Cell Tiss. Res. 184, 121-132 (1977)
Cell and Tissue Research 9 by Springer-Verlag 1977
Electron Microscopical Study on the Development of the Nerve Supply of the Pituitary Pars intermedia of the Mouse* Rune Jarsk/ir Department of Zoology,Universityof Gothenburg, Gothenburg, Sweden
Summary.The development of the nerve supply of the pituitary pars intermedia (PI) of C 3H mice was studied by electron microscopy. Nerve fibres and terminal structures, most probably adrenergic, first appear in the newborn. The adult innervation pattern is achieved by the end of the first postnatal week. In the adult animal two types of nerve terminals were distinguished; type A (peptidergic or neurosecretory) and type B (adrenergic). The peptidergic fibres were scarce and exhibited no synapse-like contacts. It is suggested that they are of secondary importance in a d i r e c t nervous hypothalamic control of PI function. Type B terminals were found throughout the PI. They formed synapse-like contacts with the glandular cells, indicating that the primary innervation is exerted by adrenergic neurons. An autonomous differentiation of the glandular cells and in the adult a combined direct nervous and neurohumoral control of PI function is suggested.
Key words: Pars intermedia - Mouse - Growth and development Ultrastructure.
Introduction It is well established that the pars intermedia (PI) of the hypophysis synthesizes melanocyte stimulating hormone (MSH). In lower vertebrates MSH is involved in the dispersion of melanin granules within the dermal melanophores. Pharmacological and surgical experiments have shown that the hypothalamic regulation of the amphibian PI activity is inhibitory, exerted by a direct aminergic innervation (see Terlou et al., 1974, for review). Send offprint requests to: RuneJarsk/ir, Departmentof Zoology,Fack, S-40033 Gothenburg 33, Sweden
* This investigation was supported by grant No B 2180-026 from the Swedish Natural Science Research Council. The skilful technical assistance of Mrs Ulla Wennerbergis gratefullyacknowledged
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In addition to M S H also adrenocorticotropic hormone (ACTH) (Moriarty, 1973; Naik, 1973; Stoeckel et al., 1973; Moriarty and Moriarty, 1975) and a corticotropin-like intermediate lobe peptide (CLIP) (see Lowry and Scott, 1975) have been demonstrated in the mammalian PI. As m a m m a l s do not possess effectual melanophores, it has been suggested that the PI is involved in functions other than regulation of pigmentation. Its possible connection with the sebaceous, preputial and pineal glands, adrenal cortex and reproductive system have been considered (for review, see Howe, 1973; Moriarty, 1973; Baker, 1974). The exact functional significance of the PI in mammals remains, however, to be clarified. Several investigations have shown that the adult m a m m a l i a n PI is innervated, e.g. in the rat (Ziegler, 1963, Rat; for lit. cf. Diepen, 1962). Application of the FalckHillarp technique has revealed the presence of monoaminergic nerve fibres in different species including the mouse (Fuxe, 1964; Dahlstr6m and Fuxe, 1966; Odake, 1967; Belenky et al., 1970; Baumgarten et al., 1972; Weman and Nobin, 1973). On the basis of their morphology, different types of nerve terminals contacting the parenchymal cells of the PI have been described by electron microscopy (Bargmann et al., 1967; Wittkowski, 1967; Howe and Maxwell, 1968; Vincent and Anand Kumar, 1969; Belenky et al., 1970; Cameron and Foster, 1971 ; Baumgarten et al., 1972; Naik, 1972a, b; Ooki et al., 1973; Weman and Nobin, 1973; Anand K u m a r and Vincent, 1974; Chatterjee, 1974; Lawzewitsch and Monastirsky, 1974). Experimental data indicate that both direct innervation (Hadley et al., 1975; Tilders and Mulder, 1975; Tilders et al., 1975) and hypothalamic inhibitory and/or releasing factors (Taleisnik et al., 1972; Kastin et al., 1973; Schally et al., 1973) are implicated in the regulation of PI function in mammals. As the presence of nerve fibres appears to be a prerequisite for a functioning PI, it is of interest to study their ontogenetic appearance and the establishment of contacts with the parenchymal cells. Electron microscopical studies on this subject are scarce (rabbit, Chatterjee, 1974). The present investigation was undertaken to study the development in mice.
Material and Methods Mice of the C3H strain wereused. Males werecaged with femalesfrom 8 to 8.45 a.m.(Rugh, 1968,p. 45). This day was designatedas day 1 of pregnancy.Accordingto this classificationthe animalswere born on the 20th day of gestation and termed "newborn". The following stages were investigated(number of animals within brackets): 19-dayold embryo (6), newborn (7), 1 day old (4), 2 (3), 3 (3), 4 (4), 5 (4), 6 (4), 7 (4), 14 (3), 21 (3), 28 (3), adults (6). No distinction was made between males and females. Mice were killed by decapitation, adult animals under ether anaesthesia, between 9 and 11 a.m. to avoid possible diurnal variations. Within 2 min the pituitary region was exposed and ice-cold glutaraldehyde(seebelow)cautiouslypoured over the tissue. The median eminence/pituitaryregionwas excised as a unit and immersed for two hours in ice-cold 3 70 glutaraldehyde, purified by activated charcoal (Gillett and Gull, 1972),in 0.1 M cacodylatebuffer with CaC12added, pH 7.2-7.4. All specimens were rinsed in the fixativevehicle for 3 x 10 rain and postfixed for I h in ice-cold 1 ~ osmium tetroxide buffered to pH 7.2-7.4 with the same buffer as above, rapidly dehydrated in acetone and embedded in Epon.
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pD
Fig. 1 a and b. Schematic diagrams of the pars intermedia (PI. dotted area) and adjacent regions in the adult mouse, based on median sagittal a and transverse b sections. The region of the PI in contact with the pars nervosa (PN) is designated the juxta-neural lobe part (1) as contrasted with the surrounding free parts (2). The hypophysial cleft(C) separates the PI from the pars distalis(PD).The rostral zone (R) is not included in this investigation. PT pars tuberalis, ME median eminence, III third ventricle
For the purpose of orientation and routine histology semi-thick(0.5-1 lam) transverse and sagittal sections of different regions of the hypophysial complex were cut with glass knives and stained with toluidine blue. Diagrams of median sagittal and cross sections through the hypothalamus and pituitary gland of the adult mouse are shown in Figure 1 to illustrate the terminology used in this paper. The tissue in front of the rostral limit of the hypophysial cleft (rostral zone of the PI) was not included in this investigation. After removal of the epoxy resin (Mayor et al., 1961)semi-thick(2-4 ~tm)sections were stained with aldehyde fuchsin for the demonstration of neurosecretory substances (Stoeckel et al., 1972). Ultra-thin sections, cut with diamond knives, were mounted on uncoated 100 mesh copper grids; they were stained with uranyl acetate and lead citrate and examined in a Hitachi HS-8 electron microscope operated at 50 kV. The diameters of granular inclusions were measured (to the outside of the membrane) on micrographs enlarged 2.8 x from an initial magnification of 11,000 •
Results
Adult Pars intermedia The PI o f the adult m o u s e comprises different cell types, Most c o m m o n are the M S H - A C T H cells, c o n t a i n i n g secretory granules a n d vesicles o f variable size a n d electron density. They are delimited from the hypophysial cleft by a c o n t i n u o u s layer of flattened n o n - g r a n u l a r cells termed marginal cells. Similarly devoid o f g r a n u l a r inclusions are the stellate cells, whose n a r r o w cytoplasmic processes b r a n c h between g l a n d u l a r cells. ACTH-like cells characterized by smaller electron dense granules are occasionally f o u n d a m o n g M S H - A C T H cells. PI a n d pars nervosa (PN) are separated by a capillary plexus. A t intervals, this plexus is i n t e r r u p t e d a n d the two lobes are connected w i t h o u t i n t e r v e n i n g b a s e m e n t m e m b r a n e s . These c o n n e c t i o n s are here called contact zones. F o r a m o r e detailed account, the reader is referred to E u r e n i u s a n d Jarsk/ir (1975). Based o n m o r p h o l o g i c a l criteria, three different types o f nerve terminals can be distinguished in the PI o f the a d u l t mouse. Type A terminals (Fig. 2a) are similar to those described in the P N o f the m o u s e (Eurenius a n d Jarsk~ir, 1974). They possess electron dense granules with a n average diameter o f 135-140 nm, electron lucent vesicles m e a s u r i n g a b o u t 50 nm, a n d single m i t o c h o n d r i a . These terminals are
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Fig. 2 a-c. Pars intermedia of an adult mouse, a Type A terminals (A) with electron dense granules averaging 135-140 nm. They are scarce and exclusively found in close proximity to the parenchymal basement m e m b r a n e (BM). x 30,800. la Type B terminal (B) with electron dense granules averaging 95 n m in diameter. • 30,800. e Type C terminal (C) devoid of electron dense granules. • 30,800. PS perivascular space
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usually scarce with inter-individual variations. They are exclusively found in a juxta-neural lobe position of the PI, in close proximity to the parenchymal basement membrane (outer basement membrane of the perivascular space of the capillary plexus). Furthermore, they are often observed within the perivascular space and occasionally penetrate, for some distance, into the extensions of the connective tissue space dividing the PI into lobules. These fine structural observations correlate well with the distribution of aldehyde fuchsin-positive substances as revealed under the light microscope (not illustrated). No synapse-like contacts between this type of terminal and the glandular cells were observed. Type B terminals (Fig. 2b) contain electron dense granules, averaging 95 nm (range 75-125 nm), and electron lucent vesicles about 50 nm in diameter. Type C terminals (Fig. 2c) are similar to type B but devoid of electron dense granules. Both B and C types are found throughout the parenchyma in all parts of the PI coincident with the extension of the hypophysial cleft; this constitutes a morphological delimitation of the PI from the pars distalis. Furthermore, they are frequently seen within the perivascular space. In randomly chosen sections, a quantitative evaluation of 250 terminals reveals that types B and C occur in equal numbers. 5-10 ~ of them exhibit synapse-like contacts with glandular cells (MSHACTH as well as ACTH-Iike cells). About 80 ~ of the terminals of type B contain only 1-2 electron dense granules, making a classification somewhat arbitrary. Examination of serial sections revealed that 7 terminals originally classified as type C in the first section did contain electron dense granules in adjacent sections, thus representing type B terminals from a morphological point of view.
Fetal Stage (19-day Old Embryo) Examining the PI in the 19-day old embryo (day before birth) revealed no structures in any part of the lobe which safely could be identified as nerves. In the perivascular space, however, terminals with or without electron dense granules and electron lucent vesicles are frequently observed. Contact zones, as described in the adult animal, are present.
Postnatal Stages In most newborn animals a few axons, lying singly or in small bundles, appear in the juxta-neural lobe region of the PI, but exclusively in the basal (aboral) part where they penetrate between basal cells (prospective stellate cells, see Eurenius and Jarskfir, 1975) and glandular cells. Axonal enlargements (Fig. 3) reside in the same position. They are tentatively identified as nerve terminals as they contain small electron lucent vesicles of the same size as those in nerve terminals of the adult animal (50 nm) but devoid of electron dense granules. Axons and nerve terminals are more abundant close to the contact zones. At this stage, as in all postnatal animals examined, nerve terminals with or without electron dense granules are frequently present in the perivascular space.
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Fig. 3. Pars intermediaof a newbornmouse.At this stage the first terminal structures(7) are seen. They contain vesicular elements and penetrate between glandular cells (GC) and basal cells (BC). BM parenchymal basement membrane, x 30,800
In the 1-day old animal, an increased number of axons and nerve terminals occur in the same regions as described in the newborn. They are also found in deeper parts of the parenchyma close to the marginal cells, and occasionally in the surrounding free parts of the PI. Some terminals contain electron dense granules with an average diameter of about 85 nm. As in the previous stage, axons and nerve terminals appear to be more abundant close to the contact zones. Nerve fibres penetrating both the outer basement membrane of the perivascular space and the PI are occasionally observed. The further development is characterized by an intense penetration of nerves into all parts of the PI. The first synapse-like structures appear in the 5-day old juvenile. By day 7 (Fig. 4) they are as frequent as in adult animals. By the end of the first postnatal week the electron dense granules within the terminals increase in size. While they measured 85 nm during postnatal days 1-6, they attain the adult size (about 95 nm) at day 7 onwards. Furthermore, the frequency of terminals containing electron dense granules increases from about 25 ~ during days 3-6 to about 40 ~ on day 7, i.e. close to that seen in the adult animal (about 50 ~). As judged by the size of the granular inclusions these terminals can be classified as type B.
Terminals of type A were not found before the end of the third postnatal week.
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Fig. 4. Pars intermediaofa 7 day oldjuvenile.An axon terminal(T) liesbetweenglandularcells(GC)and makes synapse-likecontacts (arrows). x 30,800
Discussion
Nerve Types in the Adult Animal Light, fluorescence and electron microscopical investigations of the PI in mammals have revealed the presence of different types of nerve terminals (for references, see Introduction). They can be classified according to the terminology introduced by Bargmann et al. (1967) as peptidergic or neurosecretory (corresponding to type A in this paper), adrenergic (type B) or cholinergic (type C). There seem to be apparent differences between species in the occurrence of different types of nerve terminals, as well as in the density of the innervation pattern and the frequency of synapse-like contacts with the parenchymal cells. In adult mice, two (Belenky et al., 1970) or three (Naik, 1972a, b) types of terminals have been distinguished. The peptidergic terminals are only occasionally found in the PI of this strain of mice. They are variable in number from one animal to the other, being exclusively observed close to the parenchymal basement membrane. They show no synapselike structures. This is consistent with previous electron microscopical observations in the rat (Baumgarten et al., 1972). Aldehyde fuchsin-positive fibres penetrating the
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PI have never been seen in the present material. The same result has been reported for the rat (Rodriguez and Gimenez, 1972). The limited distribution of peptidergic nerve fibres in the mouse argues against their being important in a direct nervous control of PI function in this species. This, however, does not preclude an action of peptidergic nerve fibres via a vascular route. In the mouse the predominant nerve types are those described as adrenergic and cholinergic. They penetrate into all parts of the PI and sometimes make synapselike contacts with the glandular cells. The classification of these terminals based upon morphological criteria alone, and in randomly chosen sections, appears somewhat arbitrary, as already pointed out for the rat by Baumgarten et al. (1972). By using 5-hydroxydopamine as a false transmitter, these authors confirmed the catecholaminergic nature of the nerve terminals, similar to those described as adrenergic in this report. The occurrence of type C nerves in the PI of the mouse remains to be demonstrated, as examination of serial sections reveals that terminals originally classified as cholinergic do contain electron dense granules (although single or few). It is therefore felt that the innervation is predominantly adrenergic. This is strengthened by fluorescent microscopical investigations, revealing a network of adrenergic fibres in the PI of the mouse (Fuxe, 1964; Dahlstr6m and Fuxe, 1966; Odake, 1967; Bj6rklund et al., 1968; Belenky et al., 1970) and other mammals (Loizou, 1971; Baumgarten et al., 1972; Partanen and Rechardt, 1973; Weman and Nobin, 1973). In the rat, at least, these fibres contain dopamine, originating in cell bodies located in the most rostral zone of the arcuate nucleus (Bj6rklund et al., 1973). Recently it has been demonstrated that dopamine exerts an inhibitory action on the secretion of substances from the PI of mammals (Hadley et al., 1975; Tilders and Mulder, 1975; Tilders et al., 1975) in a way similar to that earlier described in amphibians (see Terlou et al., 1974, for review). In view of these findings, the discussion on the development of the nerve supply of the mouse PI, will mainly be a question of inhibitory dopaminergic nerve fibres. It has to be pointed out, however, that the regulation of the PI function might also be influenced by releasing and/or inhibiting factors (Taleisnik et al., 1972; Kastin et al., 1973; Schally et al., 1973) reaching the cells via a neurovascular route.
Fetal Stages The present findings show that thedevelopment of the nerve supply of the mouse PI is a postnatal process. This holds true also for the rat (Svalander, 1974), whereas in the rabbit (Chatterjee, 1974) nerve terminals appeared during late embryonic life. As reported in a previous paper (Eurenius and Jarsk/ir, 1975) the first granulated cells appear in the PI of the 17-day old mouse embryo. Furthermore, Enemar (1963) demonstrated melanophore-expanding activity in the 16-day old embryo, i.e. several days before birth, when the first nerve fibres become visible. The onset of production of active substances and their package into granules is thus independent of the innervation of the PI. In fact, organ culture and/or immunohistochemical techniques indicate that the cytodifferentiationof the rat adenohypophysial anlage is not influenced by the hypothalamus at all (Watanabe et al., 1973; Dupouy and
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Dubois, 1975; Chatelain et al., 1976; Nemesk6ry et al., 1976; Watanabe and Daikoku, 1976).
Postnatal Stages As the first nerve fibres and terminal structures appearing in the PI of the newborn animal contain no electron dense granules, direct classification is not possible. However, the fluorescence microscopical demonstration of nerve fibres in the PI of the newborn mice of the same strain as used in this investigation (Bj6rklund et al., 1968) suggests that they represent developing adrenergic nerves. In the rat, the first fluorescent fibres do not appear until the end of the first postnatal week (Loizou, 1971 ; Partanen and Rechardt, 1973). The reason for this discrepancy is not known, but may simply reflect technical difficulties in visualizing the few fibres present at these early stages. In the PI of the newborn animal, nerve fibres are found in close proximity to the outer basement membrane of the perivascular space. They appear simultaneously along the juxta-neural lobe region, having no preference for the rostral part, which might be expected with respect to their origin in the arcuate nucleus (Bj6rklund et al., 1973). Nerves are more abundant close to the contact zones. The functional significance of these zones during embryonic life is obscure, but in the postnatal animal they may represent a route for nerve fibres penetrating directly from the PN to the PI. On the other hand, nerve fibres and axon terminals are frequently present in the perivascular space, even in the prenatal stage. Occasionally, they can be seen piercing the parenchymal basement membrane, thus indicating an alternative way of penetration. Whether they represent fibres originally present in the perivascular space, or nerves derived from the PN, must await future clarification. Several data presented in this communication are consistent with the view that the end of the first postnatal week represents an important step in the development of the nerve supply of the PI of this strain of mice: a) Axons and terminals are found in all parts of the lobe. b) Synapse-like contacts, first observed in the 5-day old animal, occur in a frequency similar to that seen in the adult (about 6 ~). c) The size of the electron dense granules in the terminals is the same as that of the mature animal (about 95 nm). d) The frequency of nerve terminals containing electron dense granules is about 40 ~, i.e. close to the adult (about 50 ~o). Consequently, the structural basis for a direct hypothalamic influence on PI function exists.
Hypothalamic Control of PI Function As mentioned before, both direct innervation and factors transported via a neurovascular link might be implicated in the regulation of PI function. The lack of a well defined blood supply to the PI in mammals (Wingstrand, 1966; Howe, 1973; Baker, 1974; Porter et al., 1974) may indicate that the hypothalamic control is mediated by way of nerve fibres rather than via the circulation. If so, it is reasonable to assume that every secretory cell should be in close contact with nerve terminals, especially if they are inhibitory in nature.
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A review o f the literature reveals that there seem to be differences between m a m m a l i a n species so far investigated. Thus, the PI o f the cat (Bargmann et al., 1967), ferret (Vincent and A n a n d Kumar, 1969), rabbit ( C a m e r o n and Foster, 1971 ; Chatterjee, 1974) and mink ( W e m a n and Nobin, 1973) appear to be extensively invaded by different types o f nerve terminals making frequent synapse-like contacts with the secretory cells. In rat (Rodriguez and Gimenez, 1972) and m o n k e y (Anand K u m a r and Vincent, 1974), however, only a relatively small p r o p o r t i o n o f the cells seem to be innervated. This might reflect differences not only in the way o f hypothalamic influence, but also in the functioning o f the PI. A l t h o u g h no quantitative evaluation was made in the present study, it is conceivable that the PI o f the mouse is relatively poorly innervated. It is tempting to suppose that in species such as the rat, m o n k e y and mouse, besides a direct nervous influence, factors m a y be conveyed to the PI via diffusion from the capillary plexus interposed between the PI and PN. This would support the concept expressed by Rodriguez and Gimenez (1973) that, during evolution, there is a shift from a predominantly nervous control in most lower vertebrates, to a neurovascular control in mammals. A n y suggestion based u p o n morphological criteria alone is speculative and hampered by the fact that virtually nothing is k n o w n a b o u t the exact function o f the PI in mammals. F o r the adult mouse it is proposed that direct innervation m a y represent a "fast", and neurovascular transmission a "slow", mechanism regulating the PI function.
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Stoeckel, M.E., Porte, A., DeUmann, H.-D.: Selective staining of neurosecretory material in semithin epoxy sections by Gomori's aldehyde fuchsin. Stain Technol. 47, 81-86 (1972) Svalander, C.: Ultrastructure of the fetal rat adenohypophysis. Acta endocr. (Kbh.), Suppl. 188, 1-122 (1974) Taleisnik, S., Tomatis, M.E., Celis, M.E.: Role of catecholamines in the control of melanocytestimulating hormone secretion in rats. Neuroendocrinology 10, 235-245 (1972) Terlou, M., Goos, H.J.Th., van Oordt, P.G.W.J.: Hypothalamic regulation of pars intermedia activity in amphibians. Fortschr. Zool. 22, 117-133 (1974) Tilders, F.J.H., Mulder, A.H.: In-vitro demonstration of melanocyte-stimulating hormone release inhibiting action of dopaminergic nerve fibres. J. Endocr. 64, 63P-64P (1975) Tilders, F.J.H., Mulder, A.H., Sme|ik, P.G.: On the presence of a MSH-release inhibiting system in the rat neurointermediate lobe. Neuroendocrinology 18, 125-130 (1975) Vincent, D.S., Anand Kumar, T.C.: Electron microscopic studies on the pars intermedia of the ferret. Z. Zellforsch. 99, 185 197 (1969) Watanabe, Y.G., Daikoku, S.: Immunohistochemical study on adenohypophysial primordia in organ culture. Cell Tiss. Res. 166, 407-412 (1976) Watanabe, Y.G., Matsumura, H., Daikoku, S.: Electron microscopic study of rat pituitary primordium in organ culture. Z. Zellforsch. 146, 453-461 (1973) Weman, B., Nobin, A.: The pars intermedia of the mink, Mustela vison. Fluorescence, light and electron microscopical studies. Z. Zellforsch. 143, 313-327 (1973) Wingstrand, K.G. : Microscopic anatomy, nerve supply and blood supply of the pars intermedia. In: Harris, G.W., Donovan, B.T. eds., The pituitary gland, Vol. 3, pp. 1-27. London: Butterworth & Co. 1966 Wittkowski, W.: Synaptische Strukturen und Elementargranula in der Neurohypophyse des Meerschweinchens. Z. Zellforsch. 82, 434-458 (1967) Ziegler, B.: Licht-und elektronenmikroskopische Untersuchungen an Pars intermedia und Neurohypophyse der Ratte. Z. Zellforsch. 59, 486-506 (1963)
Accepted July 18, 1977
Cell and Tissue Research 9 by Springer-Verlag 1977
Electron Microscopical Study on the Development of the Nerve Supply of the Pituitary Pars intermedia of the Mouse* Rune Jarsk/ir Department of Zoology,Universityof Gothenburg, Gothenburg, Sweden
Summary.The development of the nerve supply of the pituitary pars intermedia (PI) of C 3H mice was studied by electron microscopy. Nerve fibres and terminal structures, most probably adrenergic, first appear in the newborn. The adult innervation pattern is achieved by the end of the first postnatal week. In the adult animal two types of nerve terminals were distinguished; type A (peptidergic or neurosecretory) and type B (adrenergic). The peptidergic fibres were scarce and exhibited no synapse-like contacts. It is suggested that they are of secondary importance in a d i r e c t nervous hypothalamic control of PI function. Type B terminals were found throughout the PI. They formed synapse-like contacts with the glandular cells, indicating that the primary innervation is exerted by adrenergic neurons. An autonomous differentiation of the glandular cells and in the adult a combined direct nervous and neurohumoral control of PI function is suggested.
Key words: Pars intermedia - Mouse - Growth and development Ultrastructure.
Introduction It is well established that the pars intermedia (PI) of the hypophysis synthesizes melanocyte stimulating hormone (MSH). In lower vertebrates MSH is involved in the dispersion of melanin granules within the dermal melanophores. Pharmacological and surgical experiments have shown that the hypothalamic regulation of the amphibian PI activity is inhibitory, exerted by a direct aminergic innervation (see Terlou et al., 1974, for review). Send offprint requests to: RuneJarsk/ir, Departmentof Zoology,Fack, S-40033 Gothenburg 33, Sweden
* This investigation was supported by grant No B 2180-026 from the Swedish Natural Science Research Council. The skilful technical assistance of Mrs Ulla Wennerbergis gratefullyacknowledged
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In addition to M S H also adrenocorticotropic hormone (ACTH) (Moriarty, 1973; Naik, 1973; Stoeckel et al., 1973; Moriarty and Moriarty, 1975) and a corticotropin-like intermediate lobe peptide (CLIP) (see Lowry and Scott, 1975) have been demonstrated in the mammalian PI. As m a m m a l s do not possess effectual melanophores, it has been suggested that the PI is involved in functions other than regulation of pigmentation. Its possible connection with the sebaceous, preputial and pineal glands, adrenal cortex and reproductive system have been considered (for review, see Howe, 1973; Moriarty, 1973; Baker, 1974). The exact functional significance of the PI in mammals remains, however, to be clarified. Several investigations have shown that the adult m a m m a l i a n PI is innervated, e.g. in the rat (Ziegler, 1963, Rat; for lit. cf. Diepen, 1962). Application of the FalckHillarp technique has revealed the presence of monoaminergic nerve fibres in different species including the mouse (Fuxe, 1964; Dahlstr6m and Fuxe, 1966; Odake, 1967; Belenky et al., 1970; Baumgarten et al., 1972; Weman and Nobin, 1973). On the basis of their morphology, different types of nerve terminals contacting the parenchymal cells of the PI have been described by electron microscopy (Bargmann et al., 1967; Wittkowski, 1967; Howe and Maxwell, 1968; Vincent and Anand Kumar, 1969; Belenky et al., 1970; Cameron and Foster, 1971 ; Baumgarten et al., 1972; Naik, 1972a, b; Ooki et al., 1973; Weman and Nobin, 1973; Anand K u m a r and Vincent, 1974; Chatterjee, 1974; Lawzewitsch and Monastirsky, 1974). Experimental data indicate that both direct innervation (Hadley et al., 1975; Tilders and Mulder, 1975; Tilders et al., 1975) and hypothalamic inhibitory and/or releasing factors (Taleisnik et al., 1972; Kastin et al., 1973; Schally et al., 1973) are implicated in the regulation of PI function in mammals. As the presence of nerve fibres appears to be a prerequisite for a functioning PI, it is of interest to study their ontogenetic appearance and the establishment of contacts with the parenchymal cells. Electron microscopical studies on this subject are scarce (rabbit, Chatterjee, 1974). The present investigation was undertaken to study the development in mice.
Material and Methods Mice of the C3H strain wereused. Males werecaged with femalesfrom 8 to 8.45 a.m.(Rugh, 1968,p. 45). This day was designatedas day 1 of pregnancy.Accordingto this classificationthe animalswere born on the 20th day of gestation and termed "newborn". The following stages were investigated(number of animals within brackets): 19-dayold embryo (6), newborn (7), 1 day old (4), 2 (3), 3 (3), 4 (4), 5 (4), 6 (4), 7 (4), 14 (3), 21 (3), 28 (3), adults (6). No distinction was made between males and females. Mice were killed by decapitation, adult animals under ether anaesthesia, between 9 and 11 a.m. to avoid possible diurnal variations. Within 2 min the pituitary region was exposed and ice-cold glutaraldehyde(seebelow)cautiouslypoured over the tissue. The median eminence/pituitaryregionwas excised as a unit and immersed for two hours in ice-cold 3 70 glutaraldehyde, purified by activated charcoal (Gillett and Gull, 1972),in 0.1 M cacodylatebuffer with CaC12added, pH 7.2-7.4. All specimens were rinsed in the fixativevehicle for 3 x 10 rain and postfixed for I h in ice-cold 1 ~ osmium tetroxide buffered to pH 7.2-7.4 with the same buffer as above, rapidly dehydrated in acetone and embedded in Epon.
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2
pD
Fig. 1 a and b. Schematic diagrams of the pars intermedia (PI. dotted area) and adjacent regions in the adult mouse, based on median sagittal a and transverse b sections. The region of the PI in contact with the pars nervosa (PN) is designated the juxta-neural lobe part (1) as contrasted with the surrounding free parts (2). The hypophysial cleft(C) separates the PI from the pars distalis(PD).The rostral zone (R) is not included in this investigation. PT pars tuberalis, ME median eminence, III third ventricle
For the purpose of orientation and routine histology semi-thick(0.5-1 lam) transverse and sagittal sections of different regions of the hypophysial complex were cut with glass knives and stained with toluidine blue. Diagrams of median sagittal and cross sections through the hypothalamus and pituitary gland of the adult mouse are shown in Figure 1 to illustrate the terminology used in this paper. The tissue in front of the rostral limit of the hypophysial cleft (rostral zone of the PI) was not included in this investigation. After removal of the epoxy resin (Mayor et al., 1961)semi-thick(2-4 ~tm)sections were stained with aldehyde fuchsin for the demonstration of neurosecretory substances (Stoeckel et al., 1972). Ultra-thin sections, cut with diamond knives, were mounted on uncoated 100 mesh copper grids; they were stained with uranyl acetate and lead citrate and examined in a Hitachi HS-8 electron microscope operated at 50 kV. The diameters of granular inclusions were measured (to the outside of the membrane) on micrographs enlarged 2.8 x from an initial magnification of 11,000 •
Results
Adult Pars intermedia The PI o f the adult m o u s e comprises different cell types, Most c o m m o n are the M S H - A C T H cells, c o n t a i n i n g secretory granules a n d vesicles o f variable size a n d electron density. They are delimited from the hypophysial cleft by a c o n t i n u o u s layer of flattened n o n - g r a n u l a r cells termed marginal cells. Similarly devoid o f g r a n u l a r inclusions are the stellate cells, whose n a r r o w cytoplasmic processes b r a n c h between g l a n d u l a r cells. ACTH-like cells characterized by smaller electron dense granules are occasionally f o u n d a m o n g M S H - A C T H cells. PI a n d pars nervosa (PN) are separated by a capillary plexus. A t intervals, this plexus is i n t e r r u p t e d a n d the two lobes are connected w i t h o u t i n t e r v e n i n g b a s e m e n t m e m b r a n e s . These c o n n e c t i o n s are here called contact zones. F o r a m o r e detailed account, the reader is referred to E u r e n i u s a n d Jarsk/ir (1975). Based o n m o r p h o l o g i c a l criteria, three different types o f nerve terminals can be distinguished in the PI o f the a d u l t mouse. Type A terminals (Fig. 2a) are similar to those described in the P N o f the m o u s e (Eurenius a n d Jarsk~ir, 1974). They possess electron dense granules with a n average diameter o f 135-140 nm, electron lucent vesicles m e a s u r i n g a b o u t 50 nm, a n d single m i t o c h o n d r i a . These terminals are
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Fig. 2 a-c. Pars intermedia of an adult mouse, a Type A terminals (A) with electron dense granules averaging 135-140 nm. They are scarce and exclusively found in close proximity to the parenchymal basement m e m b r a n e (BM). x 30,800. la Type B terminal (B) with electron dense granules averaging 95 n m in diameter. • 30,800. e Type C terminal (C) devoid of electron dense granules. • 30,800. PS perivascular space
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usually scarce with inter-individual variations. They are exclusively found in a juxta-neural lobe position of the PI, in close proximity to the parenchymal basement membrane (outer basement membrane of the perivascular space of the capillary plexus). Furthermore, they are often observed within the perivascular space and occasionally penetrate, for some distance, into the extensions of the connective tissue space dividing the PI into lobules. These fine structural observations correlate well with the distribution of aldehyde fuchsin-positive substances as revealed under the light microscope (not illustrated). No synapse-like contacts between this type of terminal and the glandular cells were observed. Type B terminals (Fig. 2b) contain electron dense granules, averaging 95 nm (range 75-125 nm), and electron lucent vesicles about 50 nm in diameter. Type C terminals (Fig. 2c) are similar to type B but devoid of electron dense granules. Both B and C types are found throughout the parenchyma in all parts of the PI coincident with the extension of the hypophysial cleft; this constitutes a morphological delimitation of the PI from the pars distalis. Furthermore, they are frequently seen within the perivascular space. In randomly chosen sections, a quantitative evaluation of 250 terminals reveals that types B and C occur in equal numbers. 5-10 ~ of them exhibit synapse-like contacts with glandular cells (MSHACTH as well as ACTH-Iike cells). About 80 ~ of the terminals of type B contain only 1-2 electron dense granules, making a classification somewhat arbitrary. Examination of serial sections revealed that 7 terminals originally classified as type C in the first section did contain electron dense granules in adjacent sections, thus representing type B terminals from a morphological point of view.
Fetal Stage (19-day Old Embryo) Examining the PI in the 19-day old embryo (day before birth) revealed no structures in any part of the lobe which safely could be identified as nerves. In the perivascular space, however, terminals with or without electron dense granules and electron lucent vesicles are frequently observed. Contact zones, as described in the adult animal, are present.
Postnatal Stages In most newborn animals a few axons, lying singly or in small bundles, appear in the juxta-neural lobe region of the PI, but exclusively in the basal (aboral) part where they penetrate between basal cells (prospective stellate cells, see Eurenius and Jarskfir, 1975) and glandular cells. Axonal enlargements (Fig. 3) reside in the same position. They are tentatively identified as nerve terminals as they contain small electron lucent vesicles of the same size as those in nerve terminals of the adult animal (50 nm) but devoid of electron dense granules. Axons and nerve terminals are more abundant close to the contact zones. At this stage, as in all postnatal animals examined, nerve terminals with or without electron dense granules are frequently present in the perivascular space.
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Fig. 3. Pars intermediaof a newbornmouse.At this stage the first terminal structures(7) are seen. They contain vesicular elements and penetrate between glandular cells (GC) and basal cells (BC). BM parenchymal basement membrane, x 30,800
In the 1-day old animal, an increased number of axons and nerve terminals occur in the same regions as described in the newborn. They are also found in deeper parts of the parenchyma close to the marginal cells, and occasionally in the surrounding free parts of the PI. Some terminals contain electron dense granules with an average diameter of about 85 nm. As in the previous stage, axons and nerve terminals appear to be more abundant close to the contact zones. Nerve fibres penetrating both the outer basement membrane of the perivascular space and the PI are occasionally observed. The further development is characterized by an intense penetration of nerves into all parts of the PI. The first synapse-like structures appear in the 5-day old juvenile. By day 7 (Fig. 4) they are as frequent as in adult animals. By the end of the first postnatal week the electron dense granules within the terminals increase in size. While they measured 85 nm during postnatal days 1-6, they attain the adult size (about 95 nm) at day 7 onwards. Furthermore, the frequency of terminals containing electron dense granules increases from about 25 ~ during days 3-6 to about 40 ~ on day 7, i.e. close to that seen in the adult animal (about 50 ~). As judged by the size of the granular inclusions these terminals can be classified as type B.
Terminals of type A were not found before the end of the third postnatal week.
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Fig. 4. Pars intermediaofa 7 day oldjuvenile.An axon terminal(T) liesbetweenglandularcells(GC)and makes synapse-likecontacts (arrows). x 30,800
Discussion
Nerve Types in the Adult Animal Light, fluorescence and electron microscopical investigations of the PI in mammals have revealed the presence of different types of nerve terminals (for references, see Introduction). They can be classified according to the terminology introduced by Bargmann et al. (1967) as peptidergic or neurosecretory (corresponding to type A in this paper), adrenergic (type B) or cholinergic (type C). There seem to be apparent differences between species in the occurrence of different types of nerve terminals, as well as in the density of the innervation pattern and the frequency of synapse-like contacts with the parenchymal cells. In adult mice, two (Belenky et al., 1970) or three (Naik, 1972a, b) types of terminals have been distinguished. The peptidergic terminals are only occasionally found in the PI of this strain of mice. They are variable in number from one animal to the other, being exclusively observed close to the parenchymal basement membrane. They show no synapselike structures. This is consistent with previous electron microscopical observations in the rat (Baumgarten et al., 1972). Aldehyde fuchsin-positive fibres penetrating the
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PI have never been seen in the present material. The same result has been reported for the rat (Rodriguez and Gimenez, 1972). The limited distribution of peptidergic nerve fibres in the mouse argues against their being important in a direct nervous control of PI function in this species. This, however, does not preclude an action of peptidergic nerve fibres via a vascular route. In the mouse the predominant nerve types are those described as adrenergic and cholinergic. They penetrate into all parts of the PI and sometimes make synapselike contacts with the glandular cells. The classification of these terminals based upon morphological criteria alone, and in randomly chosen sections, appears somewhat arbitrary, as already pointed out for the rat by Baumgarten et al. (1972). By using 5-hydroxydopamine as a false transmitter, these authors confirmed the catecholaminergic nature of the nerve terminals, similar to those described as adrenergic in this report. The occurrence of type C nerves in the PI of the mouse remains to be demonstrated, as examination of serial sections reveals that terminals originally classified as cholinergic do contain electron dense granules (although single or few). It is therefore felt that the innervation is predominantly adrenergic. This is strengthened by fluorescent microscopical investigations, revealing a network of adrenergic fibres in the PI of the mouse (Fuxe, 1964; Dahlstr6m and Fuxe, 1966; Odake, 1967; Bj6rklund et al., 1968; Belenky et al., 1970) and other mammals (Loizou, 1971; Baumgarten et al., 1972; Partanen and Rechardt, 1973; Weman and Nobin, 1973). In the rat, at least, these fibres contain dopamine, originating in cell bodies located in the most rostral zone of the arcuate nucleus (Bj6rklund et al., 1973). Recently it has been demonstrated that dopamine exerts an inhibitory action on the secretion of substances from the PI of mammals (Hadley et al., 1975; Tilders and Mulder, 1975; Tilders et al., 1975) in a way similar to that earlier described in amphibians (see Terlou et al., 1974, for review). In view of these findings, the discussion on the development of the nerve supply of the mouse PI, will mainly be a question of inhibitory dopaminergic nerve fibres. It has to be pointed out, however, that the regulation of the PI function might also be influenced by releasing and/or inhibiting factors (Taleisnik et al., 1972; Kastin et al., 1973; Schally et al., 1973) reaching the cells via a neurovascular route.
Fetal Stages The present findings show that thedevelopment of the nerve supply of the mouse PI is a postnatal process. This holds true also for the rat (Svalander, 1974), whereas in the rabbit (Chatterjee, 1974) nerve terminals appeared during late embryonic life. As reported in a previous paper (Eurenius and Jarsk/ir, 1975) the first granulated cells appear in the PI of the 17-day old mouse embryo. Furthermore, Enemar (1963) demonstrated melanophore-expanding activity in the 16-day old embryo, i.e. several days before birth, when the first nerve fibres become visible. The onset of production of active substances and their package into granules is thus independent of the innervation of the PI. In fact, organ culture and/or immunohistochemical techniques indicate that the cytodifferentiationof the rat adenohypophysial anlage is not influenced by the hypothalamus at all (Watanabe et al., 1973; Dupouy and
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Dubois, 1975; Chatelain et al., 1976; Nemesk6ry et al., 1976; Watanabe and Daikoku, 1976).
Postnatal Stages As the first nerve fibres and terminal structures appearing in the PI of the newborn animal contain no electron dense granules, direct classification is not possible. However, the fluorescence microscopical demonstration of nerve fibres in the PI of the newborn mice of the same strain as used in this investigation (Bj6rklund et al., 1968) suggests that they represent developing adrenergic nerves. In the rat, the first fluorescent fibres do not appear until the end of the first postnatal week (Loizou, 1971 ; Partanen and Rechardt, 1973). The reason for this discrepancy is not known, but may simply reflect technical difficulties in visualizing the few fibres present at these early stages. In the PI of the newborn animal, nerve fibres are found in close proximity to the outer basement membrane of the perivascular space. They appear simultaneously along the juxta-neural lobe region, having no preference for the rostral part, which might be expected with respect to their origin in the arcuate nucleus (Bj6rklund et al., 1973). Nerves are more abundant close to the contact zones. The functional significance of these zones during embryonic life is obscure, but in the postnatal animal they may represent a route for nerve fibres penetrating directly from the PN to the PI. On the other hand, nerve fibres and axon terminals are frequently present in the perivascular space, even in the prenatal stage. Occasionally, they can be seen piercing the parenchymal basement membrane, thus indicating an alternative way of penetration. Whether they represent fibres originally present in the perivascular space, or nerves derived from the PN, must await future clarification. Several data presented in this communication are consistent with the view that the end of the first postnatal week represents an important step in the development of the nerve supply of the PI of this strain of mice: a) Axons and terminals are found in all parts of the lobe. b) Synapse-like contacts, first observed in the 5-day old animal, occur in a frequency similar to that seen in the adult (about 6 ~). c) The size of the electron dense granules in the terminals is the same as that of the mature animal (about 95 nm). d) The frequency of nerve terminals containing electron dense granules is about 40 ~, i.e. close to the adult (about 50 ~o). Consequently, the structural basis for a direct hypothalamic influence on PI function exists.
Hypothalamic Control of PI Function As mentioned before, both direct innervation and factors transported via a neurovascular link might be implicated in the regulation of PI function. The lack of a well defined blood supply to the PI in mammals (Wingstrand, 1966; Howe, 1973; Baker, 1974; Porter et al., 1974) may indicate that the hypothalamic control is mediated by way of nerve fibres rather than via the circulation. If so, it is reasonable to assume that every secretory cell should be in close contact with nerve terminals, especially if they are inhibitory in nature.
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A review o f the literature reveals that there seem to be differences between m a m m a l i a n species so far investigated. Thus, the PI o f the cat (Bargmann et al., 1967), ferret (Vincent and A n a n d Kumar, 1969), rabbit ( C a m e r o n and Foster, 1971 ; Chatterjee, 1974) and mink ( W e m a n and Nobin, 1973) appear to be extensively invaded by different types o f nerve terminals making frequent synapse-like contacts with the secretory cells. In rat (Rodriguez and Gimenez, 1972) and m o n k e y (Anand K u m a r and Vincent, 1974), however, only a relatively small p r o p o r t i o n o f the cells seem to be innervated. This might reflect differences not only in the way o f hypothalamic influence, but also in the functioning o f the PI. A l t h o u g h no quantitative evaluation was made in the present study, it is conceivable that the PI o f the mouse is relatively poorly innervated. It is tempting to suppose that in species such as the rat, m o n k e y and mouse, besides a direct nervous influence, factors m a y be conveyed to the PI via diffusion from the capillary plexus interposed between the PI and PN. This would support the concept expressed by Rodriguez and Gimenez (1973) that, during evolution, there is a shift from a predominantly nervous control in most lower vertebrates, to a neurovascular control in mammals. A n y suggestion based u p o n morphological criteria alone is speculative and hampered by the fact that virtually nothing is k n o w n a b o u t the exact function o f the PI in mammals. F o r the adult mouse it is proposed that direct innervation m a y represent a "fast", and neurovascular transmission a "slow", mechanism regulating the PI function.
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Accepted July 18, 1977
