Facilitation of Luteinizing Hormone Release by Progesterone in Proestrous Rats FUMIHIKO KOBAYASHI, KATSUMI HARA AND TAMOTSU MIYAKE

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1 Facilitation of Luteinizing Hormone Release by Progesterone in Proestrous Rats FUMIHIKO KOBAYASHI, KATSUMI HARA AND TAMOTSU MIYAKE Shionogi Research Laboratory, Shionogi & Co., Ltd., Fukushima-ku, Osaka Synopsis In order to elucidate the facilitation of ovulatory release of luteinizing hormone (LH) and subsequent ovulation, subcutaneous injection or intrahypothalamic implantation of progesterone was carried out on the morning of proestrus in 4-day cyclic rats. By injecting progesterone on the morning or early afternoon of proestrus, an approximate 3 hr advancement of ovulatory release of LH was demonstrated by confirming the occurrence of ovulation on the next morning after timed hypophysectomy performed on the afternoon of proestrus. Ovulation also occurred about 3 hr earlier in the estrous morning when progesterone had been given on the morning of proestrus. The minimum effective dose of progesterone to facilitate LH-release was somewhere between 0.1 and 0.625mg per rat. Facilitation of LH-release by steroid administration on the morning of proestrus is not entirely specific for progesterone. Norethisterone, medroxyprogesterone acetate, and desoxycorticosterone(doc) also induced the advanced release of LH. Facilitation of ovulatory release of LH was induced also in the rats having progesterone crystals implanted in the median eminence-arcuate region of the hypothalamus on the morning of proestrus. These results indicate that a neural timing factor regulating the onset of LHrelease in proestrus is labile to a certain extent, and that it operates about 3 hr in advance when it is exposed to progesterone given on the morning of proestrus, and that the appearance of facilitatory action of progesterone depends on the pre-existing estrogen level in the circulating blood. Received for publication April 8, 1970 It is well known that progesterone has a biphasic effect on ovulation and, according to the conditions, it can either stimulate or inhibit the release of luteinizing hormone (LH) (for a review see Everett, 1964; Sawyer, 1964; Rothchild, 1965; Davidson, 1969). A lack of spontaneous ovulation during pregnancy or pseudopregnancy is evidence of the inhibitory effect on LH-release of endogenous progesterone, the secretion of which is increased during gestation. Exogenous progesterone also inhibits or delays spontaneous ovulation in rats and in many other species. The stimulating effect of progesterone on ovulation has been shown under several conditions, e. g. in persistent estrous rats(everett, 1940), in normal 5-day cyclic rats(everett, 1948; Brown- Grant, 1967) and in immature rats primed with pregnant mare's serum gonadotropin(mc- Cormack and Meyer, 1963 and 1965). With regard to the ovulation induction in adult rats, it was previously suggested by Everett(1948) that the appearance of stimulating action of progesterone depends on the pre-existing estrogen level in the circulating blood. In our previous work, progesterone administered into 4-day cyclic rats induced a delayed ovulation only when injected before 2 a. m. proestrus, by which time estrogen secretion had not attained the maximum(hori et al., 1968), and it accounted for uterine distension and uterine weight increase on the day of proestrus though it was not enough for the release of LH on the same day (Kobayashi

2 et al., 1969b and d). Recently, Uchida et al. (1969c) in our laboratory demonstrated that progesterone given on the morning of proestrus caused an increase in ovarian progesterone secretion one hr earlier than the time of its spontaneous increase seen at 5: 30 p. m. proestrus, suggesting the accerelation of LHrelease by exogenous progesterone. Zeilmaker (1966) also reported the advanced release of ovulating hormone by the injection of progesterone on the morning of proestrus in 4-or 5-day cyclic rats. The present experiments were designed to elucidate the stimulatory effect of progesterone, given either by subcutaneous injection or by intrahypothalamic implantation on the day of proestrus, on the release of LH and the subsequent ovulation in 4-day cyclic rats. Materials and Methods Adult female rats of the Wistar strain weighing approximately 200g were used. They were housed in an air-conditioned room and exposed to light from 8 a.m. to 8 p. m. Vaginal smears were examined every morning at least 2 weeks before experiment and the rats showing regular 4-day estrous cycles were employed. For the subcutaneous injection of progesterone and other steroid, the compounds were dissolved or suspended in sesame oil and administered into the neck with a volume of 0.2ml per injection. To investigate whether progesterone had any activity for advancing the time of LH-release, which spontaneously occurred between 5 and 7p. m. proestrus under our lighting schedule (Kobayashi et al., 1968), subcutaneous injection or intrahypothalamic implantation of the steroid was carried out on the day of proestrus. The occurrence of ovulatory release of LH was confirmed by ovulation which was identified in the following morning after the treatment by the presence of ova in the dilated ampullae of the oviducts as described in the previous report(kobayashi and Miyake, 1960). Experiment 1. Progesterone was injected subcutaneously at 9a.m., 11a.m. or 1p.m. proestrus at a single dose of 10 mg per rat. During the afternoon on the same day, rats from a group of 5 to 10 animals were hypophysectomized, hourly from 1p.m. till 7 p.m., by the Tanaka's trans-auricular method (Tanaka, 1955) under methylhexabital sodium(125mg/kg, i.p.) anesthesia. They were autopsied the following morning to inspect ovulation, and the time of LH-release accounting for the ovulation was ascertained. Experiment 2. Following treatment with varying doses of progesterone or with 10mg each of the other steroids listed in Table 2 at 11a.m. proestrus, rats were hypophysectomized at 4 p.m. on the same day and autopsied the next morning for the confirmation of ovulation. Experiment 3. Progesterone was injected subcutaneously at 11 a.m. proestrus with a dose of 10mg per rat. Rats from a group of 5 to 10 animals were autopsied, hourly between 11 p.m. proestrus and 7 a.m. estrus, in order to determine the time of ovulation. Experiment 4. Crystalline progesterone was tapped into the tips of stainless steel tubes with an inner diameter of 0.2mm and an outer diameter of 0.5mm. The outer surface of the tube was carefully scraped and cleaned with ether under a dissecting microscope. Bilateral implantations of progesterone were stereotaxically placed in the anterior hypothalamic area, the medial basal hypothalamus, or the anterior pituitary, before 11:30 a.m. on the morning of proestrus under methylhexabital sodium anesthesia and the tubes were fixed on the skull with dental cement. All the operated rats were anesthetized again with pentobarbital sodium, 40mg/kg, at 4 p.m. on the same day to prevent later release of LH. As a control experiment, crystalline cholesterol was implanted in the same hypothalamic or hypophysial region by the same experimental procedures. Ovulation was confirmed the following morning as described above. Brains were carefully dissected out after perfusion, initially with saline and subsequently with 10% formalin, fixed in 10% formalin for at least 1 week, sectioned by a freezing method and stained with thionin. The locations of implantation sites were ascertained by histological examination. Results Experiment 1. Effect of progesterone on ovulatory release of LH in proestrous rats In order to determine whether the time of ovulatory release of LH is affected by progesterone given at 9a. m., 11a. m. or 1p. m. proestrus with a dose of 10mg per rat, hypophysectomy was performed at intervals of one hr on the afternoon of the same day and autopsy was done on the following morning for inspecting ovulation. The results are presented

3 PROGESTERONE FACILITATION ON LH-RELEASE Table 1. Effects of progesterone treatment and hypophysectomy on ovulation in proestrous rats') a) Ovulation was inspected in the following morning after treatment. b) No. of rats ovulated/no. of rats treated. (): Average No. of ova per rat. in Table 1. In the controls, ovulation did not occur at all on the morning of estrus in the rats hypophysectomized before 4 p. m. proestrus but did occur in the rats operated on at 5 p. m. or later. In the rats given progesterone at 9 a. m. proestrus, 4 out of 10 rats hypophysectomized at 2 p. m., more than a half of the rats hypophysectomized at 3 or 4 p. m. and all of the rats operated on at 5 p. m. proestrus ovulated on the morning of estrus. When the rats had been treated with progesterone at 11 a. m. proestrus, ovulation was confirmed on estrus in nearly all the rats hypophysectomized at 3 p. m. proestrus and all of the rats ovulated when operated on at 4 or 5 p. m. proestrus. Full ovulation was observed also in the rats given progesterone at 1 p. m. proestrus and hypophysectomized 4 hr later. These results clearly indicate that progesterone, when given on the morning or on the early afternoon of proestrus, advances the time of ovulatory release of LH, the "critical period" for LHrelease, by 2 or 3 hr. Regardless of the timing of progesterone injection tried so far, ovulation was obtained in many cases even in the rats hypophysectomized at 2 or 3 p. m. on the same day, suggesting that the advancement of the "critical period" for LH-release, if stimulated by progesterone, is limited in time for about 3 hr. Experiment 2. Effects of progesterone and several other steroids on ovulatory release of LH in proestrous rats To find out the minimum effective dose of progesterone to advance the "critical period" for LH-release in proestrus, rats were treated with varying doses of progesterone at 11 a. m. proestrus and were hypophysectomized at 4 Table 2. Effects of progesterone and other steroids on ovulation in proestrous ratsa) a) Rats were hypophysectomized at 4 p.m. proestrus and the occurrence of ovulation was inspected in the following morning after treatment. b) Steroid was injected subcutaneously at 11 a.m. proestrus. MAP: Medroxyprogesterone acetate. DOC: 11-Desoxycorticosterone.

4 KOBAYASHI et al. Endocrinol. April 1970 Japon. p. m. on the same day. As indicated in Table 2, all of the rats given mg progesterone per rat showed ovulation on the morning of estrus, whereas the rats treated with 0.1mg progesterone failed to ovulate, suggesting that the minimal effective dose was in the range of 0.1 to mg. The effects of several other steroids, 10 mg per rat, on the ovulatory release of LH are also presented in Table 2. Among the steroids tested, norethisterone, medroxyprogesterone acetate, and desoxycorticosterone (DOC) showed the same activity as progesterone in facilitating ovulatory release of LH; norethynodrel showed an activity similar to or less than those of the above compounds; testosterone was quite inactive in this study. These results indicate that facilitation of ovulatory release of LH by the steroid administration on the morning of proestrus is not entirely specific for progesterone. Experiment 3. Effect of progesterone given at proestrus on the time of ovulation at estrus To investigate whether the time of ovulation normally occurring on the morning of estrus is advanced or not by the injection of progesterone at 11 a. m. proestrus, ovulation was checked hourly by inspecting oviducts during the period between 11 p. m. proestrus and 7 a. m. estrus. In the control rats, spontaneous ovulation was initially observed in 3 out of 5 rats at 4 a. m. estrus and all of the rats had ovulated by 7 a. m. estrus, being just 12 hr after the peak of LH-release on the afternoon of proestrus under our lighting schedule (Kobayashi et al., 1968). In the rats treated with 10 mg progesterone at 11 a. m. proestrus, earliest ovulation was observed at 2 a. m. estrus in 4 out of 10 rats and all of the rats showed ovulation by 3 a. m., being also approximately 12 hr after the advanced release of LH (cf. Table 1). These results indicate that progesterone, when given on the morning of proestrus, advances by 2 or 3 hr not only the time of LH-release on the afternoon of proestrus but also the time of ovulation in the rats on the next morning. Experiment 4. Effect of progesterone implantation in the hypothalamus on ovulatory release of LH in proestrous rats Crystalline progesterone tapped into stainless steel tubes was implanted bilaterally in the anterior hypothalamic area, the medial basal hypothalamus, or the anterior pituitary of the cyclic rats on the morning of proestrus to investigate the localization of progesterone facilitating ovulatory release of LH. The results presented in Table 4 demonstrate that the Table 4. Effects of bilateral progesterone implantsa) on ovulation in proestrous ratsb) Table 3. Effects of progesterone given at 11 a.m. proestrus on the timing of ovulation in the rats a) No. of rats ovulated/no. of rats treated. (): Average No. of ova per rat. a) Bilateral implantation of progesterone or cholesterol was carried out before 11:30 a.m. on the morning of proestrus. b) Rats were anesthetized with sodium pentobarbital at 4 p.m. proestrus and were inspected for ovulation in the following morning aftei treatment. c) No. of rats ovulated/no. of rats treated. (): Average number of ova per rats ovulated. AHA: Anterior hypothalamic area. MBH: Medial basal hypothalamus. AP: Anterior pituitary.

5 PROGESTERONE FACILITATION ON LH-RELEASE facilitation of ovulation was observed only in the rats having progesterone implanted in the medial basal hypothalamus. Implantation of progesterone in the anterior hypothalamic area and in the anterior pituitary was less effective, and intrahypothalamic or intrahypophysial implantation of crystalline cholesterol, as a control, was not effective, in advancing ovulatory release of LH in proestrous rats. In addition, as can be seen from Figure 1, advancement of ovulatory release of LH was only manifested in the rats receiving proges- terone implantation in the median eminencearcuate region of hypothalamus. Implantation of progesterone in the ventromedial hypothalamus was entirely ineffective in advancing ovulatory release of LH. These results indicate that the median eminence-arcuate region of hypothalamus is the site of stimulatory action of progesterone in facilitating ovulatory release of LH in proestrous rats. Discussion Fig. 1. Parasagittal diagram of rat hypothalamus illustrating localization of bilateral progesterone implants in proestrous rats. Solid circles represent the implants that advanced LH release; open circles indicate the implants that failed to advance LH release. Abbreviations:ah= anterior hypothalamic area; arc= arcuate nucleus; dmh = dorsomedial nucleus; mm= medial mammillary nucleus; pm= dorsal premammillary nucleus pv= paraventricular nucleus; sch= suprachiasmatic nucleus; so supraoptic nucleus; vmh ventromedial nucleus; AP= Anterior pituitary; CC= corpus callosum; CA= commissura anterior; CHO= optic chiasma; FX fornix; PP= posterior pituitary. The present experiments clearly demonstrate that progesterone facilitates the time of ovulatory release of LH by about 3 hr when administered on the morning or early afternoon of proestrus in 4-day cyclic rats. This confirms the results of Zeilmaker (1966), who claimed that injection of progesterone on the morning of proestrus resulted in the advancement of LH-release by about 3 hr in 4- or 5- day cyclic rats. Recently, Uchida et al. (1969c) in our laboratory demonstrated that progesterone given subcutaneously on the day of proestrus between 3 a. m. and 1 p. m. increased progesterone secretion into the ovarian venous blood collected at 4 p. m. on the same day. Since the preovulatory increase in progesterone secretion depends upon the pituitary LH (Uchida et al., 1969a and b), the advanced increase in progesterone secretion in the proestrous afternoon caused by the injection of progesterone on the morning of proestrus is considered as the result of advancement of LH-release induced by the exogenous progesterone, which is clearly shown in the present study. The advanced release of LH was induced also by synthetic progestins and DOC given on the morning of proestrus, suggesting that the facilitation of LH-release was not entirely specific for progesterone. This agrees with the result of our previous experiment (Kobayashi et al., 1969c), in which a 48-hr delay of ovulation induced by progesterone treatment on diestrus was restored by 24 hr with synthetic progestins given appropriately.

6 Endocrinol. April 1970 Japon. Similar action of synthetic progestin was demonstrated in immature rats by McCormack and Meyer (1963 and 1965). Compared with the dose of progesterone which accelerated spontaneous ovulation, a huge amount of progesterone was required to restore delayed ovulation (Kobayashi et al., 1969a). Further, norethynodrel, which failed to restore delayed ovulation in our previous study (Kobayashi et al., 1969c), facilitated LH-release in 3 out of 6 rats in this study. These results suggest that spontaneous ovulation is a much more sensitive and favorable indicator than delayed ovulation for analyzing the facilitatory action of the steroid. The time of ovulation on the early morning of estrus was also advanced by 2 to 3 hr when progesterone had been given on the morning of proestrus. This means that progesterone administered on the morning of proestrus not only facilitates LH-release on the afternoon of proestrus but also accelerates the subsequent ovulation occurring the next morning without changing the interval between LH-release and ovulation. It is presumed, therefore, that progesterone does not affect the process of follicular development in the period between LH-release and ovulation. There are many evidences demonstrating that the time of ovulatory release of LH always shifts with an exact 24-hr interval in rats whenever delay or advancement of ovulation occurs after the treatment with ovarian steroids or other pharmacological agents (Everett, 1964; Rothchild, 1965; Markee et al., 1952). Also our previous experiment substantiated that the delayed ovulation induced by progesterone was restored by the injection of estrogen or additional progesterone, and that the LH concentration in the circulating blood always reached a peak at the same time, 7 p. m., as the spontaneous LHrelease in proestrus was at a maximum (Kobayashi et al., 1969a). The present study, however, clearly demonstrates that the ovulatory release of LH advanced about 3 hr when progesterone was given on the morning of proestrus. Accordingly, the previous conclusion should be corrected, as the timing mechanism in the central nervous system, which regulates the onset of LH-release under a certain lighting schedule, can be modified daily, as well as hourly, by the action of progesterone. The facilitatory effect of progesterone on LH-release and subsequent ovulation manifests itself only when the steroid is given within a certain time period, during the morning and early afternoon of proestrus; the injection of progesterone before 2 a. m. proestrus completely suppresses spontaneous ovulation (Kobayashi et al., 1969a ; Brown-Grant, 1967). In addition, it was demonstrated that the estrogen secreted through the afternoon of diestrus till about 2 a. m. proestrus accounted for the uterine distension and the increase of uterine weight in proestrus (Kobayashi et al., 1969b). It is suggested, therefore, that the facilitatory effect of progesterone on LHrelease is manifested only when enough estrogen to induce the uterine changes has been secreted. This concept agrees with a recent finding of Brown-Grant (1969), who demonstrated that ovulation in 4-day cyclic rats could be induced in the expected proestrus by a sequential injection of progesterone on diestrus following estradiol benzoate in metestrus. Progesterone crystals implanted bilaterally in the median eminence-arcuate region of hypothalamus on the morning of proestrus produced advancement of ovulatory release of LH. This confirms the recent report of DOcke and DOrner (1969), who indicated that when progesterone had been implanted bilaterally in the ventromedial-arcuate region of hypothalamus on the day of proestrus in cyclic rats primed with progesterone two days earlier to induce ovulation delay, the percentage of ovulation in the expected estrus was significantly increased. In the present experiment, however, progesterone implanted in the ventromedial hypothalamus failed to facilitate ovulatory release of LH. The difference between the function of the ventromedial hypo-

7 Vol.17, No.2 PROGESTERONE FACILITATION ON LH-RELEASE thalamus and the median eminence-arcuate region in the mechanism of gonadotropin secretion was demonstrated by electro-physiological studies in cats and rats (Kawakami and Saito, 1967; Terasawa et al., 1969). It is possible that progesterone implanted in the median eminence-arcuate region of hypothalamus acts on the anterior pituitary by transport through the hypophysial portal circulation. This direct action on the pituitary is excluded, however, by the finding that bilateral intrapituitary implantation of progesterone was completely inefective in facilitating LH-release. The anterior hypothalamic region, which was initially considered as a stimulatory feedback site of progesterone (Barraclough et al., 1964), is also excluded as a progesterone feedback site facilitating the ovulatory release of LH. The results obtained from the present study suggest, therefore, that the median eminence-arcuate region of hypothalamus is the primary site of progesterone action in facilitating LH-release. References Barraclough, C. A., S. Yrarrazaval and R. Hatton (1964). Endocrinology 75, 838. Brown-Grant, K. (1967). J. Physiol. 190, 101. Brown-Grant, K. (1969). J. Endocrinol. 43, 553. Davidson, J. M. Frontiers in Neuroendocrinology, Ganong, W. F. and Martini, L. (ed.). Oxford Univ. Press, London and Tronto, p. 343 (1969). DOcke, F. and G. DOrner (1969). Neuroendocrinology 4, 139. Everett, J. W. (1940). Endocrinology 27, 681. Everett, J. W. (1948). Ibid. 43, 389. Everett, J. W. (1964). Physiol. Rev. 44, 373. Hori, T., M. Ide and T. Miyake (1968). Endocrinol. Japon. 15, 215. Kawakami, M. and H. Saito (1967). Japan. J. Physiol. 17, 466. Kobayashi, F. and T. Miyake (1960). Ann. Rept. Shionogi Res. Lab. 10, 213. (1968). Endocrinol. Japon. 15, 313. (1969a). Ibid. 16, 251. (1969b). Ibid. 16, 261. (1969c). Ibid. 16, 493. (1969d). Ibid. 16, 501. Markee, J. W., J. W. Everett and C. H. Sawyer (1952). Rec. Progr. Hormone Res. 7, 139. McCormack, C. E. and R. K. Meyer (1963). Gen. Comp. Endocr. 3, 300. McCormack, C. E. and R. K. Meyer (1965). Fertil. Steril. 16, 384. Rothchild, I. (1965). Vitamins and Hormones 23, 209. Sawyer, C. H. Gonadotropins. Cole, H. H. (ed.). Freeman, San Francisco and London, p.113 (1964). Tanaka, A. (1955). Ann. Rept. Shionogi Res. Lab. 5, 154. Terasawa, E., D. I. Whitmoyer and C. H. Sawyer (1969). Amer. J. Physiol. 217, Uchida, K., M. Kadowaki and T. Miyake (1969a). Endocrinol. Japon. 16, 227. Uchida, K., M. Kadowaki and T. Miyake (1969b). Ibid. 16, 239. Uchida, K., M. Kadowaki and T. Miyake (1969c). Ibid. 16, 485. Zeilmaker, G. H. (1966). Acta Endocrinol. (Kbh.) 51, 461.