The Effects of Quinestrol As a Contraceptive in Mongolian Gerbils (Meriones Unguiculatus)

Exp. Anim. 60(5), 489–496, 2011

—Original— The Effects of Quinestrol as a Contraceptive in Mongolian Gerbils (Meriones unguiculatus)

Xiao-Hui LV and Da-Zhao SHI

College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193, China

Abstract: The contraceptive effects of quinestrol in Mongolian gerbils were examined. The results showed that body weight significantly increased after quinestrol treatment, except in the group that received the highest dose. The gonadosomatic index of ovaries decreased, whereas that of uteri increased, and uterine edema appeared after quinestrol treatment. Histological examination revealed that the ovaries had a lack of mature follicles and corpora lutea and that the myometrium and endometrium of the uteri became thin after quinestrol treatment. Persistent estrous appeared after quinestrol treatment, and time to persistent estrous shortened with increasing doses of quinestrol. Serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels decreased, whereas estradiol (E2) and progesterone (P4) levels increased after quinestrol treatment, and the effects were dose-dependent. During gestation, the serum E2 levels in the different treatment groups were not significantly different. During gestation in the control groups, the serum P4 levels from days 0 to 15 were higher than in the quinestrol-treated groups; however, they did not show significant differences from days 18 to 24. Doses of 0.1 to 2.7 μg/g quinestrol over 6 days completely inhibited fertility. Birth time was prolonged with increasing doses of quinestrol. The findings suggest that quinestrol has marked estrogenic effects in Mongolian gerbils and may inhibit follicle maturation and ovulation through lowered gonadotropin levels. Uterine edema and abnormal E2 and P4 levels during gestation are important causes of pregnancy failure in quinestrol-treated Mongolian gerbils. Quinestrol causes prolonged inhibition of fertility in Mongolian gerbils. Key words: fertility control, Mongolian gerbils, quinestrol

Introduction They are the main reservoir host of Yersinia pestis, which causes plague [20]. Therefore, Mongolian gerbil The Mongolian gerbil (Meriones unguiculatus, Milne populations are important to control. Additionally, the Edwards, 1867) belongs to the subfamily Gerbillinae Mongolian gerbil has been extensively used as an and is mainly distributed across the arid steppes, experimental animal model in neuroscience, physiology, semideserts and adjacent farming-pastoral areas of North reproduction, and behavioral research [28]. China, Mongolia, and the Baikal Lake region of Russia Control of fertility in rodent populations was first sug- [20, 28]. Mongolian gerbils cause serious damage to gested by Knipling [17], with use of chemosterilants for crops in agricultural areas when present in large numbers. rodent control suggested later by Davis [3]. The

(Received 30 March 2011 / Accepted 4 June 2011) Address corresponding: D.-Z. Shi, College of Agriculture and Biotechnology, China Agricultural University, No. 2 Yuanmingyuan West Road, Haidian District, Beijing 100193, China 490 X.-H. Lv and D.-Z. Shi advantage of chemosterilants over conventional ment period. These smears were considered to represent poisoning has been shown theoretically in the prevention estrous (cornified cells) [29]. Five randomly selected of rat reproduction [18]. Synthetic estrogens had been gerbils per group were sacrificed by ether inhalation on studied for control of rodent populations [9, 13, 22]. the first day after quinestrol administration. Blood Quinestrol is a synthetic estrogen with marked estrogenic samples (0.5–0.6 ml) were collected via orbital venous effects and prolonged activity [10, 24, 25], and it is an puncture following light ether anesthesia before eutha- effective contraceptive in women. Additionally, nasia. The ovaries and uteri were weighed and col- quinestrol has been shown to reduce the fertility of rats lected for histological analysis. The remaining half of [2, 11, 27]. To our knowledge, the application of the virgin female gerbils were paired with experienced quinestrol for fertility control in Mongolian gerbils has males and examined by vaginal smear the next morning. not been previously reported, nor have the effects of The first day sperm was found in a vaginal smear was quinestrol in Mongolian gerbils been studied. The designated as day 0 of gestation. The male was removed objectives of this study were to confirm whether when the female was confirmed pregnant. The parturi- quinestrol inhibits fertility in Mongolian gerbils and to tion day was regarded as the last day of gestation. Blood study its effects as a contraceptive in this species. samples (0.2–0.3 ml) were taken every 3 days during gestation. The birth time (the period from the day sperm Materials and Methods was first detected in vaginal smears to the day of parturition) and litter size were recorded. Animals The Mongolian gerbils used in this study were from Histological study of sex organs a domesticated colony bred from animals captured in the Ovaries and uteri were fixed in 4% paraformaldehyde, Xilinguole League of Inner Mongolia. The gerbils were gradually dehydrated in ethanol and embedded in paraf- maintained at 23 ± 1°C, with automatically controlled fin, sectioned at 5μ m and stained with eosin and hema- lighting from 0700 to 2100 h (14 h light : 10 h dark). toxylin for histological examination. The sections were The Mongolian gerbils were provided with a food mix- observed by light microscopy. ture containing equal parts of corn and sunflower seeds, and they were given water ad libitum. Fifty virgin fe- Hormone assays male, 4-month-old gerbils (55–65 g) with regular estrous Serum was separated by centrifugation at 1,000 × g cycles, as confirmed by vaginal smear [29], were used. for 20 min at 4°C and stored at –80°C until assayed. The study was conducted according to Guidelines for Concentrations of FSH and LH were measured using rat Animal Experiments and approved by the Animal Care ELISA kits (EIAab Science Co., Ltd., Wuhan, China), and Use Committee at the China Agricultural Univer- as described and validated previously [30]. The mini- sity. mum detectable dose was 0.078 mIU/ml for FSH and 0.195 mIU/ml for LH, respectively. The intra- and in- Experimental design terassay variations for FSH and LH were 4.8 and 7.4%, Quinestrol (Zizhu Medicine Co., Ltd., Beijing, China) respectively. Serum E2 and P4 levels were determined was dissolved in peanut oil. Fifty virgin female gerbils by a chemiluminescence immunoassay (CLIA) [21] us- were randomly divided into five groups. The gerbils ing CLIA kits (Furui Biotechnology Co., Ltd., Beijing, were given quinestrol intragastrically daily at single China). The minimum detectable dose was 1.50 pg/ml doses of 0, 0.1, 0.3, 0.9, and 2.7 μg/g body weight (BW) for estradiol and 0.05 ng/ml for progesterone. The intra- for 6 days [32]. The control group was given peanut oil. and interassay variations for both were less than 10% The feeding habits of all experimental groups were ob- and 15%, respectively. served. The body weight of all animals was measured daily. Vaginal smears were also taken daily to assess the Statistical analysis effect of quinestrol on the estrous cycles during the treat- The data were analyzed by one-way ANOVA with QUINESTROL EFFECTS IN MONGOLIAN GERBILS 491

Table 1. Effects of quinestrol on the body weights and gonadosomatic indices of Mongolian gerbils Gonadosomatic index Initial body Final body Dose (μg/g) N (gonad weight/body weight × 1000) weight (g) weight (g) Ovaries Uteri 0 5 59.72 ± 2.55 61.44 ± 2.46 0.43 ± 0.11 2.21 ± 0.45 0.1 5 58.72 ± 1.95 64.57 ± 1.51 0.28 ± 0.03 16.81 ± 2.17* 0.3 5 57.37 ± 1.01 60.42 ± 1.64 0.28 ± 0.03 14.72 ± 2.08* 0.9 5 56.53 ± 1.88 59.86 ± 2.44 0.47 ± 0.03 15.24 ± 1.96* 2.7 5 59.01 ± 2.02 59.00 ± 1.33 0.25 ± 0.01 6.89 ± 0.79 * Significant compared with the control groups (P<0.05).

Tukey’s test for post hoc multiple comparison analysis. quinestrol treatment (Table 1). The gonadosomatic in- Pearson correlation coefficients were calculated. Values dices of the uteri from groups treated with 0.1–0.9 μg/g were considered statistically significant at P<0.05 and quinestrol were significantly higher than those of the highly significant at P<0.01. The analyses were per- groups treated with 0 and 2.7 μg/g quinestrol (P<0.05). formed using SPSS 16.0 for Windows. Data are pre- The correlation coefficients between the quinestrol sented as means ± SEM. doses and the gonadosomatic indices of uteri were not significant. The percentages of uterine edema were 60, Results 100, 100, and 80% after quinestrol treatment at 0.1, 0.3, 0.9, and 2.7 μg/g, respectively. Histologic examination Effects of quinestrol on body weight and sex organs of uteri treated with quinestrol showed thinning of the The feeding habits of gerbils from all experimental myometrium and endometrium (Fig. 1G–J). Addition- groups were unaltered during the course of the study. ally, the endometrium showed reduced epithelial hyper- There was no significant difference in initial and final plasia, few profound glands, hyalinized stroma, and body weights for the gerbils in the different groups shedding cilia. (Table 1). However, each group exhibited a significant increase in final body weight compared with initial body Effects of quinestrol on estrous cycles weight (P<0.05), except for the group treated with the The regular estrous cycle of Mongolian gerbils was dose of 2.7 μg/g quinestrol. There was a significant interrupted treatment with 0.1–2.7 μg/g doses of quin- negative correlation between dosage and the change in estrol. Persistent estrous appeared in quinestrol-treated body weight (r=–0.392, P<0.01). gerbils. The control group had normal estrous cycles. The gonadosomatic index of ovaries after quinestrol Time to persistent estrous decreased gradually with the treatment decreased, except in the group treated with increasing doses of quinestrol. The times to persistent 0.9 μg/g (Table 1). There were no significant differ- estrous were 4.50 ± 0.34, 4.1 ± 0.43, 4.00 ± 0.33, and ences in the gonadosomatic indices of the different 3.75 ± 0.41 days after treatment with 0.1, 0.3, 0.9, and groups. The correlation between the dose of quinestrol 2.7 μg/g doses of quinestrol. There was no significant and the gonadosomatic index of the ovaries was not sig- difference between the times to persistent estrous. There nificant. Histologic examination showed that the ovaries was no significant correlation between the time to per- of the gerbils in the control group contained different sistent estrous and the dose of quinestrol. stages of follicles and corpora lutea (Fig. 1A). The ovaries of the quinestrol-treated groups contained only Effects of quinestrol on serum hormonal levels growing follicles and lacked mature follicles and cor- Serum FSH and LH levels gradually decreased 1 day pora lutea (Fig. 1B–E). after the various quinestrol doses were administrated to The gonadosomatic indices of uteri increased after Mongolian gerbils (Fig. 2A and 2B). Serum FSH in the 492 X.-H. LV AND D.-Z. SHI

Fig. 1. Histological features of ovaries (A–E) and uteri (F–J) 1 day after quinestrol administration in Mongolian gerbils. A and F, B and G, C and H, D and I, and E and J were treated with quinestrol at doses of 0, 0.1, 0.3, 0.9, and 2.7 μg/g, respectively. Sections were stained with hematoxylin and eosin. Original magnifi cation: ×100 for A–E, ×200 for F, and ×400 for G–J.

Fig. 2. Effects of quinestrol on serum FSH (A), LH (B), E2 (C), and P4 (D) levels 1 day after quinestrol treatment in Mongolian gerbils. Data are means ± SEM (n=5). Bars with different superscripts are signifi cantly different (P<0.05). QUINESTROL EFFECTS IN MONGOLIAN GERBILS 493

Fig. 3. Effects of quinestrol on serum E2 (A) and P4 (B) levels during gestation in Mongolian gerbils. Data are means ± SEM (n=5). *Significant compared the other groups (P<0.05).

control group (9.59 ± 0.24 mIU/ml) was significantly Mongolian gerbils (Fig. 2C and 2D). The serum E2 higher than in the quinestrol-treated groups (P < 0.05). level in the 2.7 μg/g quinestrol group (121.97 ± 39.49 The serum FSH levels were 5.91 ± 0.61, 5.02 ± 0.78, pg/ml) was significantly higher than in other groups 4.49 ± 0.78, and 4.00 ± 0.78 mIU/ml after quinestrol (P<0.05). The serum E2 levels were 18.68 ± 2.17, 17.18 treatment at the doses of 0.1, 0.3, 0.9, and 2.7 μg/g, re- ± 5.67, 21.85 ± 0.79, and 37.38 ± 3.22 pg/ml after quin- spectively; there was no significant difference between estrol treatment at doses of 0, 0.1, 0.3, and 0.9 μg/g, these groups. There was a significant negative correla- respectively; there was no significant difference between tion between quinestrol dose and serum FSH (r=–0.532, groups. The levels of serum P4 in the 0.9 μg/g (15.01 P<0.05). Serum LH in the control group (13.98 ± 0.48 ± 0.86 ng/ml) and 2.7 μg/g groups (18.67 ± 0.97 ng/ml) mIU/ml) was significantly higher than that in the quin- were significantly higher than in the control (5.45 ± 0.75 estrol-treated groups (P<0.05). The serum LH levels ng/ml), 0.1 μg/g (4.68 ± 1.30 ng/ml), and 0.3 μg/g groups were 9.17 ± 0.83, 8.87 ± 1.11, 6.14 ± 1.31, and 5.00 ± (6.98 ± 1.01 ng/ml, P<0.05). There was a significant 1.44 mIU/ml after quinestrol treatment at doses of 0.1, positive correlation between the quinestrol dose and the 0.3, 0.9, and 2.7 μg/g, respectively; there was no sig- serum E2 (r=0.838, P<0.01) and serum P4 levels nificant difference between these groups. There was a (r=0.902, P<0.01). significant negative correlation between the dose of The serum E2 levels in the various treatment groups quinestrol and serum LH levels (r=–0.648, P<0.01). during gestation were not significantly different (Fig. Serum E2 and P4 levels gradually increased 1 day after 3A). The serum E2 levels in the quinestrol-treated the various doses of quinestrol were administrated to groups were higher than in the control group at day 0 494 X.-H. Lv and D.-Z. Shi

Table 2. Effects of quinestrol treatment on the fertility of Mongolian gerbils Dose (μg/g) N Birth time (d) Litter size No. females No. males 0 5 24.40 ± 0.25 5.80 ± 0.37 3.00 ± 0.55 2.80 ± 0.37 0.1 5 39.33 ± 5.36 3.80 ± 0.58 2.00 ± 0.32 1.80 ± 0.37 0.3 5 66.00 ± 6.43* 3.00 ± 0.91 1.50 ± 0.29 1.50 ± 0.65 0.9 5 69.67 ± 9.14* 4.75 ± 1.03 2.50 ± 0.65 2.25 ± 0.48 2.7 5 86.67 ± 5.78* 6.00 ± 0.58 3.67 ± 0.33 2.33 ± 0.33 Birth time represents the period from the day sperm was first detected in vaginal smears to the day of parturition. *Significant compared with the control groups (P<0.05).

(107.98 ± 11.40 pg/ml) and day 3 (111.13 ± 5.34 pg/ml) Discussion of gestation. However, the serum E2 levels in the quinestrol-treated groups were lower than in the control Both antifertility and marked estrogenic activities of group at day 18 (159.54 ± 15.56 pg/ml), day 21 (168.12 quinestrol have been observed in rats [5, 26]. Quinestrol ± 15.55 pg/ml), and day 24 (148.22 ± 13.54 pg/ml) of has been considered as a possible rodent chemosterilant gestation. The serum P4 levels in the control group from [27, 32]. However, quinestrol activity varies among day 0 to day 15 of gestation were higher than in the species based on the dose given and the route of admin- groups treated with quinestrol (Fig. 3B). The serum P4 istration. The normal estrous cycle of the Mongolian levels during gestation in the control group at day 6 (9.89 gerbil is 4–6 days [23, 29]. The duration of quinestrol ± 0.77 ng/ml), day 9 (10.31 ± 0.67 ng/ml), and day 12 treatment in the present study covered one Mongolian (11.56 ± 1.02 ng/ml) were significantly higher than in gerbil estrous cycle. The treatment period in this study the quinestrol-treated groups (P<0.05). The serum P4 was different from that in a previous report [32], where- levels from day 18 to day 24 during gestation were not as the dose was similar. The results of the present study significantly different between groups. indicate that quinestrol, when administrated for one estrous cycle in doses from 0.1 to 2.7 μg/g, has contracep- Effects of quinestrol on the fertility tive effects in Mongolian gerbils. The fertility of Mongolian gerbils was completely The body weight and gonadosomatic indices of uteri inhibited after treatment with 0.1 to 2.7 μg/g of quinestrol increased, whereas the gonadosomatic indices of ovaries for 6 days. The restorable percentage of littering females decreased after quinestrol treatment in Mongolian ger- was 100% after the various different doses of quinestrol bils. This finding is similar to what was observed in rats treatment. All females eventually produced litters. The and mice [5, 7, 8, 11, 26]. However, there was a sig- birth time was prolonged with increasing doses of quin- nificant negative correlation between the dosage and the estrol. The birth times in the 0.3, 0.9, and 2.7 μg/g change in body weight in Mongolian gerbils. A uterine quinestrol-treated groups were significantly longer than growth-stimulating activity of orally administered estro- in the control and 0.1 μg/g quinestrol-treated groups genic material has been observed [19, 25]. Persistent (P<0.05). There was a significant positive correlation estrous appeared after quinestrol treatment, and the time between birth time and quinestrol dose (r=0.79, P<0.01). to persistent estrous shortened with increasing doses of The mean litter sizes and mean numbers of females and quinestrol; this finding is consisted with findings in pre- males in the litters are presented in Table 2. There was vious investigations [7, 8, 11, 27]. Estrogen has the no significant correlation between the quinestrol dose capacity to produce cornification of vaginal epithelial and the mean litter sizes and mean numbers of females cells associated with estrous in rodents [12]. The ap- and males in the litters. pearance of persistent estrous may be caused by the marked estrogenic effects of quinestrol in Mongolian gerbils. Blocked gonadotropin release may be the major reason QUINESTROL EFFECTS IN MONGOLIAN GERBILS 495 why the ovaries only had growing follicles and lacked patterns of E2 and P4 levels are correlated with the re- mature follicles and corpora lutea after quinestrol treat- productive stages of Mongolian gerbils [21]. A higher ment in Mongolian gerbils; this phenomenon is also P4 level during the first half of gestation is important for observed in rats [4, 14]. The potent gonadotropin-inhib- successful pregnancy in Mongolian gerbils [15, 21]. iting properties of quinestrol could be related to its stor- However, the P4 levels during the first half of gestation age in the brain and its marked estrogenic effects, which in quinestrol-treated Mongolian gerbils were lower than would be consistent with previous findings [7, 24]. The those in the control group. A rise in E2 and a decline in significant negative correlation between quinestrol dose P4 are required for parturition initiation in Mongolian and gonadotropin levels in the present study also could gerbils [21]. However, in the present study, the E2 lev- be a consequence of these properties. The ability of els in the quinestrol-treated groups were lower than in quinestrol to prevent ovulation in rats and women has the control group from day 18 to 24 during gestation. been previously shown [1, 26]. However, the reasons The female Mongolian gerbils became pregnant after for increases in serum E2 and P4 levels in quinestrol- different lengths of time, demonstrating a return to fertil- treated Mongolian gerbils are not clear. Uterine edema ity after different doses of quinestrol treatment, which was observed in Mongolian gerbils in the present study is in agreement with results found in the literature [26]. after treatment with various doses of quinestrol, and this Although the mean litter size decreased in the quinestrol- finding is similar to one reported for rats [2, 11]. The treated gerbils, the ratio of females to males in the litters reasons for uterine edema in quinestrol-treated Mongo- remained close to 1:1, except in the highest dose group. lian gerbils are not clear. The uterine edema may be These findings were similar to those in other reports [27, related to the abnormal E2 and P4 levels. The effects of 32]. The restoration of fertility after quinestrol treatment quinestrol on reproductive hormone (FSH, LH, E2, and in the Mongolian gerbils has also been observed in rats P4) levels are dose-dependent in a positive and/or nega- [27]. The birth time was prolonged with increasing tive manner in Mongolian gerbils. However, the gona- doses of quinestrol, and there was a significant positive dosomatic indices of ovaries and uteri were different correlation between the birth time and dosage. These among the treatment groups, and there was no correlation findings sufficiently display the prolonged inhibitive with the doses of quinestrol. The phenomenon in which fertility effects of quinestrol in Mongolian gerbils. the gonadosomatic indices of uteri decreased at higher In summary, quinestrol has marked estrogenic effects doses of quinestrol treatment was also observed in mice in Mongolian gerbils. It likely inhibits follicle matura- and rats [6, 31]. This may be related to the uterine es- tion and ovulation through lowered gonadotropin levels trogen receptor levels [16]. There is no good explanation in Mongolian gerbils. The uterine edema and abnormal for the finding that the gonadosomatic indices of ovaries E2 and P4 levels during gestation caused by quinestrol increased in the group treated with 0.9 μg/g quinestrol. are important reasons for pregnancy failure following The reason for this discrepancy between reproductive quinestrol treatment. The fertility of Mongolian gerbils hormone levels and gonadosomatic index in Mongolian is profoundly inhibited by quinestrol, and it is restorable. gerbils needs further investigation. Although the precise action mode of quinestrol as a con- The normal pregnancy of the Mongolian gerbil is 24 traceptive in Mongolian gerbils requires further inves- to 26 days [23]. None of the female Mongolian gerbils tigation, this study provides information regarding its in this study were pregnant after quinestrol treatment in contraceptive effects. the normal pregnancy time. Uterine edema and abnormal E2 and P4 levels during gestation are causes of preg- Acknowledgments nancy failure. Uterine edema is not conducive to sperm penetration and transport. A thin myometrium and thin We thank Dr. Yongsong Cao and Dr. Deping Han for endometrium are also not favorable to embryo implanta- reviewing this manuscript and their critical comments. tion and growth. Estradiol and progesterone are impor- This study was supported by grants from the National tant reproductive hormones during gestation. Changing Key Basic Research and Development Program of Chi- 496 X.-H. Lv and D.-Z. Shi na (No. 2007CB109105) and the New Technologies 100: 1252–1259. Research for Farmland Rodent Control of the Ministry 17. Knipling, E.F. 1959. Sterile male method of population control. Science 130: 902–904. of Agriculture of China. 18. Knipling, E.F. and McGuire, J.U. 1972. Potential role for sterilization for suppressing rat populations, a theoretical References appraisal. pp. 1–27. 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