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Effect of Gonadotropic Hormones on the Rat Pineal Gland. A Fluorescence Histochemical and Biochemical Study*. A. R. Smith, P. Pevet, L. van de Kar, and R. v.
Journal of Neural Transmission 36, 217--226 (1975) @ by Springer-Verlag 1975

Effect of Gonadotropic Hormones on the Rat Pineal Gland A Fluorescence Histochemical and Biochemical Study* A. R. S m i t h , P. P e v e t , L. v a n d e Kar, and R. v. O o s t e r o m Netherlands Central Institute for Brain Research, Amsterdam, The Netherlands With 4 Figures Received March 21, 1975

Research, though toilsome, is easy; Imagination, though delightful, is difficult. Summary Changes in the number of autofluorescent and serotonin-containing pinealocytes have been studied atter gonadotropic hormone administration using fluorescence histochemistry. Moreover, the 5-HT levels in the pineal gland were determined biochemically under the same experimental conditions. The results suggest that pineal 5-HT levels are not influenced by increased gonadotropin levels, while the amount of autofluorescent pinealocytes increases affer gonadotropin injections. It has been postulated that differences in androgenic hormone levels could not cause the changes in the number of autofluorescent pinealocytes. The reaction of the pineal gland to increased gonadotropin levels seemed important in view of the regulatory function of the epiphysis on the hypothalamo-hypophyseo-gonadal axis.

Introduction

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It has earlier been shown that the pineal gland probably exerts antigonadotropic activity via the hypothalamo-hypophyseo-

* This paper is dedicated to Prof. Dr. J. Ari~ns Kappers, who has been our teacher for several years.

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gonadal system (AriFns Kappers et al., 1974). Several investigators examined the influence of gonadectomy on the pineal gland in different respect (AriFns Kappers et al., 1974; Satodate et al., 1970). It has been demonstrated that gonadectomy causes an increase in gonadotropin (Gay and Midgley, 1964; Yamato et al., 1970) blood levels and that the ultrastructure of the gland (Satodate et al., 1970), as well as pineal substrate levels (Pevet and Smith), are also influenced by this operation. It has been assumed that the changes mentioned occurring in the gland a~er gonadectomy are caused by increased hormone levels (viz., of FSH and LH). In previous investigations (AriFns Kappers et aI., 1974; Smith and Ari~ns Kappers, 1973) we observed the presence of a yellow autofluorescent protein in the pineal gland of rat and rabbit also occurring in the parvocelIular, the arcuate and ventromedial hypothalamic nuclei. Evidence has been presented that the amount of this substance changes during castration, in the pineal gland as well as in the hypothalamus (Ari~ns Kappers et al., 1974; Smith and Ari(ns Kappers, 1973; Smith, 1974; Smith and Ari(ns Kappers, 1975). It is, therefore, of interest to study the direct effect of increased gonadotropin levels, produced by injections of HCG, PMSG and FSH, on the autofluorescent material and on the 5-HT levels in the pineal gland. In this paper we report investigations on the changes of the autofluorescent material and the 5-HT levels during acute and longterm treatment with gonadotropins. The present findings are discussed in respect to the previous ones.

Materials and Methods

For this study 166 male Wistar rats (TNO, Zeist), weighing approximately 200 g, were used. Eighty-one animals were used for the 5-HT determination and 86 for the fluorescence microscopical investigation. All rats were kept in individual cages at 25 ~ and constant humidity. They received tap water and rat pellets ad Iibiturn. All animals were kept on a 12 hours light/12 hours darkness artificial lighting scheme, light being turned on at 7 a.m. The animals were killed between 9 and 10 a.m. by decapitation to prevent the influence of the circadian rhythm of the compounds concerned. The pineal glands were removed within 3 min aRer decapitation. For the fluorescence microscopical study they were frozen in Freon-12 cooled to -180 ~ by liquid nitrogen, while for the 5-HT determination they were put in ice-cold perchloric acid.

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Administration of PMSG, HCG, and Bovine-FSH; Fluorescence Microscopy 1. Of 55 rats 33 were i.m. injected with 25 I.U. PMSG (Gestyl, Organon), whereas 22 animals were used as controls receiving 0.1 ml of 0.9 % NaC1. The animals injected with PMSG were sacrificed in groups of 3 rats on day 1 up till day 7, on day 14, day 21 and day 28. The control rats were killed in groups of two rats on day 1 up till day 7, on day 14, day 21 and day 28, respectively. 2. Of 16 rats 12 were used for injections with gonadotropins during 4 days. Three groups of 4 rats each were injected with 25 I.U. PMSG, H C G (Pregnyl, Organon) and FSH-Bovine, respectively. The control animals were injected with the solvent only (0.1 ml of 0.9 ~ NaC1). 3. Of 15 rats 10 were used for the injection with gonadotropins during 1 week. Five animals received 25 I.U. PMSG i.m. while 5 received 25 I.U. H C G i.m. The remaining 5 rats served as controls and were injected with the solvent only.

Fluorescence Histochemistry A modification of the classic histochemical fluorescence method (Falck et al., 1962), as described by Heene (1968) and Smith et aI. (1972), was used. This method allows the alternating comparison of frozen-dried sections with formaldehyde-treated sections of the same piece of hypothalamic tissue. Unfixed, unthawed, unstained, serial, transverse pineal sections (15 #) were cut on a cryostat ( - 1 8 ~ Alternating sections were placed in two vacuum tubes, one containing paraformaldehyde powder equilibrated in air of about 50 ~ humidity (Merck), the other devoid of this powder. The sections in both tubes were frozen-dried at - 3 0 ~ for 20 rain. During this period pressure was decreased to 0.01 mm Hg. The tubes containing the sections were then brought to room temperature within 15 rain in vacuo. APcer freeze-drying, the tubes (still under vacuum), were placed for 1 hour in an oven ( + 6 0 ~ (Corrodi and Jonsson, 1965; Hamberger, 1967; Hamberger et aI., 1965). The sections in the tube containing the paraformaldehyde powder were treated with formaldehyde vapour, while the sections in the other tube were used as blancs, in order to demonstrate autofluorescence. For microscopic examination, the sections were stretched on object glassed with xylene and mounted with fluormount (Merck). For the demonstration of fluorescence, a Leitz Orthoplan microscope equiped with a vertical illuminator was used. This was provided with a revolver containing a TAL 405 interference filter (Schott and Gen., Mainz, Germany), which served as an excitation filter having about 50 % transmittance at 405 nm. The filter was used in combination with a dichroic mirror (for further details see Ploem, 1971). A Schott fluorescence selection filter SAL 530 was set up in the light path above the vertical illuminator as a secondary filter. As a light source, a high-pressure mercury lamp HBO 100 (Osram, Berlin, Germany) was used. This lamp was operated on a stabilized direct current supply (Leitz, Wetzlar, Germany).

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Quantification Method For the quantification of serotonin-containing and autofluorescent ceils, 40 transverse serial sections were cut from the pineal of each animal. Twenty sections were frozen-dried only, while 20 alternating sections were frozen-dried and treated with formaldehyde vapour. In each section, the number of autofluorescent and serotonin-containing cells was determined by counting these cells in 4 areas, chosen at random, present in an eyefield measuring 0.03 mm -~. In each animal the mean number of cells (n) present in each pineal was calculated per eyefield, as well as the variance and the standard error of the mean. The results of the individual animals were statistically combined according to De Jonge (1963).

Serotonin Determination For this determination 81 rats were used. Twenty-seven rats were injected with 25 I.U. HCG (pregnyl) and 27 animals with PMSG (gestyi) while 27 rats served as controls receiving the solvent only (0.1 ml of 0.9 % NaC1). Eigtheen rats were sacrificed 4 days after the first injection, 18 other rats af[er 7 days and the remaining 18 animals were killed a&er 3 weeks. The control rats were sacrificed simultaneously with the experimental animals in groups of 9 rats. In order to obtain sufficient 5-HT per determination, 3 pineaIs had to be pooled for each. The results were expressed in ),/g pineal tissue. Isolation of 5-HT was based on Amberlite column chromatography. Pineal glands were removed, pooled and extracted in 10 ml ice-cold 0.4M perchloric acid containing 0.1 ml ascorbic acid (2 %) and 0.2 ml EDTA (10 %). The extracts were neutralized with 5 N K2Co to pH 5, centrifuged and then the supernatant was applicated on Amberlite CG 50 columns (4.5 • 50 nm) with a flow rate not exceeding 0.5 ml/min. The columns were washed out with 0.02 M phosphate buffer (10 ml at pH 6.5) containing 0.2 0/0 EDTA. Elution was performed with 1 N HC1, and serotonin was determined fluorimetricatly (And~n and Magnusson, 1967).

Results

Experiment i. During PMSG injections for 28 days (Fig. 1), no significant increase can be observed in the amount of 5-HT-containing pinealocytes at the different times the rats were killed. On the other hand, a slight, but significant decrease could be shown during that time. In contrast, a large increase was found in the number of autofluorescent cells after 7 days (approx. 300 % of the control value). Interestingly, the amount of autofluorescent material returns gradually to its control level between day 7 and day 14, then practically

Pineal 5-HT and Gonadotropins

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Fig. 1. Diagram showing the influence of PMSG (Gestyl) on the number of autofluorescent (AF) and serotonergic (5HT) pinealocytes during 28 days of administration in adult male Wistar rats. Results are expressed in percentage of the control values. Shaded values represent the control animals

remaining constant. The results of the administration of gonadotropic hormones (HCG, PMSG and FSH) on the number of 5-HTcontaining pinealocytes during 4 days (Fig. 2), are similar to those illustrated in figure 1. No effect could also be found a~er 4 days of gonadotropin administration when serotonin was measured biochemically (Fig. 3). After administration of gonadotropins during one week no effect could be observed at all, neither regarding the amount of 5-HT-containing pinealocytes (Fig. 4), nor in the 5-HT levels determined biochemically. Experiment 2. AfLer administration of gonadotropins during 4 days, the amount of autofluorescent cells was significantly increased only aflcer PMSG injections (Fig. 2), while H C G and FSH did not influence their number significantly (Fig. 2). Journal of Neural Transmission 36/3-4

17

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A.R. Smith, P. Pevet, L. van de Kar, and R. v. Oosterom:

Experiment 3. Aider 7 days of gonadotropin administration onty aRer PMSG administration (Fig. 4) a significant increase of autofluorescent cells was found corresponding with the first experiment (Fig. 1). No effect could be found atter HCG administration.

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Fig. 4. Diagram showing the influence of gonadonopic hormone injections during 7 days on the number of autofluorescent (AF) and serotonergic (5HT) ceils in the pineal gland of adult male rats

Discussion

It appeared that after 4 days of administration of PMSG and H C G and FSH practically no significant changes occur in the amount of 5-HT-containing pinealocytes, a slight decrease of 5-HT after injection of PMSG after 7 days excepted. This is not in agreement with the biochemical findings, in the respect that PMSG does not seem to lower the 5-HT level significantly after 7 days. This discrepancy between the biochemical and fluorescence microscopical findings can possibly be explained by the different ways of measuring the serotonin concentration. With the fluorescence histochemical method the evaluation of 5-HT-containing cells is revealed by subtraction of the yellow fluorescing ceils and the yellow autofluorescent cells, whereas, in the biochemical study, exact concentrations of serotonin can be measured. Therefore, this method is the' one of preference which means that the result obtained with the subtraction methods appears to be less valuable, at least for the serotonin content of the pineal. The question why, after 4 days, no significant effect occurs in experiment I whereas a significant increase does occur in the number of autofluorescent cells after PMSG injections in experiment II, is puzzling. The most plausible explanation for this result may be the different sensitivity of the rats to PSMG in both experimental groups, although the animals were of the same weight and same age, and 17"

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A. R. Smith, P. Pevet, L. van de Kar, and R. v. Oosterom"

were kept in the same experimental conditions. Perhaps the same effect started earlier in group II than in group I. Trying to interprete these results it is known that gonadotropin levels increase a~er gonadectomy (Gay and Midgley, 1964; Yarnato et aI., 1970). It is also known that after gonadectomy an increase of yellow autofluorescent pinealocytes is observed (AriFns Kappers et al., 1974; Smith and Ari~ns Kappers, 1973; Smith, 1974; Pevet et al., 1975), while no changes could be demonstrated in the amount of 5-HT in the pineal as either determined by fluorescence microscopy or by biochemical methods (Pevet et at., 1975). Thus it appears that identical changes can be observed in the 5-HT and autofluorescent material content after gonadectomy and injections of PMSG exclusively, as observed in the present investigation. The results suggest that there exists no relation between pineal 5-HT levels and gonadal hormones, while significant alterations in the amount of yellow autofluorescent pineatocytes occur after castration (Pe.vet et al., 1975) and PMSG injection. The results show that PMSG produces a significant effect on the autofluorescent pinealocytes, while PCG and pure FSH bovine injections do not give rise to significant alterations in this cell population. The question why pure FSH does not produce the same changes as PMSG still cannot be answered, tt seems, however, evident that a small amount of LH in combination with a larger amount of FSH produces significant alterations only. The increased level of androgens after gonadotropin administration could also be the cause of the increased amount of yellow autofluorescent pinealocytes. However, also afLer orchidectomy, which induces a decrease of androgen blood levels, an increase of these pinealocytes can be observed (And~n and Magnusson, 1967). Thus, the alterations in the number of autofluorescent cells seems independent of the level of androgens. In concluding it seems (1) that there is no influence of increased gonadotropin levels on the 5-HT levels in the pineal gland of the male rat; (2) that changes in the amount of yellow autofluorescent cells in the pineal are exclusively caused by PMSG injections and not by androgenic hormon changes.

Acknowledgements The authors gratefully acknowledge the excellent technical assistance of Mr. J. I. Oberink. The generous gii~ of gonadotropic hormones used in this study by the Organon Company, is gratefully acknowledged.

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References And~n, N.-E., and T. Magnusson: An improved method for the fluorimetric determination of 5-hydroxytryptamine in tissues. Acta physiol, scand. 69, 87--94 (1967). Ari~ns Kappers, J., A. R. Smith, and R. A. C. de Vries: The mammalian pineal gland and its control of hypothalamic activity. Progr. Brain Res. 4/, 149--174 (1974). Corrodi, H., and G. Jonsson: Fluoreszenzmethoden zur histochemischen Sichtbarmadlung yon Monoaminen. 5. Identifizierung des fluoreszierenden Produktes aus Modeltversuchen mit 5-Methoxytryptamin und Formaldehyd. Acta Histochem. 22, 247--258 (1965). Falck, B., N. A. Hitlarp, G. Thieme, and A. Torp: Fluorescence of catechotamines and related compounds condensed with formaldehyde. J. Histochem. Cytochem. 10, 348--354 (1962). Gay, V. L., and A. R. Midgley: Response of the adult rat to orchidectomy and ovariectomy as determined by LH-Radio-immuno-assay. Endocrinology 84, 359--1364 (1964). Hamberger, B.: Reserpine-resistant uptake of catecholamines in isolated tissue of the rat. Acta physiol, scand. Suppl. 295 (1967). Hamberger, B., T. Malmfors, and G. Sachs: Standardization of paraformaldehyde and of certain procedures for the histochemical demonstration of catecholamines. J. Histochem. Cytochem. 13, I47 (t965), Heene, R.: Histochemischer Nachweis yon Katechotaminen und 5-Hydroxytryptaminen am Kryostatschnitt. Histochem. t4, 324--327 (1968). Jonge, H. de: Inleiding tot de medische statistiek, Vot. 1 en 2. Leiden: Inst. v. prevent geneesk. 1963. Pevet, P., and A. R. Smith (not published). Pevet, P., A. R. Smith, L. v. d. Kar, and H. v. Bronswi~k: Effect of castration on the rat pineal gland; a fluorescence histochemical and biochemical study. (Submitted to Experientia, 1975.) Ploem, J. S.: The microscopic differentiation of the colour of formaldehyde induced fluorescence. In: Histochemistry of Nervous Transmission, pp. 27--38. Progr. Brain Res..34 (Erlink~5, 0., ed.). Amsterdam: Elsevier. 1971. Satodate, R., K. S. Hsieh, and M. Ota: Morphological changes in the pineal gland of the albino rat by hypophysectomy and ovariectomy. Experientia 26, 638--640 (1970). Smith, A. R.: Over de invloed van de glandula pineatis op de hypothalamus, een histochemisch onderzoek. Ned. Tijdschr. v. Geneesk. 118, 14t8 (t974). Smith, A. R., and J. Ariens Kappers: Effect of rat pineal gland on the hypothalamo-hypophyseo-gonadal axis. VI. Internat. Symposium on Neurosecr. 323 (Knowles, Sir Ft., and L. Vollrath, eds.). 1973. Smith, A. R., and J. Ari~ns Kappers: Effect of pinealectomy, gonadectomy, pCPA and pineal extracts on the rat parvocellular neurosecretory hypothalamic system; a fluorescence histochemical investigation. Brain Researc~ 86, 353--371 (1975).

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Smith, A. R., J. Ari~ns Kappers, and J. F. Jongkind: Distribution and quantification of serotonin-containing and autofluorescent cells in the rabbit pineal organ. Gem Comp. End. 18, 364--371 (1972). Yamato, M., N. D~ NiebeI, and E. M. Bogdanove: Analysis of initial and delayed e[ffectsof orchidectomy and ovariectomy on pituitary and serum LH levels in adult and immature rats. Endocrinology 86, l l 0 2 - - t i l l (1970). Author's address: Dr. A. R. Smith, The Netherlands Central Institute for Brain Research, Ijdijk 28, Amsterdam-O, The Netherlands.