J Toxicol Pathol 2006; 19: 169–177 Original Changes in the Reproductive Organs Depending on Phases of Reproductive Cycle and Aging in Female Cynomolgus Monkeys Dai Watanabe1,2, Toru Hoshiya1, Junko Sato1,#, Yuko Yamaguchi1, Kohsuke Horiguchi1, Yoshikazu Nagashima1, Azusa Okaniwa1, and Hiroyasu Yoshikawa2 1Kannami Laboratory, Bozo Research Center Inc., 1308 Kuwahara-Sanbonmatsu, Kannami-cho, Tagata-gun, Shizuoka 419–0101, Japan 2Department of Veterinary Pathology, School of Veterinary Medicine and Animal Science, Kitasato University, Towada-shi, Aomori-ken 034–8628, Japan #Present: Mitsubishi Chemical Safety Institute Ltd., 14 Sunayama, Kamisu-shi, Ibaraki-ken 314–0255, Japan Abstract: In order to prepare background data for toxicity studies, reproductive organs of a total of 106 purpose-bred female cynomolgus monkeys 2.0 to 9.0 years of age were surveyed for the sequence of events during reproductive cycles. Based on histological features, these animals were classified into groups according to the six phases of the reproductive cycle in addition to a group at the immature stage. Follicular phases were characterized by development of follicles and luteal phases by development of functional corpus luteum. Proliferation of endometrial glands started at the follicular phase and reached the maximum at the luteal phase. Features in the menstrual phase were almost the same as those in the follicular phases except for the shedding of superficial surface of endometrium. The percentage of animals in the phases of follicular development was 51%, and those in luteal development 46%, indicating roughly equal duration of both phases of the reproductive cycle. Absolute and relative weights of the ovaries and uterus were lowest in early follicular phase and gradually increased and reached the maximum in the early luteal phase, then decreased to the level of the early follicular phase. The difference in weight between the bigger and smaller ovaries, remarkable between 5.0 to 5.5 years of age, was at the maximum in the early luteal phase. The absolute weight of the ovaries and uterus increased rapidly and reached its peak between 5.0 to 5.5 years of age and remained unchanged thereafter, although the body weight gradually increased up to 9.0 years of age. (J Toxicol Pathol 2006; 19: 169–177) Key words: reproductive cycle-related changes, cynomolgus monkeys, ovaries, uterus Introduction of each phase of the reproductive cycle by means of various in-life observation and examinations is impractical in routine Considering that few detailed descriptions about toxicity studies. Because the reproductive organs are subject normal cyclic changes of the female reproductive organs are to remarkable changes during the reproductive cycle, available1, the present survey was undertaken to elucidate abundant background data are essential for precise sequential changes in the weight and histology of the major assessment of findings on the reproductive organs. In reproductive organs associated with reproductive cycles and addition, cynomolgus monkeys used in toxicological aging in female cynomolgus monkeys kept in an animal research are frequently at the age of adolescence and laboratory environment at our research facilities. We therefore their reproductive organs are functionally as well concentrated on identification of each phase of the as morphologically in the course of development. reproductive cycle by means of morphology and weight changes in the major reproductive organs, as determination Materials and Methods Animals and histological specimens Received: 19 May 2006, Accepted: 12 October 2006 The present study examined a total of 106 female Mailing address: Dai Watanabe, Kannami Laboratory, Bozo Research cynomolgus monkeys 2.0 to 9.0 years of age. Seventy-three Center Inc., 1308 Kuwahara-Sanbonmatsu, Kannami-cho, Tagata-gun, per cent of them were between 3 and 5.5 years of age. The Shizuoka 419–0101, Japan TEL: 81-559-78-7851 FAX: 81-559-78-7898 number of animals and body weight for each age-group are E-mail: [email protected] shown in Table 1. These animals were purpose-bred and 170 Reproductive Organs of Female Cynomolgus Monkeys Table 1. Body Weight of Monkeys in Each Age group Table 2. Major Morphological Characteristics of Reproductive Organs in Immature Stage and in Each Phase of Age group No. of Range Mean Median Reproductive Cycle (years) Animals Stage and phases Major characteristics 2.0–3.0 6 2.27–3.11 2.73 2.83 3.0–3.5 16 2.20–3.65 2.77 2.65 Immature Thickness of endometrial layer about 1/2 of that in 3.5–4.0 18 2.13–3.93 2.88 2.78 follicular phase; cornification of vaginal epithelial 4.0–4.5 20 2.12–5.83 3.43 3.37 layer incomplete, primordial follicles conspicuous. 4.5–5.0 12 2.57–5.51 3.33 3.13 Menstrual Hemorrhage and shedding of superficial surface of 5.0–5.5 11 2.51–5.90 3.48 3.35 (Early follicular) endometrial layer, and menstrual corpus luteum 5.5–6.0 9 2.53–4.12 3.16 3.02 with vacuolated luteal cells. 6.0–6.5 9 2.73–5.95 4.22 4.04 Early follicular Endometrial layer: thinner than that in follicular 6.5–9.0 5 3.17–7.62 4.34 3.68 phase and a few endometrial glands running straight. Development of many follicles, some of Unit: kg them are undergoing atresia, and vacuolation of luteal cells is marked in the ovaries. Follicular Thickened endometrial layer, but endometrial glands only slightly coiled. imported from the People’s Republic of China and Pre-ovulation Thickened endometrial layer, and a large maturing Nafovanny, Vietnam, subject to quarantine in accordance follicle. with national regulations on import quarantine. The animals Early luteal Corpus luteum with central cavern and hemorrhage. were kept at our facility and were housed in individual cages Beginning of coiling and dilation of endometrial (750 × 750 × 700 mm) under routine controlled conditions: glands prominent. 18 to 28°C; relative humidity, 30 to 80%; lighting, 12 hours Luteal Highly developed functional corpus luteum, and per day. They were provided a pellet diet (restricted feeding markedly coiled endometrial glands. 150 g/animal/day, PS, Oriental Yeast Co. Ltd., Tokyo, Japan), and tap water ad libitum per an automatic supply system. All animals were non-treated animals collected He compared primate menstrual cycles with estrous cycles in from control groups used in toxicological studies during the other species. Our classification of phases of reproductive years 1996 to 2002. All experimental procedures were done cycles, however, is not necessarily identical with current in accordance with the animal welfare guidelines of Bozo functional classification consisting of follicular and luteal Research Center Inc. All the histological specimens of the phases. For example, the phase of menstrual hemorrhage, ovaries, uterus and vagina were hematoxylin eosin stained usually included in the early follicular phase, is dealt with preparations prepared according to routine laboratory separately in the present study because of its morphological procedures. Prior to preparation, the ovaries (each side characteristics. separately) and uterus were weighed. Presentation of data Identification of each phase of the reproductive cycle Morphological characteristics of the major reproductive The sign of the phase of the reproductive cycle organs used as criteria for identification of the immature discernible from routine cage-side observation is menstrual stage and each phase of the reproductive cycle are hemorrhage. Definite periodic menstrual cycles, however, summarized in Table 2. Each individual classified according could be recognized only in 29 out of 106 animals in the to the above criteria was examined for (1) the relationship present study, and they ranged between 26 days and 43 days between each phase and the weights of the ovaries and with 31 days on average with 2 exceptional animals (90 and uterus, (2) the relationship between weights of the ovaries 99 days). In the other 74 animals, no periodic menstrual and uterus and age, and (3) the relationship between the age hemorrhages were recognized. However, these figures do and sexual cycle. All the absolute and relative organ weight not mean that about 70% animals in the present survey data per reproductive phase-group and per age-group are underwent no reproductive cycle. Because of individual expressed as range, group mean and median. The ovaries difference in the amount of menstrual hemorrhage and from each individual were regarded separately according to behavioral nature, routine cage-side observation provides no their weight, larger and smaller. precise results. Some animals licked up the menstrual hemorrhage. In fact, morphological data revealed that all the Results animals were in a certain phase of the reproductive cycle. From these circumstances, data were arranged exclusively Histological characteristics and criteria of the major based on histological pictures of the reproductive organs reproductive organs for identification of the immature representing the six phases of the reproductive cycle, stage and the six phases of the reproductive cycle (Fig. especially that of the ovaries and uterus in the present study. 1) The above description of the six phases of the reproductive Immature stage: In the ovary (Fig. 1A1), primordial cycle depends for the most part on the textbook of Banks2. follicles were conspicuous and first and secondary follicles, Watanabe, Hoshiya, Sato et al. 171 Table 3. Number of Animals in Immature Stage and in Each Phase of Reproductive Cycle in Each Age Group Age Group* Phase Immature Menstrual Early follicular Follicular Pre-ovulation Early luteal Luteal (3)** (Early follicular) (4) (34) (9) (11) (38) (7) 2 to 3 (6)** 3*** 1 2 3 to 3.5 (16) 2 1 10 3 3.5 to 4 (18) 2 6 2 1 7 4 to 4.5 (20) 6 2 1 11 4.5 to 5 (12) 1 4 1 2 4 5 to 5.5 (11) 5 1 3 2 5.5 to 6 (9) 1 2 1 5 6 to 6.5 (9) 3 1 1 2 2 6.5 to 9 (5) 1 1 1 2 *: Age in years.