Variations of Serum E2 and P4 Levels

Exp. Anim. 59(2), 231–237, 2010Variations of Serum Estradiol and Progesterone Levels during Consecutive Reproductive States in Mongolian Gerbils (Meriones unguiculatus )Xiao Hui LV and Da Zhao SHICollege of Agriculture and Biotechnology, China Agricultural University, Beijing 100193, ChinaAbstract: Serum estradiol (E2) and progesterone (P4) levels were measured by chemiluminescence immunoassay (CLIA) during consecutive reproductive states in Mongolian gerbils. The results show that E2 and P4 levels throughout the estrous cycle in Mongolian gerbils peaked at proestrus and estrus, respectively. During gestation in primiparous and multiparous gerbils, E2 levels reached a small peak on day 6 and a maximum peak on day 21, followed by a slight decline until parturition. Thereafter, they dropped sharply during the first 3 days of lactation and remained stable until lactation ended. P4 levels reached a peak on day 12 during gestation in primiparous gerbils, while they peaked on day 6 in multiparous gerbils. The levels of P4 then decreased until parturition. During lactation, P4 levels peaked on day 9 in primiparous gerbils, while they peaked on day 6 in multiparous gerbils, and then the levels declined gradually until lactation ended. The findings suggest that the variations of E2 levels during the estrous cycle, gestation and lactation in Mongolian gerbils are similar to those observed in rats and mice. Changes in E2 levels during different reproductive states were the same in the primiparous and multiparous gerbils, but the variations in P4 levels did not display this tendency. Changing patterns of E2 and P4 levels are suitable for the reproductive stages of Mongolian gerbils.Key words: estradiol, estrous cycle, gestation, lactation, progesterone(Received 31 August 2009 / Accepted 18 December 2009)Address corresponding: D.Z. Shi, College of Agriculture and Biotechnology, China Agricultural University, No. 2 Yuanmingyuan West Road, Haidian District, Beijing 100193, ChinaIntroductionThe Mongolian gerbil (Meriones unguiculatus Milne Edwards, 1867) belongs to the subfamily Gerbillinae , and is mainly distributed across the arid steppes, semi-deserts and adjacent farming-pastoral areas of North China, Mongolia, and the Baikal Lake region of Russia [17, 21, 35]. Its population is volatile and these gerbils cause serious damage to crops of farming areas when they are present in large numbers. They are also themain reservoir host of plague [17, 35]. Because of the special characteristics of the Mongolian gerbil, it has become an extensively used experimental animal, for example, in neuroscience, physiology, pharmacological, parasitological, and endocrinological research [21]. The estrous cycle of the Mongolian gerbil is 4 to 7 days, pregnancy is 24 to 26 days, lactation is 21 to 22 days, and the litter size at birth ranges from 1 to 9 offspring [11, 12, 18, 22]. We can further understand the reproduc-tive characteristics of Mongolian gerbils through studies—Original—232X.H. LV aND D.Z. SHIof reproductive hormones. However, there are only a few reports on the secretion patterns of reproductive hormones during different reproductive states in Mon-golian gerbils [6, 11, 12, 22, 24]. The aims of this study were, first, to confirm the variations of serum estradiol (E2) and progesterone (P4) levels in different reproduc-tive states of Mongolian gerbils and to analyze the dif-ferences of E2 and P4 patterns between Mongolian gerbils and other rodents such as rats and mice; second, to analyze the differences in variations of E2 and P4 levels in primiparous and multiparous gerbils; and third, to analyze the relationships between the two hormone levels and the reproductive states of Mongolian ger-bils.Materials and Methods Animals and samplingMongolian gerbils used in this study were indoor breeding individuals whose parents were captured in the Xilinguole League of Inner Mongolia. The gerbils were maintained at 20 ± 3°C with automatically controlled lighting from 0700 to 2100 h (14L:10D). They were provided with a food mixture containing equal parts of corn and sunflower seeds, and they were given water ad libitum. Eight 4-month-old virgin female gerbils (55–65 g) with regular estrous cycles of 4 days as confirmed by vaginal smear were used [22]. The virgin female gerbils were paired with experienced males in the afternoon and were examined by vaginal smear the next morning. The day on which sperm were found in the vaginal smear was designated as day 0 of gestation. The male was removed when the female was confirmed as pregnant. The parturition day was regarded as the last day of ges-tation and day 0 of lactation. The day when the pups were able to obtain food by themselves was regarded as the last day of lactation. The mean gestation period of the Mongolian gerbils in the present study was 24.1 ± 0.1 days (24 days: n=7; 25 days: n=1). The time from the end of lactation in primiparous gerbils to the estrous cycle in multiparous gerbils was about two weeks. Blood sampling from female gerbils began in the vir-gin estrous cycle, and continued through gestation and lactation of primiparous gerbils, the post-weaning es-trous cycle, and the gestation and lactation of multipa-rous gerbils. Blood samples were taken every day dur-ing the estrous cycle and every 3 days during gestation and lactation. The sampling was conducted 42 times in each gerbil on the premise of its normal survival. Blood samples (0.2–0.3 ml) were collected between 1400 and 1500 h via the orbital venous sinus following light ether anaesthesia. Serum was separated by centrifugation at 1,000 ×g for 20 min at 4°C and stored at –80°C until analysis. The gerbil handling and tests were performed according to the Guide for the Care and Use of Labora-tory animals.Hormone assaysSerum E2 and P4 levels were determined by a chemi-luminescence immunoassay (CLIa) using commercial kits for CLIa (Furui Biotechnology Co., Ltd., Beijing, China). The sensitivity was 30–2,000 pg/ml for estra-diol and 0.25–80 ng/ml for progesterone. The intra-assay variations for both were less than 10%, while both inter-assay variations were less than 15%.Statistical analysesData distributions were analyzed for normality by the one-sample Kolmogorov-Simirnov test. The hormone data were analyzed by one-way aNOV a (SPSS for Win-dows 11.5) for repeated measures. Pearson correlation coefficients were calculated between E2 and P4 levels per reproductive state. Linear regression was tested between E2 and P4 levels and the number of pups. Val-ues were considered as statistically significant at P<0.05 and highly significant at P<0.01. Data are presented as means ± SEM (n=8).ResultsHormonal variations during the estrous cycle of virgin gerbils and during gestation and lactation in primiparous Mongolian gerbils (Fig. 1)Serum E2 during the estrous cycle of virgin gerbils reached a peak at proestrus (73.12 ± 4.19 pg/ml, P<0.05) and displayed no significant differences throughout es-trus (60.82 ± 4.66 pg/ml), metestrus (59.35 ± 4.44 pg/ ml), and diestrus (58.74 ± 3.40 pg/ml). Serum P4 re-mained constant through proestrus (3.08 ± 0.49 ng/ml),233HORMONaL VaRIaTIONS aND REPRODUCTIVE STaTESmetestrus (3.07 ± 0.55 ng/ml), and diestrus (2.92 ± 0.52 ng/ml), peaking at estrus (5.08 ± 0.87 ng/ml, P <0.05).Serum E2 of primiparous gerbils was low on day 0 of gestation (61.70 ± 2.67 pg/ml) when sperm were ob-served in the vaginal smear. Then, it slowly increased during early gestation, reaching a minor peak on day 6 (79.45 ± 13.27 pg/ml), then remained stable from day 6 to day 18 of gestation (75.06 ± 6.20 pg/ml). During later gestation, a sharp increase in the E2 was seen that reached a maximum on day 21 (132.23 ± 11.68 pg/ml) after which it decreased to a relatively high level on the day of parturition (107.43 ± 5.85 pg/ml). Serum P4 was also low on day 0 of gestation (3.52 ± 0.66 ng/ml) but rapidly rose until day 6 (20.83 ± 1.83 ng/ml). Subse-quently, serum P4 continued rising and reached a maxi-mum on day 12 (25.82 ± 1.95 ng/ml). Thereafter it declined and reached its lowest point on the day of par-turition (6.79 ± 1.05 ng/ml). Serum P4 was higher dur-ing the fi rst half of gestation than during the second half of gestation in primiparous gerbils. Serum E2 levels were negatively correlated with P4 levels (r =–0.560, P <0.01) in the second half of gestation, while the cor-relation coeffi cients during other gestation periods were not signifi cant in primiparous gerbils (P >0.05).Serum E2 gradually fell from day 0 of lactation (107.43 ± 5.85 pg/ml) to day 3 (74.62 ± 6.42 pg/ml) in primiparous gerbils. Subsequently, it remained stable from day 3 to day 24 (68.90 ± 2.82 pg/ml) without sig-nifi cant changes during this period (P >0.05). Duringlactation serum P4 gradually increased from day 0 andreached a peak on day 9 (13.27 ± 3.13 ng/ml). It dropped on day 12 (8.13 ± 1.34 ng/ml) and then remained rela-tively stable until day 24 (5.54 ± 0.75 ng/ml, P >0.05). There was no signifi cant correlation between serum E2 levels and serum P4 levels during lactation in primipa-rous gerbils (P >0.05).Hormonal variations during the post-weaning estrous cycle, gestation and lactation in multiparous Mongolian gerbils (Fig. 2)During the post-weaning estrous cycle, serum E2 reached a peak at proestrus (95.81 ± 17.47 pg/ml, P <0.05), but there were no significant differences throughout estrus (63.80 ± 5.52 pg/ml), metestrus (51.02 ± 6.84 pg/ml), and diestrus (49.13 ± 5.20 pg/ml). Serum P4 remained constant through proestrus (6.57 ± 1.35 ng/ml), metestrus (5.41 ± 0.67 ng/ml), and diestrus (6.95 ± 1.12 ng/ml) before reaching its maximum at estrus (12.08 ± 2.75 ng/ml, P <0.05).In multiparous gerbils, the serum E2 level during ges-tation gradually increased from day 0 (47.24 ± 5.37 pg/ml), and reached a small peak on day 6 (68.87 ± 7.58 pg/ml). It then sharply rose to a maximum (174.70 ± 27.58 pg/ml) on day 21. Subsequently, serum E2 dis-played a slight decline from day 21 to the day of partu-rition (163.47 ± 29.03 pg/ml). Serum P4 showed a rapid rise from 5.05 ± 1.41 ng/ml on day 0 to a peak onday 6 (24.24 ± 3.19 ng/ml). after day 6, serum P4 ex-Fig. 1. Serum E2 and P4 levels during the virgin estrus cycle, gestation, and lactation in primiparousMongolian gerbils. Each point represents the mean ± SEM of 8 gerbils. (P, E, M, D=proestrus, estrus, metestrus, disestrus. Gestation: 0–21 and lactation: 0–24.)234X.H. LV aND D.Z. SHIhibited a gradual decline and reached moderate levels (12.49 ± 1.40 ng/ml) on day 12. During the second half of gestation in multiparous gerbils, the P4 level con-tinuously decreased and reached a minimum on the day of parturition (5.60 ± 0.88 ng/ml). Serum E2 levels were negatively correlated with serum P4 levels (r =–0.334, P <0.05) during gestation in multiparous gerbils.During lactation, serum E2 showed a rapid fall from day 0 (163.47 ± 29.03 pg/ml) to day 3 (70.11 ± 6.57 pg/ml). Subsequently, it remained stable from day 3 to day 24 (63.04 ± 4.24 pg/ml), without any signifi cant differ-ences (P >0.05) during this time period. Serum P4 gradually increased from day 0 (5.60 ± 0.88 ng/ml) and reached a peak on day 6 (10.99 ± 3.35 ng/ml) of lactation. It then fell and remained relatively stable from day 18 (4.65 ± 0.47 ng/ml) to day 24 (4.25 ± 0.30 ng/ml). There was no signifi cant correlation between serum E2 levels and serum P4 levels (P >0.05) during lactation in mul-tiparous gerbils.Regression between estradiol and progesterone levels and the number of littersIn each primiparous gerbil, the mean number of the litter was 6.3 ± 0.3 (5–8 pups), with 2.6 ± 0.2 females and 3.6 ± 0.3 males, while the mean number of the litter was 6.1 ± 0.4 (4–8 pups), with 2.1 ± 0.4 females and 3.9 ± 0.3 males per multiparous gerbil. There was no sig-nifi cant linear regression between the mean number of the litter for each primiparous and multiparous gerbiland the serum E2 and P4 levels at the following times: days 6, 9, 12, and 21 of gestation and days 0, 3, 6, and 9 of lactation.DiscussionHormonal variations during the estrous cycle in virgin Mongolian gerbilsThe changes of E2 and P4 levels during the estrous cycle correlated with the ovulation of Mongolian gerbils. The Mongolian gerbil is a spontaneous ovulation animal. a spontaneous ovulation animal is considered to be one that undergoes estrogen secretion earlier than progester-one release [16], while the progesterone peak is earlier than the ovulation time [19]. Most matings of Mongolian gerbils were observed from 1700 to 2000 h, and ovula-tion occurred between 6 and 10 h after mating [18, 22]. The peak of E2 at proestrus leads to an increase of go-nadotropin releasing hormone (GnRH) level triggering the luteotropin (LH) peak of preovulation. The LH peak causes P4 to increase, and P4 eventually peaks at estrus. The phase during which P4 peaks during the estrous cycle in Mongolian gerbils is different from those of rats and mice, in which the peak appears at proestrus [9, 29]. This difference may be caused by the difference in ovu-lation time. Contrary to the results of this study, other work has indicated that serum E2 reaches a peak at di-estrus while serum P4 peaks at estrus in Mongoliangerbils [6]. although the measurement method used wasFig. 2. Serum E2 and P4 levels during the post-weaning estrus cycle, gestation, and lactation in multipa-rous Mongolian gerbils. Each point represents the mean ± SEM of 8 gerbils. (P, E, M, D=proestrus, estrus, metestrus, disestrus. Gestation: 0–21 and lactation: 0–24.)235 HORMONaL VaRIaTIONS aND REPRODUCTIVE STaTESthe same, the standard error of E2 and P4 peak values high, and it was not stated whether or not the female gerbils were virgins.Hormonal variations during gestation in primiparous Mongolian gerbilsThe results obtained in the present study indicate that the changing patterns of serum E2 and P4 levels are suitable for embryo implantation, decidualization, and parturition initiation during gestation in Mongolian ger-bils. Embryo implantation occurs from the night of day 6 through the morning of day 7 during gestation in Mon-golian gerbils [5, 18, 33] and requires the joint action of E2 and P4. The decidualization, which also needs the actions of the E2 and P4 begins shortly after implanta-tion, and the weight and size of the uterus subsequently increase [1]. In Mongolian gerbils, the maternal de-cidual reaction is at its height of development two days after implantation [5]. A higher P4 level during the first half of gestation conditions the uterus environment ap-propriate for embryo development and inhibits the reac-tion of the uterus to estrogen and oxytocin. E2 reached a peak on day 21, while P4 gradually declined prior to parturition. The changing levels of E2 and P4 are re-lated to parturition initiation, which requires a rise in E2 and a decline in P4. The level of P4 declines relative to E2 to allow estrogen to stimulate the rhythmic contrac-tion of the uterus, overcome the progesterone inhibition and raise the myometrial sensitivity to oxytocin. Mean-while, the fall of the P4 level correlates with milk gen-eration and the initiation of maternal behavior [13, 14].The variation of E2 during gestation in Mongolian gerbils is similar to those in rats and mice, in which the E2 level gradually increases during early gestation, reaching a peak during embryo implantation, and then continuously increases, reaching a peak before parturi-tion [3, 15, 26, 32]. In the present study, the pattern of P4 levels during gestation was similar to those observed in previous works [11, 12], but differed from those de-tected in rats and mice. In rats and mice, P4 levels dur-ing the first half of gestation are lower than the levels during the second half of gestation [2, 3, 9, 20].Hormonal variations during lactation in primiparous Mongolian gerbilsNursing pups had a significant effect on variations of E2 and P4 levels during lactation in Mongolian gerbils, but the E2 and P4 levels had no significant linear regres-sion relationships with the mean number of pups for each gerbil on day 0, 3, 6, or 9 of lactation. The suckling stimulus inhibits GnRH release, with the degree of in-hibition related to the suckling intensity, so as to suppress gonadotropin secrection [27, 28]. The serection of es-trogen is eventually restrained, but the physiological estrogen level is stimulative for lactation maintenance. The Mongolian gerbil displays a postpartum ovulation phenomenon [18], and the suckling stimulus during early lactation allows the corpus luteum that is formed after ovulation to become functional and secrete proges-terone [7, 10, 25, 31]. Prolactin (PRL) induced by the suckling stimulus maintains the corpus luteum function and induces continuous P4 secretion [28, 34]. Because of the diminution of the suckling stimulus along with lactation, PRL levels decline, which leads to corpus lu-teum regression and a drop in the P4 level. Changes of E2 and P4 levels during lactation in prim-iparous gerbils were similar to those in rats. The E2 level is significantly high on the day of parturition, sub-sequently declines, and then remains relatively stable until lactation ends [15, 26, 30]. The P4 level is sig-nificantly low on the day of parturition, then gradually rises from early lactation to mid lactation, and declines during late lactation [7, 8, 10, 30].Hormonal variations during different reproductive states in multiparous Mongolian gerbilsVariation of E2 in different reproductive states was the same in multiparous and primiparous gerbils, but changes in P4 were different. During the post-weaning estrous cycle, E2 levels did not change much compared with those in virgin gerbils, and a similar tendency is seen in rats [4]. However, P4 during the post-weaning estrus cycle was higher than during the estrous cycle in virgin gerbils. E2 levels during gestation were not sig-nificantly different in multiparous and primiparous ger-bils, a finding which differs from those for rats. In multiparous rats, E2 during gestation is higher than in primiparous rats [23]. P4 reached a maximum on day 6236X.H. LV aND D.Z. SHIduring gestation in multiparous gerbils, which was dif-ferent from primiparous gerbils. The peak of P4 on day 6 in multiparous gerbils is consistent with the time of embryo implantation. although P4 continuously de-creased after day 6, P4 was still higher during the first half of gestation than during the second half of gestation. During lactation, the change of E2 in multiparous gerbils was similar to that observed in primiparous gerbils. P4 during lactation also reached a maximum value on day 6, earlier than in primiparous gerbils. The reasons for the earlier peak of P4 during gestation and lactation in multiparous gerbils remain unclear.Both estrogen and progesterone are important repro-ductive hormones secreted by the gonad and play im-portant roles in regulating reproduction. Variations of E2 during the estrous cycle, gestation and lactation in Mongolian gerbils and other rodents such as rats and mice are similar, but variations of P4 are not. 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