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Localization of metallothionein I-II immunoreactivity in bovine pituitary gland
Life Sciences 70 (2001) 659–667
Localization of metallothionein I-II immunoreactivity in bovine pituitary gland Paolo Zattaa,*, Pamela Zambenedettib, Werner Wittkowskic, Emilio Carpenéd a
CNR-Center on Metalloproteins, Department of Biology, University of Padova, Italy b Anatomopathology Division, General Hospital, Dolo-Venice, Italy c Institute of Anatomy, University of Münster, Münster, Germany d Department of Biochemistry, Faculty of Veterinary Medicine, University of Bologna, Ozzano, Italy Received 16 November 2000; accepted 7 June 2001
Abstract Metallothioneins belong to a family of shock proteins characterized by an unusual high content of cystein, absence of aromatic amino acids and high metal content (Zinc and Copper). Metallothioneins are ubiquitously present in a large variety of prokaryotic and eukaryotic species as well as in all mammalian organs and tissues examined thus far. To the best of our knowledge this is the first report describing the presence of metallothioneins in the pituitary gland. Metallothioneins were identified immunohistochemically and chromatographically both in the neuro and adenohypophysis of the bovine pituitary gland. Metallothioneins are highly expressed in the neurohypophyseal glial cells, and in a subpopulation of folliculo-stellate cells located in the pars intermedia of the adenohypophysis. While the specific role of these proteins in the pituitary gland remains to be established, we hypothesize that, besides their protective action against free radicals, hypophyseal metallothioneins might be involved in the regulation of metal ion homeostasis with putative implication in release of hypothalamic peptide hormones in the neurohypophysis and synthesis/release of a-MSH by POMC-cells located in the pars intermedia of the adenohypophysis. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Hypophysis; Metallothionein; Adenohypophysis; Neurohypophysis; Alzheimer’s disease
Introduction The pituitary gland lies at the base of the brain, in a depression in the sphenoid bone called the sella turcica. The hypophysis is linked to the brain by a pituitary stalk and consists of a glandular portion (adenohypophysis), with the subdivisions pars distalis, pars intermedia and pars tuberalis, and a neural portion (neurohypophysis) with infundibulum and neural * Corresponding author. Centro CNR Metalloproteine, Dipartimento di Biologia, Università di Padova, Viale G. Colombo, 3, 35121 Padova, Italy. Fax: 391049-827-6330. E-mail address: [email protected] (P. Zatta) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 4 4 4 -8
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lobe. The pars intermedia extends between pars distalis and neural lobe [9]. The adenohypophysis elaborates numerous hormones, endorphins and other substances. The neurohypophysis is structurally complex and includes axon secretory terminations distinct from other neurons in that they do not terminate on other nerve cells or effectors, but release their secretory products directly into the blood stream [9]. MT are low-molecular weight cysteine-rich cytoplasmatic proteins able to bind heavy metals. MT are ubiquitously present in a large variety of prokaryotic and eukaryotic species as well as in all mammalian organs and tissues examined thus far. Mammalian MT comprise four similar but distinct isoforms (from MT-I to MT-IV) [12]. In the brain, MT-I/II are mainly expressed in astrocytes (and microvessels), where they play relevant roles in the homeostasis of zinc and copper in the detoxification of heavy metals and possibly as scavengers of free radicals [6]. MT-III has been identified primarely in neuronal cells [28], and MT-IV is expressed in specialized epithelial tissues [22]. More than sixty years ago, Hans Selye defined the complex concept of stress, demonstrating that the neuroendocrine and immune systems interact during stress. He defined the crucial role of the adrenal-cortex-hypophyseal axis (AHP) in the stress response. Stress is now well established to be closely related to molecular and acute emotional events and is among other features, by an increased activity of the brain hypothalamic-hypophyseal region. MT are now recognized as members of the acute-phase-protein (AcPP) family able to respond nonspecifically to the immune system when infections and injury occur. Stress, inflammation and infections have all been shown to cause an increased expression of MT [17]. Marked neuroendocrine neurohypophyseal alterations have been observed in aged subjects as a physiological phenomenon, in that it occurs even in the absence of neuropathological lesions [23]. Recently, it was demonstrated that MT are highly expressed in the brain tissue of Alzheimer’s disease (AD) subjects [31], a feature that could be linked to an alteration of the neuroendocrine system that may be particularly evident in senile dementia of Alzheimer’s type [27]. Interleukins have been found within the pituitary gland, where they may regulate the pituitary hormone [1]. Treating rats with lipopolysaccharide, known as an MT inducer, results in a strong increase in the level of IL-1b mRNA in the posterior pituitary, as well as in the anterior pituitary [16].This paper reports the first histological and chromatographic identification of MT in the adult bovine hypophysis. Materials and methods Isolation and purification of metallothionein Ten cows, about six months of age, were killed at the slaughterhouse according to Italian law using a captive bullet followed by a jugulation procedure. Their hypophyses were immediately removed and fixed in buffered formalin. Portions of adenohypohysis and neurohypophysis (400 to 600 mg), were homogenized in 3 volumes of 20 mM Tris-HCl buffer, pH 8.6 in the presence and absence of 10 mM mercaptoethanol. Homogenization was carried out by using an Ultraturrax homogenizer for 30 min at 13,500 rpm. The homogenate was centrifuged for 30 min at 100,000 g, and 300 ml of resulting supernatant was applied to a Sephadex G75 column (0.6 3 22 cm) and eluted with the same buffer. Fractions were collected, and the
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concentration of Cu and Zn in each fraction was determined by atomic absorption spectroscopy. Similar tissue samples were homogenized in 3 volumes of 0.25 M sucrose in bidistilled water. The concentration of metallothionein was determined following the method of silver saturation described by Sheuhamer and Cherian [24]. Histology Immediately after removal, hypophyses were fixed in freshly prepared 10% buffered formalin for several days, and then embedded in paraffin. Six mm sections were deparaffinized and hydrated, following standard procedures. According to PAP-method sections were deparaffinized in xylene, hydrated through a graded ethanol series and equilibration in 0.1 M TrisHCl buffer (pH 7.6) for 10 min. After preincubation with 5% normal swine serum in 0.1 M Tris-HCl (pH 7.6), the primary antibody was applied in preincubation buffer with 1% normal swine serum for 22 hr at room temperature. Antibody binding was visualized by incubating sections with biotinylated secondary antibody (anti-mouse-IgG-biotin or anti-rabbit-IgGbiotin) and subsequently with avidin-biotinylated-peroxidase complex according to ABCHRP method (Vecstatin, Vector Laboratories, Burningame, CA, USA). Thereafter, the chromogen 3-39-diaminobenzidine (0.05% in 0.1 M Tris-HcL) in H2O2 (0.001%) (Sigma, Munich, Germany), was applied to the sections for 6 min. After complertion of the staining sections were dehydrated and mounted in DePeX (Serva, Heidelberg, Germany). Some sections were counterstained with hematoxylin for morphological orientation. Antibody utilized were: Primary Antibody Secondary Antibody Vimentin (dil 1:200) Biotrend, Koln, Germany PK-kit 6101 rabbit IgG, Camon GFAP (dil 1:500) Biotrend, Koln, Germany PK-kit 6101 rabbit IgG, Camon S-100 Protein (dil 1:200) Biotrend, Germany PK-kit 6101 rabbit IgG, Camon a-MSH (dil 1:2000) UCB Bioproducts, Brain-L’Alleud Belgium PK-kit 6101 rabbit IgG, Camon ACTH (dil 1:2000) UCB Bioproducts PK-kit 6101 rabbit IgG, Camon Ferritin (dil 1:1100) DAKO, Milan, Italy For metallothionein I1II (DAKO, Milan, Italy) staining, sections were incubated in 3% H2O2 in PBS for 10 min and, after washing, treated with normal goat serum for 30 min. Sections were then incubated overnight at 48C with anti-MT-I-II antibody at a dilution of 1: 50. After washing twice with PBS, sections were incubated for 30 min with biotinylated goat antimouse IgG, rinsed three times, and then processed with streptoavidin-peroxidase ABC complex (DAKO, Milan, Italy). The sections were developed using substrate DAB-chromogen system (DAKO, Milan, Italy), and then counterstained with hematoxylin. All chemical reagents were of the purest commercial grade and were purchased from Sigma (Milan, Italy), unless specified otherwise. Results Immunohistochemistry Neurohypophysis Glial cells of the bovine hypophysis show the typical differentiation and distribution as in the other mammalian species. Tanycytes and pituicytes exhibit the well-known pattern of
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vascular processes which forms part of the structure of the neurohemal regions in the infundibulum and neural lobe. Immunostaining with anti-metallothionein antibodies yields strong staining of tanycyte and pituicyte perikarya as well as their processes in the infundibulum and neural lobe. Adenohypophysis Folliculo-stellate cells are intermingled with specific glandular cells and are present in all parts of the adenohypophysis. However, interestingly, metallothionein-positive folliculo-stellate cells were found only in the folliculo-stellate cells of the the pars intermedia (Fig. 1 E–F); Pars tuberalis and Pars distalis cells were not recognized by MT antibodies. Specific glandular cells of the adenohypophysis did not react or were stained only weakly. In order to better evaluate the presence of MT in the pituitary gland, a cytosolic fraction prepared from both the adeno- and neurohypophysis were subjected to gel filtration chromatography to isolate MT. Fig. 2A reports the elution profile of adeno cytosol, where Zn-MT shows a maximum peak at fraction number 14 corresponding to the elution position of a standard MT purified from goldfish (5). The elution profile of Cu-MT was similar to that of Zn-MT, although the quantity of Zn-MT was about three times higher with respect to Cu-MT. Fig. 2B reports the elution profile of neurohypophysis cytosol. Also in this case both Cu-MT and Zn-MT eluted at the same position, close to that of the standard MT, and Zn-MT was about 3-fold more abundant than Cu-MT. Discussion MT belong to a family of shock-proteins that participate in an array of protective stress response; a direct connection between MT-I1II, but not MT-III expression, and stress have been reported by some authors [11]. MT may serve as a source of Zn for proteins that rely on this metal for functional activity. Zinc is important for many biological processes; it prevents apoptosis, and a deficiency of this metal ion generally causes this type of cell death. Therefore, as a Zn binding protein, MT may play a relevant role in regulating apoptotic phenomena [4]. This hypothesis is supported by the observation that inhibition of MT synthesis induces morphological alterations and DNA fragmentation characteristic of apoptotic cells [26]. Experimental data suggest that MT regulate a variety of physiopathological processes, such as homeostasis of essential elements (e.g., Zn and Cu); two especially important tasks of MT could be to supply Zn to Zn-requiring enzyme/transcription factors during activated cell proliferation and to protect DNA from oxidative stress [20]. It is well known that MT and Fig. 1. Immunohistochemical staining for metallothionein I1II expression in the infundibulum (A, B), neural lobe (C, D) and Pars intermedia (E, F) of bovine hypophysis. A, B: MT-positive tanycytes and pituicytes in the infundibulum forming a network of processes in the inner zone and bundles of processes in the external zone (ZE), which project into and terminate at the vascular surface of the primary portae plexus. (PT) Pars tubularis of the adenohypophysis. Original magnification: A, 3 135; B, 3 580. C, D: MT-positive pituicytes of the neural lobe, some of which exhibit long slender processes. Original magnification: C, 3 135; D, 3 580. E, F: MT-positive folliculo-stellate cells in the Pars intermedia of the adenohypophysis. Original magnification: E, 3 135; F 3 580. (Fig. 1 A–D). MT-positive vascular processes of glial cells in the infundibulum terminated at the neurovascular surface of the median eminence (Fig. 1 B). Tanycytes and pituicytes in the neurohypophysis were also recognized by antibodies against ferritin, GFAP and S-100 protein antibodies (data not reported).
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Fig. 2. Sephadex G-75 chromatographic column. The column was equilibrated and eluted with 20 mM Tris-HCl I5 0.1 M (pH 8.&), and 10 mM mercaptoethanol. Concentration of Zn (e) and copper (h) corresponding to metallothionein peak are indicated by arrows. A) Elution profile of adenohypophysis cytosol; B) Elution profile of neurohypophysis cytosol.
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Table 1a Zn and Cu content in bovine adeno and neurohypophysis (mg/g fresh tissue)
Cu
Zn
Adenohypophysis Neurohypophysis
2.13 6 0.14 2.31 6 0.34
13.05 6 1.06 13.44 6 0.05
Table 1b Metallothionein (mg/g fresh tissue) in bovine adeno and neurohypophysis Adenohypophysis Neurohypophysis
119 6 5.0 79 6 11.0
zinc concentrations in mammals reach maximum levels prior the completion of thymic growth, and decrease to minimum levels when development of the thymus has ceased, with the exception of the brain which maintains high MT levels [21]. One noteworthy is that Znintoxicated rats show elevated secretory activity in the neurosecretory nuclei of the hypothalamus along with a decline in the amount of neurosecretion in the nervous pars of the hypophysis [15]. MTs maintain essential Zn-related secretion of hypophyseal hormones [13]. The present study also demonstrates that Zn is equally distributed in adeno- and neurohypohysis, at a concentration that is one order of magnitude higher than that of copper. In contrast, the distribution of MT differs between the adeno and neurohypophysis. MT are detected mainly in the neurohypophysis, while in the adenohypophysis Mt show a peculiarly localizationb only in the folliculo-stellate cells of the pars intermedia: although this distribution pattern could have physiological relevance, at present we cannot offer an explanation for it. Folliculo-stellate cells are supposed to play a role in the regulation of hormone secretion by pituitary glandular cells especially by producing various cytokines such as IL-6 [29]. It has been shown by several investigators that folliculo-stellate cells of pituitary gland are heterogeneous with respect to ultrastructure and expression of markers [2, 14]. Comparison between pars tuberalis and pars distalis of pituitary gland shows that besides different types of folliculo-stellate cells within both these regions there are also clear differences of immunoreactivity of folliculo-stellate cells between pars tuberalis and pars distalis [14]. In the context of such a differentiation it is not surprising that a subpopulation of folliculostellate cells expresses MT and may exert special regulation influence on groups of secretory cells expecially in the pars intermedia, a part of the pituitary which consists mainly of POMC-expressing cells. Aging of the pituitary gland with decreased secretion of pituitary hormones could cause an overexpression of MT [19]. 101 autoptically examined human hypophyses revealed a gradual increase in iron deposition in the connective tissue after the fourth decade. The active cells of hypophysis may gradually be replaced by collagenous tissue [8] decreasing the physiological efficiency of this gland. In addition, high expression of transferrin, the physiological iron carrier, and its receptor have been identified in the pituitary gland [25]. Iron is one of the key elements involved in free radical production. In this connection MTs can consistently contribute to struggle against free radical production as it has been widely reported in the literature. (Recent review by Hidalgo et al. [10]. MT are both effective free radical and transitional
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metal ion scavengers. Studies in vitro have revealed that MT react directly with reactive oxygen species (ROS) including superoxide, hydroxy radicals and hydrogen peroxyde. It has been suggested that an alteration of the neuroendocrine system may particularly occur in senile dementia of Alzheimer’s type. MT play an important role in protecting brain tissue from toxic insults responsible for brain aging [19]. Nowadays, there is a general consensus that a reduction of the activity in the hypothalamus-hypophysis-thyroid axis in the elderly is associated with adaptation of hormone production [3, 18]. Recent advances have made clear that there are many connections between immunological system and hypothalamic-pituitary axis, and among them, a relationship between IL secretion and hypothalamus activity appears to be of great interest. It is well known that IL-1 stimulates the expression of MT. The effect of IL-1b on the secretion of oxytocin and vasopressin from electrically stimulated rat neurohypophysis has been demonstrated, indicating that IL-1b might be involved in the regulation of the hypophyseal oxytocin and vasopressin [7]. Differently, in the anterior pituitary a limited IL-1b gene expressed has been reported, but a marked mRNA encoding IL-1ra was observed, hypothesizing that IL-1ra may be secreted as a systemic anti-inflammatory hormone in response to IL-1b originated from multiple sources [30]. IL-6, has been identified in the folliculostellate cells of the anterior pituitary gland where we have observed the expression of MT. Thus, MT expression in the pituitary gland might represent a homeostatic factor, as it has been widely demonstrated for other tissues, controlling free Zn in primis and Cu, as well as some neuroimmuno factors, in secundis, and contributing to the physiological regulation of hypophyseal hormone production/secretion, which is deeply alterated with aging, as well as contrasting free radical negative actions. The presence of MT in the hypophysis, as reported in this paper, could thus open a new perspective in a better understanding the role of these proteins. In this connection, a comparison of the level of MT in normal and AD hypophysis as well as in other neurodegenerative diseases is currently in progress in our laboratory. For these reasons we think that it is worthwhile to furtherly explore the expression of MT in the hypophysis as a first step to better understanding its role in this strategic gland in relation to pathogenic events associated with aging processes. References 1. 2.
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Abraham EJ, Minton JE. Cytokines in the hypophysis: a comparative look at interleukin-6 in the porcine anterior pituitary gland. Comparative Biochemistry and Physiology (A) 1997; 116: 203–207. Allaerts W, Fluitsma DM, Hoefsmit EC, Jeucken PH, Morreau H, Bosman FT, Drexhage HA. Immunohistochemical, morphological and ultrastructural resemblance between dendritic cells and folliculo-stellate cells in normal human and rat anterior pituitaries. Journal of Neuroendocrinology 1996; 8: 17–29. Ayisi K, Schmiegelov P, Linder J, Sames K. Connective tissue aging in the human hypophysis-gonadal system. Pathology Research Practice 1982; 173: 294–302. Cai L, Wang GJ, Xu ZL, Denh DX, Chakrabarti S, Cherian MG. Metallothionein and apoptosis in primary human hepatocellular carcinoma (HCC) from northen China. Anticancer Research 1998; 18: 4667–4672. Carpenè E, Vasak M. Hepatic metallothioneins from goldfish (Carassius auratus L.). Comparative Biochemistry and Physiology 1989; 92B: 463–468. Cherian MG, Chan HM. Biological function of metallothioneins. In: Suzuki KT, Imura N, Nakamura M, editors. Metallothionein III. Basel: Birkhauser Verlag,1993. pp. 87–109. Christensen JD, Hansen EW, Fjalland B. Influence of interleukin-1 beta on the secretion of oxytocin and vasopressin from the isolated rat neurohypophysis. Pharmacology and Toxicology 1990; 67: 81–83. Greenberg SR. The pathogenesis of hypophyseal fibrosis in aging: its relantionship to tissue iron deposition. Journal of Gerontology 1978; 30: 531–538.
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Halmi NS. The hypophysis. In Weiss L, editor. Cell and Tissue Biology. Baltimore: Urban & Schwarzenberg, 1988. pp. 975–994. Hidalgo J, Vasak M, Zatta P, Aschner M. Metallothioneins are essential proteins for coping with Central Nervous System damage. Brain Research Bulletin 2001 (in press). Jacob ST,Goshal, Sheridan JF. Induction of metallothionein by stress and its molecular mechanism. Gene Expression 1999; 7: 301–310. Kägi JHR, KojiamaY. Chemistry and biochemistry of metallothionein. Experientia 1987; 52: 25–61. Kaswan S, Bedwal RS. Light and electron microscopic changes in the ovary zinc deficient BALB/c mice. Indian Journal of Experimental Biology 1995; 33: 469–479. Kameda Y. Differential distribution of S-100 protein and vimentin in the hypophyseal pars tuberalis of the guinea pig. Journal of Histochemistry & Cytochemistry 1996; 44: 501–510. Kozik MB, Gramza G, Pietrzak M. Neurosecretion of the hypothalamo-hypophyseal system after intragastric administration of zinc oxide. Folia Histochemica et Cytochemica 1981; 19: 115–122. Licinio J, Wong ML. Interleukin-1 receptor antagonist gene expression in rat pituitary in the systemic inflammatory response syndrome: pathophysiological implications. Molecular Psychiatry 1997; 2: 99–103. Manuel Y, Thomas Y, Pellegrini O. Metallothionein and tissue damage IARC Scientific Publ 1992 ; 118: 231–237. Messina G, Visconti N, Trinti B. Variations in the anatomy and physiology of the thyroid gland in old age. Recenti Progressi in Medicina 1997; 88: 281–286. Mocchegiani E, Giacconi R, Cipriano C, Muzzioli M, Fattoretti P, Bretoni-Freddari C, Isani G, Zambenedetti P, Zatta P. Zinc-bound metallothionein (I1II) as potentail biological marker of ageing. Brain Research Bulletin 2001 (in press). Ogra Y, Suzuki KY. Nuclear trafficking of metallothionein : possible mechanisms and current knowledge. Cell Molecular Biology 2000; 46: 357–365. Olafson RW. Thymus metallothionein: regulation of zinc-thionein in the aging mouse. Canadian Journal of Biochemistry and Cell Biology 1985; 63: 91–95. Quaife CJ, , Findlay SD, Erickson JC, Forlick GJ, Kelly EJ, ZambrowoczBP, Palmiter RD. Induction of a new metallothionein isoform (MT-IV) occurs during differentiation of stratified squamous epithelia. Biochemistry 1994; 33: 7250–7259. Schulz C,Koppersh D, Braak H, Braak E. Cytoskeletal alterations in the aged human neurohypophysis. Neuroscience Letters 1997; 237: 93–96. Sheuhamer AM Cherian MG. Quantification of metallothionein by silver saturation. Methods in Enzymology 1991; 205: 78–83. Tampanaru SA, Stefanescu L, Thapar K, Kontogeorgos G, Sumi T and K. Kovacsk O K. Transferrin and transferrin receptor in human hypophysis and pituitary adenomas. American Journal of Pathology 1998; 152: 413–422. Tsangaris GT, Tzortzatou-Stathopoulou F. Metallothionein expression prevents apoptosis: a study with antisense phosphorothioato oligodeoxynucleotides in human T cell line. Anticancer Research 1998; 18: 2423–2433. Tsuboyama GK, Gabriel SS, Davison BALawlor BA, Ware K, Davis KL, Mohs RC.Neuroendocrine dysfunction in Alzheimer’s disease: results following TRH stimulation. Biology Psychiatry 1992; 32: 195–198. Uchida Y, Takio Y, Titani K, Hihara Y Tomonaga M. The growth inhibitory factor that is deficient in the Alzheimer’s disease is a 68 amino acid metallothionein-like protein. Neuron 1991; 7: 337–347. Vankelcom H, Matgys P, Van Damme J, Heremans H, Billiau A Denef C. Immunocytochemical evidence that S-100-positive cells of the mouse anterior pituitary contain interleukin-6 immunoreactivity. Journal of Histochemistry & Cytochemistry 1993; 41: 151–156. Wong ML, Buongiorno PB, Rettori V, McCann SM, Licinio J. Interleukin 1beta, IL-1 receptor antagonist, IL10, and IL-13 gene expression in the central nervous system and anterior pituitary during systemic inflammation: pathophysiological complications. Proceedings of the National Academy of Sciences (USA) 1997; 94: 227–232. Zambenedetti P, Giordano R, Zatta P. Metallothioneins are highly expressed in astrocytes and microcapillaries in Alzheimer’s disease. Journal of Chemical Neuroanatomy 1998; 15: 21–26.
Localization of metallothionein I-II immunoreactivity in bovine pituitary gland Paolo Zattaa,*, Pamela Zambenedettib, Werner Wittkowskic, Emilio Carpenéd a
CNR-Center on Metalloproteins, Department of Biology, University of Padova, Italy b Anatomopathology Division, General Hospital, Dolo-Venice, Italy c Institute of Anatomy, University of Münster, Münster, Germany d Department of Biochemistry, Faculty of Veterinary Medicine, University of Bologna, Ozzano, Italy Received 16 November 2000; accepted 7 June 2001
Abstract Metallothioneins belong to a family of shock proteins characterized by an unusual high content of cystein, absence of aromatic amino acids and high metal content (Zinc and Copper). Metallothioneins are ubiquitously present in a large variety of prokaryotic and eukaryotic species as well as in all mammalian organs and tissues examined thus far. To the best of our knowledge this is the first report describing the presence of metallothioneins in the pituitary gland. Metallothioneins were identified immunohistochemically and chromatographically both in the neuro and adenohypophysis of the bovine pituitary gland. Metallothioneins are highly expressed in the neurohypophyseal glial cells, and in a subpopulation of folliculo-stellate cells located in the pars intermedia of the adenohypophysis. While the specific role of these proteins in the pituitary gland remains to be established, we hypothesize that, besides their protective action against free radicals, hypophyseal metallothioneins might be involved in the regulation of metal ion homeostasis with putative implication in release of hypothalamic peptide hormones in the neurohypophysis and synthesis/release of a-MSH by POMC-cells located in the pars intermedia of the adenohypophysis. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Hypophysis; Metallothionein; Adenohypophysis; Neurohypophysis; Alzheimer’s disease
Introduction The pituitary gland lies at the base of the brain, in a depression in the sphenoid bone called the sella turcica. The hypophysis is linked to the brain by a pituitary stalk and consists of a glandular portion (adenohypophysis), with the subdivisions pars distalis, pars intermedia and pars tuberalis, and a neural portion (neurohypophysis) with infundibulum and neural * Corresponding author. Centro CNR Metalloproteine, Dipartimento di Biologia, Università di Padova, Viale G. Colombo, 3, 35121 Padova, Italy. Fax: 391049-827-6330. E-mail address: [email protected] (P. Zatta) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 4 4 4 -8
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lobe. The pars intermedia extends between pars distalis and neural lobe [9]. The adenohypophysis elaborates numerous hormones, endorphins and other substances. The neurohypophysis is structurally complex and includes axon secretory terminations distinct from other neurons in that they do not terminate on other nerve cells or effectors, but release their secretory products directly into the blood stream [9]. MT are low-molecular weight cysteine-rich cytoplasmatic proteins able to bind heavy metals. MT are ubiquitously present in a large variety of prokaryotic and eukaryotic species as well as in all mammalian organs and tissues examined thus far. Mammalian MT comprise four similar but distinct isoforms (from MT-I to MT-IV) [12]. In the brain, MT-I/II are mainly expressed in astrocytes (and microvessels), where they play relevant roles in the homeostasis of zinc and copper in the detoxification of heavy metals and possibly as scavengers of free radicals [6]. MT-III has been identified primarely in neuronal cells [28], and MT-IV is expressed in specialized epithelial tissues [22]. More than sixty years ago, Hans Selye defined the complex concept of stress, demonstrating that the neuroendocrine and immune systems interact during stress. He defined the crucial role of the adrenal-cortex-hypophyseal axis (AHP) in the stress response. Stress is now well established to be closely related to molecular and acute emotional events and is among other features, by an increased activity of the brain hypothalamic-hypophyseal region. MT are now recognized as members of the acute-phase-protein (AcPP) family able to respond nonspecifically to the immune system when infections and injury occur. Stress, inflammation and infections have all been shown to cause an increased expression of MT [17]. Marked neuroendocrine neurohypophyseal alterations have been observed in aged subjects as a physiological phenomenon, in that it occurs even in the absence of neuropathological lesions [23]. Recently, it was demonstrated that MT are highly expressed in the brain tissue of Alzheimer’s disease (AD) subjects [31], a feature that could be linked to an alteration of the neuroendocrine system that may be particularly evident in senile dementia of Alzheimer’s type [27]. Interleukins have been found within the pituitary gland, where they may regulate the pituitary hormone [1]. Treating rats with lipopolysaccharide, known as an MT inducer, results in a strong increase in the level of IL-1b mRNA in the posterior pituitary, as well as in the anterior pituitary [16].This paper reports the first histological and chromatographic identification of MT in the adult bovine hypophysis. Materials and methods Isolation and purification of metallothionein Ten cows, about six months of age, were killed at the slaughterhouse according to Italian law using a captive bullet followed by a jugulation procedure. Their hypophyses were immediately removed and fixed in buffered formalin. Portions of adenohypohysis and neurohypophysis (400 to 600 mg), were homogenized in 3 volumes of 20 mM Tris-HCl buffer, pH 8.6 in the presence and absence of 10 mM mercaptoethanol. Homogenization was carried out by using an Ultraturrax homogenizer for 30 min at 13,500 rpm. The homogenate was centrifuged for 30 min at 100,000 g, and 300 ml of resulting supernatant was applied to a Sephadex G75 column (0.6 3 22 cm) and eluted with the same buffer. Fractions were collected, and the
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concentration of Cu and Zn in each fraction was determined by atomic absorption spectroscopy. Similar tissue samples were homogenized in 3 volumes of 0.25 M sucrose in bidistilled water. The concentration of metallothionein was determined following the method of silver saturation described by Sheuhamer and Cherian [24]. Histology Immediately after removal, hypophyses were fixed in freshly prepared 10% buffered formalin for several days, and then embedded in paraffin. Six mm sections were deparaffinized and hydrated, following standard procedures. According to PAP-method sections were deparaffinized in xylene, hydrated through a graded ethanol series and equilibration in 0.1 M TrisHCl buffer (pH 7.6) for 10 min. After preincubation with 5% normal swine serum in 0.1 M Tris-HCl (pH 7.6), the primary antibody was applied in preincubation buffer with 1% normal swine serum for 22 hr at room temperature. Antibody binding was visualized by incubating sections with biotinylated secondary antibody (anti-mouse-IgG-biotin or anti-rabbit-IgGbiotin) and subsequently with avidin-biotinylated-peroxidase complex according to ABCHRP method (Vecstatin, Vector Laboratories, Burningame, CA, USA). Thereafter, the chromogen 3-39-diaminobenzidine (0.05% in 0.1 M Tris-HcL) in H2O2 (0.001%) (Sigma, Munich, Germany), was applied to the sections for 6 min. After complertion of the staining sections were dehydrated and mounted in DePeX (Serva, Heidelberg, Germany). Some sections were counterstained with hematoxylin for morphological orientation. Antibody utilized were: Primary Antibody Secondary Antibody Vimentin (dil 1:200) Biotrend, Koln, Germany PK-kit 6101 rabbit IgG, Camon GFAP (dil 1:500) Biotrend, Koln, Germany PK-kit 6101 rabbit IgG, Camon S-100 Protein (dil 1:200) Biotrend, Germany PK-kit 6101 rabbit IgG, Camon a-MSH (dil 1:2000) UCB Bioproducts, Brain-L’Alleud Belgium PK-kit 6101 rabbit IgG, Camon ACTH (dil 1:2000) UCB Bioproducts PK-kit 6101 rabbit IgG, Camon Ferritin (dil 1:1100) DAKO, Milan, Italy For metallothionein I1II (DAKO, Milan, Italy) staining, sections were incubated in 3% H2O2 in PBS for 10 min and, after washing, treated with normal goat serum for 30 min. Sections were then incubated overnight at 48C with anti-MT-I-II antibody at a dilution of 1: 50. After washing twice with PBS, sections were incubated for 30 min with biotinylated goat antimouse IgG, rinsed three times, and then processed with streptoavidin-peroxidase ABC complex (DAKO, Milan, Italy). The sections were developed using substrate DAB-chromogen system (DAKO, Milan, Italy), and then counterstained with hematoxylin. All chemical reagents were of the purest commercial grade and were purchased from Sigma (Milan, Italy), unless specified otherwise. Results Immunohistochemistry Neurohypophysis Glial cells of the bovine hypophysis show the typical differentiation and distribution as in the other mammalian species. Tanycytes and pituicytes exhibit the well-known pattern of
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vascular processes which forms part of the structure of the neurohemal regions in the infundibulum and neural lobe. Immunostaining with anti-metallothionein antibodies yields strong staining of tanycyte and pituicyte perikarya as well as their processes in the infundibulum and neural lobe. Adenohypophysis Folliculo-stellate cells are intermingled with specific glandular cells and are present in all parts of the adenohypophysis. However, interestingly, metallothionein-positive folliculo-stellate cells were found only in the folliculo-stellate cells of the the pars intermedia (Fig. 1 E–F); Pars tuberalis and Pars distalis cells were not recognized by MT antibodies. Specific glandular cells of the adenohypophysis did not react or were stained only weakly. In order to better evaluate the presence of MT in the pituitary gland, a cytosolic fraction prepared from both the adeno- and neurohypophysis were subjected to gel filtration chromatography to isolate MT. Fig. 2A reports the elution profile of adeno cytosol, where Zn-MT shows a maximum peak at fraction number 14 corresponding to the elution position of a standard MT purified from goldfish (5). The elution profile of Cu-MT was similar to that of Zn-MT, although the quantity of Zn-MT was about three times higher with respect to Cu-MT. Fig. 2B reports the elution profile of neurohypophysis cytosol. Also in this case both Cu-MT and Zn-MT eluted at the same position, close to that of the standard MT, and Zn-MT was about 3-fold more abundant than Cu-MT. Discussion MT belong to a family of shock-proteins that participate in an array of protective stress response; a direct connection between MT-I1II, but not MT-III expression, and stress have been reported by some authors [11]. MT may serve as a source of Zn for proteins that rely on this metal for functional activity. Zinc is important for many biological processes; it prevents apoptosis, and a deficiency of this metal ion generally causes this type of cell death. Therefore, as a Zn binding protein, MT may play a relevant role in regulating apoptotic phenomena [4]. This hypothesis is supported by the observation that inhibition of MT synthesis induces morphological alterations and DNA fragmentation characteristic of apoptotic cells [26]. Experimental data suggest that MT regulate a variety of physiopathological processes, such as homeostasis of essential elements (e.g., Zn and Cu); two especially important tasks of MT could be to supply Zn to Zn-requiring enzyme/transcription factors during activated cell proliferation and to protect DNA from oxidative stress [20]. It is well known that MT and Fig. 1. Immunohistochemical staining for metallothionein I1II expression in the infundibulum (A, B), neural lobe (C, D) and Pars intermedia (E, F) of bovine hypophysis. A, B: MT-positive tanycytes and pituicytes in the infundibulum forming a network of processes in the inner zone and bundles of processes in the external zone (ZE), which project into and terminate at the vascular surface of the primary portae plexus. (PT) Pars tubularis of the adenohypophysis. Original magnification: A, 3 135; B, 3 580. C, D: MT-positive pituicytes of the neural lobe, some of which exhibit long slender processes. Original magnification: C, 3 135; D, 3 580. E, F: MT-positive folliculo-stellate cells in the Pars intermedia of the adenohypophysis. Original magnification: E, 3 135; F 3 580. (Fig. 1 A–D). MT-positive vascular processes of glial cells in the infundibulum terminated at the neurovascular surface of the median eminence (Fig. 1 B). Tanycytes and pituicytes in the neurohypophysis were also recognized by antibodies against ferritin, GFAP and S-100 protein antibodies (data not reported).
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Fig. 2. Sephadex G-75 chromatographic column. The column was equilibrated and eluted with 20 mM Tris-HCl I5 0.1 M (pH 8.&), and 10 mM mercaptoethanol. Concentration of Zn (e) and copper (h) corresponding to metallothionein peak are indicated by arrows. A) Elution profile of adenohypophysis cytosol; B) Elution profile of neurohypophysis cytosol.
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Table 1a Zn and Cu content in bovine adeno and neurohypophysis (mg/g fresh tissue)
Cu
Zn
Adenohypophysis Neurohypophysis
2.13 6 0.14 2.31 6 0.34
13.05 6 1.06 13.44 6 0.05
Table 1b Metallothionein (mg/g fresh tissue) in bovine adeno and neurohypophysis Adenohypophysis Neurohypophysis
119 6 5.0 79 6 11.0
zinc concentrations in mammals reach maximum levels prior the completion of thymic growth, and decrease to minimum levels when development of the thymus has ceased, with the exception of the brain which maintains high MT levels [21]. One noteworthy is that Znintoxicated rats show elevated secretory activity in the neurosecretory nuclei of the hypothalamus along with a decline in the amount of neurosecretion in the nervous pars of the hypophysis [15]. MTs maintain essential Zn-related secretion of hypophyseal hormones [13]. The present study also demonstrates that Zn is equally distributed in adeno- and neurohypohysis, at a concentration that is one order of magnitude higher than that of copper. In contrast, the distribution of MT differs between the adeno and neurohypophysis. MT are detected mainly in the neurohypophysis, while in the adenohypophysis Mt show a peculiarly localizationb only in the folliculo-stellate cells of the pars intermedia: although this distribution pattern could have physiological relevance, at present we cannot offer an explanation for it. Folliculo-stellate cells are supposed to play a role in the regulation of hormone secretion by pituitary glandular cells especially by producing various cytokines such as IL-6 [29]. It has been shown by several investigators that folliculo-stellate cells of pituitary gland are heterogeneous with respect to ultrastructure and expression of markers [2, 14]. Comparison between pars tuberalis and pars distalis of pituitary gland shows that besides different types of folliculo-stellate cells within both these regions there are also clear differences of immunoreactivity of folliculo-stellate cells between pars tuberalis and pars distalis [14]. In the context of such a differentiation it is not surprising that a subpopulation of folliculostellate cells expresses MT and may exert special regulation influence on groups of secretory cells expecially in the pars intermedia, a part of the pituitary which consists mainly of POMC-expressing cells. Aging of the pituitary gland with decreased secretion of pituitary hormones could cause an overexpression of MT [19]. 101 autoptically examined human hypophyses revealed a gradual increase in iron deposition in the connective tissue after the fourth decade. The active cells of hypophysis may gradually be replaced by collagenous tissue [8] decreasing the physiological efficiency of this gland. In addition, high expression of transferrin, the physiological iron carrier, and its receptor have been identified in the pituitary gland [25]. Iron is one of the key elements involved in free radical production. In this connection MTs can consistently contribute to struggle against free radical production as it has been widely reported in the literature. (Recent review by Hidalgo et al. [10]. MT are both effective free radical and transitional
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metal ion scavengers. Studies in vitro have revealed that MT react directly with reactive oxygen species (ROS) including superoxide, hydroxy radicals and hydrogen peroxyde. It has been suggested that an alteration of the neuroendocrine system may particularly occur in senile dementia of Alzheimer’s type. MT play an important role in protecting brain tissue from toxic insults responsible for brain aging [19]. Nowadays, there is a general consensus that a reduction of the activity in the hypothalamus-hypophysis-thyroid axis in the elderly is associated with adaptation of hormone production [3, 18]. Recent advances have made clear that there are many connections between immunological system and hypothalamic-pituitary axis, and among them, a relationship between IL secretion and hypothalamus activity appears to be of great interest. It is well known that IL-1 stimulates the expression of MT. The effect of IL-1b on the secretion of oxytocin and vasopressin from electrically stimulated rat neurohypophysis has been demonstrated, indicating that IL-1b might be involved in the regulation of the hypophyseal oxytocin and vasopressin [7]. Differently, in the anterior pituitary a limited IL-1b gene expressed has been reported, but a marked mRNA encoding IL-1ra was observed, hypothesizing that IL-1ra may be secreted as a systemic anti-inflammatory hormone in response to IL-1b originated from multiple sources [30]. IL-6, has been identified in the folliculostellate cells of the anterior pituitary gland where we have observed the expression of MT. Thus, MT expression in the pituitary gland might represent a homeostatic factor, as it has been widely demonstrated for other tissues, controlling free Zn in primis and Cu, as well as some neuroimmuno factors, in secundis, and contributing to the physiological regulation of hypophyseal hormone production/secretion, which is deeply alterated with aging, as well as contrasting free radical negative actions. The presence of MT in the hypophysis, as reported in this paper, could thus open a new perspective in a better understanding the role of these proteins. In this connection, a comparison of the level of MT in normal and AD hypophysis as well as in other neurodegenerative diseases is currently in progress in our laboratory. For these reasons we think that it is worthwhile to furtherly explore the expression of MT in the hypophysis as a first step to better understanding its role in this strategic gland in relation to pathogenic events associated with aging processes. References 1. 2.
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