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Circadian rhythm in luteinizing hormone-releasing hormone (LH-RH) content of preoptic area during the rat estrous cycle
354
Brain Research, 104 (1976) 354-358 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Circadian rhythm in luteinizing hormone-releasing hormone (LH-RH) content of preoptic area during the rat estrous cycle
SATYA P. KALRA Department of Obstetrics and Gynecology, College of Medicine, University of Florida, Gainesville, Fla. 32610 (U.S.A.)
(Accepted November 25th, 1975)
Recent studies on the anatomical distribution of luteinizing hormone-releasing hormone ( L H - R H ) in the central nervous system of the rat have shown that while L H - R H concentrations are high in the median eminence-arcuate region (ME-ARC) 1-3,13,16,1s,21,22, a substantial amount is also localized rostrally in the preoptic area (POA)l-a,13,16,1s, 22. The bulk of L H - R H activity in POA is, in turn, associated with the organum vasculosum of the lamina terminalis2, 22 and detectable activity may also be present more laterally in a few perikarya of the medial preoptic area a,16,1s. In the female rat, there is a daily neuronal signal for the release of L H from the pituitaryS, 6. The circadian 'clock' governing the daily L H release mechanism is probably resident in the medial preoptic area 14 which is subject to modifications by a variety of environmental and hormonal factors4, 5. Among the hormonal factors, ovarian steroids play a predominant role in the activation and/or facilitation of this neuronal rhythm. In continuation of the studies on the interaction of systemic concentrations of ovarian steroids with the L H release mechanism(s) 1° a daily rhythm in L H - R H activity in POA was detected. These data on daily L H - R H fluctuations during the rat estrous cycle are the subject of this communication. Sprague-Dawley female rats (200-240 g) were housed in an air-conditioned room under controlled light conditions (lights on 05.00-17.00 h). Vaginal smears were taken daily and only those rats which showed at least two consecutive 4-day cycles were selected for the experiments. Groups of rats (5 rats/group) were sacrificed at various times during the estrous cycle within a week of the initiation of the experiment. Brains of these rats were quickly dissected out and a block of brain tissue from each rat, approximately 7 m m long, bounded caudally by the optic chiasm and extending dorsally above the level of the anterior commissure and laterally 3-4 mm from the midline, was homogenized in 2 ml of 0.1 N HC1. The acid extract of this POA region was kept frozen at - - 2 0 °C until assayed for L H - R H activity a week later. Prior to the assay, acid extracts of the POA were neutralized with 1 N N a O H and centrifuged. A small aliquot of the supernatant was taken for analysis of L H - R H activity by radioimmunoassay as described by Nett e t a / . 17. Antiserum No. 42 to
355
1200-
9000n
-r II:
600-
_1
300"
c j HOURS 12 E~I'RUS
m '
21
m ' 2'2
15 DIESTRUS I
m
t ll2
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9 ]8 DIESTRUSTT
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8 16 PROESTRUS
,'2 ESTRUS
Fig. 1. Daily fluctuations in LH-RH of POA during the rat estrous cycle. Each point (except 16.00 h proestrus) represents the mean LH-RH activity in POA of 5 rats. Groups of 5 rats each were sacrificed on the hour between 14.00 and 18.00 h on proestrus. Since there was no significant difference among them, in the interest of clarity, they have been grouped and shown as a 16.00 h group. Vertical bars are 2 S.E.M. Hatched horizontal bars denote darkness between 19.00-05.00 h. L H - R H was kindly given to us by Dr. T. Nett and Dr. G. D. Niswender. Beckman L H - R H was used as reference standard and for radioiodination. The results on temporal fluctuations in L H - R H activity in POA during the rat estrous cycle are summarized in Fig. 1. As evident from the data, the lowest levels of POA L H - R H (361 4- 46.4 pg/POA) were recorded at 12.00 h of estrus, an observation consistent with a recent report 1. Thereafter, L H - R H contents of POA were raised significantly at 21.00 h of estrus (P < 0.01) and declined later at 08.00 h of diestrus I (P < 0.01). L H - R H levels increased gradually through diestrus I to attain statistically significant levels by 22.00 h (P < 0.01). By noon of the following day on diestrus II, L H - R H stores were reduced (P < 0.01) from the peak values of diestrus I. Later, at 18.00 h of diestrus II, L H - R H rose rapidly to very high values of 1213 ± 156.3 pg/POA. This was followed by a drop to 443 _-• 61.2 pg/POA at 08.00 h of proestrus. Thereafter L H - R H in POA was unaffected through the critical period till 18.00 h on proestrus. However, as on other days of the estrous cycle, a pronounced accumulation at 20.00 h (P < 0.01) was succeeded by a prompt reduction in POA L H - R H levels between 23.00 and 24.00 h of proestrus (P < 0.01). This was soon followed by a further depletion at 12.00 h of estrus to the lowest values recorded during the estrous cycle (P < 0.05). Araki e t al. 1 measured L H - R H in the anterior hypothalamus at intervals between 08.00 and 17.00 h on each day during the estrous cycle of rats maintained on a slightly different light-dark cycle (lights on 06.0020.00 h). They observed a significant depletion of L H - R H at 12.00 h from the high levels of 08.00 h of estrus. Although POA L H - R H levels at 08.00 h of estrus were not
356 examined in the present study it is apparent from these data that a reduction in POA L H - R H began sometime after 20.00 h of proestrus to reach the lowest values at noon of estrus. In general, the highest L H - R H activity in POA was found between 18.00 and 22.00 h and the lowest levels were detected between 08.00 and 12.00 h on each day of the cycle. In the understanding of the neuroendocrine regulation of reproductive cyclicity in the female rat, the physiological significance of the daily elevations in POA LHR H activity, together with the mechanisms regulating this daily rhythm, is a subject of immense importance and speculation. The specific anatomical site(s) of synthesis of L H - R H in POA is uncertain at present 1-3,16,1s,22 but it appears that a part of L H - R H activity of ME-ARC is derived from POA15, 20. According to Knigge and Scott 12 POA L H - R H is released into CSF of the third ventricle for delivery to and storage in ME-ARC. However, Mess eta[. 15, supported later by Schneider et al. 2°, are of the view that the transport of POA L H - R H to ME-ARC is accomplished in the long axons of the L H - R H synthesizing perikarya overlying the suprachiasmatic nuclei, which eventually terminate on the portal capillaries in the median eminence. We have recently noted that severing by Halfisz knife of the fiber system which converges on ME-ARC anteriorly resulted in a marked depletion of L H - R H in ME-ARC and a significant impairment in L H release from the pituitary 8. A small reduction in L H - R H activity, determined by bioassay, was reported in ME-ARC following lesions in the suprachiasmatic region 1'5,2°. In addition to this, we have found a pronounced accumulation of L H - R H in the neural tissue lying anterior to the knife cuts s. These observations clearly suggest that a substantial concentration of L H - R H in ME-ARC has an origin in the preoptic area and, furthermore, the axonal in contrast to the ventricular transport mechanism may be the predominant route of delivery of POA L H - R H to ME-ARC. In any case, it seems logical to assume that L H - R H of POA along with the amount synthesized in ME-ARC itself is involved in the tonic release of pituitary LH. This hypothesis can be extended to include POA L H - R H as an indispensable constituent of a high amplitude pulse of L H - R H discharged in the afternoon of proestrus to elicit a surge of LH 7,931. If the L H - R H rhythm in POA is a part of the 'clock' mechanism resident in POA itself 14 then it derives that either this rhythm is intrinsic to this region and is independent of environmental factors, or its activation requires a balance of external factors (light, temperature etc.) and the feedback action of ovarian steroids 4,5. It is noteworthy that under laboratory conditions of controlled light and temperature the daily elevations of POA L H - R H are coincident with the daily peaks in systemic concentration of progesterone described earlied0A 9. Considerable amounts of estradiol were also present in the peripheral circulation throughout the estrous cycle1°. On the basis of these observations, it is reasonable to suggest that the activation and/or sustenance of the daily POA-RH rhythm is dependent upon the intricate balance of circulating ovarian steroids such that progesterone constitutes a primary feedback signal on an estrogen primed background. On the other hand, it is possible that estrogen alone or in synergism with progesterone at an appropriate time during
357 the day is the m a j o r permissive factor for the circadian r h y t h m in P O A L H - R H . F u r t h e r studies in progress o n the action of o v a r i a n steroids on h y p o t h a l a m i c L H R H secretion should provide clues to the identification of factor(s) regulating the daily P O A L H - R H r h y t h m . The secretarial help o f C i n d y H a r n a g e is acknowledged. Supported by G r a n t s from the P o p u l a t i o n Council, N I H R O H D 08634-0, A and the 1974 N | H G e n e r a l Research S u p p o r t G r a n t R R 05362-13.
1 ARAKI, S., FERIN, M., ZIMMERMAN,E. A., AND VAN DE WIELE, R. L., Ovarian modulations of immunoreactive gonadotropin-releasing hormone (GN-RH) in the rat brain: evidence for a differential effect on the anterior and mid-hypothalamus, Endocrinology, 96 (1975) 644-650. 2 BAKER,B. L., DERMODY,W. C., ANDREEL,J. R., Distribution of gonadotropin-releasing hormone in the rat brain as observed with immunocyto-chemistry, Endocrinology, 97 (1975) 125-135. 3 BARRY,J., DUBOIS,M. P., AND CARETTE,B., Immunofluorescence study of the preoptico-infundibular LRF neurosecretory pathway in the normal, castrated or testosterone-treated male rat, Endocrinology, 95 (1974) 1416-1423. 4 BOGDANOVE, E. M., Hypothalamic-hypophyseal interrelationships: basic aspects. In H. BALIN AND S. GLASSER(Eds.), Reproductive Biology, Excerpta Medica, Amsterdam, 1972, pp. 5-70. 5 EVERETT,J. W., Central neural control of reproductive functions of the adenohypophysis, PhysioL Rev., 44 (1964) 373~31. 6 EVERETT, J. W., AND SAWYER, C. H., A 24-hour periodicity in the 'LH release apparatus' of female rat, disclosed by barbiturate sedation, Endocrinology, 47 (1950) 198-218. 7 KALRA,S. P., unpublished. 8 KALRA, S. P., Luteinizing hormone-releasing hormone in the preoptic area and hypothalamus, and release of gonadotropins following anterior hypothalamic deafferentation and estrogen treatment of female rats, Endocrinology, in press. 9 KALRA, S. P., Temporal changes in serum LH-releasing factor (LRF), luteinizinghormone (LH), and hypothalamic LRF following progesterone (P) treatment of estrogen-primed ovariectomised rats: effects of cycloheximide, Fed. Proc., 34 (1975) 239 (Abstract). 10 KALRA,S. P., ANDKALRA,P. S., Temporal interrelationships among circulating levels of estradiol, progesterone and LH during the rat estrous cycle: effects of progesterone, Endocrinology, 95 (1974) 1711-1717. 11 KALRA, S. P., AND MCCANN, S. M., Changes in gonadotropin-releasing factor content in the rat hypothalamus following electrochemical stimulation of anterior hypothalamic area and during the estrous cycle, Neuroendocrinology, 12 (1973) 321-333. 12 KNIGGE, K. M., AND SCOTT, O. E., Structure and function of median eminence, Amer. J. Anat. 129 (1970) 223-243. 13 KORDON, C., KERDELHUI~,B., PATTOU, E., AND JUTISZ, M., Immunocytochemical localization of LH-RH in axons and nerve terminal of the rat median eminence, Proc. Soc. exp. Biol. (N. Y.), 147 (1974) 122-127. 14 K/SvEs, K., AND HAL.g,SZ, B., Location of the neural structures triggering ovulation in the rat, Neuroendocrinology, 6 (1970) 180-193. 15 MESS, B., FRASCHINI,F., MOTTA,M., AND MARTINI,L., The topography of the neurones synthesizing the hypothalamic releasing factor. In L. MARTINI,F. FRASCHINIAND M. MOTTA(Eds.), Proc. 2nd Int. Congr. Hormonal Steroids, No. I32, Excerpta Medica, Amsterdam, 1966, pp. 10041013. 16 NAIK, D. V., Immunoreactive LH-RH neurons in the hypothalamus identified by light and fluorescent microscopy, Cell Tiss. Res., 157 (1975) 423~,36. 17 NETT, T. M., AKaAR,A. M., NISWENDER,G. D., HEDLUND,M. T., AND WHITE,W. F., A radioimmunoassay for gonadotropin releasing hormone (GN-RH) in serum, J. clin. Endocr., 36 (1973) 880-885. 18 QUIJADA, M., KRULICH, L., FAWCETT, C. P., lUNDBERG, K. K., AND MCCANN, S. M., Localization of TSH-releasing factor (TRF), LH-RF and FSH-RF in rat hypothalamus, Fed. Proc., 30 0971) 197. (Abstract.)
358 19 RAMALEY,J. A., AND BARTOSIK,D., Rhythms of progesterone and corticosterone after PMS induced puberty. Effects of ovariectomy, Endocrinology, 95 (1975) 1719-1725. 20 SCHNEIDER,H. P. G., CRIGHTON, O. B., AND McCANN, S. M., Suprachiasmatic LH-releasing factor, Neuroendocrinology, 5 0969) 271-280. 21 S~TA,L6, G., VIGH, S., SCHALLY,A. V., ARIMURA,A., AND FLERKO,B., LH-RH containing neural elements in the rat hypothalamus, Endocrinology, 96 0975) 135-142. 22 WHEATON,J. E., KRULICH, L., AND MCCANN, S. M., Localization of luteinizing hormone-releasing hormone in the preoptic area and hypothalamus of the rat using radioimmunoassay, Endocrinology, 97 (1975) 30-38.
Brain Research, 104 (1976) 354-358 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Circadian rhythm in luteinizing hormone-releasing hormone (LH-RH) content of preoptic area during the rat estrous cycle
SATYA P. KALRA Department of Obstetrics and Gynecology, College of Medicine, University of Florida, Gainesville, Fla. 32610 (U.S.A.)
(Accepted November 25th, 1975)
Recent studies on the anatomical distribution of luteinizing hormone-releasing hormone ( L H - R H ) in the central nervous system of the rat have shown that while L H - R H concentrations are high in the median eminence-arcuate region (ME-ARC) 1-3,13,16,1s,21,22, a substantial amount is also localized rostrally in the preoptic area (POA)l-a,13,16,1s, 22. The bulk of L H - R H activity in POA is, in turn, associated with the organum vasculosum of the lamina terminalis2, 22 and detectable activity may also be present more laterally in a few perikarya of the medial preoptic area a,16,1s. In the female rat, there is a daily neuronal signal for the release of L H from the pituitaryS, 6. The circadian 'clock' governing the daily L H release mechanism is probably resident in the medial preoptic area 14 which is subject to modifications by a variety of environmental and hormonal factors4, 5. Among the hormonal factors, ovarian steroids play a predominant role in the activation and/or facilitation of this neuronal rhythm. In continuation of the studies on the interaction of systemic concentrations of ovarian steroids with the L H release mechanism(s) 1° a daily rhythm in L H - R H activity in POA was detected. These data on daily L H - R H fluctuations during the rat estrous cycle are the subject of this communication. Sprague-Dawley female rats (200-240 g) were housed in an air-conditioned room under controlled light conditions (lights on 05.00-17.00 h). Vaginal smears were taken daily and only those rats which showed at least two consecutive 4-day cycles were selected for the experiments. Groups of rats (5 rats/group) were sacrificed at various times during the estrous cycle within a week of the initiation of the experiment. Brains of these rats were quickly dissected out and a block of brain tissue from each rat, approximately 7 m m long, bounded caudally by the optic chiasm and extending dorsally above the level of the anterior commissure and laterally 3-4 mm from the midline, was homogenized in 2 ml of 0.1 N HC1. The acid extract of this POA region was kept frozen at - - 2 0 °C until assayed for L H - R H activity a week later. Prior to the assay, acid extracts of the POA were neutralized with 1 N N a O H and centrifuged. A small aliquot of the supernatant was taken for analysis of L H - R H activity by radioimmunoassay as described by Nett e t a / . 17. Antiserum No. 42 to
355
1200-
9000n
-r II:
600-
_1
300"
c j HOURS 12 E~I'RUS
m '
21
m ' 2'2
15 DIESTRUS I
m
t ll2
t
2'3
9 ]8 DIESTRUSTT
z
210
8 16 PROESTRUS
,'2 ESTRUS
Fig. 1. Daily fluctuations in LH-RH of POA during the rat estrous cycle. Each point (except 16.00 h proestrus) represents the mean LH-RH activity in POA of 5 rats. Groups of 5 rats each were sacrificed on the hour between 14.00 and 18.00 h on proestrus. Since there was no significant difference among them, in the interest of clarity, they have been grouped and shown as a 16.00 h group. Vertical bars are 2 S.E.M. Hatched horizontal bars denote darkness between 19.00-05.00 h. L H - R H was kindly given to us by Dr. T. Nett and Dr. G. D. Niswender. Beckman L H - R H was used as reference standard and for radioiodination. The results on temporal fluctuations in L H - R H activity in POA during the rat estrous cycle are summarized in Fig. 1. As evident from the data, the lowest levels of POA L H - R H (361 4- 46.4 pg/POA) were recorded at 12.00 h of estrus, an observation consistent with a recent report 1. Thereafter, L H - R H contents of POA were raised significantly at 21.00 h of estrus (P < 0.01) and declined later at 08.00 h of diestrus I (P < 0.01). L H - R H levels increased gradually through diestrus I to attain statistically significant levels by 22.00 h (P < 0.01). By noon of the following day on diestrus II, L H - R H stores were reduced (P < 0.01) from the peak values of diestrus I. Later, at 18.00 h of diestrus II, L H - R H rose rapidly to very high values of 1213 ± 156.3 pg/POA. This was followed by a drop to 443 _-• 61.2 pg/POA at 08.00 h of proestrus. Thereafter L H - R H in POA was unaffected through the critical period till 18.00 h on proestrus. However, as on other days of the estrous cycle, a pronounced accumulation at 20.00 h (P < 0.01) was succeeded by a prompt reduction in POA L H - R H levels between 23.00 and 24.00 h of proestrus (P < 0.01). This was soon followed by a further depletion at 12.00 h of estrus to the lowest values recorded during the estrous cycle (P < 0.05). Araki e t al. 1 measured L H - R H in the anterior hypothalamus at intervals between 08.00 and 17.00 h on each day during the estrous cycle of rats maintained on a slightly different light-dark cycle (lights on 06.0020.00 h). They observed a significant depletion of L H - R H at 12.00 h from the high levels of 08.00 h of estrus. Although POA L H - R H levels at 08.00 h of estrus were not
356 examined in the present study it is apparent from these data that a reduction in POA L H - R H began sometime after 20.00 h of proestrus to reach the lowest values at noon of estrus. In general, the highest L H - R H activity in POA was found between 18.00 and 22.00 h and the lowest levels were detected between 08.00 and 12.00 h on each day of the cycle. In the understanding of the neuroendocrine regulation of reproductive cyclicity in the female rat, the physiological significance of the daily elevations in POA LHR H activity, together with the mechanisms regulating this daily rhythm, is a subject of immense importance and speculation. The specific anatomical site(s) of synthesis of L H - R H in POA is uncertain at present 1-3,16,1s,22 but it appears that a part of L H - R H activity of ME-ARC is derived from POA15, 20. According to Knigge and Scott 12 POA L H - R H is released into CSF of the third ventricle for delivery to and storage in ME-ARC. However, Mess eta[. 15, supported later by Schneider et al. 2°, are of the view that the transport of POA L H - R H to ME-ARC is accomplished in the long axons of the L H - R H synthesizing perikarya overlying the suprachiasmatic nuclei, which eventually terminate on the portal capillaries in the median eminence. We have recently noted that severing by Halfisz knife of the fiber system which converges on ME-ARC anteriorly resulted in a marked depletion of L H - R H in ME-ARC and a significant impairment in L H release from the pituitary 8. A small reduction in L H - R H activity, determined by bioassay, was reported in ME-ARC following lesions in the suprachiasmatic region 1'5,2°. In addition to this, we have found a pronounced accumulation of L H - R H in the neural tissue lying anterior to the knife cuts s. These observations clearly suggest that a substantial concentration of L H - R H in ME-ARC has an origin in the preoptic area and, furthermore, the axonal in contrast to the ventricular transport mechanism may be the predominant route of delivery of POA L H - R H to ME-ARC. In any case, it seems logical to assume that L H - R H of POA along with the amount synthesized in ME-ARC itself is involved in the tonic release of pituitary LH. This hypothesis can be extended to include POA L H - R H as an indispensable constituent of a high amplitude pulse of L H - R H discharged in the afternoon of proestrus to elicit a surge of LH 7,931. If the L H - R H rhythm in POA is a part of the 'clock' mechanism resident in POA itself 14 then it derives that either this rhythm is intrinsic to this region and is independent of environmental factors, or its activation requires a balance of external factors (light, temperature etc.) and the feedback action of ovarian steroids 4,5. It is noteworthy that under laboratory conditions of controlled light and temperature the daily elevations of POA L H - R H are coincident with the daily peaks in systemic concentration of progesterone described earlied0A 9. Considerable amounts of estradiol were also present in the peripheral circulation throughout the estrous cycle1°. On the basis of these observations, it is reasonable to suggest that the activation and/or sustenance of the daily POA-RH rhythm is dependent upon the intricate balance of circulating ovarian steroids such that progesterone constitutes a primary feedback signal on an estrogen primed background. On the other hand, it is possible that estrogen alone or in synergism with progesterone at an appropriate time during
357 the day is the m a j o r permissive factor for the circadian r h y t h m in P O A L H - R H . F u r t h e r studies in progress o n the action of o v a r i a n steroids on h y p o t h a l a m i c L H R H secretion should provide clues to the identification of factor(s) regulating the daily P O A L H - R H r h y t h m . The secretarial help o f C i n d y H a r n a g e is acknowledged. Supported by G r a n t s from the P o p u l a t i o n Council, N I H R O H D 08634-0, A and the 1974 N | H G e n e r a l Research S u p p o r t G r a n t R R 05362-13.
1 ARAKI, S., FERIN, M., ZIMMERMAN,E. A., AND VAN DE WIELE, R. L., Ovarian modulations of immunoreactive gonadotropin-releasing hormone (GN-RH) in the rat brain: evidence for a differential effect on the anterior and mid-hypothalamus, Endocrinology, 96 (1975) 644-650. 2 BAKER,B. L., DERMODY,W. C., ANDREEL,J. R., Distribution of gonadotropin-releasing hormone in the rat brain as observed with immunocyto-chemistry, Endocrinology, 97 (1975) 125-135. 3 BARRY,J., DUBOIS,M. P., AND CARETTE,B., Immunofluorescence study of the preoptico-infundibular LRF neurosecretory pathway in the normal, castrated or testosterone-treated male rat, Endocrinology, 95 (1974) 1416-1423. 4 BOGDANOVE, E. M., Hypothalamic-hypophyseal interrelationships: basic aspects. In H. BALIN AND S. GLASSER(Eds.), Reproductive Biology, Excerpta Medica, Amsterdam, 1972, pp. 5-70. 5 EVERETT,J. W., Central neural control of reproductive functions of the adenohypophysis, PhysioL Rev., 44 (1964) 373~31. 6 EVERETT, J. W., AND SAWYER, C. H., A 24-hour periodicity in the 'LH release apparatus' of female rat, disclosed by barbiturate sedation, Endocrinology, 47 (1950) 198-218. 7 KALRA,S. P., unpublished. 8 KALRA, S. P., Luteinizing hormone-releasing hormone in the preoptic area and hypothalamus, and release of gonadotropins following anterior hypothalamic deafferentation and estrogen treatment of female rats, Endocrinology, in press. 9 KALRA, S. P., Temporal changes in serum LH-releasing factor (LRF), luteinizinghormone (LH), and hypothalamic LRF following progesterone (P) treatment of estrogen-primed ovariectomised rats: effects of cycloheximide, Fed. Proc., 34 (1975) 239 (Abstract). 10 KALRA,S. P., ANDKALRA,P. S., Temporal interrelationships among circulating levels of estradiol, progesterone and LH during the rat estrous cycle: effects of progesterone, Endocrinology, 95 (1974) 1711-1717. 11 KALRA, S. P., AND MCCANN, S. M., Changes in gonadotropin-releasing factor content in the rat hypothalamus following electrochemical stimulation of anterior hypothalamic area and during the estrous cycle, Neuroendocrinology, 12 (1973) 321-333. 12 KNIGGE, K. M., AND SCOTT, O. E., Structure and function of median eminence, Amer. J. Anat. 129 (1970) 223-243. 13 KORDON, C., KERDELHUI~,B., PATTOU, E., AND JUTISZ, M., Immunocytochemical localization of LH-RH in axons and nerve terminal of the rat median eminence, Proc. Soc. exp. Biol. (N. Y.), 147 (1974) 122-127. 14 K/SvEs, K., AND HAL.g,SZ, B., Location of the neural structures triggering ovulation in the rat, Neuroendocrinology, 6 (1970) 180-193. 15 MESS, B., FRASCHINI,F., MOTTA,M., AND MARTINI,L., The topography of the neurones synthesizing the hypothalamic releasing factor. In L. MARTINI,F. FRASCHINIAND M. MOTTA(Eds.), Proc. 2nd Int. Congr. Hormonal Steroids, No. I32, Excerpta Medica, Amsterdam, 1966, pp. 10041013. 16 NAIK, D. V., Immunoreactive LH-RH neurons in the hypothalamus identified by light and fluorescent microscopy, Cell Tiss. Res., 157 (1975) 423~,36. 17 NETT, T. M., AKaAR,A. M., NISWENDER,G. D., HEDLUND,M. T., AND WHITE,W. F., A radioimmunoassay for gonadotropin releasing hormone (GN-RH) in serum, J. clin. Endocr., 36 (1973) 880-885. 18 QUIJADA, M., KRULICH, L., FAWCETT, C. P., lUNDBERG, K. K., AND MCCANN, S. M., Localization of TSH-releasing factor (TRF), LH-RF and FSH-RF in rat hypothalamus, Fed. Proc., 30 0971) 197. (Abstract.)
358 19 RAMALEY,J. A., AND BARTOSIK,D., Rhythms of progesterone and corticosterone after PMS induced puberty. Effects of ovariectomy, Endocrinology, 95 (1975) 1719-1725. 20 SCHNEIDER,H. P. G., CRIGHTON, O. B., AND McCANN, S. M., Suprachiasmatic LH-releasing factor, Neuroendocrinology, 5 0969) 271-280. 21 S~TA,L6, G., VIGH, S., SCHALLY,A. V., ARIMURA,A., AND FLERKO,B., LH-RH containing neural elements in the rat hypothalamus, Endocrinology, 96 0975) 135-142. 22 WHEATON,J. E., KRULICH, L., AND MCCANN, S. M., Localization of luteinizing hormone-releasing hormone in the preoptic area and hypothalamus of the rat using radioimmunoassay, Endocrinology, 97 (1975) 30-38.