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Precocious puberty and synaptogenesis in the hypothalamic arcuate nucleus in pregnant mare serum gonadotropin (PMSG) treated immature female rats
Brain Research, 129 (1977) 375-378
375
© Elsevier/North-Holland Biomedical Press
Precocious puberty and synaptogenesis in the hypothalamic arcuate nucleus in pregnant mare serum gonadotropin (PMSG) treated immature female rats
A. MATSUMOTO and Y. ARAI Department of Anatomy, Juntendo University School of Medicine, Hongo, Tokyo 113 (Japan)
(Accepted March 16th, 1977)
The onset of puberty is dependent on a complex process of neuroendocrinological events which involve the maturation of the hypothalamic-pituitary-gonadal axis. For the control of gonadotropin secretion, the medial basal hypothalamus (MBH) is thought to be a final key station between the hypothalamus and pituitary. This region is not only influenced directly by the feedback action of sex steroids, but also modulated by facilitatory and inhibitory neural inputs from the intra- and extrahypothalamic afferent systems 4,9. The functional maturation of the onset mechanism of puberty must be correlated with the growth rate of these neuronal components of the MBH and also with the establishment of the neural connections with the converging afferent systems. Recently, we have reported that the number of synapses in the arcuate nucleus (ARCN), one of the principal neuronal components of the MBH, increases remarkably up to puberty during the postnatal development in the female rats 6. In the present study, as one step to elucidate the nature of the changes in the neural mechanisms involved in initiation of puberty, semiquantitative measurements on the synaptic formation in the A R C N were performed in immature female rats treated with pregnant mare serum gonadotropin (PMSG). Twenty-eight-day-old Wistar female rats (housed under 14-10 h light-dark illumination) were injected with 20 IU PMSG (Serotropin, Teikoku Hormone Mfg. Co., Tokyo) in 0.2 ml of saline subcutaneously (Group III). Females ovariectomized at 27 days of age were also given P M S G injection the next morning (Group II). Intact controls were injected with saline solution on the same day (Group I). All experimental and control animals were sacrificed 72-74 h after the injection. Electron microscopic observations were made on coronal sections from the middle part of the ARCN. Axodendritic and axosomatic synapses were counted in a field of 18,000 sq. # m in a single section of the identical level of the A R C N in each brain. The procedures involved in the preparation of these materials and counting synapses have been described in previous papers1, 6. The results are summarized in Fig. 1. The mean number of axodendritic synapses in 5 PMSG-treated females (Group III) was 1553 -4- 110 per field of 18,000
376 V) :3 :3 1500
0 ¢" 1000 UI O ¢/I
"~
500
0
E 100
C
(z)
OVX
~
(m)
(II) i U Axodt4nd~'itk:
Fig. 1. Numbers of axodendritic and axosomatic synapses per field of 18,000 sq./zm in the ARCN of PMSG-treated immature female rats (Groups II and HI) and non-treated controls (Group I). The vertical bars indicate S.E.M. values. For axodendritic synapses, Group I or II vs Group IH is significant (P < 0.001); Group I vs II, non-signihicant. For axosomatic synapses, non-signihicant among the three groups. sq./~m and significantly greater than that of 8 intact controls (Group [, 726 ± 86) or 5 PMSG-treated ovariectomized rats (Group II, 811 ± 58). There was no significant difference in the number of axodendritic synapses between the control and P M S G treated ovariectomized animals. As for the axosomatic synapses, the incidence was very low and there were no significant differences among the three groups (see Fig. 1). In the animals of G r o u p III, axon terminals were characterized by a large: number of spherical synaptic vesicles (about 50 nm in diameter) and thickening of the synaptic membranes. Most of them were almost identical with mature types of synapses seen in the A R C N of adult animals 1,6. Large granular vesicles (about 100 nrn in diameter) were found to coexist with spherical synaptic vesicles in the presynaptic endings, but the number of this type of synapse was smaller than that of the synapses containing only spherical synaptic vesicles in the axon terminals. In the control or PMSG-treated ovariectomized females, the mature type of synaptic structures were less frequent. Ovarian and uterine weights in the animals of G r o u p III were significantly larger than those of the control females (P < 0.001). Ovaries of the PMSG-treated females (Group l i d contained numerous fresh corpora lutea. At autopsy, no ova were found in the oviducts of the intact controls (Group I). However, all of the PMSG-treated females (Group III) ovulated.
377 The results of the present study clearly indicate that acceleration of synaptogenesis in the A R C N occurred concurrently with precocious ovulation in PMSGtreated female rats. It is highly probable that ovarian estrogen played a facilitatory role in the development of synaptic organization in the A R C N in these PMSG-treated females, because P M S G treatment failed to increase the number of synapses in the A R C N in PMSG-treated ovariectomized rats. Based on electrophysiological and biochemical studiesa,l°,11, la, Timiras and her associates have postulated that administration of estrogen to infantile rats results in precocious maturation of the brain. Toran-Allerand 12 reported that the proliferation of neuronal processes of the explants from newborn mouse hypothalamic tissues was markedly accelerated by the addition of estrogen to the culture medium. Furthermore, early postnatal injections of estrogen to female rats have been found to cause a marked increase in the number of axodendritic synapses in the ARCN1, 7. These results provide supportive evidence that estrogen stimulates afferent axonal growth, terminal arborization and dendritic differentiation in the prepubertal brain. The maturation of negative and positive feedback systems has been regarded as one of the factors contributing to the onset of puberty2, s. Since the M B H is considered to be one focus of the feedback action of sex steroids, the simultaneous increase of mature synapses in the A R C N with PMSG-induced precocious ovulation implies the establishment of new neural circuits in the MBH system, which may contribute to the maturation of the feedback mechanisms. This is consistent with our previous findings that a marked increase in the number of mature-type synaptic structures in the A R C N of female rats was observed at the peripubertal stage during the normal postnatal development 1,6. Several lines of evidence have suggested that the intra- and extrahypothalamic systems exert facilitatory or inhibitory influences on the MBH mechanisms involved in regulation of the onset of puberty 5. In the present study, however, it is still not known how the newly formed synapses in the A R C N following P M S G treatment correlate with these afferent systems and how the effects of the increase of synaptic structures in this nucleus reflects upon the triggering mechanisms of the onset of puberty. Further study is required to solve this point. The authors wish to express their thanks to Dr. T. Usui, Associate Professor of our department, for his valuable suggestions. This study was partly supported by grants from the Ministry of Education of Japan.
1 Arai, Y. and Matsumoto, A., Synapse formation of the hypothalamic arcuate nucleus during postnatal development in the female rat and its modification by neonatal estrogen treatment, Psychoneuroendocrinology, 2 (1977) in press. 2 Barraclough, C. A., Sex steroid regulation of reproductive neuroendocrine processes. In R. O. Greep (Ed.), Handbook of Physiology, Sect. 7, Vol. 2, Part 1, Amer. Physiol. Soc., Washington, D.C., 1973, pp. 29-56. 3 Curry, J. J. and Timiras, P. S., Development of evoked potentials in specific brain systems after neonatal administration of estradiol, Exp. Neurol., 34 (1972) 129-139. 4 Dyer, R. G., MacLeod, N. K. and Ellendorff, F., Electrophysiological evidence for sexual di-
378
5
6 7 8 9 10 11 12
13
morphism and synaptic convergence in the preoptic and anterior hypothalamic areas of the ra~. Proc. roy. Soc. B, 193 (1976) 421-440. Gorski, R. A., Extrahypothalamic influences on gonadotropin regulation. In M. M Grumbach, G. D. Grave and F. E. Mayer (Eds.), Control of the Onset of Puberty, Wiley, New York, 1974, pp. 182-207. Matsumoto, A. and Arai, Y., Developmental changes in synaptic formation in the hypothalamic arcuate nucleus of female rats, Cell Tiss. Res., 169 (1976) 143-156, Matsumoto, A. and Arai, Y., Effect of estrogen on early postnatal developmeni of synaptic formation in the hypothalamic arcuate nucleus of the female rats, Neurosci. Lett., 2 (1976) 7%82. Ramirez, V. D., Endocrinology of puberty. In R. O. Greep (Ed.), Handbook qfPhysiolog3. , Sect. 7, Vol. 2, Part 1, Amer. Physiol. Soc., Washingon, D.C., 1973, pp. 1-28. Sawyer, C. H., First Geoffrey Harris memorial lecture. Some recent developments in b r a i n pituitary-ovarian physiology, Neuroendocrinology, 17 (1975) 97-124. Terasawa, E. and Timiras, P. S., Electrophysiological study of the limbic system in the rat at onset of puberty, Amer. J. Physiol., 215 (t968) 1462-1467. Timiras, P. S., Developmental Physiology and Aging, Macmillan, New York, 1972. Toran-Allerand, C. D., Sex steroids and the development of the newborn mouse hypotba!amus and preoptic area in vitro: implications for sexual differentiation, Brain Research, 106 (1976) 407--412. Woolley, D. E. and Timiras, P. S., The gonad-brain relationship: effects of female sex hormones on electroshock convulsions in the rat, Endocrinology, 70 (1962) 196--209.
375
© Elsevier/North-Holland Biomedical Press
Precocious puberty and synaptogenesis in the hypothalamic arcuate nucleus in pregnant mare serum gonadotropin (PMSG) treated immature female rats
A. MATSUMOTO and Y. ARAI Department of Anatomy, Juntendo University School of Medicine, Hongo, Tokyo 113 (Japan)
(Accepted March 16th, 1977)
The onset of puberty is dependent on a complex process of neuroendocrinological events which involve the maturation of the hypothalamic-pituitary-gonadal axis. For the control of gonadotropin secretion, the medial basal hypothalamus (MBH) is thought to be a final key station between the hypothalamus and pituitary. This region is not only influenced directly by the feedback action of sex steroids, but also modulated by facilitatory and inhibitory neural inputs from the intra- and extrahypothalamic afferent systems 4,9. The functional maturation of the onset mechanism of puberty must be correlated with the growth rate of these neuronal components of the MBH and also with the establishment of the neural connections with the converging afferent systems. Recently, we have reported that the number of synapses in the arcuate nucleus (ARCN), one of the principal neuronal components of the MBH, increases remarkably up to puberty during the postnatal development in the female rats 6. In the present study, as one step to elucidate the nature of the changes in the neural mechanisms involved in initiation of puberty, semiquantitative measurements on the synaptic formation in the A R C N were performed in immature female rats treated with pregnant mare serum gonadotropin (PMSG). Twenty-eight-day-old Wistar female rats (housed under 14-10 h light-dark illumination) were injected with 20 IU PMSG (Serotropin, Teikoku Hormone Mfg. Co., Tokyo) in 0.2 ml of saline subcutaneously (Group III). Females ovariectomized at 27 days of age were also given P M S G injection the next morning (Group II). Intact controls were injected with saline solution on the same day (Group I). All experimental and control animals were sacrificed 72-74 h after the injection. Electron microscopic observations were made on coronal sections from the middle part of the ARCN. Axodendritic and axosomatic synapses were counted in a field of 18,000 sq. # m in a single section of the identical level of the A R C N in each brain. The procedures involved in the preparation of these materials and counting synapses have been described in previous papers1, 6. The results are summarized in Fig. 1. The mean number of axodendritic synapses in 5 PMSG-treated females (Group III) was 1553 -4- 110 per field of 18,000
376 V) :3 :3 1500
0 ¢" 1000 UI O ¢/I
"~
500
0
E 100
C
(z)
OVX
~
(m)
(II) i U Axodt4nd~'itk:
Fig. 1. Numbers of axodendritic and axosomatic synapses per field of 18,000 sq./zm in the ARCN of PMSG-treated immature female rats (Groups II and HI) and non-treated controls (Group I). The vertical bars indicate S.E.M. values. For axodendritic synapses, Group I or II vs Group IH is significant (P < 0.001); Group I vs II, non-signihicant. For axosomatic synapses, non-signihicant among the three groups. sq./~m and significantly greater than that of 8 intact controls (Group [, 726 ± 86) or 5 PMSG-treated ovariectomized rats (Group II, 811 ± 58). There was no significant difference in the number of axodendritic synapses between the control and P M S G treated ovariectomized animals. As for the axosomatic synapses, the incidence was very low and there were no significant differences among the three groups (see Fig. 1). In the animals of G r o u p III, axon terminals were characterized by a large: number of spherical synaptic vesicles (about 50 nm in diameter) and thickening of the synaptic membranes. Most of them were almost identical with mature types of synapses seen in the A R C N of adult animals 1,6. Large granular vesicles (about 100 nrn in diameter) were found to coexist with spherical synaptic vesicles in the presynaptic endings, but the number of this type of synapse was smaller than that of the synapses containing only spherical synaptic vesicles in the axon terminals. In the control or PMSG-treated ovariectomized females, the mature type of synaptic structures were less frequent. Ovarian and uterine weights in the animals of G r o u p III were significantly larger than those of the control females (P < 0.001). Ovaries of the PMSG-treated females (Group l i d contained numerous fresh corpora lutea. At autopsy, no ova were found in the oviducts of the intact controls (Group I). However, all of the PMSG-treated females (Group III) ovulated.
377 The results of the present study clearly indicate that acceleration of synaptogenesis in the A R C N occurred concurrently with precocious ovulation in PMSGtreated female rats. It is highly probable that ovarian estrogen played a facilitatory role in the development of synaptic organization in the A R C N in these PMSG-treated females, because P M S G treatment failed to increase the number of synapses in the A R C N in PMSG-treated ovariectomized rats. Based on electrophysiological and biochemical studiesa,l°,11, la, Timiras and her associates have postulated that administration of estrogen to infantile rats results in precocious maturation of the brain. Toran-Allerand 12 reported that the proliferation of neuronal processes of the explants from newborn mouse hypothalamic tissues was markedly accelerated by the addition of estrogen to the culture medium. Furthermore, early postnatal injections of estrogen to female rats have been found to cause a marked increase in the number of axodendritic synapses in the ARCN1, 7. These results provide supportive evidence that estrogen stimulates afferent axonal growth, terminal arborization and dendritic differentiation in the prepubertal brain. The maturation of negative and positive feedback systems has been regarded as one of the factors contributing to the onset of puberty2, s. Since the M B H is considered to be one focus of the feedback action of sex steroids, the simultaneous increase of mature synapses in the A R C N with PMSG-induced precocious ovulation implies the establishment of new neural circuits in the MBH system, which may contribute to the maturation of the feedback mechanisms. This is consistent with our previous findings that a marked increase in the number of mature-type synaptic structures in the A R C N of female rats was observed at the peripubertal stage during the normal postnatal development 1,6. Several lines of evidence have suggested that the intra- and extrahypothalamic systems exert facilitatory or inhibitory influences on the MBH mechanisms involved in regulation of the onset of puberty 5. In the present study, however, it is still not known how the newly formed synapses in the A R C N following P M S G treatment correlate with these afferent systems and how the effects of the increase of synaptic structures in this nucleus reflects upon the triggering mechanisms of the onset of puberty. Further study is required to solve this point. The authors wish to express their thanks to Dr. T. Usui, Associate Professor of our department, for his valuable suggestions. This study was partly supported by grants from the Ministry of Education of Japan.
1 Arai, Y. and Matsumoto, A., Synapse formation of the hypothalamic arcuate nucleus during postnatal development in the female rat and its modification by neonatal estrogen treatment, Psychoneuroendocrinology, 2 (1977) in press. 2 Barraclough, C. A., Sex steroid regulation of reproductive neuroendocrine processes. In R. O. Greep (Ed.), Handbook of Physiology, Sect. 7, Vol. 2, Part 1, Amer. Physiol. Soc., Washington, D.C., 1973, pp. 29-56. 3 Curry, J. J. and Timiras, P. S., Development of evoked potentials in specific brain systems after neonatal administration of estradiol, Exp. Neurol., 34 (1972) 129-139. 4 Dyer, R. G., MacLeod, N. K. and Ellendorff, F., Electrophysiological evidence for sexual di-
378
5
6 7 8 9 10 11 12
13
morphism and synaptic convergence in the preoptic and anterior hypothalamic areas of the ra~. Proc. roy. Soc. B, 193 (1976) 421-440. Gorski, R. A., Extrahypothalamic influences on gonadotropin regulation. In M. M Grumbach, G. D. Grave and F. E. Mayer (Eds.), Control of the Onset of Puberty, Wiley, New York, 1974, pp. 182-207. Matsumoto, A. and Arai, Y., Developmental changes in synaptic formation in the hypothalamic arcuate nucleus of female rats, Cell Tiss. Res., 169 (1976) 143-156, Matsumoto, A. and Arai, Y., Effect of estrogen on early postnatal developmeni of synaptic formation in the hypothalamic arcuate nucleus of the female rats, Neurosci. Lett., 2 (1976) 7%82. Ramirez, V. D., Endocrinology of puberty. In R. O. Greep (Ed.), Handbook qfPhysiolog3. , Sect. 7, Vol. 2, Part 1, Amer. Physiol. Soc., Washingon, D.C., 1973, pp. 1-28. Sawyer, C. H., First Geoffrey Harris memorial lecture. Some recent developments in b r a i n pituitary-ovarian physiology, Neuroendocrinology, 17 (1975) 97-124. Terasawa, E. and Timiras, P. S., Electrophysiological study of the limbic system in the rat at onset of puberty, Amer. J. Physiol., 215 (t968) 1462-1467. Timiras, P. S., Developmental Physiology and Aging, Macmillan, New York, 1972. Toran-Allerand, C. D., Sex steroids and the development of the newborn mouse hypotba!amus and preoptic area in vitro: implications for sexual differentiation, Brain Research, 106 (1976) 407--412. Woolley, D. E. and Timiras, P. S., The gonad-brain relationship: effects of female sex hormones on electroshock convulsions in the rat, Endocrinology, 70 (1962) 196--209.