0736-5748/84 $3.00+0.00 Pergamon Press Ltd. © 1984 ISDN
Int. J. Devl. Neuroscience, Vol. 2, No. 2, pp. 113-120, 1984. Printed in Great Britain.
D E V E L O P M E N T OF I M M U N O R E A C T I V E LHRH N E U R O N S IN THE FETAL RAT H Y P O T H A L A M U S SHIGEO DAIKOKU,* Yuzo MAKI, YOSHIHITO OKAMURA, YOSHIHIRO TSURUO and SETSUJI HISANO Department of Anatomy, School of Medicine, The University of Tokushima, Tokushima 770, Japan
(Accepted 16 September 1983) Abstract--The ontogenesis of immunoreactive (ir) LHRH neurons was investigated in rats applying the double-bridge PAP method of Vacca et al. (J. Histochem. Cytochem. 2,8, 297-307, 1980). Ir LHRH was first evident in the cell bodies confined in the ventromedial surface of the anterior part of the forebrain vesicles on day 16.5 of gestation. Only one or two cells, if any, were found in the brains examined; the cells are oval showing ir brown granules within the perikarya and also within the processes extended from both poles. On day 17.5 of gestation, a few ir cells appeared in the olfactory cortex, medial septum, medial preoptic area, diagonal band of Broca, and ventro-lateral surface of the anterior hypothalamus, and, with development, their numbers increased gradually. During the prenatal period, no ir cell exists in the arcuate nucleus or in the medial-basal hypothalamus. Beaded ir fibers appeared in the organum vasculosum of the lamina terminalis (OVLT) and in the external layer of the median eminence on 18.5 and 19.5 days of gestation, respectively. The present findings ascertain that the hypothalamic regulation of hypophysial gonadotrophic function exists during fetal period in rats.
Key words: Ontogenesis, LHRH neuron, Hypothalamus, Rat, Immunohistochemistry.
In the hypothalamus, over 10 kinds of neurons containing physiologically active peptides have been identified showing definitive localizations. Immunohistochemistry demonstrated many L H R H cell bodies confined to the septal, preoptic and rostral hypothalamic regions in rats treated with colchicine, 3'19'33 Nembutal,33 steroids and/or ovariectomy,22 reserpine 37 or dehydration26 but none or only few in non-treated adult animals. 1-3,16,20,21,23-25,27,28 In the developing rat brain, the location and the stage at which L H R H cell bodies are first differentiated have not yet been determined. 8'18'38 Recently, however, we have found many ir LHRH-containing cell bodies in normal newborn rats. 10 This may suggest that, within LHRH-containing perikarya, synthesis of the peptide exceeds its destruction during the early postnatal period. The reverse may occur in adults. This being so, L H R H might be stored in the perikarya when the metabolism remains low during the prenatal period. Thus we attempted again to determine the development of ir L H R H neurons in fetal rat hypothalamus by applying a more sensitive method for immunolabeling than the original PAP method, 34 since the ontogenesis of L H R H neuron system would be essential for understanding of the development of the hypothalamic-hypophysial-gonadal axis.
MATERIALS AND METHODS Male and female Sprague-Dawley rats were mated at night. On the following morning, females with sperm in the vagina were designated as being on day 0.5 of gestation, since mating usually occurred from 10 to 11 o'clock at night. Animals were killed between 10 and 11 o'clock in the morning. Fetuses aged from 12.5 to 21.5 days of gestation were obtained from mothers killed by cervical dislocation. A minimum of five fetuses of the same gestational age were taken from different mothers and examined. The fetuses were removed from the uterine cavity and decapitated. The heads were immersed in Bouin's fixative. The brain of the embryos aged 12.5-14.5 days was dissected out as a whole with the pituitary, and immersed for 24 h in the same fixative. In fetuses aged 15.5-17.5 days, the brain, including the telencephalon and the diencephalon, was dissected out with the pituitary and fixed in Bouin's fixative, and in fetuses aged 18.5-21.5 days the diencephalon was removed and fixed. The fixed brain was embedded in paraffin, and cut serially at 4-5 Ixm in the coronal plane. Every fifth section was stained with anti-LHRH serum. * Author to whom correspondence should be addressed.
113
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The immune property of the serum has been described previously, s The staining specificity was established by negative staining when the antibody had been absorbed with the antigen. For immunostaining, the double-bridge peroxidase-antiperoxidase (PAP) method of Vacca et al. 36 was applied. Prior to the immunostaining, deparaffinized sections were exposed to 3% hydrogen peroxide for 20 min at room temperature to eliminate the endogenous peroxidase activity. After this pretreatment, the sections were incubated with (1) rabbit anti-LHRH serum (1:500) containing cerebral extract and 0.1% bovine serum albumin overnight at 4°C, (2) goat anti-rabbit IgG (Daiichi Radioisotope Institute, Tokyo, 1 : 1000) for 2 h at 32°C, (3) PAP (1 : 100) for 1 h at 32°C, (4) goat anti-rabbit IgG (1:2000) for 30 min at 32°C, and (5) PAP (1:100) for 30 min at 32°C. The antigen-antibody complex was then labeled by incubating the tissues in Tris buffer containing diaminobenzidine and hydrogen peroxide for 20-40 min at 38°C. The sections were mounted and examined by light microscopy. RESULTS Ir L H R H cells are first determined on day 16.5 of gestation in the ventromedial surface of the anterior part of the forebrain vesicle (Fig. 1). Only one or two cells are present in each fetus, and only two of the five fetuses examined reveal these cells. The cells are oval and appear to extend one or two processes from the poles. The cytoplasm surrounding the immunonegative nucleus is thin but contains various numbers of ir brown granules. On day 17.5 of gestation, the cells increase slightly in number, compared with the previous stage, but do not exceed five in each animal (Fig. 2). The cells are found in three of the six fetuses examined. The cell bodies are oval and the cytoplasm includes varying numbers of ir granules. The cytoplasmic processes prominently extend showing ir granules. The cells are confined within the olfactory cortex including olfactory tubercle, medial septum, medial preoptic area, diagonal band of Broca and lateral hypothalamic area. The cells are noticeably localized in the external surface area of the regions mentioned above. On day 18.5 of gestation, the cells further increase in number, appearing in three of the five fetuses examined (Fig. 3). The cell bodies are confined in the same regions as described for the previous stage, and the fibers appear in the OVLT but not in the external layer of the median eminence. On day 19.5 of gestation, the medial septum shows many ir cell bodies and several long beaded ir fibers running dorso-ventraUy (Fig. 4). In the diagonal band of Broca, medial preoptic area, olfactory tubercle, and the lateral hypothalamic area, the cell bodies are still few. As described in the previous paper, 8 a few ir fibers appear to terminate in the external layer of the median eminence. On day 20.5 of gestation, the cells are present in all animals examined. The cell bodies localize in the olfactory tubercle, medial septum, diagonal band of Broca, and medial preoptic area, and very sporadically in the anterior hypothalamic nucleus, lateral hypothalamic area and pericommissural area (Fig. 5). The cells increase gradually in number in these regions with development; this continues after birth.l° During the prenatal period examined, we cannot determine the ir cell body in the arcuate nucleus or in the medial-basal part of the hypothalamus. DISCUSSION This work is the first demonstrating the existence of ir LHRH-containing cell bodies in the fetal rat brain. Inasmuch as ir L H R H cell bodies are only few or lacking in the hypothalamus of normal adult rats, but become evident in the septal, preoptic, and rostral hypothalamic regions in adult animals treated with colchicine, 3'19'33 dehydration, 26 or estrogen and/or ovariectomy, 22 it was anticipated that such treatments would augment the intracellular storage of the peptide. Several authors used thick, vibratome sections to demonstrate ir L H R H perikarya in the medial preoptic area, diagonal band of Broca, septal nucleus, anterior hypothalamic area, supraoptic commissure, and olfactory tubercle in normal or colchicine-treated rats. 4,17-42 In contrast, normal newborn rats have consistently demonstrated many ir cell bodies within the regions mentioned above. 9 Estrogenized neonatal rats also showed ir cell bodies in the septum, and in the caudal and rostral preoptic areas.12 As the existence of the ir cell bodies was verified during the prenatal period, the
Fig. 1. A n ir L H R H neuron (arrow) in the ventromedial surface of the anterior part of the forebrain vesicle of a 16.5-day-old fetus (x50). The neuron is shown on the right side with a large magnification ( x 1750). lr particles are evident within the perikaryon.
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Fig. 2. lr L H R H n e u r o n s (arrows) in the anterior part of the brain of a 17.5-day-old fetus ( x 51)). The brain is cut at the level of the anterior part of the forebrain vesicle (a), of the septum (b), of the preoptic area (c), and of the optic chiasma (d). Each n e u r o n is shown on the right side with a large magnification ( × 17511).
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Fig. 3. Ir L H R H neurons (1,2) confined in the dorsal (a) and ventral (b) portions of the septum of an 18.5day-old fetus (x50). The neurons (1, 2) are shown in the middle ( x 1750).
Fig. 4. lr L H R H neurons (arrows, 1, 2) and fiber (3) in the septum of a 19.5-day-old fetus (×50~). The numbered neurons and beaded fiber are shown on the right side (× 1750). CA: commissura anterior.
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Fig. 5. Ir L H R H neurons (arrows, 2, 3) and fiber (1) in the septum of a 21.5-day-old fetus ( × 50). Beaded fiber (1) and numbered neurons (2, 3) are shown on the right side (× 1750).
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Ontogenesis of ir LHRH neurons
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idea that the hypothalamic regulation of the hypophysial-gonadal system is present during fetal period in rats 7 was then substantiated. The usage of the double-bridge PAP method of Vacca et al. 36 allowed us to demonstrate the peptide within the fetal perikarya in rats. Indeed, we were not able previously8"18'38 to determine ir L H R H cell bodies in the fetal rat hypothalamus by the original PAP method of Sternberger et al. 34 The fetal perikarya probably store the peptide after day 16.5 of gestation but the amount is so low that it is indicated as negative with the original PAP method. An immunoassay study determined L H R H in the rat hypothalamus from day 15 of gestation, 5 which corresponds to our day 16.5. Of interest is the location where L H R H cell bodies first appear. The cell bodies were found in the ventromedial surface of the anterior part of the forebrain vesicle, as has been described for fetal guinea pigs. 29 The cortex of the forebrain vesicle consists of neurons which migrated from the neuroepithelium of the ventricle; L H R H appears within the perikarya of the migrated neurons. In sequential development, ir L H R H cell bodies appeared within the peripheral brain areas, such as the septum, olfactory tubercle, diagonal band of Broca, or lateral hypothalamic area. In contrast, ACTH and its related peptides differentiate in the neuroepithelial cells lining the base of the third ventricle on 12.5 or 13.5 days of gestation in rats, 11"3°and ACTH- containing cells migrate thereafter to the arcuate nucleus. Similarly, somatostatin also appears in the neuroepithelial cells covering the third ventricle on day 12.5 of gestation and in the neuroepithelial cells lining the anterolateral wails of the third ventricle even at newborn stage (Ohtsuka et al., unpublished data). The factor(s) which induce such chronological and topographical discriminations in development of the peptides remain to be elucidated. Evidence is available to show the developmental chronology of gonadotrophs in rats; the first LH gonadotrophs appear on day 17.5 of gestation. 6'9'31'32'35'4° In rats, the portal vessels are ready on day 16.5 of gestation to transport neuropeptides from the ventral surface of the median eminence to the adenohypophysis.9 As shown in the present study, L H R H perikarya were observed in the rostral hypothalamus on day 16.5 of gestation. Thus, this could suggest that LHRH induces the development of gonadotrophs as mentioned by several authors. 5'13-15 However, this concept remains to be verified since ir L H R H fiber terminals could not be detected on the ventral surface of the median eminence before the development of gonadotrophs even at the level of the electron microscopic immunohistochemistry;18 LHRH discharged from the fiber terminals at the ventral surface of the median eminence is transported to the adenohypophysis. Consistent with the previous findings,s'18 the present study shows the ir fibers in the ventral surface of the median eminence on day 19.5 of gestation. This confirms our previous conclusion that gonadotrophs develop without hypothalamic LHRH. 39 Recently, Chatelain et al. 6 have proposed that the development of ACTH, MSH, GH, LPH and prolactin cells in the adenohypophysis does not require the presence of the fetal hypothalamus. Various authors have mentioned that LHRH is not essential for differentiation of the first ir LH cells. 41 Acknowledgements--We are grateful to Mr. I. Shimada and Mr. T. Fukunishi for preparation of tissue sections and photographs. This work was supported by a grant from the Ministry of Education, Science and Culture of Japan.
REFERENCES 1. Ajika K. (1979) Simultaneous localization of LHRH and catecholamines in rat hypothalamus. J. Anat. 128, 331-347. 2. Baker B. L., Dermody W. C. & Reel J. R. (1975) Distribution of gonadotropin-releasing hormone in the rat brain as observed with immunocytochemistry. Endocrinology 97, 125-135. 3. Barry J., Dubois M. P. & Poulain P. (1973) LRF producing cells of the mammalian hypothalamus. A fluorescent antibody study. Z. Zellforsch. 146, 351-366. 4. Bennett-Clarke C. & JosephS. A. (1982) ImmunocytochemicaldistributionofLHRHneurons and processes in therat: hypothalamic and extra-hypothalamic locations. Cell Tiss. Res. 221,493-504. 5. Chiappa S. A. & Fink G. (1977) Releasing factor and hormonal changes in the hypothalamic-pituitary-gonadotrophin and -adrenocorticotrophin systems before and after birth and puberty in male, female and androgenized female rats. J. Endocr. 72, 211-224. 6. Chatelain A., Dupouy J. P. & Dubois M. P. (1979) Ontogenesis of cells producing polypeptide hormones (ACTH, MSH, LPH, GH, prolactin) in the fetal hypophysis of the rat: influence of the hypothalamus. Cell Tiss. Res. 196, 409427. 7. Daikoku S., Watanabe Y. G. & Kinutani M. (1976) On the influence of the hypothalamic-hypophysial system on the developing Leydig cells. Anat. Embryol. 149, 209-224.
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8. Daikoku S., Kawano H. & Matsumura H. (1978) In vivo and in vitro studies on the appearance of LHRH neurons in the hypothalamus of perinatal rats. Cell Tiss. Res. 194, 433-445. 9. Daikoku S., Kawano H., Abe K. & Yoshinaga K. (1981) Topographical appearance of adenohypophysial cells with special reference to the development of the portal system. Arch. histol, jap. 44, 103-116. 10. Daikoku S., Hisano S. & Maki Y. (1982) Immunohistochemical demonstration of LHRH-neurons in young rat hypothalamus: light and electron microscopy. Arch. histol, jap. 45, 69-82. 11. Daikoku S., Chikamori M., Adachi T., Okamura Y., Nishiyama T. & Tsuruo Y. (1983) Ontogensis of hypothalamic immunoreactive ACTH cells in vivo and in vitro: role of Rathke's pouch. Devl Biol. 97 (in press). 12. Elkind-Hirsch K.. King J. C., Gerall A. A. & Arimura A. A. (1981) The luteinizing hormone-releasing hormone (LHRH) system in normal and estrogenized neonatal rats. Brain Res. Bull. 7,645-654. 13. Fink G. & Smith G. C. (1971) Ultrastructural features of the developing hypothalamt~-hypophysial axis in the rat. A correlative study. Z. Zellforsch. 119, 208-226. 14. Gross D. S. & Baker B. L. (1979) Developmental correlation between hypothalamic gonadotrophin-releasing hormone and hypophysiai luteinizing hormone. Am. J. Anat. 154, 1-10. 15. Hal~isz B., Kosaras B. & Lengvfiri I. (1972) Ontogenesis of the neurovascular link between the hypothalamus and the anterior pituitary in the rat. In Brain-Endocrine Interaction (eds Knigge, Scott & Weindl), pp. 27-34. Karger, Basel. 16. Hoffman G. E., Melnyk V., Hayes T., Bennett-Clarke C. & Fowler E. (1978) Immunocytology of LHRH neurons. In Brain-Endocrine Interaction (eds Scott, Kozlowski & Weindl), pp. 67-82. Karger, Basel. 17. Joseph S. A., Piekut D. T. & Knigge K. M. (1981) lmmunocytochemical localization of luteinizing hormone-releasing hormone (LHRH) in vibratome-sectioned brain. J. Histochem. Cytochem. 29, 247-254. 18. Kawano H., Watanabe Y. G. & Daikoku S. (1980) Light and electron microscopic observation on the appearance of immunoreactive LHRH in perinatal rat hypothalamus. Cell Tiss. Res. 213, 465474. 19. Kawano H. & Daikoku S. (1981) Immunohistochemical demonstration of LHRH neurons and their pathways in the rat hypothalamus. Neuroendocrinology 32, 179-186. 20. King J. C., Parsons J. A., Erlandsen S. L. & Williams T. H. (1974) Luteinizing hormone-releasing hormone (LH-RH) pathway of the rat hypothalamus revealed by the unlabeled antibody peroxidase-antiperoxidase method. Cell Tiss. Res. 153, 211-217. 21. King J. C., Elkind K. E., Gerall A. A. & Milar R. P. (1978) Investigation of the LH-RH system in the normal and neonatally steroid-treated male and female rats. In Brain-Endocrine Interaction (eds Scott, Kozlowski & Weindl), pp. 97-107. Karger, Basel. 22. King J. C., Tobet S. A., Snavely F. L. & Arimura A. A. (1982) LHRH immunopositivc cells and their projections to the median eminence and organum vasculosum of the lamina terminalis. J. comp. Neurol. 209,287-300. 23. Kordon C., Kerdelhu6 B., Pattou E. & Jutisz M. (1974) lmmunocytochemical localization of LHRH in axons and nerve terminals of the rat median eminence. Proc. Soc. exp. Biol. Med. 147, 122-127. 24. Kozlowski G. P. & Hostetter G. (1978) Cellular and subcellular localization and behavioural effects of gonadotropinreleasing hormone (Gn-RH) in the rat. In Brain-Endocrine Interaction (eds Scott, Kozlowski & Weindl), pp. 138-153. Karger, Basel. 25. Kozlowski G. P., Chu L., Hostetter G. & Kerdelhu6 B. (1980) Cellular characteristics of immunolabeled luteinizing hormone releasing hormone (LHRH) neurons. Peptides 1, 37-46. 26. Krisch B. (1978) The distribution of LHRH in the hypothalamus of the thirsting rat. A light and electron microscopic immunocytochemical study. Cell Tiss. Res. 186, 135-148. 27. Leonardelli J., Barry J. & Dubois M. P. (1973) Mise en 6vidence par immunofluorescence d'un constituant immunologiquement apparent6 au LH-RF dans l'hypothalamus et l'6minence mediane chez les Mammif6res. C. r. Acad. Sci., Paris 276, 2043-2046. 28. Merchenthaler I., Kov~icsG., Lov~sz G. & Sdt~il6 G. (1980) The preopticoinfundibular LH-RH tract of the rat. Brain Res. 198, 63-74. 29. SchwanzeI-Fukuda M., Robinson J. A. & Silverman A. J. (1981) The fetal development of the luteinizing hormoncreleasing hormone (LHRH) neuronal system of the guinea pig brain. Brain Res. Bull. 7,293-315. 30. Schwartzberg D. G. & Nakane P. K. (1982) Ontogenesis of adrenocorticotropin-related peptide determinants in the hypothalamus and pituitary gland of the rat. Endocrinology 110, 855-864. 31. S6tfil6 G. & Nakane P. K. (1972) Studies on the functional differentiation of cells in fetal anterior pituitary glands of rats with peroxidase-labeled antibody method. Anat. Rec. 172, 403. 32. $6t~16 G. & Nakane P. K. (1976) Functional differentiation of fetal anterior pituitary cells in the rat. Endocr. Exp. 10, 155-166. 33. Sdtfil6 G., Vigh S., Schally A. V., Arimura A. & Flerk6 B. (1976) lmmunohistological study of the origin of LH-RHcontaining nerve fibers of the rat hypothalamus. Brain Res. 103, 597--602. 34. Sternberger L. A., Hardy P. H. Jr., Cuculis J. J. & Meyer H. G. (1970) The unlabeled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidaseantihorseradish peroxidase) and its use in identification of spirochetes. J. Histochem. Cytochem. 18, 315-333. 35. Tougard C., Picart R. & Tixier-Vidal A. (1977) Cytogenesisofimmunoreactivegonadotropiccellsinthefetalrat pituitary at light and electron microscope levels. Devl Biol. 58, 148--163. 36. Vacca L. L., Abraham S. J. & Naftchi N. E. (1980) A modified peroxidase-antiperoxidase procedure for improved localization of tissue antigens: localization of substance P in rat spinal cord. J. Histochem. Cytochem. 28, 297-307. 37. Vigh S., S6t~il6 G., Schally A. V., Arimura A. & Flerk6 B. (1978) LH-RH-containing nerve fibers in the brain of rats treated with sulpiride or reserpine. Brain Res. 152,401-405. 38. Watanabe K. (1980) Regional differences in the development ofluteinizinghormone-releasinghormonenerve endings in the rat. Endocrinology 106, 139-144. 39. Watanabe Y. G. & Daikoku S. (1976) Immunohistochemical study on adenohypophysial primordia in organ culturc. Cell Tiss. Res. 166, 407-412. 40. Watanabe Y. G. & Daikoku S. (1979) An immunohistochemical study on the cytogenesis of adenohypophysial cells in fetal rats, Devl Biol. 68, 557-567. 41. Wenger T. & Nemesk6ri A. (1979) On the ontogenesis of the hypothalamic luteinizing hormone-releasing hormone (LH-RH) in the male and female rat. Neuroendocr. Lett. 1,29-34. 42. Witkin J. W., Paden C. M. & Silverman A.-J. (1982) The luteinizing hormone-releasing hormone (LHRH) systems in the rat brain. Neuroendocrinology 35,429-438.
Int. J. Devl. Neuroscience, Vol. 2, No. 2, pp. 113-120, 1984. Printed in Great Britain.
D E V E L O P M E N T OF I M M U N O R E A C T I V E LHRH N E U R O N S IN THE FETAL RAT H Y P O T H A L A M U S SHIGEO DAIKOKU,* Yuzo MAKI, YOSHIHITO OKAMURA, YOSHIHIRO TSURUO and SETSUJI HISANO Department of Anatomy, School of Medicine, The University of Tokushima, Tokushima 770, Japan
(Accepted 16 September 1983) Abstract--The ontogenesis of immunoreactive (ir) LHRH neurons was investigated in rats applying the double-bridge PAP method of Vacca et al. (J. Histochem. Cytochem. 2,8, 297-307, 1980). Ir LHRH was first evident in the cell bodies confined in the ventromedial surface of the anterior part of the forebrain vesicles on day 16.5 of gestation. Only one or two cells, if any, were found in the brains examined; the cells are oval showing ir brown granules within the perikarya and also within the processes extended from both poles. On day 17.5 of gestation, a few ir cells appeared in the olfactory cortex, medial septum, medial preoptic area, diagonal band of Broca, and ventro-lateral surface of the anterior hypothalamus, and, with development, their numbers increased gradually. During the prenatal period, no ir cell exists in the arcuate nucleus or in the medial-basal hypothalamus. Beaded ir fibers appeared in the organum vasculosum of the lamina terminalis (OVLT) and in the external layer of the median eminence on 18.5 and 19.5 days of gestation, respectively. The present findings ascertain that the hypothalamic regulation of hypophysial gonadotrophic function exists during fetal period in rats.
Key words: Ontogenesis, LHRH neuron, Hypothalamus, Rat, Immunohistochemistry.
In the hypothalamus, over 10 kinds of neurons containing physiologically active peptides have been identified showing definitive localizations. Immunohistochemistry demonstrated many L H R H cell bodies confined to the septal, preoptic and rostral hypothalamic regions in rats treated with colchicine, 3'19'33 Nembutal,33 steroids and/or ovariectomy,22 reserpine 37 or dehydration26 but none or only few in non-treated adult animals. 1-3,16,20,21,23-25,27,28 In the developing rat brain, the location and the stage at which L H R H cell bodies are first differentiated have not yet been determined. 8'18'38 Recently, however, we have found many ir LHRH-containing cell bodies in normal newborn rats. 10 This may suggest that, within LHRH-containing perikarya, synthesis of the peptide exceeds its destruction during the early postnatal period. The reverse may occur in adults. This being so, L H R H might be stored in the perikarya when the metabolism remains low during the prenatal period. Thus we attempted again to determine the development of ir L H R H neurons in fetal rat hypothalamus by applying a more sensitive method for immunolabeling than the original PAP method, 34 since the ontogenesis of L H R H neuron system would be essential for understanding of the development of the hypothalamic-hypophysial-gonadal axis.
MATERIALS AND METHODS Male and female Sprague-Dawley rats were mated at night. On the following morning, females with sperm in the vagina were designated as being on day 0.5 of gestation, since mating usually occurred from 10 to 11 o'clock at night. Animals were killed between 10 and 11 o'clock in the morning. Fetuses aged from 12.5 to 21.5 days of gestation were obtained from mothers killed by cervical dislocation. A minimum of five fetuses of the same gestational age were taken from different mothers and examined. The fetuses were removed from the uterine cavity and decapitated. The heads were immersed in Bouin's fixative. The brain of the embryos aged 12.5-14.5 days was dissected out as a whole with the pituitary, and immersed for 24 h in the same fixative. In fetuses aged 15.5-17.5 days, the brain, including the telencephalon and the diencephalon, was dissected out with the pituitary and fixed in Bouin's fixative, and in fetuses aged 18.5-21.5 days the diencephalon was removed and fixed. The fixed brain was embedded in paraffin, and cut serially at 4-5 Ixm in the coronal plane. Every fifth section was stained with anti-LHRH serum. * Author to whom correspondence should be addressed.
113
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The immune property of the serum has been described previously, s The staining specificity was established by negative staining when the antibody had been absorbed with the antigen. For immunostaining, the double-bridge peroxidase-antiperoxidase (PAP) method of Vacca et al. 36 was applied. Prior to the immunostaining, deparaffinized sections were exposed to 3% hydrogen peroxide for 20 min at room temperature to eliminate the endogenous peroxidase activity. After this pretreatment, the sections were incubated with (1) rabbit anti-LHRH serum (1:500) containing cerebral extract and 0.1% bovine serum albumin overnight at 4°C, (2) goat anti-rabbit IgG (Daiichi Radioisotope Institute, Tokyo, 1 : 1000) for 2 h at 32°C, (3) PAP (1 : 100) for 1 h at 32°C, (4) goat anti-rabbit IgG (1:2000) for 30 min at 32°C, and (5) PAP (1:100) for 30 min at 32°C. The antigen-antibody complex was then labeled by incubating the tissues in Tris buffer containing diaminobenzidine and hydrogen peroxide for 20-40 min at 38°C. The sections were mounted and examined by light microscopy. RESULTS Ir L H R H cells are first determined on day 16.5 of gestation in the ventromedial surface of the anterior part of the forebrain vesicle (Fig. 1). Only one or two cells are present in each fetus, and only two of the five fetuses examined reveal these cells. The cells are oval and appear to extend one or two processes from the poles. The cytoplasm surrounding the immunonegative nucleus is thin but contains various numbers of ir brown granules. On day 17.5 of gestation, the cells increase slightly in number, compared with the previous stage, but do not exceed five in each animal (Fig. 2). The cells are found in three of the six fetuses examined. The cell bodies are oval and the cytoplasm includes varying numbers of ir granules. The cytoplasmic processes prominently extend showing ir granules. The cells are confined within the olfactory cortex including olfactory tubercle, medial septum, medial preoptic area, diagonal band of Broca and lateral hypothalamic area. The cells are noticeably localized in the external surface area of the regions mentioned above. On day 18.5 of gestation, the cells further increase in number, appearing in three of the five fetuses examined (Fig. 3). The cell bodies are confined in the same regions as described for the previous stage, and the fibers appear in the OVLT but not in the external layer of the median eminence. On day 19.5 of gestation, the medial septum shows many ir cell bodies and several long beaded ir fibers running dorso-ventraUy (Fig. 4). In the diagonal band of Broca, medial preoptic area, olfactory tubercle, and the lateral hypothalamic area, the cell bodies are still few. As described in the previous paper, 8 a few ir fibers appear to terminate in the external layer of the median eminence. On day 20.5 of gestation, the cells are present in all animals examined. The cell bodies localize in the olfactory tubercle, medial septum, diagonal band of Broca, and medial preoptic area, and very sporadically in the anterior hypothalamic nucleus, lateral hypothalamic area and pericommissural area (Fig. 5). The cells increase gradually in number in these regions with development; this continues after birth.l° During the prenatal period examined, we cannot determine the ir cell body in the arcuate nucleus or in the medial-basal part of the hypothalamus. DISCUSSION This work is the first demonstrating the existence of ir LHRH-containing cell bodies in the fetal rat brain. Inasmuch as ir L H R H cell bodies are only few or lacking in the hypothalamus of normal adult rats, but become evident in the septal, preoptic, and rostral hypothalamic regions in adult animals treated with colchicine, 3'19'33 dehydration, 26 or estrogen and/or ovariectomy, 22 it was anticipated that such treatments would augment the intracellular storage of the peptide. Several authors used thick, vibratome sections to demonstrate ir L H R H perikarya in the medial preoptic area, diagonal band of Broca, septal nucleus, anterior hypothalamic area, supraoptic commissure, and olfactory tubercle in normal or colchicine-treated rats. 4,17-42 In contrast, normal newborn rats have consistently demonstrated many ir cell bodies within the regions mentioned above. 9 Estrogenized neonatal rats also showed ir cell bodies in the septum, and in the caudal and rostral preoptic areas.12 As the existence of the ir cell bodies was verified during the prenatal period, the
Fig. 1. A n ir L H R H neuron (arrow) in the ventromedial surface of the anterior part of the forebrain vesicle of a 16.5-day-old fetus (x50). The neuron is shown on the right side with a large magnification ( x 1750). lr particles are evident within the perikaryon.
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Fig. 2. lr L H R H n e u r o n s (arrows) in the anterior part of the brain of a 17.5-day-old fetus ( x 51)). The brain is cut at the level of the anterior part of the forebrain vesicle (a), of the septum (b), of the preoptic area (c), and of the optic chiasma (d). Each n e u r o n is shown on the right side with a large magnification ( × 17511).
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Fig. 3. Ir L H R H neurons (1,2) confined in the dorsal (a) and ventral (b) portions of the septum of an 18.5day-old fetus (x50). The neurons (1, 2) are shown in the middle ( x 1750).
Fig. 4. lr L H R H neurons (arrows, 1, 2) and fiber (3) in the septum of a 19.5-day-old fetus (×50~). The numbered neurons and beaded fiber are shown on the right side (× 1750). CA: commissura anterior.
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Fig. 5. Ir L H R H neurons (arrows, 2, 3) and fiber (1) in the septum of a 21.5-day-old fetus ( × 50). Beaded fiber (1) and numbered neurons (2, 3) are shown on the right side (× 1750).
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Ontogenesis of ir LHRH neurons
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idea that the hypothalamic regulation of the hypophysial-gonadal system is present during fetal period in rats 7 was then substantiated. The usage of the double-bridge PAP method of Vacca et al. 36 allowed us to demonstrate the peptide within the fetal perikarya in rats. Indeed, we were not able previously8"18'38 to determine ir L H R H cell bodies in the fetal rat hypothalamus by the original PAP method of Sternberger et al. 34 The fetal perikarya probably store the peptide after day 16.5 of gestation but the amount is so low that it is indicated as negative with the original PAP method. An immunoassay study determined L H R H in the rat hypothalamus from day 15 of gestation, 5 which corresponds to our day 16.5. Of interest is the location where L H R H cell bodies first appear. The cell bodies were found in the ventromedial surface of the anterior part of the forebrain vesicle, as has been described for fetal guinea pigs. 29 The cortex of the forebrain vesicle consists of neurons which migrated from the neuroepithelium of the ventricle; L H R H appears within the perikarya of the migrated neurons. In sequential development, ir L H R H cell bodies appeared within the peripheral brain areas, such as the septum, olfactory tubercle, diagonal band of Broca, or lateral hypothalamic area. In contrast, ACTH and its related peptides differentiate in the neuroepithelial cells lining the base of the third ventricle on 12.5 or 13.5 days of gestation in rats, 11"3°and ACTH- containing cells migrate thereafter to the arcuate nucleus. Similarly, somatostatin also appears in the neuroepithelial cells covering the third ventricle on day 12.5 of gestation and in the neuroepithelial cells lining the anterolateral wails of the third ventricle even at newborn stage (Ohtsuka et al., unpublished data). The factor(s) which induce such chronological and topographical discriminations in development of the peptides remain to be elucidated. Evidence is available to show the developmental chronology of gonadotrophs in rats; the first LH gonadotrophs appear on day 17.5 of gestation. 6'9'31'32'35'4° In rats, the portal vessels are ready on day 16.5 of gestation to transport neuropeptides from the ventral surface of the median eminence to the adenohypophysis.9 As shown in the present study, L H R H perikarya were observed in the rostral hypothalamus on day 16.5 of gestation. Thus, this could suggest that LHRH induces the development of gonadotrophs as mentioned by several authors. 5'13-15 However, this concept remains to be verified since ir L H R H fiber terminals could not be detected on the ventral surface of the median eminence before the development of gonadotrophs even at the level of the electron microscopic immunohistochemistry;18 LHRH discharged from the fiber terminals at the ventral surface of the median eminence is transported to the adenohypophysis. Consistent with the previous findings,s'18 the present study shows the ir fibers in the ventral surface of the median eminence on day 19.5 of gestation. This confirms our previous conclusion that gonadotrophs develop without hypothalamic LHRH. 39 Recently, Chatelain et al. 6 have proposed that the development of ACTH, MSH, GH, LPH and prolactin cells in the adenohypophysis does not require the presence of the fetal hypothalamus. Various authors have mentioned that LHRH is not essential for differentiation of the first ir LH cells. 41 Acknowledgements--We are grateful to Mr. I. Shimada and Mr. T. Fukunishi for preparation of tissue sections and photographs. This work was supported by a grant from the Ministry of Education, Science and Culture of Japan.
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