September 1990 Jpn J Anim Reprod Vol 36 (3)

Different Responsiveness to for Induction of Pseudopregnancy between the First and the Third after Vaginal Opening in Rats

Hiroshi TOMOGANE and Akira YOKOYAMA

Departmentof Animal Science,School of Agriculture,Nagoya University,Chikusa, Nagoya 464-01, Japan

(Accepted for publication May 16, 1990)

Summary. Effects of progesterone on the function of in estrous cycle were compared between the first and the third estrous cycle after vaginal opening in rats. When 2.5 mg/100 g body weight (B.W.) progesterone was administered between 07:00 and 08:00 hr on the day of estrus or on day 1 of diestrus to rats showing the first estrous cycle, the diestrous period was persisted more than 9 days in 95% of animals. In the diestrous period, plasma (PRL) level showed daily changes and corpora lutea became functional. The luteal function was enhanced by the deciduoma formed after uterine traumatization on day 3 of diestrus. However, in rats showing the third estrous cycle, 7.5 mg/100 g B.W. progesterone was required to induce similar response to those obtained in the first estrous cycle. Also, no functional corpora lutea and no daily change in PRL secretion were observed in rats injected with progesterone on day 1 of diestrus in the third estrous cycle. These results indicate that progesterone acts more effectively for induction of pseudopregnant diestrus in the first estrous cycle than in the third estrous cycle. KEYWORDS: PSEUDOPREGNANCY, PROGESTERONE, PROLACTIN, PUBERTAL RAT Jpn J Anim Reprod 36, 176-183, 1990

In adult cyclic rats, the corpus luteum has If progesterone administration is limited not become functional, unless the cervical to the early morning of day of estrus in adult stimulus is given. However, about a half of cyclic rats, the animal becomes pseudopreg- immature rats maintained leucocytic vaginal nancy (Everett, 1963; Rothchild and smears for more than 9 days after the first Schubert, 1963; Luque and Castro-Vazquez, spontaneous without the cervical 1983) and shows daily changes in PRL stimulus (Tomogane et al., 1987). We have secretion (de Greef and Zeilmaker, 1978; also reported that the nocturnal prolactin Murakami et al., 1980). In order to elucidate (PRL) surge was induced more easily in the participation of progesterone in the ovariectomized pubertal rats than in ovar- initiation of the long diestrous period in the iectomized adult rats by the implantation of pubertal rats showing the first estrous cycle, progesterone-containing tubings at the the efficiency of progesterone in inducing estrous stage (Tomogane and Yokoyama, pseudopregnancy was compared between 1989). These results suggest that circulating the first and the third estrous cycle after progesterone around the periovulatory vaginal opening in rats. stage would induce PRL secretion which Materials and Methods could relate to the occurrence of the long diestrous period after the first spontaneous Animals ovulation. Virgin female Wistar-Imamichi strain rats

176 bred in our laboratory were used in the first cutaneously into rats between 07:00 and

(32-37 days of age, 47 rats; 95-110 g, body 08:00 hr on either estrous day or day 1 of weight) and in the third (45-55 days of age, diestrus in the first and the third estrous

180-210 g body weight) estrous cycle after cycle. Uterine horns were traumatized be- vaginal opening. They were housed in indi- tween 09:00 and 11:00 hr on day 3 of vidual cages under controlled conditions of diestrus and the rats were decapitated on light (lights on at 05:00-19:00 hr) and day 9 of diestrus. The uterine horn was temperature (24•}2•Ž). Commercial diet exposed through a midventral abdominal

(CE2, Nihon CLEA, Yokohama, Japan) and incision under ether anesthesia. A suture water were provided ad libitium. In pubertal needle with a silk thread was introduced into rats, vaginal opening was checked every the uterine lumen, the thread was looped morning after 28 days of age. Vaginal through the antimesometrial surface, and smears after vaginal opening were obtained tied loosely in the form of a loop on the by vaginal lavage with physiological saline outside of the uterus. Six loops were made at every morning until the end of the third appropriate intervals on each uterine born. estrous cycle, i.e. the fourth vaginal estrous In the shamoperated group, uterine horns day. The animal room was kept quiet for at were exposed and no thread was introduced. least one hour before blood sampling. Anim- Tunk blood was collected into a chilled als were decapitated within 15 seconds after heparinized glass tube and centrifuged im- removing them from the cages. mediately for 10 min at 1500 g and 4•Ž. Plasma was stored at -20•Ž until assayed for

Experiment1. Effects of a single injection of progesterone. Uteri were removed, dep- progesterone on the length of the diestrous rived of the surrounding tissue and weighed period to the nearest 0.5 mg. Progesterone (Sigma Chemical Co., St Louis, MO, USA) was subcutaneously admi- Experiment 3. Daily changes in PRL secretion nistered (1.25, 2.5, 5.0 or 7.5 mg in 0.2 ml on day 2 of long diestrous period induced by sesame oil per 100 g body weight) between progesterone 07:00 and 08:00 h on the day of estrus in the Progesterone at the dosage of 2.5 and 7.5 first or the third estrous cycle. In a part of mg per 100 g B.W. was administered sub- animals, 2.5 mg per 100 g body weight cutaneously between 07:00 and 08:00 hr on (B.W.) progesterone was injected between either the day of estrus or on day 1 of 07:00 and 08:00 hr on day 1, 2 or 3 of diestrus in the first or the third estrous diestrus in the first estrous cycle and 7.5 mg cycles. The animals were decapitated and progesterone per 100 g B.W. was injected trunk blood was collected at 05:00, 12:00 or between 07:00 and 08:00 hr on day 1 of 18:00 hr on day 2 of diestrus. Plasma was diestrus in the third estrous cycle. The stored at -20•Ž until assayed for PRL. vehicle was administered subcutaneously to the control animal. The first day on which Measurements of Hormones leucocytic vaginal smears appeared after the Plasma concentration of progesterone was treatment was designated day 1 of diestrus. measured by a radioimmunoassay using an antiserum raised in the rabbit against prog- Experiment 2. Effects of traumatization on esterone-3-carboxymethyloxime-BSA the function of corpora lutea of pseudopreg- (Teikoku Hormone MFG. Co. Ltd, Kawasa- nant rats induced by progesterone treatment ki, Japan), [1, 2, 6, 7,-3H(N)] progesterone Progesterone at the dosage of 2.5 and 7.5 (specific activity 96.5 Ci/mmol, New England mg per 100 g B.W. was injected sub- Nuclear, Boston, MA, USA) and progester-

177 one (Sigma) as described previously (Tomo- one-way analysis of variance was used to gane and Yokoyama, 1989). Each sample assess the statistical difference at P<0.05. (2.5 or 10 ƒÊl) was assayed in duplicate. Fisher's exact probability test (one-tailed Intra-assay coefficient of variation was 9.8% test) was used to compare the statistical at the level of 150 pg/tube. The lowest difference in the ratio between the two detectable level in the assay was 12.5 pg/ groups. tube. Results Plasma PRL was determined by using the rat PRL RIA kit provided by the NIDDK, Experiment 1: Effects of a single injection of Bethesda, MD, USA. Each sample (15 ƒÊl) progesterone on the length of the diestrous was assayed in duplicate. Plasma PRL values period (Table 1). were expressed in terms of NIDDK rPRL- In control animals treated with the vehicle RP-3 preparation. The intra-assay coef- on the day of the first estrus, 13 out of 25 ficient of variation was 7.6% at the level of (52%) showed diestrous vaginal smears last- 500 pg/tube. The lowest detectable level was ing for more than 9 days with the mean of

62.5 pg/tube. All samples were assayed in 11.5•}0.6 (S.E.M., n=13) (Table 1). The one radioimmunoassay. diestrous period was extended for more than 10 days in all rats by progesterone Statistical analysis administration at the dosage level of more

Results were expressed as mean•}SEM than 2.5 mg per 100 g B.W. on the first Student's t-test or Cochran-Cox test after estrous day.

Table 1. Effects of a single injection of progesterone on the length of the diestrous period in the first and the third estrous cycle in rats

78 When progesterone (2.5 mg/100 g B.W.) values in each group were combined values was administered either on the first day of from estrous day and day 1 of diestrus, since estrus or on day 1 of diestrus in rats showing all of the animals traumatized on either days the first estrous cycle, the appearance rate of formed deciduoma. In rats treated with diestrus continuing for more than 9 days was progesterone on either the day of estrus or significantly higher than that in rats treated on day 1 of diestrus in the first estrous cycle, on day 2 or 3 of diestrus (P<0.05, Table 1). the uterine weight and the concentration of In the third estrus cycle, the administra- plasma progesterone were significantly high- tion of progesterone (7.5 mg/100 g B.W.) on er in the traumatized group than in the the day of estrus induced the long diestrous sham-operated group (P<0.05). Significant- period lasting for more than 9 days in 89.4% ly higher (P<0.05) uterine weight and the of the treated animals, but no long diestrus plasma progesterone concentration were appeared after the administration on day 1 also obtained in traumatized rats treated of diestrus. The diestrous period was within with progesterone on the day of the third 8 days in all animals. estrus than in sham-operated animals (P<0.05). When progesterone was adminis- Experiment 2: Effects of traumatization on tered on day 1 of diestrus in rats showing the the function of corpora lutea of pseudopreg- third estrous cycle, however, the mean nant rats induced by progesterone treatment vaginal diestrous period was 8 days and the (Table 2). plasma progesterone concentration was sig- In rats showing the first estrous cycle, the nificantly lower than in rats treated with

Table 2. Effects of traumatization of the uterus on concentrations of plasma progesterone and weight of the uterus on day 9 of diestrus in pseudopreg- nancy induced by the treatment with progesterone on estrous day (E) or on day 1 of diestrus (D1) in the first and the third estrous cycle in rat

179 Table 3. Daily changes in prolactin secretion on day 2 of diestrus of the estrus cycle in animals injected with progestrene on estrous day (E) or day 1 of diestrus (DI) in the first and the third estrous cycle in rats

progesterone on the day of the third estrus rats showing the third estrous cycle is the (P<0.05, Table 2). same as that used in the adult rat in the previous workers (Everett, 1963; Rothchild Experiment 3: Daily changes in PRL secretion and Schubert, 1963; de Greef and Zeilmak- on day 2 of long diestrous period induced by er, 1978; Murakami et al., 1980; Luque and progesterone (Table 3). Castro-Vaquez, 1983). In rats showing the Plasma concentrations of PRL in pubertal first estrous cycle, only one third of prog- rats injected with progesterone (2.5 mg/100 esterone dosage in the third estrous day (2.5 g B.W.) on the day of the first estrus were mg/100 g B.W.) was required for inducing higher at 05:00 and 18:00 hr than at 12:00 daily changes in PRL secretion and conse- hr (P<0.05). In the third estrous cycle, quently, for forming the functional corpus however injection of progesterone (2.5 mg/ luteum, indicating that the rats showing the 100 g B.W.) on the day of estrus or (7.5 first estrous cycle is susceptible to progester- mg/100 g B.W.) on day 1 of diestrus did not one for occurring the pseudopregnancy. induce PRL surge at 05:00 and 18:00 hr. The appearance rate of pseudopregnancy When rats showing the third estrous cycle after the administration of progesterone on were injected with 7.5 mg per 100 g B.W. of day 1 of diestrus in the rat showing the first progesterone on the day of estrus, the estrous cycle was also higher than that in the plasma PRL concentration at 05:00 and vehicle treated controls, whereas the treat- 18:00 hr were also significantly higher than ment on day 1 of diestrus in the rat showing at 12:00 hr (P<0.05). the third estrous cycle failed to increase the appearance rate (Table 1). Discussion Our previous report in pubertal rats re- The present results indicate that adminis- vealed that progesterone administered with- tration of progesterone induced pseudop- in 2 days after ovariectomy on the first regnancy more effectively in the first estrous estrous day could induce the nocturnal PRL cycle than in the third estrous cycle (Table 1, surge (Tomogane and Yokoyama, 1989). 2, 3). The dose of progesterone used in the PRL is well known to play an essential role present experiments (7.5 mg/100g BW) for for inducing the functional corpus luteum in

180 the rat (Rothchild, 1981). Exposure of the observed in the first estrous cycle subsequent newly formed corpus luteum to PRL before to pregnant mare's serum gonadotrophin noon on day 2 of diestrus is indispensable (PMSG)-induced first ovulation and the for its function (Nikitovitch-Weiner and appearance rate of long diestrous period Everett, 1958; Malven, 1969; DOhler and increased with advancing age of PMSG Wuttke, 1974). The present results, there- administration from 22 to 28 days of age fore, suggest that the susceptive period of (Tomogane and Yokoyama, 1990). Then, in the corpus luteum to PRL in pubertal rats the second estrous cycle after the first showing the first estrous cycle would be PMSG-induced estrous cycle, we never similar to the period in adult rats reported observed the estrous cycle consisting of the by previous workers (Nikitovitch-Weiner diestrous period as pseudopregnancy and Everett, 1958; Malven, 1969; DOhler (Tomogane and Yokoyama, 1990). It has and Wuttke, 1974). been demonstrated that daily changes in The present results (Table 3) are consis- PRL secretion caused by cervical stimulus tent with the previous findings demonstrat- appears firstly at 25 days of age in immature ing that pubertal rats have a high susceptibil- rats (Smith and Ramaley, 1978). The action ity of the mechanism controlling the noctur- of progesterone to induce daily changes of nal surge of PRL to progesterone (Tomo- PRL secretion was less sensitive in adult gane and Yokoyama, 1989). In addition, cyclic rats in comparison with pubertal cyclic when progesterone administration in the rats (Tomogane and Yokoyama, 1989). morning of the estrous day was able to Therefore, an appearance of the long dies- induce daily change of PRL secretion, the trus in rats showing the first estrous cycle elevation in plasma PRL level immediately could be due to the change in the respon- after the administration occurred (de Greef siveness of hypothalamus to progesterone and Zeilmaker, 1987; Murakami et al., 1980). with the maturation of the individual This immediate elevation was also induced animal. by small dosage of progesterone on estrous The present results also confirmed the day in pubertal rats than in adult rats (H. previous finding that the uterine deciduoma Tomogane, K. Mizoguchi, A. Yokoyama, formed increased the level of plasma prog- unpublished data). These results thus sug- esterone (Hashimoto and Weist, 1969; Nuti gest that the difference in the appearance et al., 1975). It is likely that PRL-like hor- rate of the progesterone-induced pseudop- mone produced by the decidual tissue would regnancy between the first and the third exist in pubertal rats as well as the adult rat estrous cycle would be ascribed to the differ- (Jayatilak et al., 1984; Herz et al., 1986) and ence in the sensitivity of the hypothalamus that the deciduoma would prevents the controlling PRL secretion in response to production of the luteolytic substance (Cas- progesterone. trancane and Shaikh, 1976; Nanes et al., The long diestrous period lasting for 1983). more than 9 days in the first estrous cycle In summary, the present results together was observed in about a half of the pubertal with previous ones (Tomogane and animal treated with the vehicle (Table 1). Yokoyama, 1989; 1990) suggest the possible This rate is the same as in pubertal animals sequence inducing the long diestrus in the receiving no treatment (Tomogane et al., first estrous cycle in the rat: the prolactin 1987) or the animals which vaginal smears secretion was initiated by smaller amount of were not taken for 3 days after vaginal progesterone compared with the adult rat opening (H. Tomogane, unpublished data). because of the high sensitivity to progester- Similar long diestrous period was also one. PRL thus stimulates further secretion

181 of progesterone from the corpus luteum, the 113: 385-390. latter hormone in turn stimulating the secre- Malven PV (1969) Luteotrophic and luteolytic response to prolactin in hypothysectomized rats. Endocrinolo- tion of PRL. gy 84: 1224-1229. Murakami N, Takahashi M, Suzuki Y (1980) Induction Acknowledgment of pseudopregnancy and prolactin surges by a We are grateful to Dr. S. Raiti, National single injection of progesterone. Biol Reprod 22: 253-258. Hormone and Pituitary Program, NIDDK, Nanes MS, Morishige WK, Rothchild I (1983) The role Bethesda, MD, USA, for supplying rPRL- of decidual tissue in the development of the RIA kit. dependency on luteinizing hormone in the rat corpus luteum. Endorinology 112: 2181-2186. References Nikitovitch-Weiner M, Everett JW (1958) Comparative study of luteotropin secretion by hypophysial auto- Castracane VD, Shaikh AA (1976) Effect of decidual transplants in the rat: Effet of site and stages of the tissue on the uterine production of prostaglandins oestrous cycle. Endocrinology 62: 522-532. in pseudopregnant rats. . Reprod Fert 46: 101-104. Nuti KM, Sridharan BN, Meyer RK (1975) Reproduc- Miler KD, Wuttke W (1974) Total blockade of phasic tive biology of PMSG-primed immature female pituitary prolactin in rats: Effect on serum LH and rats. Biol Reprod 13: 38-44. progesterone during the estrous cycle. Endocrinolo- Rothchild I (1981) The regulation of mammalian gy 94: 1595-1960. corpus luteum. Recent Prog Horm Res 37: 183-298. Everett JW (1963) Pseudopregnancy in the rat from Rothchild I, Schubert R (1963) Corpus luteum-pituitary brief treatment with progesterone: Effect of isola- relationship: induction of pseudopregnancy in the tion. Nature 4881: 695-696. rat by progesterone. Endocrinology 72: 968-972. Hashimoto I, Wiest WG (1969) Correlation of the Smith MS, Ramaley JA (1978) Development of ability to secretion of ovarian steroids with function of a initiate and maintain prolactin surge induced by single generation of corpora lutea in the immature uterine cervical stimulation in immature rats. En- rat. Endocrinology 84: 873-885. docrinology 102: 351-357. Herz Z, Khan I, Jayatilak PG, Gibori G (1986) Evidence Tomogane H, Ikeda M, Yokoyama A (1987) Daily for the secretion of decidual luteotropin: A prolac- changes in concentration of plasma prolactin dur- tin-like hormone produced by rat decidual cells. ing diestrous period of the first estrous cycle in Endocrinology 118: 2203-2209. pubertal rats. In: Kyoto Prolactin Conference, vol. Jayatilk PG, Glaser LA, Warshaw ML, Herz Z, Gruber 2 (Hoshino K ed.), Kyoto University Faculty of JR, Gibori G (1984) Relationship between luteiniz- Medicine, Kyoto, pp41-50. ing hormone and decidual luterotropin in the Tomogane H, Yokoyama A (1989) Development of maintenance of luteal steroidgenesis. Biol Reprod progesterone dependency in the appearance of 31: 556-564. nocturnal prolactin surge in immature rats. J de Greef WJ, Zeilmaker GH (1978) Serum prolactin Reprod Fertil 85: 503-509. concentration during hormonally induced Tomogane H, Yokoyama A (1990) Positive feedback pseudopregnancy in the rat. Endocrinology 105: relationship between progesterone and daily 195-199. change of prolactin secretion that develops func- Luque EH, Castro-Vazquez A (1983) Sensory mechan- tional corpora lutea in PMSG-induced first oestrous isms involved in the induction of pseudopregnancy cycle in prepubertal rats. Comp BiochemPhysiol 95A: by progesterone: Increased sensitivity to stimula- 185-188. tion of the pudendal sencory field. Endocrinology

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友金 弘 ・横 山 昭 名 古 屋 大 学 農 学 部 家 畜 繁 殖 学 教 室,〒464-01名 古 屋 市 千 種 区 不 老 町

幼 若 ラ ッ トの初 回発 情 周 期 時 に高 頻 度 で 認 め られ る偽 黄 体 は機 能 化 し,血 中 プ ロ ジ ェ ス テ ロ ン は偽 妊 娠 様 の 高 妊 娠 様 発 情 休止 期 の発 生機 序 を明 らか にす る一 端 と して, 濃 度 を示 し,脱 落 膜 腫 形 成 に よ りさ ら に上 昇 した。 一 方, 開 口後 の 初 回発 情 周 期 時 と3回 目発 情 周 期 時 との 間で腟 3回 目の発 情 周 期 時 の ラ ッ トで は 同様 の 効 果 を得 る ため プ ロ ジ ェス テ ロ ンの偽 妊娠 誘 起 効 果 を比 較 した。 の プ ロ ジ ェ ス テ ロ ン量 は増 加 し,初 回発 情 周 期 時 の3倍 初 回 発 情 周 期 を示 す 幼 若 動 物 で は体 重100g当 た り 量 を必 要 と した。 2.5mgの プ ロ ジ ェ ス テ ロ ン を発 情 日 あ る い は発 情 休 止 こ れ らの結 果 は,幼 若 動 物 の初 回発 情 周 期 時 に は プ ロ 期1日 目 に投 与 す る とすべ て の動 物 で発 情 休 止 期 は9日 ジ ェス テ ロ ンが偽 妊 娠 様 発 情 休 止 期 を効 率 よ く誘 起 す る 以 上 と な り,血 中 プ ロ ラ クチ ン濃 度 は 日内変 動 を示 した。 こ と を示 す 。

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