Immunocytochemistry of the Avian Hypothalamus and Adenohypophysis

INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 103

Immunocytochemistry of the Avian Hypothalamus and Adenohypophysis SHIN-ICHI M l K A M l

Department of Veterinary Anatomy, Faculty of Agriculture, twate University, Morioka 020, Japan 1. Introduction.. .... 11. Anatomy of the Avian Hypothalamus ....................... A. Anterior Hypothalamic (Preopticohypothalamic) Region. ... B. Midhypothalamic (Tuberal) Region. ...................... C. Posterior Hypothalamic (Mamillary) Region ... Ill. Hypothalamic Neurosecretory System. ...................... A. Magnocellular Hypothalamic Neurosecretory System ...... B. Parvocellular Hypothalamic Neurosecretory System.. ..... IV. Median Eminence V. Hypophysial Porta VI. Avian Adenohypo A . General View.. ........................................ B. Morphogenesis of the Adenohypophysis. C. Cytodifferentiation of the Pars Distalis . . . . .: . . . . . . . . . . . . . D. Cytology and lmmunocytochemistry of the Pituitary Cells.. V11. Concluding Remarks ...................................... References ...............................................

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I. Introduction The hypothalamo-hypophysial system of birds has evolved to a high degree of morphological differentiation and functional specialization. It has been the subject of extensive studies with respect to neuroanatomy and neurophysiology of the hypothalamic nuclei and its role in reproductive function. The topography of the avian hypothalamic nuclei has been described in classical neuroanatomical terms by Huber and Crosby (l929), Kappers el al. (1936), Kuhlenbeck (1937), Wingstrand (l951), Crosby and Showers (1969), and Kuenzel and van Tienhoven (1982). van Tienhoven and Juhasz (1962), Karten and Hodos (1967), and Bayle et al. (1974) have published stereotaxic atlantes of the brain of the domestic fowl, domestic pigeon, and Japanese quail. Copyright C>1986 by Academic Pre%. Inc. All right, of reproduction in anv form rererved.

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The neurosecretory material visualized by Gomori's aldehyde fuchsin method has long been a criterion for morphological studies in neuroendocrinology and was confirmed immunocytochemically to be neural lobe hormones. On the other hand, the rostra1 hypothalamus, especially its medial preoptic area, has been shown to be important in the regulation of the release of gonadotropic hormones (Szentagothai ef a / . , 1968; Barry ~t nl., 1973). The problem of localization of the hypothalamic cells that produce gonadotropin-releasing hormone (LHRH) has been the subject of numerous studies (Bons ef al.. 1978; Hoffman et nl., 1978; Sterling and Sharp, 1982; Jozsa and Mess, 1982). Recently, many kinds of neuropeptides including LHRH, thyrotropin-releasing hormone (TRH), corticotropin-releasing factor (CRF), somatostatin, substance P, and opioid peptides have been isolated from the hypothalamus and characterized biochemically. The development of highly sensitive imniunocytochemical techniques has enabled these substances to be detected in tissue sections of the central nervous system in many vertebrates. Some of these peptides are distributed in the superficial layer of the median eminence and may play important roles as neurohormones in the control of pituitary function. The median eminence of birds has distinct anterior and posterior divisions and is supplied with distinct components of axons from different areas of the hypothalamus. preoptic hypothalamic, and tuberal regions. The primary capillary plexus which covers the surface of the median eminence consists of a distinct anterior and posterior capillary plexus, corresponding to the anterior and posterior divisions of the median eminence. These two groups of capillary plexus converge into two groups. anterior and posterior, of portal vessels. The anterior group of portal vessels is mainly distributed in the sinusoids of the cephalic lobe of the pars distalis, whereas the posterior group of portal vessels mainly supplies the sinusoids of the caudal lobe of the pars distalis. The pars distalis of birds consists of well-defined cephalic and caudal lobes which are distinct in their cellular constituents. The cephalic lobe contains adrenocorticotropic hormone (ACTH) cells, thyroid-stimulating hormone (TSH) cells, prolactin (PRL) cells, and gonadotropic (GTH or FSH/LH) cells, while the caudal lobe consists of somatotropic (GH or STH) cells and gonadotropic (GTH) cells. The anatomical relationship between the median eminence and cephalic and caudal lobes suggests the possibility that the function of the cephalic lobe may be controlled by the anterior median eminence. whereas that of the caudal lobe is controlled by the posterior median eminence. Because of this possibility, and also because of the cytological and functional differentiation of these two lobes, it is important to investigate the distribution of neuropeptides in the

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median eminence and the cellular constituents of both lobes of the pars distalis. This review deals first with the immunocytochemical localization and fine structure of neuropeptide-containing neurons in the avian hypothalamus and median eminence and second with the immunocytochemistry of the adenohypophysis of the birds, especially the types of anterior pituitary cells which produce each pituitary hormone and their distributions in the gland.

11. Anatomy of the Avian Hypothalamus

The avian hypothalamus is located ventral to the thalamus and dorsal to the optic chiasma and pituitary gland. It forms the wall and floor of the third ventricle. The avian hypothalamus has been roughly divided into three regions: the anterior or preopticohypothalamic region, the midhypothalamic (tuberal) region, and the posterior hypothalamic (mamillary) region (Crosby and Showers, 1969; Kuenzel and van Tienhoven, 1982). The nomenclature used in this review is adopted from the terminology suggested by Kuenzel and van Tienhoven (1982) who identified a total 19 nuclei and 2 areas within the 3 regions.

A. ANTERIORHYPOTHALAMIC (PREOPTICOHYPOTHALAMIC) REGION Whether the preoptic area is regarded as a part of the diencephalon or is considered to be a part of the telencephalon, this area and the hypothalamus are intimately interrelated in function and must be considered together (Crosby and Showers, 1969). The preoptic area lies at a level immediately rostral to the rostral end of the optic chiasma. Its rostral border is the anterior wall of the third ventricle, while the posterior border is the level of the anterior commissure. The dorsolateral border near the rostral end is clearly marked by the septomesencephalic tract, while the ventral border is the base of the brain and, posteriorly, the optic chiasma. Within the boundary of this region, there are nine nuclei and one hypothalamic area (Kuenzel and van Tienhoven, 1982). They are as follows (Mikami et ul., 1976).

I . The nucleus preopticus medialis of the white-crowned sparrow consists of scattered nerve cells located in the preoptic area that extend between the septomesencephalic tract and the preoptic recess. These cells are larger than those of other rostral parvocellular nuclei; they are polygonal in shape and provided with distinct axons. The perikarya are

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rich in polysomes, granular endoplasmic reticulum, polymorphic mitochondria, lysosomes, and well-developed Golgi apparatus. Dense granules, 130-150 nm in diameter, are frequently observed within the Golgi area. 2. The n. preopticus dorsolateralis consists of elongated small neurons scattered along the medial side of the septomesencephalic tract. The neurons in this nucleus are not numerous but contain sparse large dense granules, 150-200 nm in diameter. 3. The n . preopticus periventricularis of the white-crowned sparrow consists of a small group of cells arranged on both sides of the rostral end of the supraoptic recess of the third ventricle. The neurons are somewhat larger than those of the suprachiasmatic nucleus but smaller than those of the supraoptic nucleus. The perikarya are round or polygonal and contain rich free ribosomes, granular endoplasmic reticulum, small rounded mitochondria. and moderately developed Golgi apparatus. Occasionally, neurons containing large, dense granules, 150-200 nm in diameter, are observed in the vicinity of the ventricle (Fig. I ) . 4. The n. magnocellularis preopticus is the most rostral cluster of the neurosecretory cells and is usually located in the preopticohypothalamic transitional area along the ventral surface of the brain. It may be the rostral extension of the n. supraopticus. 5. The n. rnagnocellularis supraopticus consists of at least three main groups of cells: ( 1 ) medial, (2) ventral, and (3) lateral divisions. In the white-crowned sparrow, the neurons of the medial division, which are scattered in the periventricular zone of the supraoptic region, are less numerous, smaller in size, and less distinct than those of the preoptic and lateral divisions. The lateral division is the most conspicuous group, consisting of aggregations of Gomori-positive neurons. These neurosecretory cells are large polygonal in form and contain well-developed granular endoplasrnic reticulum. prominent Golgi apparatus, lysosome-like dense bodies, and dense-cored neurosecretory granules, 150-220 nm in diameter. The perikarya are surrounded by fibrous glial processes, dendrites. and axons of other neurons. The axons displaying contact with perikarya contain smaller dense-cored granules 80 nm in diameter, numerous clear vesicles (50 nm in diameter), and neurofilaments. In the chicken and dove, cells having a special staining quality are scattered along the lamina of the periventricular preoptic nucleus and are often aggregated as the n . filiformis (Crosby and Showers, 1969). 6. The n. paraventricularis of birds also consists of widely scattercd neurons. The most rostral group is immediately rostral to the anterior commissure near the organum vasculosum of the lamina terminalis. The most caudal group extends into the area of the dorsomedial nucleus. The

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FIG. 1. Magnocellular neurosecretory neurons (N) in the n. preopticus periventricularis of the Japanese quail, containing well-developed endoplasmic reticulum (ER). Golgi apparatus (G), small mitochondria (M), and numerous secretory granules (Gr). 150-220 nm in

diameter. x 10,OOO.

cells of the lateral group are scattered in the medial area of the lateral forebrain bundle. The perikarya of these neurosecretory neurons contain a well-developed Golgi apparatus, granular endoplasmic reticulum, small mitochondria, and numerous neurosecretory granules, 180-220 nm in diameter. They may be grouped into ventral, median, dorsal, and lateral divisions. 7. The n. suprachiasmaticus (SCN) has been clearly identified in the rostra1 hypothalamus of passerine birds (Oksche and Farner, 1974; Mi-

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kami rt d . , 1976) and in the Japanese quail (Yamada ct d . , 1984).The n. suprachiasmaticus of the white-crowned sparrow is conspicuous as an accumulation of small neurons at each basolateral angle of the third ventricle just dorsal to the optic chiasma. This nucleus is in the same frontal plane as the lateral division of the supraoptic nucleus; however, it extends more caudally. The perikarya of the neurons of the n. suprachiasmaticus are oval or polygonal and are rich in free ribosomes and granular endoplasmic reticulum (Fig. 2). They also contain numerous small mitochondria, a well-developed Golgi apparatus. and a few lysosomes. Some of

FIG.2. Two parvocellular neurons of the suprachiasmatic nucleus of the white-crowned sparrow. showing perikarya with well-developed endoplasmic reticulum (ER). Golgi apparatus (GI. and mitochondria (M).x 10,OOO.

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these perikarya contain numerous coated vesicles and also dense-cored granules approximately 100 nrn in diameter. The neuropil is formed by myelinated and unmyelinated axons, dendrites, and glial processes. Some of the unmyelinated fibers contain dense-cored granules 90-100 nm in diameter and clear vesicles of the 50 nm class. 8 and 9. The n. filiformis and n. anterior (rostralis) hypothalami have been described and identified in Passer domesticits by Crosby and Showers (1969).

B. MIDHYPOTHALAMIC (TUBERAL) REGION The midhypothalamic region is the largest area of the three regions. The rostra1 border is marked .by the anterior comrnissure (dorsally) and optic chiasma and supraoptic decussation (ventrally). The posterior border is estimated by the appearance of the medial marnillary nucleus. The lateral borders are marked by four fiber tracts: the quinotofrontal tract, ansa lenticularis, occipitomesencephalic tract, and lateral forebrain bundle. Six nuclei and one area were found within the boundary of this region by Kuenzel and van Tienhoven (1982). They are as follows.

I . The n. ventromedialis hypothalami (VMN) has been termed the nucleus hypothalamicus posterior medialis in galliforrn birds as used in the atlas of van Tienhoven and Juhasz (1962) and as described by Sharp and Follett (1969a). Crosby and Woodburne (1940) first proposed that ventromedial hypothalamic nucleus replace the older terminology. 2. The n. periventricularis hypothalami (PHN) has been suggested to replace the stratum cellulare internum of the older terminology by Kuenzel and van Tienhoven (1982). 3. The n. inferior hypothalami (IH) may merely be a ventral and posterior continuation of the ventromedial hypothalamic nucleus: however, it is a pertinent term for use in the avian brain, because in the whitecrowned sparrow, it is a distinct nucleus containing unusually large and polygonal nerve cells with multipolar cytoplasmic processes (Mikami ef al., 1975b). In the chicken brain, the inferior hypothalamic nuclei can be distinguished from the ventromedial nuclei by light microscopy. 4. The n. dorsomedialis hypothalami as described by Crosby and Showers (1969) appears at the level of the n. inferior hypothalami in the brain of the fowl. This gray area lies between the dorsal hypothalamic area and ventromedial nucleus, and infundibular nuclei medially and lateral hypothalamic area laterally. 5. The n. infundibularis replaces the n. arcuata and n. tuberis as has already been proposed for mammals as well as for birds (Oehmke, 1968;

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Oksche and Farner, 1974; Mikami ef al., 1975b). In the white-crowned sparrow. the nerve cells in the anterior portion of the basal infundibular nucleus are large and display a well-developed endoplasmic reticulum, Golgi apparatus, large mitochondria, and numerous free ribosomes. They contain a few dense-cored granules, 120 nm in diameter. Axons containing clear 50 nm vesicles and 100-120 n m granules terminate on these cells to form axosomatic synapses (Fig. 4A). In the posterior part of the basal infundibular nucleus, there are "dark" and "clear" types of nerve cells. The large "clear" nerve cells possess a well-developed Golgi apparatus, granular endoplasmic reticulum, and many more dense-cored granules 100 nm in diameter (Figs. 3 and 4B). The coated vesicles and newly formed granules are frequently observed in the Golgi area of these cells. The "dark" neurons also contain dense-cored granules.

c. POSTERIOR HYPOTHALAMIC ( M A M I L L A R YREGION ) This region is characterized by the presence of the mamillary nuclei in birds. The rostra1 border is marked by the medial mamillary (MM) nucleus, while the posterior border is clearly shown by the supramamillary decussation (SMD) which indicates the caudal end of the hypothalamus. Five nuclei are found in the region in addition to three nuclei which are extended from the tuberal region: ( I ) n. mamillaris medialis, (2) n. mamillaris lateralis, (3) n. premamillaris, (4)n. intercalatus hypothalami, and ( 5 ) n. supramamillaris interstitialis (Kuenzel and van Tienhoven, 1982). 1 . The n. mamillaris medialis is clearly evident in birds. A mamillary body is distinct in the brain of the fully grown chicken. I t shows a secondary subdivision into a more medial nucleus consisting of medium-sized nerve cells, and a more lateral part, containing darkly stained large cells. 2. The latter part may correspond to the n. mamillaris lateralis. 3. The n. premamillaris lies dorsal to the medial mamillary nucleus, but is separated from it by fascicles of the supramamillary decussation. 4. The n. intercalatus hypothalami is a separate group of more deeply 5tained cells.

111. Hypothalamic Neurosecretory System

The concept of neurosecretion emerged largely from light and electron microscopic. histochemical, and physiological studies of magnocellular hypothalamic neurons that form a hypothalamo-hypophysial system, which was established early as the system that secretes the neural lobe

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FIG.3. Ependymal (Ep), glial (GI), and subependymal cells (N) in the posterior part of the basal infundibular nucleus of the white-crowned sparrow. The subependymal neurons (N) contain well-developed organelles and a few dense secretory granules. x 5000.

FIG.4. ( A , B ) Subependymal parvocellular neurons ( N ) in the anterior (A) and posterior

( B ) parts of the infundibular nucleus of the white-crowned sparrow. B is an enlargement of a part of Fig. 3 . These neurons contain well-developed endoplasmic reticulum ( E N . ciolgi apparatus ( G ) .lysosomes ( L ) . and dense secretory granules. 100-120 nm ( A ) or 100 nrn (8) in diameter. x l'i.000.

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hormones, vasopressin and oxytocin. On the other hand, it is now generally accepted that the functions of the anterior pituitary gland are regulated by peptidergic and aminergic elements of the hypothalamus. Recently, many kinds of neuropeptides have been isolated from the hypothalamus and characterized biochemically. The development of highly sensitive immunocytochemical techniques has enabled these substances to be detected in tissue sections. Many kinds of neuropeptides have been localized largely in the parvocellular neurons in the central nervous system of vertebrates. The hypothalamic components that regulate the hypophysial functions may be considered to be two systems: (1) classical aldehyde fushcin (AF)positive magnocellular neurosecretory elements of the hypothalamoneurohypophysial system and (2) parvocellular elements that are capable of formation of the releasing hormones that control the adenohypophysial functions.

A. MAGNOCELLULAR HYPOTHALAMIC NEUROSECRETORY SYSTEM The magnocellular system of the hypothalamus has been an important subject for the study of neurosecretory phenomena for nearly half a century. The first peptides isolated and characterized from the neural tissue were the peptide hormones of the pars nervosa, vasopressin, and oxytocin. We now know that the magnocellular neurosecretory system is synonymous with the neural lobe hormone secretory system. The magnocellular hypothalamic neurosecretory system of birds is different from that of lower vertebrates but resembles that of reptiles and mammals, in that the original preoptic nucleus (PON) has become divided into three main groups, magnocellular preoptic (PON), supraoptic (SON), and paraventricular (PVN) nuclei. The AF-positive neurosecretory cells of birds are generally rather diffusely scattered in the preoptico-hypothalamic transitional area and in the rostral hypothalamus. The localization of the magnocellular hypothalamic nuclei of birds has already been described. The distribution of the vasotocin and mesotocin system will be described below. Vasotocin and Mesotocin System The topography of the vasotocin and mesotocin system has been described in several species of birds (Goosens et al., 1977; Gabrion et al., 1978; Bons, 1980). Goosens et af. (1977) divided the supraoptic nucleus of the starling into five divisions: ( I ) the anterior division consisting almost exclusively of vasotocin cells, (2) the external division consisting of vasotocin cells, (3) the ventral division, containing the most rostral large vaso-

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tocin cells and caudally extended small vasotocin cells, (4) the internal group, consisting of vasotocin and mesotocin cells, and ( 5 ) the lateral group, consisting predominantly of mesotocin cells. and divided the paraventricular nucleus (PVN) into three groups: ( I ) the ventral group (mesotocin and vasotocin cells), (2) the median and lateral group (mesotocin cells), and (3) the external and dorsal divisions (mixed cells and scattered cells between the entopeduncular tract and lateral forebrain bundle). Bons (1980) also identified separate mesotocin- and vasotocin-producing neurons in the anterior preoptic region and at different levels of the SON and PVN of the mallard and Japanese quail. In the Japanese quail. vasotocin-immunoreactive perikarya are distributed in the SON and PVN. They are classic magnocellular neurosecretory cells stained with Gomori's AF staining. The SON consists of four main groups of cells-preoptic, medial, ventral, and lateral divisions. The preoptic division is the most rostra1 cluster of the vasotocin-reactive cells and is the same nuclei as n. magnocellularis preopticus. The vasotocin neurons of the median divisions are scattered in the periventricular zone extended from the medial preoptic area rostrally to the anterior end of the PVN caudally (Fig. 5A and B). The ventral division consists of a large aggregation of vasotocin-immunoreactive neurons located along the ventral surface of the hypothalamus between the lateral forebrain bundle and the optic chiasma and extends caudally to continue to the cell group of the entopeduncular division. The vasotocin-reactive cell groups of PVN are divided into four divisions-ventral, medial, dorsal, and lateral divisions. These vasotocin-immunoreactive perikarya project axons to the median eminence and neural lobe through the supraoptico-hypothalamic tract. Vasotocin-immunoreactive fibers pass to the neural lobe through the internal layer of the median eminence, where they branch off the fibers to the external layer of the anterior median eminence (Fig. 13B). Mesotocin-immunoreactive perikarya also occur in the SON and PVN, although they are fewer in number than vasotocin cells. Mesotocin neurons also project axons to the neural lobe, but not to the external layer of the anterior median eminence (Fig. 13A).

B. PARVOCELLULAR HYPOTHALAMIC NEUROSECRETORY

SYSTEM

The hypothalamus of birds encompasses several conspicuous nuclei with a mosaic-like arrangement. The parvocellular neurons of some of these nuclei contain secretory granules, 100-120 nm in diameter. The parvocellular neurons in the preopticohypothalamic and the infundibular nuclei are involved in hypophysiotropic function. Recently, many kinds of neuropeptides have been found in the hypothalamus. At present,

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FIG.5. (A-D) Adjacent serial sections of the preoptic area of the Japanese quail, stained immunocytochemically with anti-vasotocin (VT)(A,B) and anti-CRF sera (C,D), respectively, B is an enlargement of a part of A , showing vasotocin-immunoreactive neurons in the paraventricular nucleus. D is an enlargement of a part of C, showing CRF-immunoreactive neurons in the medial preoptic nucleus. (A,C) x80, (B) x440, (D) X800.

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neuropeptides. such as LHRH, somatostatin. TRH. CRF, vasoactive intestinal polypeptide. met- and leu-enkephalin, substance, P, CCK, glucagon, and others, have been localized largely in the parvocellular neurons in the septo-preoptico-hypothalamic system in higher vertebrates. Some of these peptides play important roles as neurohormones or releasing factors in the regulation of anterior pituitary function. This section deals with the immunocytochemical localization of hypothalamic neuropeptidecontaining neurons which terminate in the external layer of the median eminence of the Japanese quail. I . LHRH There have been numerous data to support the hypothalamic control of the pituitary gonadal axis in birds. Based on studies of various hypothalamic deafferentation and lesioning, it has been de:ermined that both preoptic and tuberal hypothalamic areas are important in regulating the gonadotropin secretion in the white-crowned sparrow, duck, cockerel. and Japanese quail (Benoit. 1962: Graber er d.,1967: Wilson, 1967; Sharp and Follett. 1969a; Stetson, 1969: Ravona et d . , 1973: Davies and Follett, 1975. 1980). Immunoreactive LHRH fibers were reported for the first time in the median eminence of the duck (Calas et (11.. 1973, 1973. Subsequently. LHRH-containing perikarya have been demonstrated in the anterior hypothalamus and preoptic area of several species of birds (McNeil et a / . , 1976: Bons er d . , 1978; Hoffman er cil., 1978; Sterling and Sharp, 1982; Joz5a and Mess, 1982). Immunoreactive LHRH perikarya have been demonstrated in the preoptico-anterior hypothalamus, septal area, and dorsal region of the infundibular nucleus of the duck by Bons et al. (1978) and of chicken and pheasant by Hoffman ei al. (1978). However, Jozsa and Mess ( 1982) reported that LHRH-immunoreactive perikarya were located in the preoptic and septal areas and in the bulbus olfactorius; however, no LHRH-immunoreactive perikarya were found in the tuberal part of the hypothalamus in the chicken. Sterling and Sharp (1982) also indicated that LHRH-reactive perikarya were thinly scattered in bilateral bands close to the third ventricle extending from the nucleus preopticus paraventricularis magnocellularis, and passing in front of the anterior commissure into the septal area. In the septal area. the perikarya tended to spread out laterally. A few LHRH perikarya were seen in the anterior portion of the nucleus paraventricularis magnocellularis but were not found in the infundibular nuclear complex. Dense accumulations of LHRH-containing fibers have been demonstrated in the external layer of the median eminence, and most of these LHRH fibers were considered to be derived from the perikarya distrib-

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uted in the preoptic-anterior hypothalamus. Bons et ul. (1978) reported that LHRH fibers, originating from the perikarya located in the preoptic nucleus, could be traced through the ventral hypothalamus down to the external layer of the rostra1 and caudal median eminence, in close vicinity to the hypophysial portal system. Sterling and Sharp (1982) indicated that LHRH fiber tracts were seen running dorsoventraliy in the preoptic area apparently associated with the lamina terminalis, and that possible fiber terminals were found in the lamina terminalis and in the external layers of the anterior and posterior divisions of the median eminence. They also found a large number of fibers distributed throughout the infundibular nuclear complex and scattered fibers close to the third ventricle in the anterior hypothalamus. Jozsa and Mess (1982) also indicated that LHRH fibers course from preoptico-septa1 areas toward the median eminence mainly along the wall of the third ventricle in the form of the periventricular network. They also mentioned the presence of two other LHRH fiber tracts, the tractus preoptico-infundibularis and the tractus preopticoterminalis. Avian LHRH is structurally different from mammalian hypothalamic LHRH, arginine in position eight being replaced by a neutral amino acid, glutamine. King and Millar (1979) have synthesized Gln8-LHRH and established that it has identical immunological and biochemical properties to the natural chicken peptide. Recently, Miyamoto et ul. (1984) isolated a second avian gonadotropin-releasing hormone, named chicken GnRH-11, from the chicken hypothalamus and used it to produce anti-chicken GnRH-I1 serum. There have been no immunocytochemical studies as yet using anti-aviari LHRH serum. These antisera have now been used in immunocytochemical studies of LHRH perikarya and fibers in the hypothalamus of the Japanese quail. In the Japanese quail, LHRH-immunoreactive perikarya occur in the nucleus preopticus, nucleus hypothalamicus anterior medialis, and nucleus septalis medialis (Fig. 6A-C). Additional perikarya also occur in the dorsal region of the nucleus infundibularis. Immunoreactive fibers are projected from these perikarya to the median eminence. In the median eminence, LHRH-immunoreactive fibers are distributed in a palisade-like arrangement in the external layer throughout the anterior and posterior median eminence (Figs. 13D and 14A). The LHRH fibers in the median eminence contain LHRH-immunoreactive elementary granules, 75-100 nm in diameter, in their axoplasm. These fibers terminate directly or indirectly on the basement membrane in intimate contact with the primary capillary of the portal vessels. Other fiber terminals are found in the vicinity of the organum vasculosum of the lamina terminalis and in the suprachiasmatic region. The LHRH-producing system of birds consists of

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FIG.6 . (A-C) LHKH-immunoreactive perikarya and fibers in the medial septa1 nucleus in frontal (A.B) and sagittal (C) sections ofrhe Japanese quail. B i s an enlargement of a part of A, \howkg the bipolar LHRH cells with long cytoplasmic procesxh. ( A ) ~ 2 0 0 (I3.C) . x 400.

two producing sites located in the preoptic-anterior hypothalamus and tuberal hypothalamus.

2 . Somatostatin ( S O M ) Somatostatin, isolated from the ovine hypothalamus, inhibits secretion of the pituitary growth hormone. Since the discovery of somatostatin (Brazeau et al., 1973), increasing data have been supplied on its immunocytochemical localization in the central nervous system. The distribution of somatostatin-containing neurons in the hypothalamus of birds has been demonstrated in various species. including duck

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(Blahser et a/., 1978), chicken (Blahser, 1980, 1983). and parakeet (Takatsuki et a/., 1981). Somatostatin neurons locate in the hypothalamus particularly in the mediobasal portion, and project the axons to the neurohema1 area of the median eminence. Somatostatin is thought to act as a neurohormone, which is released into the portal circulation to mediate the somatotropin secretion from the pars distalis. Blahser et ul. (1978) described two main accumulations of SOM cell bodies in the duck: one is extensively distributed in the supraoptic and paraventricular nuclei and the other lies just dorsal to the hypothalamo-hypophysial tract, but they reported no cells in the lateral hypothalamic region. On the other hand, Takatsuki et a/. (1981) reported in the parakeet numerous cell bodies in the lateral hypothalamus, nucleus medialis hypothalamicus posterior, and its caudal part, but not in the preoptic area and periventricular zone. Blahser (1980, 1983) and Takatsuki et al. (1981) also mentioned the dispersed localization of SOM-containing neurons in the telencephalon, thalamus, lateral mesencephalic region, and caudal brainstem. In the hypothalamus of the Japanese quail, three main groups of somatostatin-containing perikarya are observed ( 1 ) a periventricular group distributed in the periventricular area extending from the preoptic nucleus to the paraventricular nucleus, (2) a lateral hypothalamic group, and (3) an anterior infundibular group (Figs. 7A,B,D, and 8A). Widespread localization of perikarya is also found in the telencephalon, thalamus, and brainstem. In the median eminence, dense accumulations of somatostatin-reactive fibers are localized in the external layer of the anterior and posterior divisions (Figs. 7C and 14B). In the anterior division of the median eminence, the bundles of reactive fibers are coarse and protrude into the pars tuberalis, beyond the surface of the median eminence, where they terminate on the wall of the primary portal capillaries. The somatostatin-reactive fibers in the posterior median eminence are more fine and diffusely distributed in the external layer of the median eminence. There are three major pathways of somatostatin fibers: ( 1 ) extending from the lateral hypothalamus and infundibular nuclei to the median eminence, (2) projecting from the preoptic area to the median eminence, and (3) ascending from the brainstem to the infundibular nuclei and median eminence (Fig. 7C). Two types of somatostatin-immunoreactive granules, 80-100 and 150 nm in diameter, in separate axons were demonstrated in the median eminence of the Japanese quail. The localization of somatostatin-reactive perikarya in the hypothalamus overlaps that of neurons containing other peptides such as LHRH, met-enkephalin, substance P, vasotocin, and mesotocin. The coexistence of somatostatin and other peptide-containing neurons suggests a func-

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FIG.7. (A-D) Sagittal (A.B) and frontal (C,D)sections of the basal hypothalamus of the Japanese quaif rhrough the infundibular nucleus (IN) and posterior median eminence (PMEJ. stained immunocytocheniically using anti-somatostatin serum. B is an enlargement of an anterior part of the infundibular nucleu\ in A. showing somatostatin-immunoreactive cells. (A.C) ~ 1 0 0(.B ) x ? S O . ( D ) ~ 6 0 0 .

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FIG.8. (A-C) Electron micrographs of somatostatin (A) and met-enkephalin (B,C) immunoreactive perikarya in the infundibular nucleus of the Japanese quail, stained irnrnunocytochemically using antisera against somatostatin and met-enkephalin before embedding. The reaction product is seen on the granules (Gr) and endoplasmic reticulum (ER), but not on the Golgi apparatus ( G ) . (A) x 16,000, (B,C) x 12,000.

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tional correlation among them to control the function of the neurosecretory system and pituitary gland.

3. Corticotropin-Releasing Fcictor (CRF) CRF was isolated from the ovine hypothalamus and characterized biochemically by Vale and his colleagues (Spiess er al., 1981). The demonstration of the 41-amino acid sequence and synthesis of ovine CRF made possible the preparation of a specific anti-CRF serum and immunocytochemical investigation of CRF in the brain. This peptide has been known to stimulate release of ACTH from the pituitary gland (Vale et al., 1983; Rivier er ml., 1982). The localization of CRF-containing neurons has been demonstrated immunocytochemically in the brain of various mammals. Recently, a CRF-like substance has been demonstrated immunocytochemically in the hypothalamus of the domestic fowl by Jozsa et (11. (19841, who reported CRF-immunoreactive products in cell bodies in the paraventricular, preoptic and mamillary nuclei of the hypothalamus and in the extrahypothalamic area and in fibers of the external layer of the median eminence. In the Japanese quail. CRF-immunoreactive parvocellular perikarya were observed mainly in the nucleus preopticus medialis, nucleus paraventricularis, and nucleus mamillaris of the hypothalamus as well as in the extrahypothalamic nucleus accumbens, nucleus septalis lateralis, and nucleus dorsolateralis thalami. They are oval or spindle-shaped parvocellular neurons densely packed with immunoreactive material. In the SON and PVN, CRF-immunoreactive perikarya intermingle with magnocellular vasotocin and mesotocin neurons, but no CRF immunoreaction was found to coexist with the vasotocin- or mesotocin-containing system (Fig. 5C and D). CRF-immunoreactive fibers are densely located in the external layer of the anterior division of the median eminence, but not in its posterior division. In the anterior median eminence, they occur in a palisade-like arrangement in the external layer and terminate on the basement membrane of the external surface (Fig. 13C). After unilateral adrenalectomy, CRF-immunoreactive material in the external layer of the anterior median eminence decreases remarkably. These results indicate that the CRF neurosecretory system exists in the avian central nervous system and that CRF is released into the anterior group of portal vessels to regulate the release of ACTH from the cephalic lobe of the pars distalis. 4. Methiortine-Enkephalin The pentapeptides, methionine- and leucine-enkephalin first isolated from the brain by Hughes et al. (1975), have been known as modulators of

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pain transmission (Hokfelt et al., 1977; Jossell and Iversen, 1977). A wide distribution of met-enkephalin in the avian central nervous system was recently reported by Blahser and Dubois (1980), De Lanerolle et al. (1981), Mikami (1983), and Blahser (1983). The presence of perivascular enkephalin fiber terminals in the median eminence suggests an involvement of the enkephalin system in the neuroendocrine function of the hypothalamo-hypophysial system. Met-enkephalin-immunoreactive neurons comprise a wide scattered system in the avian central nervous system. In the hypothalamus of the Japanese quail, a large number of met-enkephalin-reactive perikarya are scattered widely from the medial preoptic area to the ventrocaudal part of the paraventricular nucleus (Fig. 9A and B). The perikarya in the ventral part of the paraventricular nucleus are intermingled with the magnocellular neurons of this nucleus. Other groups of met-enkephalin-reactive perikarya are observed in the nucleus hypothalamicus posterior medialis and posterior part of the nucleus infundibularis. Numerous enkephalin-reactive fibers are distributed in the preoptic area, paraventricular nucleus, and infundibular nucleus, where they form a network of fibers surrounding other parvocellular neurons (Fig. 8B and C). In the median eminence, dense accumulations of met-enkephalin-containing fibers are observed in the external layer of both anterior and posterior divisions of the median eminence (Fig. 14C). The met-enkephalin fibers are thicker in the external layer of the anterior median eminence than the posterior division. The presence of perivascular enkephalin fiber terminals in the median eminence implies that enkephalin may be released from the median eminence into the portal circulation and involved in the neuroendocrine function of the hypothalamo-hypophysial system. Deyo et al. (1979) reported that enkephalin increases prolactin (PRL) release by inhibiting dopamine release from nerve terminals in the median eminence, while Romagnano et al. (1982) have postulated that enkephalin fibers in the median eminence can influence pituitary hormone release by interacting directly with fibers containing a releasing factor of pituitary hormone. 5. Substance P Substance P, detected originally by von Euler and Gaddum (1931), has been isolated from the bovine hypothalamus and determined to be an undecapeptide by Chang and Leeman (1970). Substance P-immunoreactive neurons were demonstrated immunohistochemically in the hypothalamus of the primates (Hokfelt et al., 1977), rat (Cuello and Kanazawa, 1978; Ljungdahl el al., 1978), mouse (Stoeckel et al., 1982), opossum and fowl (Ho and De Palatis, I980), pigeon (Reiner et al., 1983), and Japanese quail (Mikami, 1983). In the avian species, Ho and DePalatis (1980) demonstrated substance P in the median eminence of the fowl, and Reiner et

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F I G .9. ( A . B ) Met-enkephalin-immunoreactive perikarya and fibers in the nucleus of 3tria terminalis ( A ) and paraventricular nucleus t B ) of the Japanese quail. (C.D) Substance P-immunoreactive perikarya in the paraventricular nucleus tC) and inferior hypothalamic nucleus (D). ( A ) x 120. (B-D) ~ 6 0 0 .

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al. (1983) in the paleostriatal complex, mainly the lobus parolfactorius, and their projection from these regions to the midbrain tegmentum via the substance P-positive fiber bundles in the medial forebrain bundle. However, they did not mention any substance P-immunoreactive neurons in the hypothalamus. In the Japanese quail, substance P-immunoreactive perikarya are distributed in the lobus parolfactorius, medial portion of the paleostriatum augmentatum, and nucleus of the stria terminalis. In the hypothalamus, substance P-immunoreactive neurons occur in the dorsal portion of the nucleus paraventricularis and nucleus hypothalamicus inferior (Fig. 9C and D). Substance P-reactive perikarya are bipolar or multipolar parvocellular neurons and are scattered dorsoventrally on both sides of the third ventricle. Dense concentrations of substance P-immunoreactive fibers are distributed in the medial forebrain bundle and striohypothalamicus medialis and external layer of the posterior median eminence. In the ventral hypothalamus, substance P fibers are distributed around immunoreactive perikarya and extend to the external layer of the median eminence. In the median eminence, substance P-reactive fibers show a palisadelike arrangement and terminate on the wall of the primary portal capillaries on the external surface (Fig. 14D). These fibers are more abundant in the posterior division of the median eminence than in the anterior division. The distribution of substance P-immunoreactive elements overlaps with that of met-enkephalin in the preoptic area, paraventricular nucleus, tuberal hypothalamus, and median eminence. These facts suggest the possibility that substance P may act on the enkephalin neuronal system and/or act independently on a third neuronal components such as LHRH, somatostatin, VIP, and vasotocin or mesotocin components, as a neurotransmitter or a neuromodulator.

6 . Vasoactive Intestinal Polypeptide (VIP) Vasoactive intestinal polypeptide (VIP) isolated from the porcine intestine (Said and Mutt, 1970) has been widely detected in the central and peripheral nervous system (Larsson et al., 1976; Fuxe et al., 1977). Among avian species, VIP has also been shown to occur in the gastrointestinal tract and pancreas of the turkey (Vaillant ef al., 1980) and in the central nervous system of the Japanese quail (Yamada and Mikami, 1982). In the Japanese quail, numerous VIP-immunoreactive perikarya are distributed in the caudal portion of the nucleus infundibularis and nucleus mamillaris lateralis (Fig. IOA); they are sparse in the preoptic area, nucleus supraopticus, and nucleus paraventricularis. Dense localization of immunoreactive VIP fibers is observed in the

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external layer of the median eminence, in close contact with the primary portal capillaries (Fig. IOA). The main origins of these fiber terminals are VIP-immunoreactive perikarya of the nucleus infundibularis. These neurons are spindle or bipolar and extend one process to the ventricular surface and the other to the external layer of the median eminence (Fig. 10B and C). They are cerebrospinal fluid (CSF)-contacting neurons and

FIG. 10. < A ) Medial basal hypothalamus of the Japanese quail. showing VIP-imrnunoreactive neurons in the nucleus infundibularis ( I N ) and dense accumulation of VIP-reactii c fibers in the external layer o f the posterior median eminence ( P M E ) . ( B . C ) Enlargement of the parts of A . showing VIP neurons. C shows a CSF-contacting neuron extending a process to the third ventricle. ( A ) x 180. (B.CI ~ 8 0 0 .

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apparently constitute the tuberohypophysial tract that links the third ventricle and the hypophysial portal vessels. VIP-reactive neurons in the nucleus mamillaris lateralis also project axons to the external layer of the median eminence, constituting the posterior bundle of the tuberohypophysial tract. Numerous VIP-immunoreactive perikarya also occur in the nucleus accumbens pars posterior close to the lateral ventricle. They are also CSF-contacting neurons extending a process to the lateral ventricle. There are moderate distributions of VIP reactive fibers in the area ventralis and area septalis. Ultrastructurally, the VIP-immunoreactive products are found in the elementary granules, 75-1 15 nm in diameter, within the nerve fibers in the median eminence. 7. Glircugon Gut-type glucagon-like immunoreactivity has been reported in the rat hypothalamus immunohistochemically (Loren et ul., 1979) and radioimmunoassay (Hatton et al., 1982). In the Japanese quail, glucagon-immunoreactive perikarya were demonstrated in the nucleus accumbens in the medial wall of the lateral ventricle. The perikarya,are located in the subependymal layer and project their processes toward the lateral ventricle, seeming to be CSF-contacting neurons (Fig. 1 IA and B). In the hypothalamus, a few immunoreactive perikarya are distributed in the nucleus infundibularis (Fig. 1 1C). They are round- or spindle-shaped parvocellular neurons. They extend processes to the wall of the third ventricle and to the median eminence. Numerous glucagon-like immunoreactive fibers are observed in the preoptic area and in the external layer of both the anterior and posterior median eminence, showing palisade-like arrangements to terminate on the external surface of the median eminence (Fig. IlD). The distribution of the glucagon-like immunoreactive structure overlaps that of VIP-containing neuronal elements. Therefore, it strongly suggests functional correlations between VIP and glucagon in the avian hypothalamus. The presence of glucagon immunoreactivity in the median eminence suggests an involvement of the glucagon system in the neuroendocrine system. The distribution of peptidergic neurons containing vasotocin, mesotocin, LHRH, somatostatin, CRF, met-enkephalin, substance P, VIP, and glucagon in the hypothalamus and their fiber tracts to the median eminence are summarized in Fig. 12, in which peptide neurons in the sagittal plane of the hypothalamus of the Japanese quail are mapped.

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F I G . 1 1 , ( A . B ) A frontal section through the nucleus accumbens of the Japanese quail, showing glucagon-immunoreactive perikarya. which are localized in the subependymal layer and protruding the process into the lateral ventricle (LV). (C) Glucagon-reactive perikarya in the infundibular nucleus. tD) A sagitral section through the median eminence (ME). shohing glucagon-reactive fibers in the external layer. ( A ) ~ 6 2 . 5(.8 ) x 1200. t C ) '* 1000. (D) x 120.

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FIG.12. A sagittal section ofthe brain of the Japanese quail. showing the distributions of eight kinds of peptidergic neurons containing vasotocin (VT). mesotocin (MT), LHRH. somatostatin (SOM), VIP, enkephalin (ENK). substance P (SP), and CRF.

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IV. Median Eminence The median eminence is the ventral region of the floor of the third ventricle and lies between the optic chiasma and infundibular stalk. The median eminence is defined as the part of the postoptic hypothalamic wall that is coextensive with the primary capillary plexus of the hypophysial portal vessels and is often covered by the juxtaneural part of the pars tuberalis. In many birds, the pars tuberalis extends far outside the eminentia and sulcus tubero-infundibularis. The median eminence consists of three layers: an inner ependymal layer, an intermediate fiber layer, and an outer (superficial) palisade layer. The thickness of the median eminence varies considerably among the species. It is rather thin in genera such as Columba and Passerine, and contains a few internal glial cells and the capillaries running on its smooth surface. However, in Anser, it is thick, surrounded by a deep tubero-infundibular sulcus, and contains many free glial cells or pituicytes. In this case the capillaries are buried within deep furrows. The median eminence of birds has distinct anterior and posterior divisions. The anterior division forms a hemispheric hillock in the center of the ventral surface of the hypothalamus between the caudal border of the optic chiasma and a narrow furrow that separates it from the posterior division. The posterior division forms a flat hillock that continues caudad into the infundibulum (Figs. 13A-D and 14A-D). In transverse sections of the median eminence three component layers can be detected. They are referred to as the glandular, fiber, and ependyma1 layers after Wingstrand (1951). Oksche (1962) distinguished two principal zones, an internal zone comprising ependymal and fiber layers and an external zone including reticular and palisade layers. The ependymal cells are nonciliated and constitute a simple epithelial lining of the ventricle. Ependymal processes which are directed toward the external surface traverse the fiber layer, ramify in the palisade layer, and terminate in the conical vascular podia on the basement membrane of the external surface. The palisade or glandular layer contains component fibers of the tuberohypophysial tract, neurosecretory axons, processes from the ependymal and neuroglial cells of the fiber and ependymal layers. The median eminence of birds is supplied with two components of axons. Via the hypothalamo-hypophysial tract it receives axons, more or less extensively stainable by the Gomori method, from the magnocellular neurosecretory cells of the supraoptic. paraventricular, and preoptic regions. I n addition it receives a component of axons from the tuberohypophysial tract. largely, at least, from the nucleus infundibularis, which appears to be the homolog of the arcuate nucleus of mammals.

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FIG. 13. (A-D)Successive sagittal sections of the median eminence of the Japanese quail, showing the distributions of nerve terminals containing mesotocin (A), vasotocin (B), CRF (C), and LHRH (D).AME, Anterior median eminence; PME, posterior median eminence. x90.

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FIG. 14. (A-D) Successive sagittal sections of the median eminence of the Japanese quail, showing the distributions of nerve terminals containing LHRH (A). somatostatin (B). met-enkephalin (C). and substance P (D). respectively. ~ 9 0 .

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The anterior median eminence receives a rich supply of Gomori’s AFpositive fibers from the anterior hypothalamus and some fibers from the tuberal complex, whereas the posterior median eminence receives very few Gomori-positive terminals but a dense tract of fibers from the infundibular nucleus (Oksche and Farner, 1974). Many nerve fibers and glial fibers terminate on the surface of the outer palisade layer of the-median eminence to form its irregular contour. The surface of the posterior median eminence is more irregular in its outline than that of the anterior division. It is invested by a definite basement membrane (Fig. 15). The median eminence of birds presents immunocytochemically a mosaic distribution of peptide hormones and amines (Figs. 13A-D and 14 AD). In the internal zone, vasotocin, mesotocin, and neurophysin fibers pass to the neural lobe. A number of vasotocin fibers branch off from the

FIG.IS. Electron micrograph of the superficial layer of the anterior median eminence of the Japanese quail, showing several kinds of nerve terminals containing different sizes of secretory granules. x 10,OOO.

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internal zone of the anterior median eminence toward the palisade layer of the anterior median eminence and terminate on its surface contacting with the portal capillaries. Vasotocin fibers never appear in the external zone of the posterior median eminence. A number of mesotocin fibers pass through the internal zone to the neural lobe but never branch off to the external zone of the median eminence. In the Japanese quail, CRF-containing fibers are distributed only in the external zone of the anterior median eminence and terminate at the primary capillary plexus of the anterior median eminence. The decrease of CRF from the median eminence after adrenalectomy indicates that CRF may be involved in ACTH secretion from the pars distalis. TRH terminals also appear to be located primarily in the external zone of the anterior median eminence. These distributions suggest a discharge of hormones into the anterior group of portal vessels and appear to support the functional interrelation between the anterior median eminence and the cephalic lobe of the pars distalis. The fibers immunoreactive to enkephalin, substance P, VIP, glucagon, LHRH, and somatostatin are present in the external zone of both the anterior and posterior median eminence. The role of enkephalin and other peptides such as substance P. VIP. and glucagon in the median eminence has not been clarified, but these substances may be discharged into both anterior and posterior groups of the portal vessels and transported to both the cephalic and caudal lobes of the pars distalis. V. Hypophysial Portal Vessels

The anatomy of the vascular system of the avian pituitary has been described by Green (1951). Wingstrand (1951), and Vitums et al. (1964). Vitums et 01. (1964) found that the hypophysial portal vessels of the white-crowned sparrow consist of distinct anterior and posterior groups which drain, respectively. the separate anterior and posterior primary capillary plexus of the median eminence and supply separately the sinusoids of the cephalic or caudal lobe, respectively. This arrangement of the portal vessels has been demonstrated by Sharp and Follett (l969b) in the Japanese quail. and in 12 additional species in 5 orders of birds by Dominic and Singh (1969).The occurrence of distinct anterior and posterior groups of hypophysial portal vessels is regarded as typical of avians by Duvernoy e l a!. (1969). In the birds. the infundibular artery arising from the internal carotid arteries is the sole supply to the primary capillary plexus of the median eminence and to the neural lobe. The primary capillary plexus of the median eminence is largely independent of the neural lobe and also almost entirely isolated from the vascular bed of the rest of the hypothalamus. A

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direct blood supply to the pars distalis does not occur. The median eminence of birds has distinct anterior and posterior divisions and is covered by a very dense capillary plexus that is supplied by the branches of the infundibular arteries. The primary capillary plexus consists of distinct anterior and posterior capillary plexus, corresponding to the anterior and posterior divisions of the median eminence, respectively. The differentiation of the primary capillary plexus correlates well with the arrangement of the neural components in the anterior and posterior divisions of the median eminence. The anterior and posterior capillary plexuses are almost independent of each other and converge into two groups, anterior and posterior, of portal vessels. The anterior group of portal vessels is mainly distributed into the sinusoids of the cephalic lobe of the pars distalis, whereas the posterior group of portal vessels mainly supplies the sinusoids of the caudal lobe of the pars distalis. Therefore, there is good anatomical evidence to support a point to point supply between the median eminence and the pars distalis, so that the blood from the anterior part of the primary capillary plexus of the median eminence passes to the cephalic lobe of the pars distalis, while the blood from the posterior median eminence passes to the caudal lobe of the pars distalis. The anatomical relationship between the median eminence and the cephalic and caudal lobes suggests the possibility that the function of the cephalic lobes may be controlled by the anterior part of the median eminence, whereas that of the caudal lobe is controlled by the posterior median eminence, suggesting point to point regulation. Electron microscopic studies of the avian hypophysial portal vessels were performed by Mikami et al. (1970) in the white-crowned sparrow, which has distinct anterior and posterior divisions of the median eminence and anterior and posterior groups of the portal vessels. They found that the endothelial cells of the portal vessels often protrude into the vascular lumen to give the appearance of a valve-like structure and are invested by a definite basement membrane and by the pericytes which are oriented spirally to the longitudinal axis of the vessels. The presence of the endothelial protrusions and pericytes suggests that they might have a functional role in the regulation of blood flow rate of the portal vessels.

VI. Avian Adenohypophysis A. GENERALVIEW

The hypophysis in all vertebrates consists of an adenohypophysis derived from Rathke’s pouch of the stomodeal ectoderm and a neurohypophysis derived from the infundibular process of the brain floor. In the

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majority of higher vertebrates the adenohypophysis consists of a pars distalis, pars tuberalis, and pars interrnedia, whereas all avian species lack a morphologically distinct pars intermedia. The pars tuberalis in birds, which is derived from the lateral lobe of the original Rathke's pouch. forms a bridge between the median eminence of the hypothalamus and the pars distalis. The pars tuberalis of adult birds consists mostly of chrornophobic cells arranged in a sheath one to four cells thick, through which the portal vessels pass from the surface of the median eminence to the pars distalis. The pars distalis of the avian adenohypophysis consists of well-defined cephalic and caudal lobes which are distinct in their cellular constituents. These two lobes originate from the oral and aboral divisions of Rathke's pouch, respectively, and are histologically distinct. This bilobed nature of the avian pars distalis was described first by Rahn in 1939 in the domestic fowl and then confirmed by Rahn and Painter (1941) and by Wingstrand (1951) in an extensive series of species. In early investigations on pigeons, Schooley and Riddle (1936, 1938) identified only three types of cells. basophils, acidophils, and chromophobes in the pars distalis. By correlation of cytological features with the reproductive cycle, they ascribed gonadotropic activity to the basophils and prolactin secretion to the acidophils. Rahn (1939) differentiated two types of acidophils. deep-staining acidophils and light-staining acidophils, each distributed in the cephalic and caudal lobes, respectively. Rahn and Painter (1941) described the cephalic lobe as containing "chromophobes. basophils, and usually light-staining acidophils." and the caudal lobe as containing "chromophobes, basophils. and deep-staining coarsely granulated acidophils." Payne (1942. 1944) concluded that the two kinds of acidophils, A l and A2, are two distinct types of cells. In 1951. Wingstrand published a monograph on the avian pituitary gland in which he classified the glandular cells of the pars distalis into four types, chromophobes. basophils, dark-staining acidophils (A1 cells). and light-staining acidophils (A2 cells). Wingstrand ( 195 I ) pointed out that mutual mingling of the cells of the two lobes occurs occasionally, but the regional patterns are demonstrable under different physiological conditions and at all ages in all species of birds. The boundary between the cephalic and caudal lobes is indicated only by a restricted distribution of the different types of cells. The boundary between the lobes may be placed arbitrarily as an oblique plane extending from the dorsocentral region of the pars distalis. where it is in contact with the pars tuberalis, in a ventrocaudal direction to the site of contact of the intercarotid anastomosis with the pars distalis. Between the two lobes, there is a shallow furrow in which the cerebral carotid artery and intercarotid anastomosis lie.

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In 1954, Mikami (1954) and Matsuo (1954) found a new type of amphophilic "V" cells which have an affinity for both acid and basic dyes and are PAS positive, and distributed exclusively in the cephalic lobe of the chicken pars distalis. In 1958, Mikami pointed out that the two lobes are different not only in the distribution of two types of acidophils but also in function, 'because the thyroidectomy cells and adrenalectomy cells develop only in the cephalic lobe in the domestic fowl, after thyroidectomy or adrenalectomy, respectively. Tixier-Vidal et al. (1966) confirmed this physiological difference in the domestic mallard under various conditions. Further confirmation came from Brash and Betz (1971) on the basis of transplants of cephalic, middle, and caudal regions of cockerels into chick embryos. On the other hand, Herlant et al. (1960) classified the pituitary cells of the Pekin duck into six types, (Y (GH), 71 (PRL), E (ACTH), p (FSH), y (LH), and 6 (TSH), and suggested their secretory functions. Tixier-Vidal et ul. (1962, 1967, 1968), using Herlant's tetrachrome staining, have differentiated seven types of secretory cells in the hypophysis of male duck and Japanese quail: a (GH) and y (LH) cells in the caudal lobe; /3 (FSH), E (ACTH), and q (PRL) cells in the cephalic lobe; and 6 (TSH) and K (MSH) cells in both lobes. The nomenclature of pituitary cell type had become confusing because of terms based on different terminologies. The introduction of a Greek alphabetical nomenclature was an attempt to avoid such difficulties, but led to further confusion. In 1963, an international committee recommended that the nomenclature for pituitary cells should be functional, and that each cell should be named according to its secretion. Since then, this functional nomenclature has generally been used.

B. MORPHOGENESIS OF THE ADENOHYPOPHYSIS Since Wingstrand (1951) published a detailed monograph on the structure and development of the avian pituitary gland, there have been relatively a few investigations on the development of this gland (Aronsson, 1952; Wilson, 1952; Grignon, 1955; Thommes and Russo, 1959: Hammond, 1970; Mikami ef al., 1973b; Daikoku ef ul., 1974; Franco ef (11.. 1974; Betz and Jarskar, 1974). The hypophysis in all vertebrates consists of two components, an adenohypophysis arising from Rathke's pouch which extends out from the stomodeal ectoderm and a neurohypophysis arising from the infundibular process occurring from the neural ectoderm. In the early state of development, Rathke's pouch is visible as an invagination of the stomodeal epithelium in the midline, and its anterior wall is firmly adherent to the prospective infundibular wall (Fig. 16A and B). Hammond (1970), who studied the early hypophysial development in chick embryos, indicated that the prechordal mesoderm induces the for-

FIG. 16. (A-F) Sagittal sections through the hypophysis of chicken embryos at the fourth ( A . B ) . fifth (C.D). and sixth (E.F) embryonic day, which were stained immunocytochemically using anti-chicken LH serum. ( A ) Rathke’s pouch extended from the stomodeal ectoderm to the base of the infundibulum has a definite posterior process (pp) stained with anti-chicken LH serum. (B) Enlargement of a part of A. showing LH-immunoreactive

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mation of Rathke’s pouch, which is influenced to differentiate through a vascular supply arising from the pial plexus of the infundibular region. Wingstrand (1951) mentioned that at a certain stage of development (6day embryo in Gallus) it is possible to distinguish the following fundamental structures: (1) an oral and an aboral lobe and corresponding dilation of the lumen with the constriction between them, (2) an epithelial stalk, and (3) lateral lobes, the lumen of which is continuous with the oral lumen. The caudal lobe is formed by the proliferation of cells from the aboral lobe, but the pars intermedia does not develop from a contact zone with the infundibular process. The cephalic lobe is formed by the massive proliferation of the oral lobe raising an anterior diverticulum. The pars tuberalis is developed by the proliferation of the lateral lobes which form a layer of an epithelial tissue on the surface of the median eminence. The epithelial stalk is reduced rapidly, sometimes forming residual cysts. The general morphogenesis of the hypophysis of the chicken embryo is as follows. In the 5-day embryo, the floor of the diencephalon consists of 6-1 I layers of ovoid or columnar cells; a ventrad evagination forms the infundibular recess. The boundary between the median eminence and pars nervosa is as yet unclear. Rathke’s pouch extends to the tip of the infundibular process as an elongated structure with a central cavity running through the cell mass. (Fig. 16C). In sagittal sections, Rathke’s pouch shows the form of an anterocaudally elongated leaf and has a narrow constriction between the oral and aboral lobes which develop into cephalic and caudal lobes, respectively. The dorsal surface of the oral and aboral lobes is separated from the diencephalon by a thin layer of mesenchymal cells and by primitive blood vessels. In the 6-day embryo, the presumptive pars distalis is seen as a flat tubular process with its long axis at right angles to the floor of the diencephalon. At this stage, the axis of Rathke’s pouch develops a right-angle bend; thus the aboral lobe lies parallel with the floor of the diencephalon and the oral lobe is perpendicular to it (Fig. 16E and F). In the 8-day embryo, the pars nervosa is apparent and is more compact. During this stage, the oral lobe of Rathke’s pouch develops extensively and takes its position just anterior to the aboral lobe. The cavity of Rathke’s pouch disappears, but both oral and aboral lobes contain broad lumina. There are many periglandular vessels around the pars distalis; some of them form primitive portal vessels on the dorsal side of the gland. The pars distalis displays considerable cellular epithelial cells of the posterior process. (C) Oral (01) and aboral (al) lobes of Rathke’s pouch are separated from each other by a narrow constriction. The epithelial cells of the oral lobe show LH-immunopositive reaction. (D) Enlargement of a part of C. showing LH-immunopositive oral lobe. (E,F) Cephalic (cp) and caudal (cd) lobes of the pars distalis. The cephalic lobe contains many LH-immunoreactive cells. (A,C,E) X80, (B.D) ~ 3 2 0 (F) , x 160.

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FIG.17. (A-F) Sagittal sections through the hypophysis ofchick embryos at the seventh ( A , B ) and ninth (C-F) embryonic day stained immunocytochemically with anti-chicken LH ( A X ) ,anti-porcine ACTH (B,D), anti-rat PRL (E). and anti-rat TSH (F) sera. respectively. y80.

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proliferation after the eighth embryonic day. The oral lobe is more ventrally extended. In the 9-day embryo, the cellular masses of the pars distalis become more compact and lobular (Fig. 17C-F). The glandular cells of the pars distalis usually lie perpendicular to the surface of the lobe and some contain very faint granules. Mitotic figures are very common at this stage. In the 11-day embryo, the caudal lobe of the pars distalis is almost completed in form and is similar to that of the newly hatched chick; it contains many deep-staining acidophils and some basophils. The cephalic lobe is also fully formed and contain acidophils or amphophils in addition to a large number of chromophobic cells.

C. CYTODIFFERENTIATION OF THE PARSDISTALIS The cytodifferentiation of the pars distalis has been studied mainly in the chick embryo but the resuits have varied with the methods and authors. The reported time of appearance of differentiated grandular cells using light microscopy varies for the 6-11 days of incubation, and is somewhat different among investigators and in accordance with the method employed. The earliest, apparently definite, morphologic indication of cytodifferentiation observed by light microscopy seems to be the argirophilic cells reported by Wingstrand (1951) in the oral and aboral lobes of the pars distalis of the 6-day chick embryo. This is consistent with the demonstration of PAS-positive cells on day 6 (Aronsson, 1952; Grignon, 1955). Generally, it has been reported in the chick embryo that acidophils and basophils appear at day 10-11 (Rahn, 1939; Wingstrand, 1951; Grignon, 1953, although the differentiation of acidophils occurs somewhat later-day 15 or at hatching. By day 12 or 14 there are two types of basophils. Electron microscopy has been used for the investigation of the developing pars distalis to establish more precisely the temporal pattern of cytodifferentiation and the onset of secretory activity by Guedenet et al. (1970), Mikami ef al. (1973b), Daikoku el al. (1974), Franco et al. (1974), and Betz and Jarskar (1974). Guedenet et al. (1970) described Golgi apparatus with saccules and detached vesicles containing glycoprotein in the adenohypophysis of the chick embryo as early as day 5 of incubation, and glycoprotein granules in the cytoplasm on day 8. Mikami el al. (1973b)found the first membranebound secretory granules in the cytoplasm of occasional cells in the cephalic lobe of the pars distalis at the seventh day of incubation and many granules variable in form and size in most of the cells in both the cephalic and caudal lobes of 8-day embryos. On the ninth day at least two types of glandular cells are distinguished in the cephalic and in the caudal lobes,

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respectively, and differentiation of acidophils and basophils occurs in the 1 I-day embryo. They observed that the cells of cephalic and caudal lobes are morphologically distinct from their first appearance and concluded that these two lobes develop independently and differently from an early stage of ontogenesis. Immunocytochemical studies on the pituitary cells of the developing chick embryo have been performed by many authors (Ferrand ef af., 1974; Fellman e t al., 1975; Jozsa et al., 1979; Gasc and Sar, 1981; Thommes et al., 1983; Wood et a / . , 1985). However, the data concerning the date of first appearance of hormone-containing cells are very divergent. Also, the accurate distribution of respective types of pituitary cells has not been thoroughly studied, particularly at the early phase of their cytodifferentiation. The earliest sign of functional and morphological differentiation has generally been reported at day 7 of incubation in the chick embryo. Jozsa et trl. (1979) detected prolactin-containing cells on the day 6 of incubation, ACTH-containing cells by day 7, and GH-containing cells on the day 12 in the pituitary of the chick embryo. Recently, Thommes et cil. (1983) demonstrated TSH cells first in the pars distalis of t h e chick embryo on day 6.5 of incubation. As to the differentiation of ACTH cells, Ferrand et al. (1974), using antisera against purified porcine ACTH, offered direct morphological evidence about the appearance of ACTH-containing cells on the ninth day of incubation. However, Fellman et al. ( 1975) demonstrated ACTHimmunopositive cells in the cephalic segment on the eighth embryonic day, using a fluorescent-antibody method. Jozsa et af. (1979) detected ACTH immunoreactivity on the seventh day of incubation using specific monovalent ACTH,-18 antiserum. Gasc and Sar (1981) detected no immunoreactive cells before day 7 of incubation, using antisera raised against ACTH1-24or ACTH7-24.In our unpublished data on the chick embryo, ACTH-immunoreactive cells were first found in small cell groups restricted to the anterior process of the oral (cephalic) lobe on the seventh day of incubation (Fig. 17B). After the ninth day of incubation, the number of ACTH-immunoreactive cells was rapidly increased in the cephalic lobe (Fig. 17D). but no immunoreactive cells were observed in the caudal lobe. Thommes et t i l . ( 1983) demonstrated TSH-immunoreactive cells first in the pars distalis of the chick embryo on day 6.5 of incubation, using antibovine TSH-/3 and anti-human TSH-/3 sera. By day I 1.5, when two lobes of the pars distalis were easily recognized. their TSH-reactive cells were confined exclusively to the rostra1 lobe. In our recent study, immunoreactive TSH cells occurred first in the ventral part of the oral lobe of chick embryo as early as day 7 of incubation. These TSH cells were located in

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the ventral part of the oral lobe and increased gradually in number from day 8 to 10 (Fig. 17F). There was marked increase in the number of immunoreactive TSH cells in late embryonic day, but they were confined exclusively to the cephalic lobe. As to the first appearance of gonadotropes, very little information is available. Gasc and Sar (1981) demonstrated immunoreactive LH cells in the epithelium on the posterior aspect of the Rathke’s pouch in 4-day embryo (stage 23 of Humberger and Hamilton). Wood et a/. (1985) also demonstrated immunoreactive LH cells in the adenohypophysis of both male and female embryos as early as day 4.5 of incubation. They also noted a marked increase in number of LH-producing cells from day 12.5 to 18.5. In our study on chick embryo, LH-irnmunoreactive cells appeared first in the epithelium of the process on the posterior aspect of Rathke’s pouch at fourth day of incubation (Fig. 16A and B.).This process on the caudal aspect of the pouch may correspond to “Diverticolo medio” described by Bruni (1914). On the fifth day of incubation, groups of LH-immunoreactive cells encircled the pouch and appeared in the epithelial folds of the anterior processes of the oral lobe of the pouch (Figs. 16C and D). By day 6 of incubation LH-immunoreactive cells were restricted to the oral lobe (Fig. 16E and F), while on the seventh day of incubation, they increased in number and were located mainly in the oral (cephalic) lobe and a few in the aboral (caudal) lobe (Fig. 17A). On day 8 of incubation, LH-immunoreactive cells increased remarkably in the caudal lobe more than in the cephalic lobe and increased further in both lobes after day 9 of incubation (Fig. 17C). There have been only a few descriptions about the cytodifferentiation of avian FSH cells, because the highly specific avian FSH antiserum has not been available. Jozsa et a / . (1979) tried to demonstrate LH- and FSHcells in the developing chick embryo, but failed to differentiate between LH- and FSH-reactive cells. Wood et al. (1985) demonstrated immunocytochemically LH- and FSH-producing cells in the pars distalis of the chick embryo on day 4.5 of development. They observed LH-immunoreactive cells in the caudal lobe and FSH-imrnunoreactive cells in both lobe of the pars distalis, the greatest concentration of FSH cells in the extreme rostral part of the cephalic lobe. The number of LH and FSH cells increased markedly on day 12.5. In our recent study, using highly specific avian FSH antiserum, FSH-immunoreactive cells appeared first in the ventral aspect of the cephalic and caudal lobes of the %day chick embryo and increased remarkably in the caudal lobe on the tenth day of incubation (Fig. 18E and F). The distribution of FSH-irnmunoreactive cells was very similar to that of LH-reactive cells but not the same. LH-reactive cells were more numerous than FSH-reactive cells before the tenth day of

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incubation, and some of LH cells did not react to the anti-chicken FSH serum. However, after thirteenth day of incubation, the same cells seem to react to both FSH- and LH-antisera. Jozsa ef NI. (1979) identified PRL-immunoreactive cells on the sixth day of incubation, using an antibody against purified chicken PRL. Their PRL-immunoreactive cells showed further increase in number on the fifteenth day of incubation, equally distributed in both lobes of the pars distalis. However, they also noted that their antiserum crossreacts with the other pituitary hormones, except for ACTH, then pituitary cells containing other hormones could also be stained. In our study, using antiturkey PRL serum and anti-rat PRL serum, PRL-immunoreactive cells were first found in antero-dorsal area of the cephalic lobe on the ninth day of incubation (Fig. 17E). However, comparative observation of two adjacent sections revealed that PRL-reactive cells are also reactive to antiACTH serum. Therefore, we could not observe any PRL cells during embryonic day. There have been published no other immunocytochemical data on the onset of PRL-producing cells of the embryonic avian pituitary. Immunoreactive STH(GH) cells are not identifiable in the early embryonic period; it is only after the twelfth day of incubation that a possible reaction is visible in the caudal segment of the gland (Jozsa el ul., 1979). In our recent study, however, GH-immunoreactive cells occurred first in the ventral portion of the caudal lobe on the eighth day of incubation, increased remarkably in later embryonic period and occupied the caudal lobe nearly completely in the newly hatched chicken. N o GH-reactive cells could be found in the cephalic lobe.

D. CYTOLOGY A N D IMMUNOCYTOCHEMISTRY OF

THE

PITUITAR CELLS Y

Knowledge of the cytology of the adenohypophysis and its functional implication has developed rapidly in recent years. Our understanding o f the relationship between the structure and function of the adenohypophysis has been based on the interpretation of results obtained by light and electron microscopic investigations of both normal glands and those from animals subjected to a variety of experimental procedures designed to alter secretory function. The investigation of the function of cell types of the pars distalis inevitably includes two problems. The first is the empirical identification of cell types. The second is the necessarily indirect approach to the identification of function, because the effect of a n y experimental manipulation is never confined to a single cell type. This, of cour-je, opened the way to differences in interpretation and conflicts in opinion.

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However, the recent development of immunocytochemical techniques

for localization of tissue antigens has resulted in great progress in the identification and characterization of the various cell types of the adenohypophysis by means of direct demonstration of hormonal molecules in each type of cell. This section deals with recent progress in adenohypophysial cytology and immunocytochemistry, especially in the types of cells that produce

each of the pituitary hormones and their distribution in the gland (Fig. 18A-F).

FIG. 18. (A-F) Mid-sagittal sections of the hypophysis of the Japanese quail, showing the distribution of pituitary cells, stained immunocytochemically with antisera against porcine ACTH (A), rat PRL (B), rat TSH (C). chicken GH (D), chicken LH (E), and chicken FSH (F), respectively. X30.

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1. ACTH Cell (Cor-ticwtropic Cell)

The cells that secrete ACTH in the avian pars distalis have been localized in the cephalic lobe of the chicken adenohypophysis by Mikami (1956, 1958) by adrenalectomy, as a basis for identification of the ACTH cell. This localization was confirmed in other species of birds by many investigators: e.g., in the duck by Tixier-Vidal (1963) and Tixier-Vidal et a/. (1962), in Japanese quail by Tixier-Vidal et a/. (1968), and in the whitecrowned sparrow by Mikami et 01. (1969) and Haase and Farner (1969, 1971). using classical tinctorial or cytochemical methods. Mikami (19%) called this amphophilic type of cells “V cell,” which is identical with the E cell described later by Tixier-Vidal et (I/. (1968) in the Japanese quail following treatment with metapirone, and with the amphophilic cell of Matsuo et a i . (1969) and butylcholinesterase cell of Haase and Farner (1969. 1971) in the white-crowned sparrow. Electron microscopic studies on ACTH cells have confirmed that they are amphophilic cells containing secretory granules. 250-300 nm in diameter (Harrison, 1978; Mikami. 1969).

lmmunocytochemical studies on avian ACTH cells were performed in the chicken by Dubois (1973), in chick embryos by Ferrand et ul. (19741, Fellman ef (11. ( 1975), and Jozsa er nl. (1979). and in the duck by Marchand et (11. (1974) and Iturriza et a / . (1980), confirming its localization in the cephalic lobe of the pars distalis. Dubois (1973) showed that in Gallus cfomesricirs cells of the cephalic region of the adenohypophysis reacted only with the anti-ACTHI->.,antibody. This was confirmed by Marchand et at. (1974) in Cairinu moschuta; here a single cell type of the cephalic lobe reacted with anti-ACTHI-24as well as with the anti-ACTHI7-3’)antibody . Marchand et ul. (1974) demonstrated that the ACTH cells of Barbary duck. revealed by immunosera anti-ACTH17-19 and anti-ACTHl-24,occur in the cephalic lobe of the pars distalis; these round-shaped cells are cyanophilic, deep PAS positive. but alcian-blue negative and correspond to the F cells of the Pekin-drake and quail described by Tixier-Vidal et ul. (1966, 1968) and ACTH cells in the white-crowned sparrow by Mikami et ul. (1969). lturriza et NI. (1980) established that anti-ACTH1-24.antiACTH,T-,~, and anti-aMSH antibodies label exactly the same cell type located in the rostroventral zone of the cephalic lobe of a duck, Anus p l n t y r h ~ n c ~ k obut s , they never obtained imrnunopositive cells with antibovine aMSH serum. In the Japanese quail, cells binding with anti-porcine ACTHI-39serum are found exclusively in the cephalic lobe. using the PAP complex unlabeled antibody method on preembedding tissue slices and paraffin sections (Fig. 1A). These cells are amphophils stained purple by the ttichrome method in adjacent serial sections (Fig. 19A and B). They are

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round, oval, or columnar in shape and arranged in cell cords along the sinusoid. With regard to morphology, distribution in the gland, and staining affinities, these reacted cells are identical to the V cells, cephalic amphophils, and ACTH cells in the chicken and Japanese quail described by Mikami (1958, 1969) and Mikami et al. (1975a). Immunocytochemical staining with anti-porcine ACTH serum on ultrathin sections reveals that the stain is on the secretory granules, 250300 nm in diameter, in the form of the fine granules or clumps (Fig. 20B). Staining intensity is almost the same in all granules. The staining is not observed on the mitochondria, endoplasmic reticulum, Golgi apparatus, or nuclei, nor on the secretory granules of the other types of cells. The ACTH reactive cells, which are identified in the serial thick sections stained immunocytochemically (Fig. 20A), are characterized by the presence of a large amount of dense, spherical secretory granules ranging in diameter from 250 to 300 nm, less developed endoplasmic reticulum, and small rounded mitochondria. The Golgi apparatus is usually moderately developed and scattered around the nucleus (Fig. 20B). After adrenalectomy, the cells reacting with the anti-ACTH serum are more widely dispersed and less intensely stained than those in control birds. These cells increase in size and number and are transformed into chromophobic adrenalectomy cells, which contain reduced secretory granules and rich granular endoplasmic reticula. The intensity of the immunocytochemical reaction to anti-ACTH serum is more or less reduced in adrenalectomy cells, because of the reduction of secretory granules. The Golgi apparatus is prominent and extremely enlarged, showing a horseshoe shape. These changes in adrenalectomy cells correspond well with our previous observations in adrenalectomized quail (Mikami et a / ., 1975a).

2 . PRL Cell (Prolactin Cell} On the basis of light and electron microscopic studies of the pituitary glands from “lactating pigeons,” Tixier-Vidal and Follett (1973) pointed out that the 7) cells or cephalic acidophils are probably PRL-secreting cells. Mikami et al. (1969) designated a cephalic lobe acidophil as the presumptive PRL cell in the pars distalis of Zonotrichia leucophrys gambelii. Its designation as the PRL cell is strongly supported by the conspicuous increase in the activity and the degranulation of cells of this type in the incubating and brooding female Zonotrichia leucophrys pugetensis. In the Japanese quail, Mikami et al. (1975a) differentiated the PRL cell in the cephalic lobe on the basis of tinctorial properties and its similarities in fine structure to PRL cells described in 2. 1. pugetensis by Mikami et al. ( I973a).

FIG.19. (A-H) Paraffin sections of the cephalic lobe of the pars distalis of the Japanese quail. showing the ACTH cells (A,B), PRL cells (C,D), thyroidectorny cells (E.F). LH cells ( G ) . and TSH cells (HI.B, I),and F are from the sections adjacent to. and matched with those of A, C , and E, respectively. and stained by the trichrome (TC) method. G and H are the same area in the adjacent serial sections. showing that the TSH reactive cells ( H ) also show LH-positive reaction. x IOOO.

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FIG.20. Light (A) and electron (B) microphotographs of ACTH cells in sernithin (A) and thin (B) sections of the cephalic lobe, stained with anti-porcine ACTH serum by the PAP method. The same cells in both sections show ACTH-positive reaction. A, ACTH cell: P, PRL cell; L, LH cell. (A) x 1000, (B) x4000.

Using an anti-ovine PRL antibody, McKeown (1972) revealed PRLpositive cells throughout the cephalic lobe of the pigeon, tending to be more numerous on the periphery of the cephalic lobe. These cells, after staining with Herlant’s tetrachrome, assumed a faint rose color. McKeown (1972) identified these immunoreactive cells with the 7 cells in the nomenclature of Tixier-Vidal and Assenmacher (1966). Also in Colrrtnba livia, Hansen and Hansen (1977) revealed cells reacting with an anti-ovine PRL antibody to be located throughout the cephalic lobe. There was no

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difference in the number of immunoreactive cells between males and females and these cells also exist in the hypophysis of immature pigeons. In ordinary light microscopy, these cells are stained light red or rose by Brooke’s stain and correspond to the q cells of Tixier-Vidal and Assenmacher (1966). Hansen and Hansen (1977) confirmed these observations by an immunocytochernical investigation in electron microscopy; the PRL cells possess either rounded or oval granules of 80-100 nm or rod-shaped granules of 80-500 nm diameter. The reaction product from diaminobenzidine is deposited both in the secretory granules and in the periphery of the cytoplasm. In the Barbary duck (Cairinu nzoscltaru), Marchand et al. (1975). using a synthetic anti-PRL antibody, demonstrated a large number of immunoreactive cells located exclusively in the cephalic lobe of the brooding duck, but histochemical study of these cells revealed that the PRL cells are PAS positive. In the chicken embryo, Jozsa et al. (1979) were able to identify immunoreactive cells in the sixth day of incubation, using an antibody against purified chicken PRL: these roughly granulated cells. showing an intensive immunostaining, were observed along Rathke’s pouch. In the Japanese quail, the PRL-immunoreactive cells are confined exclusively to the cephalic lobe (Fig. 18B). These cells are large, oval, or columnar in shape and are grouped in cell cords surrounded by sinusoids. The round or ovoid nucleus is usually eccentrically situated. The cells contain large granules which are more or less intensely stained with acid fuchsin by the trichrorne method (Fig. 19C and D). These cells contain large, spherical or polymorphic, dense granules, 400-600 nm in diameter (Fig. 21A and C). The granular endoplasmic reticulum is extremely well developed in the form of packed, regularly parallel lamellae close to the plasma membrane. The Golgi apparatus is distinct with a prominent lamellae and vacuolar system. Mitochondria are well developed and elongated. Immunocytochemical staining with anti-rat PRL serum on the ultrathin sections reveals that the stain is on the secretory granules. about 400 nm in diameter. The staining is not observed on the other cytoplasmic organelles. This cell type is very similar in form, granulation. and ultrastructure to the PRL cells described by Mikami er ul. (1973a, 1975a) in the white-crowned sparrow and in the Japanese quail. 3 . TSH Cell (Thyrotropic Cell) Cytological studies of TSH secretory cells in avian hypophysis were made first by Payne (1944). who found hypertrophied thyroidectomy (T) cells at the anterior end and in the ventral portion of the cephalic lobe of thyroidectomized chickens. On the other hand, Morris (1953) suggested that the acidophils are involved in the secretion of TSH, and that T cells

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FIG. 21. (A-C) Light (A,B) and electron (C) micrographs of the same area of the cephalic lobe of the Japanese quail, showing PRL-reactive cells (A), LH-reactive cells (B), and their fine structure (C). PRL cells (P) and LH cells (L) are independent cells showing different distribution and fine structure. (A,B) X 1000, (C) X4000.

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are considered as modified acidophils. Mikami (1955, 1958, 1969) demonstrated that the thyroidectomy cells develop directly from some of the cephalic basophils and are confined to the cephalic lobe of thyroidectomized domestic fowls. In tinctorial and histochemical investigations on another galliform species. Cotiirnix coturnix, Tixier-Vidal et d.(1967, 1968, 1972) identified basophilic TSH cells or 6 cells in both the cephalic and caudal lobes, primarily on the basis of their reaction to thyroxin and thiourea. However, the investigations by Marchand and Bugnon (1972, 1973) on hybrid ducks with thyroidectomy and treatment with propylthiourea led to the conclusion that the TSH cells occur only in the cephalic lobe. lmmunocytochemical study in the avian TSH cells was performed by Sharp et a / . (1979) and Chiasson el d.(1979) who found cells that bind anti-bovine TSH serum exclusively in the cephalic lobe of the pars distalis of the drake. They also found that the immunocytochemically stained cells are more closely packed in the cephalic lobe and seem to be larger but less intensely stained in drakes fed methimazole than in control birds. Recently. Thommes rt al. (1983) demonstrated TSH cells in the cephalic lobe of the chick embryo, using anti-bovine TSH-p and anti-human TSHp sera. In the Japanese quail, the cells that bind anti-rat TSH serum occur exclusively in the cephalic lobe and are designated thyrotropes. They are comparatively large cells with an oval or polygonal shape and are usually located adjacent to the sinusoids. These cells have been identified as cephalic basophils by the comparison of adjacent serial sections stained by the trichrome method. The TSH cells are observed relatively infrequently in the cephalic lobe of normal birds (Figs. 18C and 19H). The ultrastructure of TSH cells. identified in adjacent semithin sections stained immunocytochemically with anti-rat TSH serum, has been studied in adjacent ultrathin sections. The TSH cells contain extremely small (100- I50 nm) dense secretory granules scattered throughout the cytoplasm, small mitochondria, sac-like endoplasmic reticulum, and slightly developed Golgi apparatus. They often contain many lysosomes. In the cephalic lobe of the quail thyroidectomized for 7 days, cells reacting with the anti-rat TSH serum are extremely enlarged and develop into thyroidectomy cells. They are more widely dispersed and occur most abundantly in the peripheral part of the cephalic lobe, often grouping together to form enlarged lobules. The thyroidectomy cells maintain the same intensity of immunoreaction to anti-rat TSH serum, but in the sections stained by the trichrome method they contain vacuolated cytoplasm (Fig. 19E and F). The distribution and response to thyroidectomy of TSHreactive cells are well correlated with the results of Marchand and

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Bugnon (1972, 1973) on hybrid ducks and of Mikami (1958, 1969) and Mikami et al. (1975a) in the fowl and Japanese quail. 4. STH Cell (Somatotropic Cell) Mikami el al. (1969, 1973a, 1975a) designated caudal lobe acidophils as putative STH cells in the white-crowned sparrow, chicken, and Japanese quail, on the basis of comparisons with the light microscopic investigations reported previously. They appear to be identical with ordinary acidophils (deep-staining acidophil of Rahn, 1939; A1 cell of Payne, 1942, and Wingstrand, 1951) which are stained with orange G or azocarmine and are generally considered to produce GH. Ultrastructurally, the “STH cells” of Mikami are similar to the a or somatotropic cells described by TixierVidal et al. (1966, 1968) in the mallard, pigeon, and Japanese quail and to the STH cells described by Danciisiu and Ciimpeanu (1970) and type-VII cells by Harrisson (1978) in the Chinese quail. Tixier-Vidal and Follett (1973), in their review on the adenohypophysis in birds, pointed out that the caudal lobe acidophils or a cells are probably GH-secreting cells, but convincing evidence had not been established. Immunocytochemical studies on the avian STH cells or somatotropes were performed by Marchand et al. (1975, 1976) in the duck, Cairiria moschata, with an anti-human GH antibody, Hansen and Hansen (1977) in the pigeon, Columba liuia, with an anti-bovine GH antibody, and Tai and Chadwick (1977) in the fowl, Callus domesticus, with an anti-chicken GH antibody. Marchand et al. (1975) identified orange G-positive, PASnegative somatotropic cells in the caudal lobe of ducks. Hansen and Hansen (1977) concluded that cells reacting with anti-bovine GH serum are restricted to the caudal lobe of the pigeon and are stained orange with Brooke’s trichrorne stain like the A1 or a cells. Their electron microscopic findings demonstrate that the secretory granules of anti-bovine GH-positive cells measure 200-300 nm. Jozsa et al. (1979) demonstrated immunoreactive somatotropic cells in the chick embryo with an antichicken GH antibody. They are not identifiable in the early embryonic period; it is only after day 12 of incubation that a positive reaction is visible in the area of the caudal segment of the gland. In the Japanese quail, cells binding anti-chicken GH serum are found exclusively in the caudal lobe and are designated as somatotropes (STH cells) (Fig. 18D). They are large, round, or oval in shape and grouped in cell cords surrounded by sinusoids. The STH cells contain large granules, which are intensely stained with orange G by the trichrome method. In electron microscopy of the ultrathin section, STH cells contain dense, spherical granules ranging in diameter from 250 to 300 nm (Fig. 22A and B). The endoplasmic reticulum appears as a series of large dilated sac- or

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FIG. 72. Adjacent serial ultrathin sections of the caudal lobe of the Japanese quail, stained immunocytochemically with anti-chicken G H herum ( A ) or with uranyl acetate-lead citrate ( B ) . S . STH cell; GT. gonadotrope. ~ 4 0 0 0 .

thread-like profiles. The mitochondria are usually large, spherical, or elongated. The Golgi apparatus is not prominent in the granulated cells. Imrnunocytochemical staining with anti-chicken GH serum on the ultrathin section reveals that the stain is on the secretory granules but not on the other organelles. These cells are correlated with caudal acidophils in the domestic fowl and Japanese quail described by Mikami (1969) and

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24 1

Mikami et al. (1975a), the somatotropes in the duck by Marchand et al. (1976), and in the pigeon by Hansen and Hansen (1977). 5. FSH and LH Cell (Gonadoiropic Cell) It has been well established that the secretion of gonadotropic hormones in birds is effected by PAS-positive, basophilic cells (Bhattacharyya and Sarkar, 1969; Mikami, 1955, 1958; Tixier-Vidal et al., 1962). However, the problem of the basophilic gonadotropes is enigmatic. Tixier-Vidal and her associates have produced evidence that there are two types of gonadotropic cells: cephalic lobe beta, the putative FSH cell, and caudal lobe gamma, the putative LH cell. However, the results of this investigation indicate the necessity for some cautions since Mikami ( 1958, 1969) and Mikami et al. (1969, 1975a) found no clear differences between the gonadotropes of the cephalic and caudal lobes in their ultrastructure and physiological responses to the gonadectomy. Mikami et al. (1969, 1975a) have tentatively identified two types of gonadotropic cells, each occurring in both lobes of the pars distalis in the white-crowned sparrow, domestic fowl, and Japanese quail, by their ultrastructure, on the basis of experiments involving castration and photoperiodic stimulation, although, it was not possible to identify kinds of gonadotropic activity by the types of gonadotropic cells. Immunocytochemical identification of gonadotropic cells in birds has been the subject of a small number of works. In 1973, Ravona et al. found that an anti-HCG antibody labeled the PAS-positive cells present in the two lobes of the chicken adenohypophysis. These cells have been identified as gonadotropes (Perek ef al., 1957). In the duck, Marchand ef al. (1975) observed that an anti-HCG antibody bound to cells dispersed in the two lobes of the gland; these cells are of large size, rounded, often in groups, and more numerous in all castrates than in the control. In histochemistry these cells are PAS negative but react intensely with alcian blue. In the Japanese quail, Wada and Asai (1976) identified the LHproducing cells immunocytochemically using anti-chicken LH serum. They described the LH cells as being PAS negative, alcian-blue positive and occurring only in the caudal lobe. In the cephalic lobe, they noted two types of basophils: PAS positive gonadotropic cells, presumably FSH cells, and alcian-blue-positive TSH cells reacting immunocytochemically to anti-chicken LH serum. However, in the Barbary drake, Marchand and Sharp (1977) demonstrated LH immunoreactive cells throughout both lobes using an indirect immunofluorescence technique and an antichicken LH serum. These immunofluorescence cells are alcian-blue-positive, PAS-negative basophils, and contain spherical granules with variable densities and diameters ranging between 40 and 280 nm in the

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cephalic lobe, and between 60 and 260 nm in the caudal lobe. They also mentioned that the anti-LH serum binds to cells that have been classified by other authors in the Pekin duck. quail, and pigeon as TSH producing delta cells in the cephalic lobe. N o immunocytochemical studies have been performed on FSH-producing cells, because avian FSH had not been available. lshii and Sakai (1980) isolated FSH from the chicken adenohypophysis and used this to raise rabbit anti-chicken FSH serum. I have had the opportunity to employ this anti-chicken FSH serum, as well as anti-chicken LH serum, for immunocytochemical studies of the adenohypophysis of the Japanese quail. In the Japanese quail. the cells that bind anti-chicken FSH serum also bind anti-chicken LH serum. The immunocytochemical method shows that antigens reacting with anti-chicken FSH and anti-chicken LH antibodies coexist in the cytoplasm of the same cells. The experimental techniques used in this study did not permit a distinction between FSH- and LH-producing cells (Fig. 18E and F). Therefore, these cells are simply designated as gonadotropes. They are distributed throughout the cephalic and caudal lobes and a few are in the pars tuberalis. Comparative observations of three successive sections treated with anti-chicken FSH. antichicken LH. and anti-rat TSH sera, respectively, show that the antichicken FSH and anti-chicken LH sera are bound to both gonadotropes and TSH cells, while the anti-rat TSH serum only reveals TSH cells located in the cephalic lobe (Fig 19G and H). Thus, this procedure can distinguish between gonadotropes and TSH cells. This fact corresponds to the results obtained by Wada and Asai (1976) and Marchand and Sharp (1977) using anti-chicken LH serum and anti-bovine TSH serum in the Japanese quail and drake. respectively. These FSHILH-reactive cells are basophils stained pale blue by the trichrome method. Under electron microscopy, the FSH/LH-reactive cells contain spherical granules with variable densities and a diameter between 120 and 200 nm (Fig. 21B and C). By the enzyme-labeled antibody (indirect) method on tissue slices of the pars distalis before embedding, secretory granules, 120-200 nm in diameter, react positively to anti-chicken FSH and antichicken LH sera. This corresponds to Mikami’s previous results in the electron microscopic studies on the adenohypophysis of the chicken and the Japanese quail (Mikami, 1969; Mikami et a/., 1975a). After castration, the FSHILH-reactive cells in both lobes increase in size and number and develop into castration cells, which reveal more or less weak immunoreactivity to anti-chicken FSH and anti-chicken LH sera, because of the reduction of secretory granules. These hypertrophied castration cells contain many large vacuoles due to dilation of the cister-

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nae of the endoplasmic reticulum, a large number of mitochondria, welldeveloped Golgi apparatus, and a few dense secretory granules (120-200 nm). There are no noticeable differences in structure of castration cells between the cephalic and caudal lobes. The cytological changes of the FSH/LH-reactive cells after castration further support that they are gonadotropic in function.

VII. Concluding Remarks

The localization of immunoreactive neuropeptides such as vasotocin, mesotocin, LHRH, somatostatin, CRF, met-enkephalin, substance P, VIP, and glucagon in the ventral hypothalamus has been studied by means of immunocytochemistry. Each peptide-producing neuron system represents its own peculiar distribution pattern in the hypothalamus, while many nuclei of the hypothalamus, for example, the medial preoptic, paraventricular, and infundibular nuclei, contain many kinds of neuropeptide-producing neuron systems which show an overlapping or mosaic-like distribution. Also, many kinds of peptidergic fibers form dense networks surrounding other types of parvocellular neurons and are closely intermingled with each other in many nuclei of the hypothalamus. Comparative mapping studies show that gross overlaps occur in the distribution of substance P, enkephalin, somatostatin, and vasotocin neurons, while these overlaps are not due to costorage of the peptides in the same neurons: each peptide occurs in separate neurons and fibers. The structural relationships between different peptidergic systems suggest the presence of functional correlations or interactions a m ~ n g systems. Therefore, the area of overlapping must be considered as a nodal point of information exchange within the central nervous system. In the median eminence, many kinds of peptide-containing fibers show mosaic-like distribution. In the internal zone, vasotocin and mesotocin fibers pass to the neural lobe, forming the supraoptico-hypophysial tract. In the external zone, vasotocin and CRF-containing fibers are distributed exclusively in the external layer of the anterior median eminence. However, other peptidergic fibers containing LHRH, somatostatin, enkephalin, substance P , a d glucagon are distributed in the external layer of both the anterior and posterior median eminence. These peptide-containing fibers terminate on the basement membrane of the external surface of the median eminence in intimate contact with the primary capillary plexus of the portal vessels. Therefore, these peptides must be released into the portal vessels and transported to the pars distalis to control the pituitary

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function. However. some neuropeptides may act as a neuromodulator in the median eminence to control the discharge of other peptides. The pars distalis of birds consists of well-defined cephalic and caudal lobes which are distinct in their cellular constituents. The cephalic lobe contains ACTH cells, TSH cells, PRL cells, and GTH cells, while the caudal lobe consists of GH (STH) cells and GTH cells. The anatomical relationship between the median eminence and cephalic and caudal lobes suggests the possibility of the presence of a "point to point regulation system" in the avian hypothalamo-adenohypophysial system. REFERENCES Aronsson. J. (1952). "Studies on the Cell Differentiation in the Anterior Pituitary of the Chick Embryo by Means of the PAS Reaction." Gleerup. Lund. Barry. J.. Dubois. M. P.. and Poulain. P. (1973). Z. ZelfJbrsch. 146, 351. Bayle. J . D.. Rarnade. F.. and Oliver. J . (1974). J. Pkysiol. (Puris) 68, 219. Benoit. J . ( 1962). Gerr. Corrrp. Endoc~riciol.Sitppl. 1, 154. Betz. T. W.. and Jarskar. R . (1974).Gcn. Cotrip. Eirdocrinol. 22, 366. Bhattacharvya. T. K.. and Sarkar. M. (1969). Acitr Morpkol. A w d . Hw7y. 17. 113. Bllhser. S. (1980). Verlr. Ancrr. G r s . 7 4 775. Blahser. S. ( 1983). I n "Avian Endocrinology. Environmental and Ecological Perspectives" (S. Mikarni. K. Honma. and M. Wada. eds.). pp. 11-24. Jpn. Sci. Soc. Press, Tokyo; Springer-Verlag. Berlin and New York. Blahser. S.. and Dubois. M. P. (1980). Cell Tis.trrr, Reu. 213. 53. Blahher. S.. Fellman. D.. and Bugnon. C. (1978). Cell Tissrre Rrs. 195, 183. Bons. N . (1980). Cell Tissrtr Res. 213, 37. Bons. N.. Kerdelhue. B.. and Assenrnacher. I . (1978). Cell Tissrtr R ~ J188, . 99. Brasch. M.. and Betz, T. W . (1971). Gen. Camp. Endocrinol. 16, 241. Brazeau. P.. Vale. W.. Burgus, R.. Ling. N.. Butcher, M.. River. J.. and Guillemin. R. (1973). Sciet7c.r 179. 77. Bruni. A. C. (1914). I n r . Monnr.tsc.hr..f. Ancrt. P/rysio/. 31, 129. Bugnon. C.. Fellrnann. 0 . . Gouget A,. and Cardot. J. (1982). Nertrosci. Lerr. 30, 2 5 . Calas. A.. Kerdelhue. B.. Assenrnacher. I.. and Julisz. M. (1973). C. R. Acrid. Sci. Paris Ser. D 227, 2765. Calas. A.. Dubois. M. P.. and Assenrnacher. I. (1975). J . Physiol. (Paris) 70, 10 B. Chang, M. M.. and Leeman. S. E. (1970). J . B i d . C/rem. 245. 4784. Chiaszon. R. B.. Radke. W. J.. Sharp. P. J.. El Tounsy. M. M.. and Klandorf. H. (1979). Fed. Proc., Fed. A m . Soc. Exp. B i d . 38,983. Crosby. E. D.. and Showers, M. J . (1969). In "The Hypothalamus" ( W . Haymaker. E. Anderson. and W . J. Nauta. eds.). pp. 61-135. Thomas, Springfield. Illinois. Croshy. E. D.. and Woodburne, R. T. (1940). Res. Ptthl. Assoc.. Rrs. Nrrv. Menr. Dix. 20, 52. Cuello. A. C.. and Kanazawa. I. (1978). J. Conip. Ncrtrol. 178, 129. Daikoku. S., Ikeuchi. C.. and Nakagawa. H. (1974). Gen. Courp. Endocrinol. 23, 256. Dancssiu. M., and Campeanu. L. (1970). Rev. Roum. Endocrinol. 7, 129. Davies. D. T.. and Follett, B. K. (1975). Proc. R . Soc. London Ser. B 191, 28S, 303. Davies. D. T.. and Follett. B. K. (1980). Gen. fotnp. Endocrinctl. 40. 220.

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