Brain Research, 154 (1978) 209-213 © Elsevier/North-Holland Biomedical Press
209
Organum vasculosum laminae terminalis and reflex ovulation, in the rabbit
H. LESCURE, B. DUFY, J. LEONARDELLI and CL. BENSCH
Department of Physiology, Laboratoryof Neurophysiology andPhysiopathology, University of Bordeaux II, 33076 Bordeaux and Departmentof Histology, Faculty of Medicine, 59045 Lille (France) (Accepted May 1lth, 1978)
The organum vasculosum of the lamina terminalis (OVLT) is in neural contactS, 6 with that part of the rostral hypothalamus known to control gonadotropin secretion s, 15,19and sexual behaviour t5 in many species. Recent immunohistochemical experiments have shown neurons and fibres immunologicaUy reactive to the luteinizing hormonereleasing hormone (LH-RH) in the rostral hypothalamus and OVLT3,4,13,19. However, very little is known of the role of the OVLT in reproductive behavior and neuroendocrine regulation. The present paper reports experiments designed to further examine the function of this area in reflex ovulation. The experiments were conducted on female rabbits. This animal was chosen for two reasons. Firstly, the OVLT is particularly well developed in that species 6; secondly, the female rabbit is a reflex ovulator requiring coital stimuli to trigger the surge of LH, thus providing a fully reliable model, in which the neuroendocrine response (ovulation) as well as the stimulus which triggers it (coitus) can be checked and time-locked. The first experiments sought to examine the disposition of the LH-RH immunoreactive system at the level of the OVLT. The second study sought to analyze the effects of electrolytic destruction of the OVLT on the sexual receptivity of the female rabbit to the male on the one hand, and on the neuroendocrine response leading to ovulation on the other. For these studies, conducted between February and April, 14 mature female rabbits of the 'Fauve de Bourgogne' strain were used. The animals, 4-6 months old, weighing 3.5-4.0 kg, were maintained on food and water ad libitum, and under natural lighting and temperature conditions. Two animals were used for the immunohistochemical study of the LH-RH immunoreactive material of the OVLT. Each hypothalamus was fixed in Bouin-Hollande's solution, embedded in paraffin, and cut in sagittal serial sections of 10/,m. An indirect immunofluorescence method was performed 12, using antisera prepared and characterized by M. P. DuboisS, 4. The lamina terminalis of the female rabbit is very rich in material immunoreactive to LH-RHL The hormone is observed primarily in nerve terminals. These are seen as slightly varicose thin fibres which are either isolated or arranged in a network (Fig. 1). Most of the immunoreactive isolated fibres run in a concave dorsocaudal direction.
210
Fig. 1. Photomicrographs of frontal sections (10/~m thick) of rabbit's hypothalamus showing an exanapie of location and extent of OVLT electrolytic destruction. A and B : low magnification of the rostral (A) and caudal (B) extensions in the same animal (frontal sections). C: higher magnification of the same areas. Dark bar: 200/~m. OC, optic chiasm.
The organization was similar to that of the external fibres of the medial fasciculus of the lamina terminalis. Their caudal projection followed a course just dorsal to the optic chiasm and directed towards the tuber cinereum. The immunoreactive material arranged in a network of fibres was largely restricted to the lower half of the lamina terminalis. This second group of L H - R H immunoreactive nerve fibres made contact with the vascular loops of the primary and secondary plexus of the OVLT. Finally, although immunoreactive nerve fibres and endings were plentiful in our preparations, only a total of 5 (3 from one rabbit and 2 from the other) immunoreactive perikarya were observed. Furthermore, these cell bodies were only weakly fluorescent. They were found in the subependymal layer, against the lateral faces of the secondary vascular plexus. Electrolytic lesions of the OVLT area were made in the remaining 12 rabbits. The animals were anaesthetized with sodium pentobarbitone (Nembutal, 25 mg/kg) and a monopolar electrode stereotaxically implanted in the OVLT. The stereotaxic coordinates for the electrode tip were AP ---0.0, lateral --frO, depth 14.0 (from the atlas prepared by Sawyer et a!.14). A 100 mA high-frequency current was administered through the electrode for 3 see by a Massiot-Philipps Neurocoagulix H F apparatus. Ten days after surgery the animals were placed in the cage of a sexually vigorous buck for 10 min. Copulation occurred during this period. Twenty-four hours later the animals ~were sacrificed with an overdose of pentobarbitone, and the ovaries were inspected for evidence of ruptured follicles. The brains were removed, cut and stained according to
211
Fig. 2. Parasagittal sections of the inferior (A) and superior (B) parts of the OVLT; with histoimmunofluorescent LH-RH positive features. White star: third ventricle; thick white arrow: an isolated positive neuron; thin white arrows: various sections of vascular loops of the OVLT secondary vascular plexus; white bar: 100/~rn. classical histological procedures. The location and extent of the lesioned area was determined from these sections. All the female rabbits which were lesioned in the anterior hypothalamus copulated when placed with a male. In addition, it was likely that ovulation occurred 24 h after copulation, since all the animals had newly ruptured ovarian follicles. In 7 animals the location of the lesion was either too high or too lateral, but in 5 animals the O V L T was totally destroyed (Fig. 2). Our results clearly indicate that the L H - R H immunoreactive system of the O V L T is not involved in the neuroendocrine mechanisms of ovulation in the female rabbit, either directly or indirectly (humoral waylS). This finding is in clear contrast to reports in the rat that anterior disconnection interferes with ovulationg,10,17. However, they do agree with studies done in rhesus monkeys, in which similarly placed lesions had no appreciable influence on pre-ovulatory L H release 11. It should be noted, too, that Dyer
212 et al. 7 showed very recently that, in rats with bilateral p r e o p t i c lesions, the d u r a t i o n o f the p r e o v u l a t o r y L H secretion was shortened, b u t p e a k levels were unchanged. Thus, in the r a b b i t the O V L T system does n o t seem to be essential for the preo v u l a t o r y r e g u l a t i o n o f o v u l a r i a n function. W e suggest t h a t the L H - R H i m m u n o r e a c t ive units, l o c a t e d in diverse areas 1-4 m a y be elements o f different peptidergic systems, a n d hence involved in a variety o f functions, a l t h o u g h the nature o f these various activities remains to be elucidated. It is j u s t possible, too, t h a t h i s t o i m m u n o l o g i c a l techniques trace materials which differ chemically a n d physiologically, b u t which have c o m m o n antigenic properties. W e t h a n k Profs. L. C. K r e y (Rockefeller University) a n d R. D y e r ( C a m b r i d g e ) for their helpful advice, a n d M r s M. Cazalis for her technical assistance. This w o r k was s u p p o r t e d by grants f r o m D G R S T (74.7.0516), C N R S ( E . R . A . 493), I N S E R M (U. 176) a n d the University o f B o r d e a u x II.
1 Araki, S., Ferin, M.,Zimmerman, E. A. and Van de Wiele, R. L., Ovarian modulation of immunoreactive gonadotropin-releasinghormone (Gn-RH) in the rat brain : evidence for a differential effect on the anterior and mid hypothalamus, Endocrinology, 96 (1975) 644-650. 2 Barry, J., Characterization and topography of LH-RH neurons in the rabbit, Neurosci. Lett., 2 (1976) 201-205. 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 Barry, J. et Dubois, M. P., l~.tudeen immunofluorescence de la diff6renciation pr6natale des cellutes hypothalamiques 61aboratrices de LH-RH et de la maturation de la voie neuros6cr6trice pr6opticoinfundibulaire chez le cobaye, Brain Research, 67 (1974) 103-113. 5 Duvernoy, H. et Koritke, J. G., Les fibres nerveuses de la lame terminale et leurs rapports avec rorgane vasculaire de la lame terminale, C.R. Ass. Anat., 48 (1963) 562-576. 6 Duvernoy, H. et Koritke, J. G., Contribution b. l'6tude de l'angioarchitectonie des organes circumventriculaires, Arch. Biol. (Lidge), 75, Suppl. (1964) 849-904. 7 Dyer, R. G., ter Haar, M. B. and Saphier, D., Preovulatory secretion of gonadotropins and TSH in cyclic rats with small lesions in the preoptic area, J. Reprod. Fert., in press. 8 Flerko, B., Hypothalamic mediation of neuroendocrine regulation of hypophysial gonadotrophic functions. In R. O. Greep (Ed.), MTP International Review of Science, Physiology, Series 1, Vol. 8, Reproductive Physiology, Butterworth, London, 1974, pp. 1-32. 9 Halgtsz, B. and Gorski, R. A., Gonadotrophic hormone secretion in female rats after partial or total interruption of neural afferents to the medial basal hypothalamus, Endocrit,ology, 80 (1967) 608-622. 10 Kaasjager, W. A., Woodbury, D. M., Van Dieten, J. A. M. J. and Van Rees, G. P., The role played by the preoptic region and the hypothalamus in spontaneous ovulation and ovulation induced by progesterone, Neuroendocrinology, 7 (1971) 54-64. 11 Krey, L. C. ,Butler, W. R. and Knobil, E., Surgical disconnection of the medial basal hypothalamus and pituitary function in the Rhesus monkey. I. Gonadotropin secretion, Endocrinology, 96 (1975) 1073-1087. 12 Leonardelli, J., Barry, J. et Dubois, M. P., Mise en 6vidence par immunofluorescence d'un constituant immunologiquement apparent6 au LH-RF darts l'hypothalamus et l'Eminence M~.diane chez les mammif6res, C. R. Acad. Sci. (Paris), 276 (1973) 2043-2046. 13 Mazzuca, M., Immunocytochemical and ultrastructural identification of luteinizing hormone-releasing hormone (LH-RH) containing neurons in the vascular organ of the lamina terminalis (OVLT) of the squirrel monkey, Neurosci. Lett., 5 (1977) 123-127. 14 Sawyer, C. H., Everett, J. W. and Green, J. D., The rabbit diencephalon in stereotaxic coordinates, J. comp. NeuroL, 101 (1954) 801-824.
213 15 Sawyer, Ch.H., Regulatory mechanisms of secretion of gonadotrophic hormones. In W. Haymaker, E. Anderson and W. J. H. Nauta (Eds.), The Hypothalamus, Thomas, Springfield, II1., 1969, pp. 389-430. 16 Schneider, H. P. G., Crighton, D. B. and McCann, S. M., Suprachiasmatic LH-releasing factor, Neuroendocrinology, 5 (1969) 271-380. 17 Tejasen, T. and Everett, J. W., Surgical analysis of the preoptic-tuberal pathway controllingovulatory release of gonadotrophins in the rat, Endocrinology, 81 (1967) 1387-1396. 18 Zimmerman, E. A., Localization of hypothalamic hormones by immunocytochemical techniques. In L. Martini and W. F. Ganong (Eds.), Frontiers in Neuroendocrinology, Vol. 4, Raven Press, NewYork, 1976, pp. 25-62. 19 Zimmerman, E. A., Hsu, K. C., Ferin, M. and Kozlowski, G. P., Localization of gonadotropinreleasing hormone (Gn-RH) in the hypothalamus of the mouse by immunoperoxidase technique, Endocrinology, 95 (1974) 1-8.
209
Organum vasculosum laminae terminalis and reflex ovulation, in the rabbit
H. LESCURE, B. DUFY, J. LEONARDELLI and CL. BENSCH
Department of Physiology, Laboratoryof Neurophysiology andPhysiopathology, University of Bordeaux II, 33076 Bordeaux and Departmentof Histology, Faculty of Medicine, 59045 Lille (France) (Accepted May 1lth, 1978)
The organum vasculosum of the lamina terminalis (OVLT) is in neural contactS, 6 with that part of the rostral hypothalamus known to control gonadotropin secretion s, 15,19and sexual behaviour t5 in many species. Recent immunohistochemical experiments have shown neurons and fibres immunologicaUy reactive to the luteinizing hormonereleasing hormone (LH-RH) in the rostral hypothalamus and OVLT3,4,13,19. However, very little is known of the role of the OVLT in reproductive behavior and neuroendocrine regulation. The present paper reports experiments designed to further examine the function of this area in reflex ovulation. The experiments were conducted on female rabbits. This animal was chosen for two reasons. Firstly, the OVLT is particularly well developed in that species 6; secondly, the female rabbit is a reflex ovulator requiring coital stimuli to trigger the surge of LH, thus providing a fully reliable model, in which the neuroendocrine response (ovulation) as well as the stimulus which triggers it (coitus) can be checked and time-locked. The first experiments sought to examine the disposition of the LH-RH immunoreactive system at the level of the OVLT. The second study sought to analyze the effects of electrolytic destruction of the OVLT on the sexual receptivity of the female rabbit to the male on the one hand, and on the neuroendocrine response leading to ovulation on the other. For these studies, conducted between February and April, 14 mature female rabbits of the 'Fauve de Bourgogne' strain were used. The animals, 4-6 months old, weighing 3.5-4.0 kg, were maintained on food and water ad libitum, and under natural lighting and temperature conditions. Two animals were used for the immunohistochemical study of the LH-RH immunoreactive material of the OVLT. Each hypothalamus was fixed in Bouin-Hollande's solution, embedded in paraffin, and cut in sagittal serial sections of 10/,m. An indirect immunofluorescence method was performed 12, using antisera prepared and characterized by M. P. DuboisS, 4. The lamina terminalis of the female rabbit is very rich in material immunoreactive to LH-RHL The hormone is observed primarily in nerve terminals. These are seen as slightly varicose thin fibres which are either isolated or arranged in a network (Fig. 1). Most of the immunoreactive isolated fibres run in a concave dorsocaudal direction.
210
Fig. 1. Photomicrographs of frontal sections (10/~m thick) of rabbit's hypothalamus showing an exanapie of location and extent of OVLT electrolytic destruction. A and B : low magnification of the rostral (A) and caudal (B) extensions in the same animal (frontal sections). C: higher magnification of the same areas. Dark bar: 200/~m. OC, optic chiasm.
The organization was similar to that of the external fibres of the medial fasciculus of the lamina terminalis. Their caudal projection followed a course just dorsal to the optic chiasm and directed towards the tuber cinereum. The immunoreactive material arranged in a network of fibres was largely restricted to the lower half of the lamina terminalis. This second group of L H - R H immunoreactive nerve fibres made contact with the vascular loops of the primary and secondary plexus of the OVLT. Finally, although immunoreactive nerve fibres and endings were plentiful in our preparations, only a total of 5 (3 from one rabbit and 2 from the other) immunoreactive perikarya were observed. Furthermore, these cell bodies were only weakly fluorescent. They were found in the subependymal layer, against the lateral faces of the secondary vascular plexus. Electrolytic lesions of the OVLT area were made in the remaining 12 rabbits. The animals were anaesthetized with sodium pentobarbitone (Nembutal, 25 mg/kg) and a monopolar electrode stereotaxically implanted in the OVLT. The stereotaxic coordinates for the electrode tip were AP ---0.0, lateral --frO, depth 14.0 (from the atlas prepared by Sawyer et a!.14). A 100 mA high-frequency current was administered through the electrode for 3 see by a Massiot-Philipps Neurocoagulix H F apparatus. Ten days after surgery the animals were placed in the cage of a sexually vigorous buck for 10 min. Copulation occurred during this period. Twenty-four hours later the animals ~were sacrificed with an overdose of pentobarbitone, and the ovaries were inspected for evidence of ruptured follicles. The brains were removed, cut and stained according to
211
Fig. 2. Parasagittal sections of the inferior (A) and superior (B) parts of the OVLT; with histoimmunofluorescent LH-RH positive features. White star: third ventricle; thick white arrow: an isolated positive neuron; thin white arrows: various sections of vascular loops of the OVLT secondary vascular plexus; white bar: 100/~rn. classical histological procedures. The location and extent of the lesioned area was determined from these sections. All the female rabbits which were lesioned in the anterior hypothalamus copulated when placed with a male. In addition, it was likely that ovulation occurred 24 h after copulation, since all the animals had newly ruptured ovarian follicles. In 7 animals the location of the lesion was either too high or too lateral, but in 5 animals the O V L T was totally destroyed (Fig. 2). Our results clearly indicate that the L H - R H immunoreactive system of the O V L T is not involved in the neuroendocrine mechanisms of ovulation in the female rabbit, either directly or indirectly (humoral waylS). This finding is in clear contrast to reports in the rat that anterior disconnection interferes with ovulationg,10,17. However, they do agree with studies done in rhesus monkeys, in which similarly placed lesions had no appreciable influence on pre-ovulatory L H release 11. It should be noted, too, that Dyer
212 et al. 7 showed very recently that, in rats with bilateral p r e o p t i c lesions, the d u r a t i o n o f the p r e o v u l a t o r y L H secretion was shortened, b u t p e a k levels were unchanged. Thus, in the r a b b i t the O V L T system does n o t seem to be essential for the preo v u l a t o r y r e g u l a t i o n o f o v u l a r i a n function. W e suggest t h a t the L H - R H i m m u n o r e a c t ive units, l o c a t e d in diverse areas 1-4 m a y be elements o f different peptidergic systems, a n d hence involved in a variety o f functions, a l t h o u g h the nature o f these various activities remains to be elucidated. It is j u s t possible, too, t h a t h i s t o i m m u n o l o g i c a l techniques trace materials which differ chemically a n d physiologically, b u t which have c o m m o n antigenic properties. W e t h a n k Profs. L. C. K r e y (Rockefeller University) a n d R. D y e r ( C a m b r i d g e ) for their helpful advice, a n d M r s M. Cazalis for her technical assistance. This w o r k was s u p p o r t e d by grants f r o m D G R S T (74.7.0516), C N R S ( E . R . A . 493), I N S E R M (U. 176) a n d the University o f B o r d e a u x II.
1 Araki, S., Ferin, M.,Zimmerman, E. A. and Van de Wiele, R. L., Ovarian modulation of immunoreactive gonadotropin-releasinghormone (Gn-RH) in the rat brain : evidence for a differential effect on the anterior and mid hypothalamus, Endocrinology, 96 (1975) 644-650. 2 Barry, J., Characterization and topography of LH-RH neurons in the rabbit, Neurosci. Lett., 2 (1976) 201-205. 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 Barry, J. et Dubois, M. P., l~.tudeen immunofluorescence de la diff6renciation pr6natale des cellutes hypothalamiques 61aboratrices de LH-RH et de la maturation de la voie neuros6cr6trice pr6opticoinfundibulaire chez le cobaye, Brain Research, 67 (1974) 103-113. 5 Duvernoy, H. et Koritke, J. G., Les fibres nerveuses de la lame terminale et leurs rapports avec rorgane vasculaire de la lame terminale, C.R. Ass. Anat., 48 (1963) 562-576. 6 Duvernoy, H. et Koritke, J. G., Contribution b. l'6tude de l'angioarchitectonie des organes circumventriculaires, Arch. Biol. (Lidge), 75, Suppl. (1964) 849-904. 7 Dyer, R. G., ter Haar, M. B. and Saphier, D., Preovulatory secretion of gonadotropins and TSH in cyclic rats with small lesions in the preoptic area, J. Reprod. Fert., in press. 8 Flerko, B., Hypothalamic mediation of neuroendocrine regulation of hypophysial gonadotrophic functions. In R. O. Greep (Ed.), MTP International Review of Science, Physiology, Series 1, Vol. 8, Reproductive Physiology, Butterworth, London, 1974, pp. 1-32. 9 Halgtsz, B. and Gorski, R. A., Gonadotrophic hormone secretion in female rats after partial or total interruption of neural afferents to the medial basal hypothalamus, Endocrit,ology, 80 (1967) 608-622. 10 Kaasjager, W. A., Woodbury, D. M., Van Dieten, J. A. M. J. and Van Rees, G. P., The role played by the preoptic region and the hypothalamus in spontaneous ovulation and ovulation induced by progesterone, Neuroendocrinology, 7 (1971) 54-64. 11 Krey, L. C. ,Butler, W. R. and Knobil, E., Surgical disconnection of the medial basal hypothalamus and pituitary function in the Rhesus monkey. I. Gonadotropin secretion, Endocrinology, 96 (1975) 1073-1087. 12 Leonardelli, J., Barry, J. et Dubois, M. P., Mise en 6vidence par immunofluorescence d'un constituant immunologiquement apparent6 au LH-RF darts l'hypothalamus et l'Eminence M~.diane chez les mammif6res, C. R. Acad. Sci. (Paris), 276 (1973) 2043-2046. 13 Mazzuca, M., Immunocytochemical and ultrastructural identification of luteinizing hormone-releasing hormone (LH-RH) containing neurons in the vascular organ of the lamina terminalis (OVLT) of the squirrel monkey, Neurosci. Lett., 5 (1977) 123-127. 14 Sawyer, C. H., Everett, J. W. and Green, J. D., The rabbit diencephalon in stereotaxic coordinates, J. comp. NeuroL, 101 (1954) 801-824.
213 15 Sawyer, Ch.H., Regulatory mechanisms of secretion of gonadotrophic hormones. In W. Haymaker, E. Anderson and W. J. H. Nauta (Eds.), The Hypothalamus, Thomas, Springfield, II1., 1969, pp. 389-430. 16 Schneider, H. P. G., Crighton, D. B. and McCann, S. M., Suprachiasmatic LH-releasing factor, Neuroendocrinology, 5 (1969) 271-380. 17 Tejasen, T. and Everett, J. W., Surgical analysis of the preoptic-tuberal pathway controllingovulatory release of gonadotrophins in the rat, Endocrinology, 81 (1967) 1387-1396. 18 Zimmerman, E. A., Localization of hypothalamic hormones by immunocytochemical techniques. In L. Martini and W. F. Ganong (Eds.), Frontiers in Neuroendocrinology, Vol. 4, Raven Press, NewYork, 1976, pp. 25-62. 19 Zimmerman, E. A., Hsu, K. C., Ferin, M. and Kozlowski, G. P., Localization of gonadotropinreleasing hormone (Gn-RH) in the hypothalamus of the mouse by immunoperoxidase technique, Endocrinology, 95 (1974) 1-8.
