10 and 20 mg/kg for the repeated dose toxicity study for 2 or 4 weeks, and at 0, 5, 10, and 20 mg/kg for the female fertility study from 2 weeks prior to mating to Day 7 of pregnancy. In the repeated dose toxicity study, increases in large sized atretic follicles, atrophy of corpora lutea were observed in the 20 mg/kg group in the 4-week study by the histopathological examination of the ovaries. There were no drug-related changes in the ovary in the 2-week study. In the female fertility study, the numbers of implantation were slightly decreased and the corpora lutea of pregnancy was not observed in the 20 mg/kg group. The dose-dependent increase in the incidence of post-implantation loss was observed, and no abnormalities were observed in the estrus cycle and mating in all treated groups. From these findings, the histopathological changes in the ovary are important endpoints for the evaluation of drug-induced ovarian damage as well as caesarean section. In conclusion, a 4-week administration period is sufficient to detect the ovarian toxicity of CP in the repeated dose toxicity study.

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The main focus of this study was to determine the optimal administration period concerning the toxic effects on ovarian morphological changes in the repeated dose toxicity study. In order to assess the morphological and functional changes induced in the ovary by cyclophosphamide (CP), the compound was administrated to female rats at dose levels of 0, 5, 10 and 20 mg/kg for the repeated dose toxicity study for 2 or 4 weeks, and at 0, 5, 10, and 20 mg/kg for the female fertility study from 2 weeks prior to mating to Day 7 of pregnancy. In the repeated dose toxicity study, increases in large sized atretic follicles, atrophy of corpora lutea were observed in the 20 mg/kg group in the 4-week study by the histopathological examination of the ovaries. There were no drug-related changes in the ovary in the 2-week study. In the female fertility study, the numbers of implantation were slightly decreased and the corpora lutea of pregnancy was not observed in the 20 mg/kg group. The dose-dependent increase in the incidence of post-implantation loss was observed, and no abnormalities were observed in the estrus cycle and mating in all treated groups. From these findings, the histopathological changes in the ovary are important endpoints for the evaluation of drug-induced ovarian damage as well as caesarean section. In conclusion, a 4-week administration period is sufficient to detect the ovarian toxicity of CP in the repeated dose toxicity study.

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The present study was designed to confirm whether or not the ovarian toxicity of di(2-ethylhexyl)adipate (DEHA), which is known to have effects on female fertility, could be evaluated by the new method of histopathological examination of the ovaries in repeated dose toxicity. DEHA was orally administered to Crl:CD(SD) female rats at the doses of 0, 200, 1,000 and 2,000 mg/kg for 2 or 4 weeks in repeated dose toxicity study and for 2 weeks before mating, throughout mating and until Gestation Days 7 in female fertility. In the repeated dose toxicity studies, increase in atresia of large follicle, decrease in currently formed corpus luteum and follicular cyst were observed in the 1,000 mg/kg and above groups, suggesting that DEHA disturbed ovulation and large follicle growth. In the fertility study, a significant increase in mean estrus cycle length and post-implantation loss rate were observed in the 1,000 mg/kg and above groups, and a significant decrease in implantation rate and number of live embryos and a significant increase in pre-implantation loss rate were observed in the 2,000 mg/kg group. The histopathological changes of ovary observed in the repeated dose toxicity studies were correlated with the result that DEHA affected the estrus cycle in the female fertility study. In conclusion, a 2-week administration period is sufficient for detection of the ovarian toxicities following treatment with DEHA by new histopathological examination of the ovaries.

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The main purpose of this collaborative work is to determine the optimal administration period required to detect toxic effects in evaluation of ovarian morphological changes in repeated-dose toxicity studies. To assess the morphological and functional changes induced in the ovaries by di-(2-ethylhexyl) phthalate (DEHP), two repeated-dose toxicity studies (repeated dose for 2 or 4 weeks) of DEHP administrated to female rats at dose levels of 0, 300, 1,000 and 3,000 mg/kg were conducted in collaboration with a female fertility study at the same dosages from 2 weeks prior to mating to Day 7 of pregnancy. Histopathology of the ovaries in both repeated-dose toxicity studies showed vacuolation of stromal cells in the groups receiving 300 mg/kg or more and an increase of large atretic follicles in groups at 1,000 mg/kg or more. In the 4-week study, a decrease in new corpora lutea was observed in the 3,000 mg/kg group. In the female fertility study, the 3,000 mg/kg group showed prolongation of the mean estrous cycle and irregular estrous cycles. Cesarean section revealed a decrease of pregnancy rate in the 3,000 mg/kg group. No effects on fertility or early embryonic development were found in groups at 1,000 mg/kg or less. These findings indicate that histopathological changes in the ovary are important endpoints for the evaluation of drugs which induce ovarian damage. In conclusion, for a repeated-dose toxicity study, a 2-week administration period is sufficient to detect ovarian toxicity caused by DEHP.

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The objective of this study was to determine the optimal period of administration for detection of ovarian toxicity in rat repeated-dose toxicity studies. A well-known ovarian toxicant, ethylene glycol monomethyl ether (EGME), was administered to female rats at dose levels of 0, 30, 100, or 300 mg/kg for 2 or 4 weeks (repeated-dose toxicity studies). The same doses were administered to female rats for 2 weeks prior to mating, during mating, and until Day 6 of pregnancy (fertility study). In the repeated-dose toxicity studies, continuous diestrus was observed at ≥ 100 mg/kg regardless of period of administration. The alterations of ovarian morphology observed at ≥ 100 mg/kg after 2 or 4 weeks of administration were characterized by hypertrophy of the corpora lutea with decreased cellular debris indicating apoptosis, and increased proliferating cell nuclear antigen (PCNA)-negative large atretic follicles. The finding that newly-formed basophilic corpora lutea were scarce in affected animals exhibiting continuous diestrus suggested suppression of ovulation due to hypertrophic corpora lutea. In the fertility study, irregular estrous cycles, prolonged mating periods, lower pregnancy rates and decreased corpora lutea of pregnancy were observed at ≥ 100 mg/kg. The irregularities of estrous cycle observed in some animals at 30 mg/kg were minimal. The ovarian histopathological changes in repeated-dose toxicity studies correlated well with impairment of female fertility found in the fertility study. It is concluded that a repeated-dose toxicity study with a treatment period for 2 weeks or longer is sufficient for evaluation of ovarian toxicity induced by EGME.

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2-week and 4-week general toxicity studies of indomethacin, a nonselective inhibitor of cyclooxygenase 1 and 2, were performed using rats. A female fertility study was also conducted to compare the results to those of ovarian histopathological findings. The main purposes of the present studies are to assess whether a precise histopathological examination, taking the morphological changes the female reproductive organs undergo during each estrus phases into account, can evaluate toxicity to the ovaries, and to determine the optimal administration period for detecting ovarian toxicity. Indomethacin was administered on a daily basis to female Sprague-Dawley rats at doses of 0, 0.4, 1.3, or 4 mg/kg in the both the general toxicity studies and the female fertility study. In the general toxicity studies, unruptured follicles or luteinized cysts were observed histopathologically in the 4 mg/kg group in both the 2-week and 4-week studies. In addition, follicular cysts were found in the 4 mg/kg group in the 4-week study. Estrous cyclicity was not disturbed in both studies. There were no histopathological changes in the ovaries of the 1.3 mg/kg group in general toxicity studies. In the female fertility study, no toxic effects on female fertility parameters were detected in the 0.4 and 1.3 mg/kg group treated with indomethacin, but 8 of 10 rats in the 4 mg/kg group died or were sacrificed before completion of the dosing period. These results demonstrated that 2 weeks of indomethacin treatment is sufficient to detect unruptured follicles or luteinized cyst in the ovary. In addition, 4 weeks of dosing maybe required for induction of follicular cysts, although we could not clearly show that these histopathological changes would affect female fertility functions. These present studies suggest that a precise histopathological examination may be able to predict the effect of test articles on female reproductive functions.

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The main focus of this study was to determine the optimal dosing period in a repeated dose toxicity study based on toxic effects as assessed by ovarian morphological changes. To assess morphological and functional changes induced in the ovary by a peroxisome proliferator-activated receptor (PPAR) α/γ dual agonist, the compound was administered to female rats at dose levels of 0, 4, 20, and 100 mg/kg/day in a repeated dose toxicity study for 2 or 4 weeks, and from 2 weeks prior to mating to Day 7 of pregnancy in a female fertility study. In the repeated dose toxicity study, an increase in atresia of large follicles, a decrease in corpora lutea, and an increase in stromal cells were observed in the treated groups. In addition, the granulosa cell exfoliations into antrum of large follicles and corpora lutea with retained oocyte are morphological characteristics induced by this compound, and they might be related with abnormal condition of ovulation. In the female fertility study, the pregnancy rate tended to decrease in the 100 mg/kg/day group. At necropsy, decreases in the number of corpora lutea, implantations and live embryos were noted in the 20 and 100 mg/kg/day group. No changes were observed in animals given 4 mg/kg/day. These findings indicated that histopathological changes in the ovary are important endpoints for evaluation of drugs inducing ovarian damage. In conclusion, a 2-week administration period is sufficient to detect ovarian toxicity of this test compound in the repeated dose toxicity study.

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To investigate the optimal administration period for evaluating ovarian toxicity that reflects abnormal female fertility in the repeated dose toxicity study, atrazine, a potent herbicide with endocrine-disrupting activity, was administered to female Sprague-Dawley (Slc:SD) rats for two or four weeks at doses of 3, 30 or 300 mg/kg for the repeated dose toxicity study, and at doses of 3, 30 or 100 mg/kg for the female fertility study from two weeks before mating to Day 7 of gestation. In the two-week repeated dose toxicity study, prolongation of diestrus and histopathological findings such as loss of the currently formed corpora lutea, decrease in the numbers of previously formed corpora lutea, increase in large-sized atretic follicles, and swelling of the previously formed luteal cells were observed in the 300 mg/kg group, suggesting that atrazine had an anovulatory effect through suppression of the luteinizing hormone surge. In the female fertility study, copulation failure caused by prolongation of diestrus was observed in one animal in the 100 mg/kg group, which could be due to the anovulatory effect of atrazine. It is demonstrated that the effect of atrazine on female fertility can be assessed by detailed histopathological examination of ovaries in a two-week repeated dose toxicity study, provided the appropriate dose levels are selected.

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The main focus of this study is to determine the optimal administration period concerning toxic effects on ovarian morphological changes in a repeated-dose toxicity study. To assess morphological and functional changes induced in the ovary by bromocriptine, the compound was administered to female rats at dose levels of 0, 0.08, 0.4 and 2 mg/kg for the 2- or 4-week repeated-dose toxicity study, and for the female fertility study from 2 weeks prior to mating to day 7 of gestation. In the 2-week repeated-dose toxicity study, increase of ovarian weights was observed at 2 mg/kg. In the 4-week repeated-dose toxicity study, ovarian weights were increased at 0.4 and 2 mg/kg. The number of corpora luteum was increased in the 0.4 and 2 mg/kg groups of the 2- and 4-week repeated-dose toxicity studies by histopathological examination of the ovaries. Bromocriptine did not affect estrous cyclicity in 2- and 4-week repeated dosing. In the female fertility study, although animals in any groups mated successfully, no females in 0.4 and 2 mg/kg groups were pregnant. There were no adverse effects on reproductive performance in the 0.08 mg/kg group. Based on these findings, the histopathological changes in the ovary are considered important parameters for evaluation of drugs including ovarian damage. We conclude that a 2-week administration period is sufficient to detect ovarian toxicity of bromocriptine in a repeated-dose toxicity study.

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In order to examine potential ovarian toxicity in 2 weeks or 4 weeks repeated-dose studies and a fertility study, chlorpromazine hydrochloride (CPZ) was administered orally to Crl:CD(SD) female rats at dosage levels of 0, 3, 10 and 30 mg/kg/day. In the repeated-dose studies, ovarian weights were decreased at ≥ 10 mg/kg in the 4 weeks study and an increase in large atretic follicles was observed histopathologically at ≥ 3 mg/kg and ≥ 10 mg/kg in the 2 and 4 weeks studies, respectively. In addition, decreased uterine weights and/or atrophic findings in the uterus and vagina at 30 mg/kg and ≥ 10 mg/kg, mucification in the vaginal epithelium and alveolar hyperplasia in the mammary gland at ≥ 3 mg/kg and ≥ 10 mg/kg were seen in the 2 and 4 weeks studies, respectively. Irregular estrous cycles were seen at ≥ 3 mg/kg and ≥ 10 mg/kg in the 2 and 4 weeks studies. The no-observed-adverse-effect level (NOAEL) for the 2 and 4 weeks studies was considered to be less than 3 mg/kg and 3 mg/kg, respectively. The fertility study with dosing from 2 weeks before mating to day 6 of gestation showed irregular estrous cycles at ≥ 10 mg/kg and prolonged copulatory intervals and a reduced fertility index at 30 mg/kg; the NOAEL was therefore considered to be 3 mg/kg, which was higher than that in the 2 weeks study. These results showed that oral CPZ treatment induced ovarian toxicity with 2 weeks or longer treatment and changed the fertility parameters and was therefore concluded that a 2 weeks administration period is adequate to detect the ovarian toxicity of CPZ.

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To find the appropriate dosing period to detect ovarian toxicity, sulpiride, a D2 antagonist was orally dosed to female rats at dose levels of 1, 10, and 100 mg/kg/day daily for 2 or 4 weeks in repeated-dose toxicity studies. In addition, sulpiride at the same dose levels was given to female rats daily during the pre-mating period, mating period, and Days 0-7 of gestation to assess its effect on fertility. In ovarian histology in the 2-week study, increases in atretic follicle were seen at 1 mg/kg or more and increases in follicular cysts at 10 mg/kg or more. In the 4-week study, these findings were seen at 1 mg/kg or more, and a decrease in large follicles was seen at 10 mg/kg or more. Increased body weight gain was observed at 10 mg/kg or more in the 2- and 4-week studies. The females in these groups exhibited development of mammary alveolus by sulpiride-induced hyperprolactinemia. In the fertility study, sulpiride-treated females showing persistent diestrus resulted in successful mating, and almost all females got pregnant. However, increased implantation loss was observed at 10 mg/kg or more, which was considered to be caused by the adverse effect of sulpiride on oocyte development. From these results, sulpiride-induced ovarian toxicity was seen at 1 mg/kg or more in the 2- and 4-week repeated-dose toxicity studies, and the observed ovarian changes were considered to be related to adverse effects on female fertility.

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Identification of ovarian toxicity is very important for safety assessment of drugs and other environmental chemicals. The detection of interference with ovarian function is very hard without a thorough understanding of the normal ovarian morphology based on reproductive physiology. The focus of the present study was therefore a practical analysis in each stage of the estrous cycles using ovaries obtained from 143 rats demonstrating normal cycling. Transversely dissected maximum areas in the ovaries were examined microscopically for the two major features, follicles and corpora lutea (CL). Classification of growing follicles was in reference to Pedersen and Peters (1968), and functionally divided into follicular stimulating hormone (FSH)-independent and dependent categories. The former, small and medium-sized follicles, respectively primordial/primary and preantral follicles, could be readily detected by immunohistochemical staining for proliferating cell nuclear antigen (PCNA). The large antral and Graafian follicles and large sized atretic follicles showed sequential changes depending on the estrous cycle stage. CL could be divided into currently and previously formed examples. Currently formed CL underwent remarkable changes in their appearance with the cycle, reflecting ovulation and progesterone production. Thus morphological analysis that is synchronized the large antral follicle changes with recently formed CL ones allows the ovary to be classified into the each estrous cycle stage. Morphological deviation from any synchronized combination provides a first pointer of ovarian toxicity. PCNA immunohistochemical staining is also useful to detect small follicles.

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