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Forebrain structures involved in ovulation and release of serum LH and FSH in proestrous rats
Brain Research, 191 (1980) 99-108 © Elsevier/North-Holland Biomedical Press
99
FOREBRAIN STRUCTURES INVOLVED IN OVULATION AND RELEASE OF S E R U M L H A N D F S H I N P R O E S T R O U S RATS
MASAZUM! KAWAKAM! and SHINTARO ANDO 2nd Department of Physiology, Yokohama City University School of Medicine, 2-33 Urafune-cho, Minami-ku, Yokohama (Japan)
(Accepted November 8th, 1979) Key words: ovulation - - forebrain - - serum LH - - FSH - - proestrous rat - - diagonal band of Broca septum - - medial preoptic area - - hypothalamus -
-
SUMMARY To investigate in detail the localization of the extrahypothalamic neural structures indispensable for ovulation and serum gonadotropin release, small electrolytic lesions were produced in the forebrain under acute experimental conditions with a platinum electrode at 11.30-13.30 h on the day of proestrus. Blood sampling was performed using ether anesthesia on the afternoon of proestrus and the number of ova in the ampulla were verified with a microscope the next morning. Lesions in the diagonal band of Broca (DBB) or the supracommissural area inhibited spontaneous ovulation and proestrous surges of serum L H and FSH. On the other hand, full or partial ovulation occurred in animals with lesions outside the DBB-supracommissural area, including the medial septum (m-SEPT) and the subcommissural periventricular part of the medial preoptic area (MPO). In this experiment, the lesions which inhibited spontaneous ovulation were limited to the DBB including the supracommissural area. Electrochemical stimulation of the DBB in pentobarbital-blocked proestrous rats was effective in inducing ovulation and serum gonadotropin release. The stimulatory effects of DBB stimulation on the ovulatory response were interrupted by bilateral lesions in the mediobasal preoptic area including the suprachiasmatic nucleus (SCH). The present study indicates that the DBB-supracommissural area is indispensable for acute ovulatory gonadotropin release, and that the DBB efferents for ovulation are conducted to the mediobasal hypotbalamus (MBH) by way of the SCHmediobasal MPO.
100 INTRODUCHON In 1964, Everett et al. 7 postulated that a diffuse septal-preopticotuberal pathway was responsible for ovulatory LH release in rats. From the results of chronic neural transections with a small knife, Halgsz and Gorski 9 suggested that the diffusely organized neural afferents required for cyclic ovulation converge toward the median eminence area. In order to search for the origin of the critical afferents for ovulation, K6ves and Halgsz 16 observed the occurrence of ovulation in rats with long-term bilateral interruption of the anterior and superior connections to the preoptic-anterior hypothalamic area and concluded that the neurogenic stimulus responsible for ovulation may arise at least from the preoptic-anterior hypothalamic area. From the results of acute neural transection, on the other hand, direct or indirect inputs from limbic structures to the medial preoptic and tuberal region of the hypothalamus have been assumed to govern the preovulatory LH surge 12,21. Recent experiments in our laboratory have suggested that areas anterior and superior to the preoptic area, including the diagonal band of Broca (DBB), septum (SEPT) and bed nucleus of stria terminalis (BST), are indispensable for the induction of the LH surge 14. The present experiments were designed to investigate in more detail the extrahypothalamic forebrain structures which are indispensable for ovulatory gonadotropin release in acute experimental conditions. MATERIALS AND METHODS Four-day cyclic adult female rats of Wister strain were used. They were housed in a light-controlled room (5.00-19.00 h light, 19.00-5.00 h dark). Vaginal smears were examined every morning for at least two consecutive cycles before use of the rats as experimental animals on the day of proestrus.
Lesion experiment Electrolytic lesions of the forebrain were made by using a platinum electrode insulated with epoxylite except for the tip. The electrode was stereotaxically placed in the forebrain structure, and lesions were produced by passing an anodal DC current (3 mA for 5 sec) through the electrode. Sham operations were performed in 8 animals in which the electrode was inserted but no current was passed. Surgical operations were performed using ether anesthesia between 11.30 and 13.30 h on the day of proestrus. Blood sampling was performed by cardiac puncture using ether anesthesia for the determination of serum gonadotropin levels (1) just before operation, (2) at 14.30 h and (3) at 18.30 h after operation. Occurrence of ovulation was evaluated in animals showing vaginal cornification on the following morning. The oviducts were compressed between two slide glasses and the number of ova in the ampulla counted with a microscope.
Stimulation experiment The effects of electrochemical stimulation of the DBB on the ovulatory response
101 and serum gonadotropin release were examined in proestrous rats anesthetized with pentobarbital sodium. An anodal DC current of 100/~A for 60 sec was passed through the center wire of a bipolar concentric stainless steel electrode which was stereotaxically placed in the DBB. In the experimental animals, electrolytic lesions in the mediobasal preoptic area including the SCH were produced immediately prior to the subsequent DBB stimulation, by passing an anodal D C current of 3 mA for 10 sec through a platinum electrode. In sham-lesioned animals, a platinum electrode was inserted into the mediobasal preoptic area, without passing an anodal D C current, prior to the DBB stimulation. In each group, surgical procedures were carried out ,between 14.30 and 15.30 h in rats anesthetized with pentobarbital sodium (30 mg/kg) injected at 13.45 h. Blood sampling was performed by cardiac puncture (1)just before operation, (2) at 90 min and (3) at 180 min after DBB stimulation. On the following morning vaginal smear and ovulatory response were examined as in the preceeding lesion experiment. Serum LH and FSH levels were measured following the radioimmunoassay methods described by Niswender et al. 17 and by N I A M D D instructions, respectively. The concentrations of LH and FSH were expressed in terms of NIH-LH-S 1 and NIHFSH-S1, respectively. Student's t-test and Fisher exact probability test were used for statistical analysis. Frozen sections of the brain were stained with cresyl violet and examined with a microscope to ascertain the extent of lesions, based on the brain atlas of Kfnig and KlippeP 5.
(A)
{B)
'~'
C,A\ Th
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Group Ill (No OvuL)
( ~ ) GroupI (Full Ovut.) "'-" Groupil (Partial Ovul.)
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Fig. 1. Schematic drawing of the sites of the forebrain lesions on sagittal sections. A: solid-line circles represent the sites of the lesions which were found in the non-ovulated rats of group III. B: solid-line circles represent the sites of the lesions which were found in the fully-ovulated rats of group I. Mean number of ova observed in the ampulla was 10.9. Broken-line circles represent the sites of the lesions which were found in the partially ovulated rats of Group II. Mean number of ova observed in the ampulla was 2.8. A, nucleus accumbens; AHA, anterior hypothalamic area; ARC, arcuate nucleus; CA, commissura anterior; CC, corpus cailosum; CO, optic chiasma; DBB, diagonal band of Broca; Fx, fornix; MPO, medial preoptic area; SCH, suprachiasmatic nucleus; SL, lateral septum; SM, medial septum; Th, thalamus.
102 RESULTS
Eff~wts of forebrain lesions on ovulation and serum gonadotropin release Ovulation. Thirty-two electrolytic lesions were located in the forebrain in loci such as the DBB, SEPT, medial preoptic area (MPO) and the area dorsal to the commissura anterior (CA). The sites of lesions which inhibited spontaneous ovulation are shown in Fig. I A, whereas those illustrated in Fig. 1B were ineffective. Ovulation was significantly inhibited following the DBB lesions in 13 of 19 animals (P < 0.01). Rats with sham lesions ovulated fully. Ovulation failed to occur in all 7 animals with lesions in the posterior part of the DBB, the typical site of these lesions is represented in Fig. 3. On the contrary, half of the 12 animals with lesions in the anterior part of the DBB ovulated (Fig. 1A, B). Unexpectedly, ovulation also failed to occur in all 4 animals subjected to lesions in the paracentral area dorsal to the CA (tentatively termed as 'supracommissural area') (Fig. 1A). Thus the lesions which inhibited spontaneous ovulation were limited to the DBB-supracommissural area (Figs. 1A and 2), although
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Fig. 2. Schematic drawing of the sites of the DBB-supracommissural lesions on serial frontal sections. Solid-line circles represent the sites of the lesions which were found in the non-ovulated rats of group III. See caption to Fig. l for definitions of abbreviations.
103
Fig. 3. A photographic representation of one of the typical DBB lesions. Arrow indicates the site of the lesion. the c o m m o n a r e a o f lesions was obscure in inhibiting s p o n t a n e o u s ovulation. O n the o t h e r hand, s p o n t a n e o u s o v u l a t i o n was n o t inhibited in a n y o f 9 animals subjected to lesions outside the D B B - s u p r a c o m m i s s u r a l area. O v u l a t i o n occurred in all 3 animals with lesions in the m e d i a l S E P T ( m - S E P T ) a n d in all 4 a n i m a l s with lesions in the subc o m m i s s u r a l periventricular p a r t o f the M P O (Fig. 1B). LH (ng/m[) 15--
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15 15 17,1
Full Ovul. Partial Ovu[. No Ovul. (Group I ) (Group II) (Group 111)
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Fig. 4. Effects of lesions in several parts of the forebrain on the release of serum LH and FSH in proestrous rats. Bars represent means and the lines, standard errors. The number of rats is given in (or above) the bars. See Fig. 1 for location of the forebrain lesions in each group.
104
Serum gonadotropin release. According to the number o f ova observed in the ampulla, animals employed in this experiment were divided into 3 groups - - group l (full ovulation, _--__8 ova), group II (partial ovulation, _< 7 ova) and group I I I (no ovulation) (Fig. 1A, B). Preovulatory surges o f serum L H and F S H were observed following the lesions in animals o f group I as well as in sham-lesioned animals. In animals of" g r o u p If, the release o f serum L H was significantly suppressed although that of serum F S H was not. In animals o f group III, all o f whose lesions were found to be located in the DBB-supracommissural area, the release o f serum L H was eliminated and that o f serum F S H was significantly suppressed (Fig. 4). EfJects of DBB stimulation on ovulation and serum gonadotropin release Ovulation. Full ovulation was induced following DBB stimulation in all 6 shamlesioned animals pretreated with pentobarbital sodium. The expected ovulation was not induced following the DBB stimulation in any of 8 animals with bilateral lesions in the S C H including parts o f the mediobasal M P O , the preoptic suprachiasmatic area
st im. Th , •
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,"11117 , /l~
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SCH
CO A?,O Fig. 5. Schematic drawing of the sites of the bilateral SCH lesions which inhibited the DBB stimulationinduced ovulation in pentobarbital-blocked proestrous rats. Parts of the MPO, POSC and AHA were involved in the SCH lesions. The stimulation sites in the DBB are represented as open circles on sagittal section. See caption to Fig. 1 for definition of abbreviations.
105 LH ( ng/ml )
F(p Sg Hm l )l
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1.0"
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5- ~ a )
(7)~'I(s)
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~(a)
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0
90
180 rain.
100rain.
- - - Sham control o ~ o Incomplete or unilateral SCH lesion -- -- B i l a t e r a l 5CH lesion p -: 0.05 vs sham control at each time
Fig. 6. Effects of the bilateral SCH lesions on the release of serum LH and FSH induced by DBB stimulation in pentobarbital-blocked proestrous rats. Each point represents mean and vertical bar, standard error. The number of rats is given in parentheses. (POSC) and the anterior hypothalamic area (AHA) (Fig. 5). There appeared to be no extensive damage to the optic tract upon histological observation. On the other hand, the expected ovulation was not inhibited in animals with incomplete or unilateral SCH lesions. Serum gonadotropin release. The DBB stimulation effectively induced release of serum L H and F S H with the peaks at 90 min and 180 rain, respectively (Fig. 6). In animals with bilateral SCH lesions, the values of serum L H at both 90 min and 180 min were significantly reduced and those of serum F S H at 180 min were significantly reduced. However, in animals with incomplete or unilateral SCH lesions, the releases of serum L H and F S H were induced in the same manner as those in sham-lesioned animals. DISCUSSION
Spontaneous ovulation and serum gonadotropin release were inhibited by small lesions produced in the DBB at 11.30-13.30 h on the day of proestrus, which led us to assume that the forebrain rostral to the preoptic area may be involved in triggering the ovulatory gonadotropin release. It has been observed that acute horizontal interruption of the limbic projection to the medial hypothalamus by preoptic r o o f cuts inhibited spontaneous ovulation12,20, 21, although regular estrous cyclicity was restored in animals with long-term preoptic-roof section10, 21. In the present experiment, the lesions in the posterior part of the DBB resulted in inhibition of spontaneous ovulation. Such lesions probably did not extend caudally far enough to include the preoptic-anterior hypothalamic area which was severed from the forebrain structures in the work by K6ves and Hal~sz 16.
106 On the other hand, none of these lesions in the DBB was placed dorsal to the preoptic roof. Recently, Kimura and Kawakami '4 made deafferentations ventroposteriorly and dorso-anteriorly to the DBB, of which the former inhibited spontaneous ovulation while the latter did not. Most of the DBB lesions in the present study were located very medially within these two boundaries. It has been assumed that the septal complex, when stimulated electrically or electrochemically, can readily induce the release of serum gonadotropin v,la. Everett et al. 7 have postulated that the nerve fiber array responsible for ovulation is dispersed in the area rostral and dorsal to the preoptic region, including the medial septal nuclei, the nucleus accumbens and the medial parolfactory area. In the present study, spontaneous ovulation was not inhibited following lesions produced in parts of the m-SEPT nor in parts of the anterior part of the DBB. Therefore, it may be suggested that ovulatory gonadotropin release is not so much affected by lesions in these two brain areas as by lesions in the posterior part of the DBB or in the supracommissural area. This is in good agreement with the 'diffuse fiber system' throughout the septal complex postulated by Everett et al. 7. Spontaneous ovulation was also inhibited by lesions in the supracommissural area. Since the sites of the DBB-supracommissural lesions were vertically overlapping each other toward the preoptic area, a neural pathway for ovulatory gonadotropin release appears to arch to the preoptic area by way of the precommissural region. This could be partly supported by the previous study in which spontaneous ovulation was inhibited by horizontal transections placed at each level between the CA and the suprachiasmatic area 14. In the present experiment, the DBB stimulation-induced ovulatory response was interrupted by SCH-mediobasal MPO lesions, suggesting that the DBB efferents for ovulation may be conducted to the MBH by way of the mediobasal part of the preoptic area. One should note here that the supracommissural lesions were placed quite dorsal to the level of the preoptic roof in the present study. It has been assumed that the preoptic roof, particularly the precommissural region, is of great importance for spontaneous ovulation 1%2°,21 and ovulation induced by estrogen r~,2~. Therefore the supracommissural area, as well as the amygdaloid complex, might be one of the regions which trigger or conduct the neural inputs to the preoptic area ['or ovulatory gonadotropin release. Greet 8 reported that the lesions in persistent-estrous rats were located ventral to the paraventricular nucleus. Recently, Barraclough et al. 1 and Brown-Grant and Raisman 4 have observed that rats with SCH lesions including the surrounding structures entered persistent-estrus. Clemens et al. 6 have also demonstrated, from chronic experiments with massive lesions in the preoptic area, that the SCH-AHA, not the MPO, may exert an important regulatory influence on reproductive cyclicity. On the other hand, Halfisz and Gorski 9 and Butler and Donovan ~ reported that rats with anterior cuts rostral to the SCH entered persistent vaginal estrus. These findings suggest that not only the SCH but also the anterior inputs to the SCH are necessary for triggering the hypothalamic mechanisms for cyclic ovulation. In addition, L H - R H producing cells are reported to be widely scattered in the paramedian rostral hypothalamus such as the DBB, SEPT and preoptic area 2,a,19. From these areas, L H - R H
107 containing axons are assumed to project to the arcuate nucleus-median eminence (ARC-ME), where LH-RH activity has been detected 11,18,22. Thus forebrain structures, including the DBB, may be assumed to have intimate relations with cyclic ovulation. It has been observed that animals with interruption of anterior and dorsal inputs to the preoptic area resume estrous cycles within several weeks10,16, 21. This might be caused by functional compensation of the preoptic-hypothalamic area, since LH release was induced following estradiol implantation into the MPO in animals with the MPO-roof section but not in intact rats 1°. The present study suggests that the DBB may contain the main neural pathway of preoptic afferents from limbic structures such as the lateral septum or medial amygdala, although the regulatory influence of the DBB on estrous cyclicity remains to be resolved. It may be concluded that the DBB-supracommissural area is part of an indispensable pathway for acute ovulatory gonadotropin release and that the DBB efferents for ovulation are conducted to the MBH by way of the SCH-mediobasal MPO. ACKNOWLEDGEMENT
This study was supported by grants from the Ministry of Education, Japan.
REFERENCES 1 Barraclough, C. A., Yarrazaval, S. and Hatton, R., A possible hypothalamic site of action of progesterone in the facilitation of ovulation in the rat, Endocrinology, 75 (1964) 838-845. 2 Barry, J., Dubois, M. P. and Poulain, P., LRH producing cells of the mammalian hypothalamus, Z. Zellforsch., 146 (1973) 351-366. 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 guinea pig, Endocrinology, 95 (1974) 1416-1423. 4 Brown-Grant, K. and Raisman, G., Abnormalities in reproductive function associated with the destruction of the suprachiasmatic nuclei in female rats, Proc. roy. Soc. B., 198 (1977) 279-296. 5 Butler, J. E. M. and Donovan, B. T., The effect of surgical isolation of the hypothalamus on reproductive function in the female rat, J. Endocr., 49 (1971) 293-304. 6 Clemens, J. A., Smalstig, E. B. and Sawyer, B. D., Studies on the role of the preoptic area in the control of reproductive function in the rat, Endocrinology, 99 (1976) 728-735. 7 Everett, J. W., Radford, H. M. and Holsinger, J., Electrolytic irritative lesions in the hypothalamus, and forebrain area: effect on luteinizing hormone release and the ovarian cycle. In L. Martini (Ed.), Steroid Biochemistry, Pharmacology, and Therapeutics, Academic Press, New York, 1964, p. 251. 8 Greer, M. A., The effect of progesterone on persistent vaginal estrus produced by hypothalamic lesions in the rat, Endocrinology, 53 (1953) 380-390. 9 Hal~isz,B. and Gorski, R. A., Gonadotrophic hormone secretion in female rats after partial or total interruption of neural afferents to the medial basal hypothalamus, Endocrinology, 80 (1967) 608-622. 10 Kawakami, M. and Kimura, F., Limbic-preoptic responses to estrogens and catecholamines in relation to cyclic LH secretion. In F. Naftolin, K. J. Ryan and J. Davies (Eds.), Subcellular Mechanisms in Reproductive Neuroendocrinology, Elsevier, Amsterdam, 1976, p. 423. 11 Kawakami, M., Negoro, H., Kimura, F., Higuchi, T. and Asai, T., Neural control of LH release in anterior periventriculo-median eminence-pituitary system, Neuroendocrinology, 19 (I 975) 137-149. 12 Kawakami, M. and Terasawa, E., Acute effect of neural deafferentation on timing of gonadotropin secretion, Endocr.jap., 19 (1972) 449-459. 13 Kawakami, M., Terasawa, E., Kimura, F. and Wakabayashi, K., Modulating effect of limbic structures on gonadotropin release, Neuroendocrinology, 12 (1973) 1-16.
108 14 Kimura, F. and Kawakami, M., Re-analysis of the preoptic afferents and efferents involved in the surge of LH, FSH and prolactin release in the proestrous rat, Neuroendocrinology, 27 (1978) 74-85. 15 K6nig, J. D. and Klippel, R. A., The Rat Brain, ,4 Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins Co., Baltimore, 1963. 16 K6ves, K. and HaIfisz, B., Location of the neural structures triggering ovulation in the rat, Neuroendocrinology, 6 (1970) 180~193. 17 Niswender, G. D., Midgley, A. R., Jr., Monroe, S. E. and Reichert, L. E., Jr., Radioimmunoassay for rat luteinizing hormone with antiovine LH serum and ovine LH131-I, Proc. Soc. exp. Biol. Med., 128 (1968) 807-811. 18 S6t~il6, G., Vigh, S., Schally, A. V., Arimura, A. and Flerk6, B., LH-RH containing neural elements in the rat hypothalamus, Endocrinology, 96 (1975) 135 142. 19 S6tfil6, G., Vigh, S., Schally, A. V., Arimura, A. and Flerk6, B., Immunohistological study of the origin of LH-RH-containing nerve fibers of the rat hypothalamus, Brain Research, 103 (1976) 597-602. 20 Van Rees, G. P., Control of ovulation by the anterior pituitary gland, In J. A. Kappers and J. P. Schad6 (Eds.), Topics in Neuroendoerinology, Progress in Brain Research, Vol. 38, Elsevier, Amsterdam, 1972, p. 193. 2 l Velasco, M. E. and Taleisnik, S., Effects of the interruption of amygdaloid and hippocampal afferents to the medial hypothalamus on gonadotropin release, J. Endocr., 51 (1971) 41-55. 22 Wheaton, J. E., Krulich, L. and McCann, S. M., Localization of luteinizing hormonereleasing hormone in the preoptic area and hypothalamus of the rat using radioimmunoassay, Endocrinology, 97 (1975) 30 38.
99
FOREBRAIN STRUCTURES INVOLVED IN OVULATION AND RELEASE OF S E R U M L H A N D F S H I N P R O E S T R O U S RATS
MASAZUM! KAWAKAM! and SHINTARO ANDO 2nd Department of Physiology, Yokohama City University School of Medicine, 2-33 Urafune-cho, Minami-ku, Yokohama (Japan)
(Accepted November 8th, 1979) Key words: ovulation - - forebrain - - serum LH - - FSH - - proestrous rat - - diagonal band of Broca septum - - medial preoptic area - - hypothalamus -
-
SUMMARY To investigate in detail the localization of the extrahypothalamic neural structures indispensable for ovulation and serum gonadotropin release, small electrolytic lesions were produced in the forebrain under acute experimental conditions with a platinum electrode at 11.30-13.30 h on the day of proestrus. Blood sampling was performed using ether anesthesia on the afternoon of proestrus and the number of ova in the ampulla were verified with a microscope the next morning. Lesions in the diagonal band of Broca (DBB) or the supracommissural area inhibited spontaneous ovulation and proestrous surges of serum L H and FSH. On the other hand, full or partial ovulation occurred in animals with lesions outside the DBB-supracommissural area, including the medial septum (m-SEPT) and the subcommissural periventricular part of the medial preoptic area (MPO). In this experiment, the lesions which inhibited spontaneous ovulation were limited to the DBB including the supracommissural area. Electrochemical stimulation of the DBB in pentobarbital-blocked proestrous rats was effective in inducing ovulation and serum gonadotropin release. The stimulatory effects of DBB stimulation on the ovulatory response were interrupted by bilateral lesions in the mediobasal preoptic area including the suprachiasmatic nucleus (SCH). The present study indicates that the DBB-supracommissural area is indispensable for acute ovulatory gonadotropin release, and that the DBB efferents for ovulation are conducted to the mediobasal hypotbalamus (MBH) by way of the SCHmediobasal MPO.
100 INTRODUCHON In 1964, Everett et al. 7 postulated that a diffuse septal-preopticotuberal pathway was responsible for ovulatory LH release in rats. From the results of chronic neural transections with a small knife, Halgsz and Gorski 9 suggested that the diffusely organized neural afferents required for cyclic ovulation converge toward the median eminence area. In order to search for the origin of the critical afferents for ovulation, K6ves and Halgsz 16 observed the occurrence of ovulation in rats with long-term bilateral interruption of the anterior and superior connections to the preoptic-anterior hypothalamic area and concluded that the neurogenic stimulus responsible for ovulation may arise at least from the preoptic-anterior hypothalamic area. From the results of acute neural transection, on the other hand, direct or indirect inputs from limbic structures to the medial preoptic and tuberal region of the hypothalamus have been assumed to govern the preovulatory LH surge 12,21. Recent experiments in our laboratory have suggested that areas anterior and superior to the preoptic area, including the diagonal band of Broca (DBB), septum (SEPT) and bed nucleus of stria terminalis (BST), are indispensable for the induction of the LH surge 14. The present experiments were designed to investigate in more detail the extrahypothalamic forebrain structures which are indispensable for ovulatory gonadotropin release in acute experimental conditions. MATERIALS AND METHODS Four-day cyclic adult female rats of Wister strain were used. They were housed in a light-controlled room (5.00-19.00 h light, 19.00-5.00 h dark). Vaginal smears were examined every morning for at least two consecutive cycles before use of the rats as experimental animals on the day of proestrus.
Lesion experiment Electrolytic lesions of the forebrain were made by using a platinum electrode insulated with epoxylite except for the tip. The electrode was stereotaxically placed in the forebrain structure, and lesions were produced by passing an anodal DC current (3 mA for 5 sec) through the electrode. Sham operations were performed in 8 animals in which the electrode was inserted but no current was passed. Surgical operations were performed using ether anesthesia between 11.30 and 13.30 h on the day of proestrus. Blood sampling was performed by cardiac puncture using ether anesthesia for the determination of serum gonadotropin levels (1) just before operation, (2) at 14.30 h and (3) at 18.30 h after operation. Occurrence of ovulation was evaluated in animals showing vaginal cornification on the following morning. The oviducts were compressed between two slide glasses and the number of ova in the ampulla counted with a microscope.
Stimulation experiment The effects of electrochemical stimulation of the DBB on the ovulatory response
101 and serum gonadotropin release were examined in proestrous rats anesthetized with pentobarbital sodium. An anodal DC current of 100/~A for 60 sec was passed through the center wire of a bipolar concentric stainless steel electrode which was stereotaxically placed in the DBB. In the experimental animals, electrolytic lesions in the mediobasal preoptic area including the SCH were produced immediately prior to the subsequent DBB stimulation, by passing an anodal D C current of 3 mA for 10 sec through a platinum electrode. In sham-lesioned animals, a platinum electrode was inserted into the mediobasal preoptic area, without passing an anodal D C current, prior to the DBB stimulation. In each group, surgical procedures were carried out ,between 14.30 and 15.30 h in rats anesthetized with pentobarbital sodium (30 mg/kg) injected at 13.45 h. Blood sampling was performed by cardiac puncture (1)just before operation, (2) at 90 min and (3) at 180 min after DBB stimulation. On the following morning vaginal smear and ovulatory response were examined as in the preceeding lesion experiment. Serum LH and FSH levels were measured following the radioimmunoassay methods described by Niswender et al. 17 and by N I A M D D instructions, respectively. The concentrations of LH and FSH were expressed in terms of NIH-LH-S 1 and NIHFSH-S1, respectively. Student's t-test and Fisher exact probability test were used for statistical analysis. Frozen sections of the brain were stained with cresyl violet and examined with a microscope to ascertain the extent of lesions, based on the brain atlas of Kfnig and KlippeP 5.
(A)
{B)
'~'
C,A\ Th
• .,'-.,...'
S C H ~ L=0.24
(~
Group Ill (No OvuL)
( ~ ) GroupI (Full Ovut.) "'-" Groupil (Partial Ovul.)
t
_
t
Fig. 1. Schematic drawing of the sites of the forebrain lesions on sagittal sections. A: solid-line circles represent the sites of the lesions which were found in the non-ovulated rats of group III. B: solid-line circles represent the sites of the lesions which were found in the fully-ovulated rats of group I. Mean number of ova observed in the ampulla was 10.9. Broken-line circles represent the sites of the lesions which were found in the partially ovulated rats of Group II. Mean number of ova observed in the ampulla was 2.8. A, nucleus accumbens; AHA, anterior hypothalamic area; ARC, arcuate nucleus; CA, commissura anterior; CC, corpus cailosum; CO, optic chiasma; DBB, diagonal band of Broca; Fx, fornix; MPO, medial preoptic area; SCH, suprachiasmatic nucleus; SL, lateral septum; SM, medial septum; Th, thalamus.
102 RESULTS
Eff~wts of forebrain lesions on ovulation and serum gonadotropin release Ovulation. Thirty-two electrolytic lesions were located in the forebrain in loci such as the DBB, SEPT, medial preoptic area (MPO) and the area dorsal to the commissura anterior (CA). The sites of lesions which inhibited spontaneous ovulation are shown in Fig. I A, whereas those illustrated in Fig. 1B were ineffective. Ovulation was significantly inhibited following the DBB lesions in 13 of 19 animals (P < 0.01). Rats with sham lesions ovulated fully. Ovulation failed to occur in all 7 animals with lesions in the posterior part of the DBB, the typical site of these lesions is represented in Fig. 3. On the contrary, half of the 12 animals with lesions in the anterior part of the DBB ovulated (Fig. 1A, B). Unexpectedly, ovulation also failed to occur in all 4 animals subjected to lesions in the paracentral area dorsal to the CA (tentatively termed as 'supracommissural area') (Fig. 1A). Thus the lesions which inhibited spontaneous ovulation were limited to the DBB-supracommissural area (Figs. 1A and 2), although
A9./~
"'.~...:I~.l.t.. ,.--""..'
A 7.0
Fig. 2. Schematic drawing of the sites of the DBB-supracommissural lesions on serial frontal sections. Solid-line circles represent the sites of the lesions which were found in the non-ovulated rats of group III. See caption to Fig. l for definitions of abbreviations.
103
Fig. 3. A photographic representation of one of the typical DBB lesions. Arrow indicates the site of the lesion. the c o m m o n a r e a o f lesions was obscure in inhibiting s p o n t a n e o u s ovulation. O n the o t h e r hand, s p o n t a n e o u s o v u l a t i o n was n o t inhibited in a n y o f 9 animals subjected to lesions outside the D B B - s u p r a c o m m i s s u r a l area. O v u l a t i o n occurred in all 3 animals with lesions in the m e d i a l S E P T ( m - S E P T ) a n d in all 4 a n i m a l s with lesions in the subc o m m i s s u r a l periventricular p a r t o f the M P O (Fig. 1B). LH (ng/m[) 15--
"-r-
[ ~ Before Operation 14:30 18:30
1057 8 FSH (,ug/ml)
r==~
Sham
J
5 5 6.
15 15 17,1
Full Ovul. Partial Ovu[. No Ovul. (Group I ) (Group II) (Group 111)
p
Fig. 4. Effects of lesions in several parts of the forebrain on the release of serum LH and FSH in proestrous rats. Bars represent means and the lines, standard errors. The number of rats is given in (or above) the bars. See Fig. 1 for location of the forebrain lesions in each group.
104
Serum gonadotropin release. According to the number o f ova observed in the ampulla, animals employed in this experiment were divided into 3 groups - - group l (full ovulation, _--__8 ova), group II (partial ovulation, _< 7 ova) and group I I I (no ovulation) (Fig. 1A, B). Preovulatory surges o f serum L H and F S H were observed following the lesions in animals o f group I as well as in sham-lesioned animals. In animals of" g r o u p If, the release o f serum L H was significantly suppressed although that of serum F S H was not. In animals o f group III, all o f whose lesions were found to be located in the DBB-supracommissural area, the release o f serum L H was eliminated and that o f serum F S H was significantly suppressed (Fig. 4). EfJects of DBB stimulation on ovulation and serum gonadotropin release Ovulation. Full ovulation was induced following DBB stimulation in all 6 shamlesioned animals pretreated with pentobarbital sodium. The expected ovulation was not induced following the DBB stimulation in any of 8 animals with bilateral lesions in the S C H including parts o f the mediobasal M P O , the preoptic suprachiasmatic area
st im. Th , •
,%
°=
SCH
0.24
CC
,"11117 , /l~
A 5.8
'
SCH
CO A?,O Fig. 5. Schematic drawing of the sites of the bilateral SCH lesions which inhibited the DBB stimulationinduced ovulation in pentobarbital-blocked proestrous rats. Parts of the MPO, POSC and AHA were involved in the SCH lesions. The stimulation sites in the DBB are represented as open circles on sagittal section. See caption to Fig. 1 for definition of abbreviations.
105 LH ( ng/ml )
F(p Sg Hm l )l
10-
1.0"
(a)
5- ~ a )
(7)~'I(s)
0m5 "
~(a)
(5)
0
90
180 rain.
100rain.
- - - Sham control o ~ o Incomplete or unilateral SCH lesion -- -- B i l a t e r a l 5CH lesion p -: 0.05 vs sham control at each time
Fig. 6. Effects of the bilateral SCH lesions on the release of serum LH and FSH induced by DBB stimulation in pentobarbital-blocked proestrous rats. Each point represents mean and vertical bar, standard error. The number of rats is given in parentheses. (POSC) and the anterior hypothalamic area (AHA) (Fig. 5). There appeared to be no extensive damage to the optic tract upon histological observation. On the other hand, the expected ovulation was not inhibited in animals with incomplete or unilateral SCH lesions. Serum gonadotropin release. The DBB stimulation effectively induced release of serum L H and F S H with the peaks at 90 min and 180 rain, respectively (Fig. 6). In animals with bilateral SCH lesions, the values of serum L H at both 90 min and 180 min were significantly reduced and those of serum F S H at 180 min were significantly reduced. However, in animals with incomplete or unilateral SCH lesions, the releases of serum L H and F S H were induced in the same manner as those in sham-lesioned animals. DISCUSSION
Spontaneous ovulation and serum gonadotropin release were inhibited by small lesions produced in the DBB at 11.30-13.30 h on the day of proestrus, which led us to assume that the forebrain rostral to the preoptic area may be involved in triggering the ovulatory gonadotropin release. It has been observed that acute horizontal interruption of the limbic projection to the medial hypothalamus by preoptic r o o f cuts inhibited spontaneous ovulation12,20, 21, although regular estrous cyclicity was restored in animals with long-term preoptic-roof section10, 21. In the present experiment, the lesions in the posterior part of the DBB resulted in inhibition of spontaneous ovulation. Such lesions probably did not extend caudally far enough to include the preoptic-anterior hypothalamic area which was severed from the forebrain structures in the work by K6ves and Hal~sz 16.
106 On the other hand, none of these lesions in the DBB was placed dorsal to the preoptic roof. Recently, Kimura and Kawakami '4 made deafferentations ventroposteriorly and dorso-anteriorly to the DBB, of which the former inhibited spontaneous ovulation while the latter did not. Most of the DBB lesions in the present study were located very medially within these two boundaries. It has been assumed that the septal complex, when stimulated electrically or electrochemically, can readily induce the release of serum gonadotropin v,la. Everett et al. 7 have postulated that the nerve fiber array responsible for ovulation is dispersed in the area rostral and dorsal to the preoptic region, including the medial septal nuclei, the nucleus accumbens and the medial parolfactory area. In the present study, spontaneous ovulation was not inhibited following lesions produced in parts of the m-SEPT nor in parts of the anterior part of the DBB. Therefore, it may be suggested that ovulatory gonadotropin release is not so much affected by lesions in these two brain areas as by lesions in the posterior part of the DBB or in the supracommissural area. This is in good agreement with the 'diffuse fiber system' throughout the septal complex postulated by Everett et al. 7. Spontaneous ovulation was also inhibited by lesions in the supracommissural area. Since the sites of the DBB-supracommissural lesions were vertically overlapping each other toward the preoptic area, a neural pathway for ovulatory gonadotropin release appears to arch to the preoptic area by way of the precommissural region. This could be partly supported by the previous study in which spontaneous ovulation was inhibited by horizontal transections placed at each level between the CA and the suprachiasmatic area 14. In the present experiment, the DBB stimulation-induced ovulatory response was interrupted by SCH-mediobasal MPO lesions, suggesting that the DBB efferents for ovulation may be conducted to the MBH by way of the mediobasal part of the preoptic area. One should note here that the supracommissural lesions were placed quite dorsal to the level of the preoptic roof in the present study. It has been assumed that the preoptic roof, particularly the precommissural region, is of great importance for spontaneous ovulation 1%2°,21 and ovulation induced by estrogen r~,2~. Therefore the supracommissural area, as well as the amygdaloid complex, might be one of the regions which trigger or conduct the neural inputs to the preoptic area ['or ovulatory gonadotropin release. Greet 8 reported that the lesions in persistent-estrous rats were located ventral to the paraventricular nucleus. Recently, Barraclough et al. 1 and Brown-Grant and Raisman 4 have observed that rats with SCH lesions including the surrounding structures entered persistent-estrus. Clemens et al. 6 have also demonstrated, from chronic experiments with massive lesions in the preoptic area, that the SCH-AHA, not the MPO, may exert an important regulatory influence on reproductive cyclicity. On the other hand, Halfisz and Gorski 9 and Butler and Donovan ~ reported that rats with anterior cuts rostral to the SCH entered persistent vaginal estrus. These findings suggest that not only the SCH but also the anterior inputs to the SCH are necessary for triggering the hypothalamic mechanisms for cyclic ovulation. In addition, L H - R H producing cells are reported to be widely scattered in the paramedian rostral hypothalamus such as the DBB, SEPT and preoptic area 2,a,19. From these areas, L H - R H
107 containing axons are assumed to project to the arcuate nucleus-median eminence (ARC-ME), where LH-RH activity has been detected 11,18,22. Thus forebrain structures, including the DBB, may be assumed to have intimate relations with cyclic ovulation. It has been observed that animals with interruption of anterior and dorsal inputs to the preoptic area resume estrous cycles within several weeks10,16, 21. This might be caused by functional compensation of the preoptic-hypothalamic area, since LH release was induced following estradiol implantation into the MPO in animals with the MPO-roof section but not in intact rats 1°. The present study suggests that the DBB may contain the main neural pathway of preoptic afferents from limbic structures such as the lateral septum or medial amygdala, although the regulatory influence of the DBB on estrous cyclicity remains to be resolved. It may be concluded that the DBB-supracommissural area is part of an indispensable pathway for acute ovulatory gonadotropin release and that the DBB efferents for ovulation are conducted to the MBH by way of the SCH-mediobasal MPO. ACKNOWLEDGEMENT
This study was supported by grants from the Ministry of Education, Japan.
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108 14 Kimura, F. and Kawakami, M., Re-analysis of the preoptic afferents and efferents involved in the surge of LH, FSH and prolactin release in the proestrous rat, Neuroendocrinology, 27 (1978) 74-85. 15 K6nig, J. D. and Klippel, R. A., The Rat Brain, ,4 Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins Co., Baltimore, 1963. 16 K6ves, K. and HaIfisz, B., Location of the neural structures triggering ovulation in the rat, Neuroendocrinology, 6 (1970) 180~193. 17 Niswender, G. D., Midgley, A. R., Jr., Monroe, S. E. and Reichert, L. E., Jr., Radioimmunoassay for rat luteinizing hormone with antiovine LH serum and ovine LH131-I, Proc. Soc. exp. Biol. Med., 128 (1968) 807-811. 18 S6t~il6, G., Vigh, S., Schally, A. V., Arimura, A. and Flerk6, B., LH-RH containing neural elements in the rat hypothalamus, Endocrinology, 96 (1975) 135 142. 19 S6tfil6, G., Vigh, S., Schally, A. V., Arimura, A. and Flerk6, B., Immunohistological study of the origin of LH-RH-containing nerve fibers of the rat hypothalamus, Brain Research, 103 (1976) 597-602. 20 Van Rees, G. P., Control of ovulation by the anterior pituitary gland, In J. A. Kappers and J. P. Schad6 (Eds.), Topics in Neuroendoerinology, Progress in Brain Research, Vol. 38, Elsevier, Amsterdam, 1972, p. 193. 2 l Velasco, M. E. and Taleisnik, S., Effects of the interruption of amygdaloid and hippocampal afferents to the medial hypothalamus on gonadotropin release, J. Endocr., 51 (1971) 41-55. 22 Wheaton, J. E., Krulich, L. and McCann, S. M., Localization of luteinizing hormonereleasing hormone in the preoptic area and hypothalamus of the rat using radioimmunoassay, Endocrinology, 97 (1975) 30 38.