Changes in FSH, LH and Prolactin Secretion During Estrous Cycle in Rats KAZUYOSHI TAYA AND MASAO IGARASHI Department of Obstetrics & Gynecology, School of Medicine, Gunma University, Maebashi Synopsis Serum and pituitary FSH, LH and prolactin contents during the normal rat estrous cycles were assayed every two hours in adult female rats of Holtzman strain (lights on 5 a.m. to 7 p.m.), using NIAMD rat FSH, LH and prolactin radioimmunoassay kits. The serum LH surge was observed from 6:00 p.m. to 7:00 p.m. on the proestrous day. The serum FSH surge was observed from 7:00 p.m. on the proestrous day to 5:00 a.m. on the estrous day. The serum prolactin concentrations also began to rise at 1:00 p.m. on the proestrous day, and remained at high levels until 10:00 a.m. on the estrous day. The reduction in pituitary FSH, LH and prolactin content was simultaneously observed from 7:00 p.m. to 11:00 p.m. on the proestrous day. These results not only comfirmed the data reported by Daane & Parlow (1971), but also demonstrated no diurnal rhythm on the first and second diestrous day. Dissociation between FSH and LH patterns seems to suggest the possibility that FSH and LH secretion may be controlled independently by the two distinct"releasing factors, FRF and LRF". Cyclic changes in FSH, LH and prolactin secretion during the rat estrous cycle have been given attention and speculated. However, it has not been easy to assay the blood FSH and LH levels using bioassay, because of low sensitivity of the bioassay methods. Schwartz and Bartosik (1962), Ramirez and McCann (1964), Schwartz and Caldarelli (1965), Igarashi (1967), Kobayashi et al. (1968), McClintock and Schwartz (1968) and Kamioka (1970) reported on the cyclic changes in rat pituitary or blood LH secretion using the ovarian ascorbic acid depletion (OAAD) bioassay (Parlow, 1961). Gans et al.(1964), Igarashi (1967), Caligaris et al.(1967), Mc- Clintock and Schwartz (1968), Goldman and Mahesh (1968), Fawke and Brown (1970) and Kamioka (1970) reported on the cyclic Received for publication March 15, 1973. changes in rat pituitary or blood FSH secretion using bioassay methods. Quite recently Monroe et at.(1969), Gay et al.(1970) and Daane and Parlow (1971) reported on the cyclic changes in blood FSH and LH, using rat FSH and LH radioimmunoassay. In this paper, the changes in more detail every two hours in serum and pituitary FSH, LH and prolactin levels were investigated than in the Monroe et al.'s, Gay et al.'s and Daane and Parlow's reports were investigated in order to clarify the diurnal rhythm of these pituitary hormones in the 4-day estrous cycle of the rat. Materials and Methods Virgin female Holtzman rats were maintained for
TAYA AND IGARASHI 3weeks at controlled room temperature (25 }2 Ž) and lighting (lights on 5:00a.m. to 7:00p.m.) prior to initiation of vaginal smearing. Only those rats demonstrating 2 or more regular 4-day cycles were used. Blood samples were obtained from the jugular vein under ether anesthesia, allowed to clot at room temperature, and then centrifuged. Serum was stored at-20 Ž until assayed. Anterior pituitary glands, removed immediately after sacrifice, were weighed and homogenized in saline. The extracts were stored at-20 Ž until assayed. The sera or pituitary extracts from five rats were pooled in each same group. FSH, LH and prolactin concentrations in the sera and pituitary extracts were determined, using the NIAMD rat FSH, LH and prolactin radioimmunoassay method according to the Daane and Parlow (1971) method slightly modified by Wakabayashi et al.(1972). All oviducts were examined under a dissecting microscope for the presence of ova in the distended ampullary portion, as described by Rowlands (1942). Results Changes in serum LH The sequential changes in serum LH levels during the estrous cycle were summarized in Figure 1. Serum LH levels began to rise at 3:00 p.m. on the proestrous day and showed a remarkable elevation of serum LH levels, the so called"lh surge", between 6:00p.m. and 7:00p.m. on the same day. Thereafter, serum LH levels decreased sharply, but remained at relatively high levels until 11:00 p.m. on the proestrous day. Therefore serum LH levels remained low until the next rise in the afternoon on the proestrous day. 7:00 p.m. on the estrous day, anterior pituitary LH content remained at relatively lower values. Minimal anterior pituitary LH content was observed at 9:00 p.m. on the proestrous day and maximal anterior pituitary LH content was observed at 4:00 p.m. on the proestrous day. Changes in serum FSH The changes in serum FSH during the normal estrous cycle in rats were summarized in Figure 1. Serum FSH levels began to rise at 4:00p.m. on the proestrous day, and attained the peak at 7:00 p.m. on the same day. This maximal elevation in serum FSH persisted until 5:00a.m. on the estrous day. Thereafter, serum FSH levels began to decrease slowly from 7:00 a.m. on the estrous day, and returned to the basic level at 1:00 p.m. on the estrous day. On the first and second diestrous day, serum FSH levels were lower than those observed on the afternoon of the proestrous day and the morning of the estrous day. Changes in anterior pituitary FSH The sequential changes in anterior pituitary FSH content during the estrous cycle were summarized in Figure 2. A sharp decline in anterior pituitary FSH content was observed from 7:00p.m. to 11:00p.m. on the proestrous day. At 1:00a.m. on the estrous day, the FSH content in pituitary returned to the basal level, although the FSH peak in serum still persisted. At the other stages of the estrous cycle, anterior pituitary FSH content showed no diurnal rhythmic change. Changes in anterior pituitary LH The changes in anterior pituitary LH content were summarized in Figure 2. The reduction in anterior pituitary LH content was observed from 7:00p.m. to 11:00p.m. on the proestrous day. Thereafter, from 1:00 a.m. on the estrous day, anterior pituitary LH content began to rise slowly. However, until Changes in serum prolactin The sequence of changes in serum levels of prolactin during the estrous cycle was summarized in Figure 1. Serum prolactin levels also began to rise slowly, at 1:00 p.m. on the proestrous day. These high levels of serum prolactin persisted until 10:00a.m. on the estrous day. Thereafter serum prolactin levels declined slowly to basic levels. At the
FSH, LH, PROLACTIN IN RAT ESTROUS CYCLE Fig. 1. Concentrations of LH, FSH and prolactin in rat sera during normal estrous cycle expressed in terms of NIAMD-RAT-LH-RP-I, NIAMD-RAT-FSH-RP-I and miu/ml. Five rats were used in the each group.
TAYA AND IGARASHI Fig. 2. Concentrations of LH, FSH and prolactin in rat anterior pituitary during normal estrous cycle, expressed in terms of NIAMD-RAT-LH-RP-I, NIAMD-RAT- FSH-RP-I and miu/pit. Five anterior pituitaries were pooled in the same group.
FSH, LH, PROLACTIN IN RAT ESTROUS CYCLE Table 1. Time of occurrence of ovulation on the morning of estrus other stages of the estrous cycle, serum prolactin concentration remained at relatively low levels. Changes in anterior pituitary prolactin A sharp decline in anterior pituitary prolactin content was observed from 7:00p.m. to 11:00p.m. on the proestrous day, as shown in LH and FSH secretion. Thereafter, a gradual recovery of anterior pituitary prolactin content occurred on the morning of the estrous day, reaching a maximal level at 9:00 p.m. on the estrous day. Minimal anterior pituitary prolactin content was shown at 9:00p.m. on the proestrous day, while the serum prolactin level showed a maximal value at the same time. Time of occurrence of ovulation on the morning of the estrous day The results were shown in Table 1. In our colony, under our lighting conditions, tubal ova were found in 80% of the rats examined from 5:00a.m. to 5:50a.m. on the morning of the estrous day. Discussion The present experiments demonstrated the sequence of changes in serum and anterior pituitary LH, FSH and prolactin concentrations during the estrous cycle of rats. The changes in the content of anterior pituitary LH during the estrous cycle were, for the most part, in agreement with previously reported observations obtained by bioassay (Schwartz and Bartosik, 1962; Anderson and McShan, 1966; Igarashi, 1967; Kobayashi et al., 1968; Kamioka, 1970) and by radioimmunoassay (Monroe et al., 1969; Gay et al., 1970; Daane and Parlow, 1971). Some earlier investigators using bioassay techniques found elevated serum LH concentrations on the afternoon of the proestrous day and the morning of the proestrous day (Schwartz and Caldarelli, 1965; Ramirez and McCann, 1964; Anderson and McShan, 1966; Igarashi, 1967 ; Kobayashi et al., 1968; Kamioka, 1970). Monroe et al.(1969), Gay et al.(1970) and Daane and Parlow (1971), using radioimmunoassay, observed that high concentrations of serum LH were detected only on the afternoon and evening of the proestrous day. In our studies, high concentrations of serum LH were observed only on the afternoon and evening of the proestrous day. The sequence of changes in serum LH concentration during the estrous cycle of rats in our report is similar to that in previous reports obtained by radioimmunoassay. Some of the slight differences between this and earlier reports obtained by bioassay may be attributed to the lack of sensitivity of bioassay techniques as discussed by Monroe et al. (1969). Some earlier investigators using bioassay techniques observed that anterior pituitary FSH contents significantly fell on the afternoon of the proestrous day (Gans et al., 1964; Igarashi, 1967; Caligaris et al., 1967; McClintock and Schwartz, 1968; Kamioka, 1970). More recently, Daane and Parlow (1971), using radioimmunoassay, observed similar results. The present results were in agreement with previously reported observations obtained by bioassay and radioimmunoassay. However, Goldman and Mahesh (1968) observed that anterior pituitary FSH contents decreased significantly during the day of proestrus in 5-day cycle rats, but not significantly in 4-day cycle rats. Fawke and Brown (1970) did not observe the reduction in anterior pituitary FSH contents on the after-
TAYA AND IGARASHI noon of the proestrous day. Some of the differences between this. and present report may also be a result of variability in the strain of rat studied and difference of the time of autopsy. The changes in the concentration of serum FSH during the proestrus and estrus as measured by radioimmunoassay were, for the most part, in agreement with Daane and Parlow's data (1971). However, the present results not only confirmed the data reported by Daane and Parlow (1971) but also demonstrated no diurnal rhythm on the first and second diestrous days as reported by Gay et al.(1970). Some earlier investigators using bioassay found elevated serum FSH concentrations on the afternoon of the proestrous day but these reporters did not observe the changes in concentrations of serum FSH during the morning of the estrous day (Igarashi, 1967; McClintok and Schwartz, 1968; Kamioka, 1970). Some earlier investigators found that anterior pituitary prolactin content of rats and guinea pigs was higher during proestrus and estrus than during diestrus (Reece and Turner, 1937; Reece, 1939; Sar and Meites, 1967). In the present report, anterior pituitary prolactin content was sharply decreased from 7:00 p.m. to 11:00 p.m. on the proestrous day as shown in LH and FSH secretion. Kwa and Verhofstad (1967), Niswendar et al.(1.969), Grindeland et al.(1969), Gay et al.(1970) and Neill and Reichert (1971) reported that the highest levels of serum prolactin occurred on the afternoon of the day of proestrus, whereas Llerena et al. (1969) observed an elevation on the afternoon of the day of estrus. Amenomori et al.(1970) observed the highest serum prolactin concentration on the day of estrus, although it was not significantly higher than on the day of proestrus and metaestrus. An elevation of serum prolactin levels was observed in our studies from 1:00p.m. on the day of proestrus to 10:00a.m. on the day of estrus. As above-mentioned, the anterior pituitary LH, FSH and prolactin contents fell sharply at the same time, from 7:00p.m. to 11:00 p.m. on the day of proestrus, but there was a big difference in the time of accelerated secretion among LH, FSH and prolactin in serum. Discrepancy between serum FSH and LH patterns seems to suggest that FSH and LH secretion may be controlled by two distinct "Releasing factors", FRF and LRF, although Arimura et al.(1972) and Guillemin (1971) are claiming that LH-RH, decapeptide, is the only or the principal FSH-RH. Acknowledgment The authors are grateful to the National Institute of Arthritis and Metabolic Diseases for providing the rat FSH, LH and prolactin radioimmunoassay kits. These experiments were partially supported by Grant M 71-165 given to Igarashi from the Population Council, N. Y.. References Anderson, R. R. and W. H. McShan (1966). Endocrinology 78, 976. Amenomori, Y., C. L. Chen and J. Meits (1970). Ibid. 86, 506. Arimura, A., L. Debeljuk and A. V. Schally (1972). Ibid. 91, 529. Caligaris, L., J. J. Astrada and S. Taleisnik (1967). Ibid. 81, 1261. Daane, T. A. and A. F. Parlow (1971). Ibid. 88, 653. Fawke, L. and P. S. Brown (1969). J. Reprod. Fert. 21, 303. Gans, E., S. E. de Jongh, G. P. van Rees. J. J. van der Werff ten Bosch and 0. L. Wolthuis (1964). Acta Endocrinologica 45, 335. Gay, V. L., A. R. Midgley, J. R. and G. D. Niswender (1970). Federation Proc. 29, 1880. Goldman, B. D. and V. B. Mahesh (1968).
FSH, LH, PROLACTIN IN RAT ESTROUS CYCLE Endocrinology 83, 97. Grindeland, R. E., W. A. McCulloch and S. Ellis (1969) Program of the Fifty first Meeting of the Endocrine Society (1969) p.44 (Abstract). Guillemin, R.(1971). Prog. VII World Congr. Fertility and Sterility, Tokyo, 1971. lgarashi, M.(1967). An invited Lecture on a pending question. Japanese Obstetrical and Gynecological Society. Kwa, H. G. and F. Verhofstad (1967). J. Endocr. 39, 455. Kobayashi, F., K. Hara and T. Miyake (1968). Endocrinol. Japan. 15, 313. Kamioka,J.(1970). Acta Obst, et Gynaec, Jap. 17, 168. Llerena, L., A. Molina and O. H. Pearson (1969). Program of the Fifty first Meeting of the Endocrine Society p. 45 (Abstract). McClintock, J. A. and N. B. Schwartz (1968). Endocrinology 83, 433. Monroe, S. E., R. W. Rebar, V. L. Gay and A. R. Midgley, J. R.(1969). Ibid. 85, 720. Niswender, G. D., C. L. Chen, A. R. Midgley, J. R., J. Meites and S. Ellis (1969). Proc. Soc. Exp. Biol. Med. 130, 793. Neill, J. D. and L. E. Reichert, J. R.(1971). Endocrinology 88, 548. Parlow, A. F.(1961). In Albert, A.(ed.), Human Pituitary Gonadotropins, Charles C. Thomas, Springfield, Illinois, p. 300. Reece, R. P.(1939). Proc. Soc. Exp. Biol. Med. 42, 54. Reece, R. P. and C. W. Turner (1937). MO. Agr. Exp. Sta. Res. Bull. 266. Rowlands, I. W.(1942). Nature, London 150, 267. Ramirez, V. D. and S. M. McCann (1964). Endocrinology 74, 814. Schwartz, N. B. and D. Bartosik (1962). Ibid. 71, 756. Schwartz, N. B. and D. Caldarelli (1965). Proc. Soc. Exp. Biol. Med. 119, 16. Sar, M. and J. Meites (1967). Ibid. 125, 1018. Wakabayashi, K. J. Antunes-Rodrigues, B. Tamaoki and S. M. McCann (1972). Endocrinology 90, 690.