Acta histochem. 89, 37-56 (1990) Gustav Fischer Verlag lena

Department of Anatomy, School of Medicine University of Missouri, Columbia, Missouri

Morphological, developmental and immunohistochemical observations on the opossum pituitary with emphasis on the pars intermedia By D. M. SHERMAN and WILLIAM J. KRAUSE With 21 Figures (Received October 20, 1989)

Abstract Development of the pituitary in Didelphis follows the general pattern of that described for most mammalian species. The dorsal region of a multichambered pituitary vesicle, which forms from Rathke's pouch, comes to lie adjacent to the presumptive infundibulum by the IOV2 d of gestation. The epithelial wall of this vesicle consists of spindle-shaped cells. The dorsal wall of the upper chamber of the pituitary vesicle forms the pars intermedia; the ventral wall of this chamber gives rise to cells of the pars distalis. Corticotropes, somatotropes, and lactotropes are seen in the presumptive adenohypophysis of the II Vz d embryo. Gonadotropes and thyrotropes appear about I d later, shortly after birth. By the 2 postnatal week, the adult distribution of all 5 cell types within the pars distalis appears to have been established. The wall bounding the pituitary cleft in the adult represents an epithelial continuum limited by a basal lamina and corresponds to the upper chamber of the original pituitary vesicle. Ultrastructurally, the limiting walls of the pituitary cleft consists of stellate (marginal) cells, large, bulbous cells, and granulated cells. The latter correspond to the various endocrine cell types normally associated with the pars distalis. Non-granular folliculostellate cells also are obsef\Ced within the epithelial cords of the pars distalis. Key words: pituitary, pars intermedia, development, ultrastructure, immunohistochemistry, opossum

1. Introduction The morphological features of pituitary development have been described in a number of eutherian species (NELSON 1933; SANO and SASAKI 1969; SCHECHTER 1970, 1971; FINK and SMITH 1971; DUBOUY and MAGRE 1973; SVALANDER 1974; WEMAN 1974) including man (ANDERSON et aL 1971; IKEDA et aL 1988). The developmental chronology of pituitary cell types has been studied in eutherians by means of immunohistochemistry and the vast majority of these studies have focused on common laboratory rodents, primarily the rat (Dupouy and DUBOIS 1975; TOUGARD et aL 1977; CHATELAIN et aL 1979; DAIKOKU 1973; BEGEOT et aL 1981; CHILDS et aL 1981,1982; CHILDS 1983; SCHWARTZBERG and NAKOME 1982; HEMMING et aL 1983; DIHL et aL 1988; KUROSUMI and TOSAKA 1988; NEMESKERI et aL 1988), and man (ELLIS et aL 1966; BAKER and JAFFE 1975; BAKER and Yu 1979; DUBOIS et aL 1978; BEGEOT et aL 1979) due to the development of appropriate antibodies for these species. The majority of such studies indicate that in the rat ACTH-immunoreactive cells are the first pituitary cells to differentiate (CHATELAIN et aL 1979; WATANABE and DAIKOKU 1979; HEMMING et aL 1983; NEMESKERI et aI., 1988) followed by GH-, PRL-, TSH-, FSH-, and LH-immunoreactive cells which appear at about the same time. The latter are reported to appear on the same day in the rat (15 d) by NEMESKERI et aL (1988). However, the time of appearance may vary as much as 1 to 3 d according to several other studies (TOUGARD et aL 1977; CHATELAIN et al. 1979; WATANABE and DAIKOKU 1979).

38

D. M. SHERMAN and W. J. KRAUSE

There are relatively few descriptive accounts on the morphology of the pituitary in adult marsupials and these have concentrated on macropodids (ORTMAN and GRIESBACH 1958; PURVES and SIRELT 1959; FARMER und PAPKOFF 1974; LEATHERLAND and RENFREE 1982, 1983a). The epithelial component of the adenohypophysis as well as the neurohypophysis has been described in Didelphis virginiana (DAWSON 1938; BODIAN 1963; ROTH and LUSE 1964). Likewise, studies on pituitary development in marsupials are limited to a few Australian species (PARKER 1917; SHOREY 1968; HALL 1977; LEATHERLAND and RENFREE 1983b; WALKER and GEMMELL 1983; HALL and HUGHES 1985; GEMMELL and NELSON 1988) and only one has focused on the North American opossum (WHEELER 1943). Epithelial cells forming the pars distalis of Didelphis are reported to be agranular and undifferentiated at birth. Cells comprising the adenohypophysis of newborn from several Australian marsupial forms, having varying gestational times, contain secretory granules. These cells exhibit additional ultrastructural features suggesting they are active in the synthesis of secretory products (HALL and HUGHES 1985; GEMMELL and NELSON 1988). LEATHERLAND and RENFREE (1983b) have demonstrated several specific cell types in the pituitary of the newborn Tammer wallaby utilizing immunocytochemistry. Such data suggests that pituitary cells of Australian marsupials are capable of elaborating secretory product despite abbreviated gestation periods. This may be important in signaling parturition in marsupials as has been suggested for eutherian species (LIGGINS et al. 1967, 1973; Bosc et al. 1974; CHALLIS et al. 1977; HEAP et al. 1977; RAWLINGS and WARD 1978; THORBURN and CHALLIS 1979) as well as prividing the impetus for migration to the pouch. The present study-re-examines the adult morphology and the development of the adenohypophysis in Didelphis virginiana and extends these observations to the ultrastructural level. The study focuses primarily on the pars intermedia and the cells lining the pituitary cleft. An attempt has been made to identify specific cell types present in the adult as well as those present immediately before and after birth using immunohistochemical probes.

2. Material and Methods Animals: 45 opossums (Didelphis virginiana) divided into the following groups were examined in this study: prenatal animals consisted of lOY" II y" and 12 d embryos; postnatal animals consisted of newborns (known to be less then 6 h old), and animals 4.5, 7.0, 14, 28, 35, 47, and 105 d old. Each developmental stage examined consisted of at least 3 animals. The pituitaries of 6 adults (3 females and 3 males) also were examined. The approximate ages of the prenatal stages used were determined from timed pregnancies. Female opossums were stripped of their litters and placed in breeding pens with continuous access to males. A sperm-positive date was determined by examining smears taken from the urogenital sinus each morning. At specified times after this date, embryos were collected by removing one uterus. The mother was permitted to survive to a pre-determined date when the remaining uterus and embryos were collected. The heads of the prenatal stages were removed and fixed by immersion after first removing some skin to enhance fixative penetration. Pituitaries of the younger postnatal stages were flooded with the appropriate fixative immediately after killing by cervical dislocation and removal of the brain. After a brief period, the pituitaries were dissected free and fixed by immersion for an additional 4 h. Opossums 28 d and older were anesthetized with ether and perfused via the left ventricle with 0.1 molll phosphate buffered saline (PBS) until most of the blood was removed from the animal. The animals were then perfused with either Bouin's solution, 10% buffered formalin or 3.0% glutaraldehyde in phosphate buffer. Following perfusion, the skull cap and brain were removed. The pituitaries were dissected free and placed in the appropriate fixative for a least 4 h. Preparation for light and electron microscopy: Heads of embryos and pituitaries were fixed in toto in either 10% buffered neutral formalin or Bouin's solution for light microscopy and immunohistochemistry. These were processed routinely for paraffin embedding and sections cut at about 6 /-lm. Some sections were stained with either hematoxylin and eosin or Masson's Trichrome. Tissues taken for electron microscopy were fixed in 3.0% glutaraldehyde in 0.1 molll phosphate buffer, pH = 7.4, for 4 h at 4 DC. Following fixation, they were transferred to 0.1 molll phosphate buffer for 2 h and then post-fixed for 2 h in 1.0 % OS04 in 0.1 molll phosphate buffer. After further washes in buffer, the tissues were dehydrated by passing them through a graded ethanol series followed by propylene oxide and then embedded in Epon 812. Thick sections (0.3 to 2.0 /-lm) were cut, stained with toluidine blue, and examined with the light

39

Development of opossum pituitary

microscope. Thin sections of selected regions of this material were then cut for electron microscopic examination. The thin sections (0.5 x 1.0 mm and 60 to 70 nm thick) were picked up on large slot grids with the section being supported in the slot by the surface tension of the water droplet. These sections were stained by transferring the grids to droplets of 2 % uranyl acetate for 1 h, washed by transferring through a series of water droplets, and then stained with Reynold's lead citrate for 3 min. After further washing, the grids supporting the stained sections were placed over carbon-coated formvar films and the sections allowed to dry down on the films prior to examination in a Zeiss 10 CR or Phillips 300 electron microscope operated at 60 kV. Preparation of carbon-coated formvar films: Pre-cleaned (with ethanol) glass slides were placed in 0.25 % formvar in dichloroethane for 20 sec, dried, and lightly coated with carbon in a Denton Vacuum evaporator. The films were immediately floated off onto distilled water, picked up on plexiglass slides into which a series of 5 mm diameter holes had been drilled, and allowed to dry. Grids supporting sections were placed on the films over the holes. The water droplet that supported the section in the grid slot evaporated allowing the Epon section to tightly adhere to the underlying film. This technique allowed large areas of tissue to be viewed ultrastructurally without the interference of grid bars. Immunohistochemistry: Serial paraffin sections of Bouin's fixed material, cut 6 [lm thick and mounted on acidcleaned slides were used for this portion of the study. The sections were deparaffinized and rehydrated to water prior to incubation in I % HzO z to remove endogenous peroxide. After blocking with normal goat serum, sections were incubated with primary antiserum for 24 h at 4°C. Immunohistochemical visualization was carried out using the Vecta-Iab "elite" kit. This method utilized the avidin-biotin-peroxidase complex (ABC) procedure (Hsu et al. 1981). Specific antisera for pituitary hormones used are shown in Table I. Table 1. Antiserum used Anteriserum raised to*

Dilution

Source

human-Growth Hormone (h-GH) rat-Growth Hormone (r-GH) human-Follicle Stimulating (h-FSH) human-Luteinizing Hormone (h-~LH) human-Thyroid Stimulating Hormone

1: 10,000 1: 10,000 I: 10,000 1: 10,000 1: 5,000

DAKO NIDDK DAKO NIDDK NIDDK

1: 5,000 1: 5,000 1: 10,000

NIDDK NIDDK NIDDK

(h-~TSH)

human-prolactin (h-PRL) rat-prolactin (r-PRL) human-Adrenocorticotropic Hormone (h-ACTH)

*All antisera were raised in rabbits except that against rat growth hormone which was raised against monkey. A formalin fixed, paraffin embedded human pituitary also was stained with each antiserum and served as a positive control. Additional controls utilized were omission of primary antisera and pre-absorption of primary antiserum with the corresponding antigen. Following staining, most sections were dehydrated, cleared with xylene and mounted. Some sections were lightly counterstained with Mayer's hematoxylin and then covers1ipped for examination. Serial sections of entire embryos and early postnatal stages, mounted and stained with hematoxylin and eosin, from previous studies (KRAUSE and CUTTS 1985, 1986), were used also. In this instance, selected slides were soaked in xylene to facilitate removal of the coverslips and the sections decolorized in acid-alcohol (0.5 %) for 40 min. They were then stained immunohistochemically using the ABC technique as described above.

3. Results 3.1. Adult Morphology The pituitary of Didelphis consists of the usual epithelial sub-divisions of the adenohypophysis (pars distalis, pars tuberalis, pars intermedia) and an infundibular process [(pars nervosa); (Fig. 1)]. The pituitary is connected to the overlying hypothalamic area by a slender stalk which contains a portion of the infundibular recess. The pars nervosa is somewhat pear-shaped and deeply embedded in the central region of the pars distalis. It extends in an anteroventral direction.

40

D. M.

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KRAUSE

INFUNDIBULAR PROCESS PARS DIS TALIS

(PARS NERVOSA)

. . A TtRIOR

POSTERIOR . .

""""'l.......-

EXTERNAL WAll

I TERNAL WALL (PARS I TERMEDIA) PITUITARY CLEFT

1 Fig. I. A diagram of a mid-sagittal section through the pituitary of an adult opossum illustrates the relationship between the limiting internal and external walls of the pituitary cleft, the pars nervosa, and the pars distalis.

A fairly wide vascular connective tissue space lies between the pars intennedia and the pars nervosa (Figs. 2, 3). The central region of the pars nervosa consists of numerous unmyelinated nerve fibres and represents those fibres of the hypothalamohypophyseal tract. Nerve tenninals originating from these fibres are organized into distinct, well-defined lobules by the surrounding connective tissue and vasculature. They are particularly well developed at the periphery of the pars nervosa (Figs 2, 3). Ultrastructurally, nerve tenninals at the periphery of each lobule are filled with electron-dense neurosecretory granules. A thin pars intennedia envelopes the pars nervosa with the exception of a small posterodorsal area and is separated from the pars distalis by a conspicuous but narrow pituitary cleft. The apical portions of cells comprising the pars intermedia often appear to abut directly against a layer of cells, associated with the pars distalis, that bounds the lateral (external) extent of the pituitary cleft (Fig. 3). The latter consists of an epithelial layer, 1 to 2 cells thick, that lies on a distinct basal lamina and is separated from the remainder of the pars distalis by a narrow, but continuous, vascular space (Fig. 3). Epithelial cells that comprise this particular layer appear morphologically similar to the majority of those observed in the pars intennedia. Numerous granular cells similar to those present in the pars distalis are present in this layer also. Thus, the lumen of the pituitary cleft is bounded centrally by the pars intennedia and laterally by a layer of cells closely associated with the pars distalis. Epithelial cells comprising the pars distalis proper are organized into irregular cords and/or clumps by the surrounding connective tissue and vascular elements. The pars tuberalis, although continuous with the pars distalis is poorly developed in Didelphis and lacks the prominent vascular channels that are usually associated with this component in other species. The pars intennedia consists of a gjngle layer of cells that is pseudostratified in character (Fig. 3). It consists of 2 distinct populations of cells: granular cells and non-granular cells. 2 types of non-granular cells are observed. The 1st of these is a large, bulbous, light-staining cell that spans the entire width of the pars intennedia (Fig. 3). Ultrastructurally, this cell type is characterized by an electron lucent cytoplasm and a large, round euchromatic nucleus. The cytoplasm contains only scant amounts of rough endoplasmic reticulum, scattered free ribosomes and numerous

Development of opossum pituitary

41

Fig. 2. A low power micrograph of a horizontal section through the pituitary of an adult opossum shows the lumen of the hypophyseal cleft (arrows), the pars distalis (D) and pars nervosa (N). Connective tissue septa divide the periphery of the neurohypophysis into numerous well organized lobules. Hematoxylin and eosin. x 10. Fig. 3. Increased magnification of a region of the pituitary cleft taken from an adult perfused with 3.0% glutaraldehyde demonstrates that the pituitary cleft (large arrow) is often very narrow and that cell apices comprising its internal (pars intermedia) and external walls often abut one another. The large, bulbous, light-staining cells (B) of the internal wall often occur in small groups. The extent of the external wall into the pars distalis is demonstrated by the small arrows. 2 lobules (L) of the neurohypophysis are shown at the upper right. Note the amount of vascular connective tissue that separates the lobules of the pars nervosa from the pars intermedia. Epon 812. Toluidine blue. x 400.

mitochondria which tend to be polarized toward the apical and basal regions of the cell (Figs. 4, 5). This particular cell type is further characterized by numerous elongate microvilli and occasional cilia that project into the lumen of the pituitary cleft (Figs. 4, 5). The 2nd type of nongranular cell is stellate in shape, more abundant than the former and, like the bulbous form, spans the entire width of the pars intermedia. Nuclei are irregular in outline and show an abundance of heterochromatin (Figs. 4, 5). The cytoplasm exhibits considerable electron density and contains only a few, poorly developed organelles. The mitochondria observed in this cell are randomly scattered throughout the cytoplasm. The apices of the stellate cell are complex and may expand to form a thin border along the lumen of the pituitary cleft. Both bulbous and stellate cells are united at their apices by tight junctions (Fig. 5). Scattered desmosomes are observed between lateral cell membranes of both cell types and on occasion the plasmalemma shows infolding and some interdigitation with that of neighbouring cells. The basal plasmalemma of each type is relatively smooth and lies on a distinct basal lamina. The granular cell type, like the non-granular forms, lies on the basal lamina associated with cells of the pars intermedia and on occasion may extend to the lumen of the pituitary cleft. The

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Development of opossum pituitary

43

granular cells appear randomly scattered within the pars intermedia and are encountered much less frequently than the non-granular cell types. They are characterized by large numbers of electrondense secretory granules scattered throughout the cytoplasm (Fig. 5). The secretory granules vary in size from one granular cell type to another and are limited by a membrane. The cytoplasm also exhibits scattered profiles of rough endoplasmic reticulum, cisternae of which may be distended with a homogeneous, colloid-appearing material. Golgi complexes and mitochondria are scattered within the perinuclear cytoplasm and other organelles, although present, are few in number and less well developed. The nucleus of this cell type is usually round or oval in shape and shows an abundance of euchromatin. What heterochromatin is present is usually concentrated near the nuclear envelope. I or 2 nucleoli may be present also. Granular cells of the pars distalis as well as those found in the epithelial layer that lines the lateral (external) border of the pituitary cleft are usually round or oval in shape with a central nucleus characterized by an abundance of euchromatin (Fig. 6). Prominent nucleoli are present in some cells. Generally, the cytoplasm of most cells that comprise the pars distalis exhibit scattered profiles of rough endoplasmic reticulum, small Golgi complexes and a moderate number of mitochondria. Secretory granules characterize the majority of epithelial cells and range in size from about 80 nm to 400 nm in diameter. They vary considerably in electron density. Some of these cells exhibit a granular size and ultrastructural features that are similar to the granular celltypes observed in the pars intermedia. Scattered non-granular stellate cells also are observed in the pars distalis of the opossum pituitary at the ultrastructural level. They are observed near the external wall of the pituitary cleft as well as throughout the pars distalis. In both regions they are seen at the periphery of extravascular channels but also appear to extend into the central regions of the epithelial cords to lie between granulated cells. The cell bodies are relatively small, irregular in shape and exhibit extensive cytoplasmic processes that course between adjacent granulated cells. The cytoplasmic processes often appear to expand into thin, sheet-like extensions of the stellate cell that pass between adjacent granulated cells. The cytoplasm contains a moderate number of mitochondria and scattered free ribosomes. Only occasional profiles of granular endoplasmic reticulum are seen in the cytoplasm. The stellate cell type of the pars distalis appears ultrastructurally very similar to the stellate cells (marginal cells) that line the margin of the pituitary cleft. 3.2. Development The presumptive pars intermedia first becomes apparent during the 10th d of gestation and forms from the anterior dorsal wall of Rathke's pouch. At this stage of development, Rathke's pouch appears as an irregular shaped, multi-chambered vesicle with upper, middle, and lower cavities. A portion of the wall bounding the upper cavity comes to lie in close association with the presumptive infundibular process, also in the formative stages of development, projecting from the floor of the diencephalon. The presumptive pars intermeqia consists of an simple epithelial layer 2 to 3 cells thick (Fig. 7). Component cells for the most part appear undifferentiated, are spindle or columnar in shape and contain large oval nuclei. Cell apices, which border the interior margin of the upper cavity, are united by tight junctions (Fig. 8). Nuclei of these cells are characterized by an abundance of euchromatin and the heterochromatin present appears limited to

Fig. 4. A montage of electron micrographs illustrating the internal (pars intermedia; I) and external (E) walls bounding the pituitary cleft. Large, bulbous, electron lucent cells (B) with cilia projecting into the lumen (L) of the cleft as well as stellate (5) and granulated cells (G) are seen in the pars intermedia. The external wall consists of stellate and granular cells. Cells comprising both the internal and external layers that bound the pituitary cleft are united at their apices by tight junctions and basally are limited by a distinct basal lamina. The outer boundary of the external layer is shown by the arrows. Adult. X ) ,200.

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Development of opossum pituitary

45

the region of the nuclear envelope. Nucleoli are frequently observed. The surrounding cytoplasm is characterized by an abundance of free ribosomes, although scattered profiles of rough endoplasmic reticulum and a few mitochondria also are seen (Fig. 8). With continued growth of the infundibular process and the apparent upward migration of Rathke's pouch (due to differential growth of this region), the 2 lower cavities of the latter are compressed laterally but remain interconnected by a narrow median passage. As the lower cavities progressively flatten, the dorsal wall of the uppermost cavity expands its intimate association with the forming infundibular process. With continued development and growth of the infundibular process concomitant with its continued association with the dorsal wall of the upper cavity of Rathke's pouch, expansion of the presumptive pars intermedia is extensive (Figs. 10, 11, 12). It eventually completely invests the forming pars nervosa with the exception of a small dorsal lateral portion. Thus, the majority of the dorsal wall of the upper cavity retains a close association with the forming pars nervosa and subsequently differentiates into the pars intermedia. At this time, the walls of the pituitary vesicle are uniformly thin, however, regions of the anterior and posterior ventral wall begin to thicken as a result of proliferation by epithelial cells in these regions (Figs. 10, 11, 12). As cells continue to proliferate in these regions to form the pars distalis, the middle and lower cavities of Rathke's pouch are compressed and eventually obliterated. The lumen of the uppermost cavity thins considerably due to growth of the pars distalis and becomes the pituitary cleft observed in older animals. Thus, epithelial cells that line the medial and lateral sides of the pituitary cleft are derived from the wall of the upper cavity of Rathke's pouch. Both epithelial regions of the original pituitary vesicle (pars intermedia and the external wall of the pituitary cleft) continue to be limited by a distinct basal lamina and a vascular connective tissue space, separating each from either the pars nervosa or the pars distalis, respectively. The boundaries of the external wall becomes obscured as development continues due to the proliferation of the various granular cells in the forming pars distalis. By the postnatal 4th d, the pars intermedia consists of an pseudostratified epithelial layer approximately 2 to 3 cells thick. Ultrastructurally, cells comprising the pars intermedia appear similar to those forming the lateral wall. Most cells appear undifferentiated, although an occasional granular cell containing a small number of cytoplasmic granules is observed. During the first 2 postnatal weeks, growth of both the neuro- and adenohypophysis is substantial. Asa result of differential growth in both portions, the pars nervosa comes to lie within the center of the adenohypophysis. The pituitary cleft continues to be well delineated and clearly separates both major subdivisions of the hypophysis. The pars intermedia thins considerably by this stage of development and consists of an simple epithelial layer approximately 2 cells thick. Component cells appear undifferentiated and tight junctions continue to unite all apices that border the pituitary cleft throughout the extensive length of the pars intermedia. Junctional complexes are observed as well between cell apices lining the lateral wall of the pituitary cleft but are less well defined. Large, light staining cells surrounded by smaller, dark-staining cells characterize the pars intermedia by the end of the 5th postnatal week. These large, cuboidal cells are thought to be analogous to the large, electron lucent, bulbous cells observed in the mature animal (Fig. 9). In

Fig. 5. Increased magnification details the 3 cells types normally associated with the pars intermedia: stellate cells (S), electron lucent bulbous cells (B), granulated cells (G). The lumen (L) of the pituitary cleft is shown at the upper right. Cell apices of the stellate and bulbous cells are united by tight junctions (arrows). Adult. X 5,000. Fig. 6. Increased magnification of the external wall illustrates in greater detail stellate (S) and granulated cells (G). Like the internal wall, cells of this wall share a common basal lamina (slrUlll arrows) and stellate cells are united at their apices by tight junctions (large arrow). The lumen (L) of the pituitary cleft is shown at the upper right. Adult. X 5,000.

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Fig. 7. A photomicrograph illustrates the association between Rathke's pouch (R) and the primordia of the infundibulum (I). A portion of the oral plate (P) and oropharyngeal membrane (M) also are shown. Epon 812. Toluidine blue. I~ d opossum embryo. x 250. Fig. 8. Epithelial cells from a region of Rathke's pouch are united at their apices by tight junctions (large arrows) and lie on a thin delicate basal lamina (small anmvs). The epithelial cells appear columnar in shape and show a definite polarity with the nucleus located in the basal cytoplasm. The cytoplasm is characterized by numerous free ribosomes. A mitotic figure (M) is shown within the epithelium near the upper center of the electron micrograph. I ~ d opossum embryo. x 7,000.

Development of opossum pituitary

47

Fig. 9. Epithelial cells forming the pars intermedia remain largely undifferentiated (with the exception of granular cells) and are columnar in shape in the 5 week old opossum. Cells continue to lie on a distinct basal lamina and cell apices that border the pituitary cleft of both the internal and external walls are united by tight junctions (arrows). X

5,000.

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D. M. SHERMAN and W. J. KRAUSE

addition, scattered regions of the pars intermedia also show the stellate (marginal) cells and granular cells. The external wall lining the pituitary cleft has narrowed to an epithelial layer I to 2 cells thick and continues to be separated from the remainder of the pars distalis by a distinct basal lamina and a well defined vascular space. The external wall consists primarily of stellate cells that extend from a limiting basal lamina to the lumen of the pituitary cleft. Their apices are united by typical tight junctions. The apices of this cell type, like those observed in the pars intermedia, often expand to form a thin border that lines the lateral margin of the pituitary cleft. Unlike the pars

lli

Fig. 10. Sagittal section through the developing adenohypophysis of a d embryo incubated with rabbit antihuman ACTH shows numerous immunoreactive cells (small arrows). A portion of the IIIrd ventricle (3 V), the infundibulum (I), and optic chiasm (0) also are shown. The region of the forming pars intermedia is shown at the large arrow. Cartilage of the basosphenoid (B) is shown at the bottom of the figure. x 100. Fig. II. A sagittal section through the developing pituitary of an opossum 7 d after birth stained for ACTH. Numerous ACTH-immunoreactive cells are seen in the anterior region of the forming pars distalis (large arrow). A definite pars intermedia (small arrow) is established and intimately associated with the developing infundibular process (I). The pars intermedia contains several ACTH-immunoreactive cells. X 100. Fig. 12. A sagittal section through the developing pituitary of Didelphis 2 weeks after birth shows continued expansion and growth of the pars distalis. Note that the ACTH-immunoreactive cells continue to be concentrated in the anterior portion (arrows). The infundibular process (I) continues its expansion into the central region of the forming pars distalis. x 100. Fig. 13. A section through the developing pituitary of a II~ d opossum embryo incubated with anti-human somatotropin demonstrates occasional, scattered immunoreactive cells (arrows). The region of the infundibulum (IF) is shown near the top left of the micrograph. X 250. Fig. 14. A portion of the developing pars distalis (D) of a newborn opossum incubated with anti-human somatotropin illustrates several immunoreactive cells (arrows). The lumen of the pituitary cleft (L) is shown at the center left. x 250. Fig. IS. A region of developing pituitary from a 2 week old opossum incubated with anti-human thyrotropin illustrates only a few scattered immunoreactive cells (small arrows). Portions of the infundibulum (I), pars intermedia (PI), and lumen of the pituitary cleft (L) also are shown. X 250. Fig. 16. A region of developing pituitary from a 7 d old opossum incubated with anti-human luteotropin shows only occasional immunoreactive cells (arrows). The lumen of the pituitary cleft (L), pars intermedia (PI), and infundibulum (I) are shown at the right. x 250. Fig. 17. The developing pituitary of a 2 week old opossum incubated with anti-human luteotropin shows a relatively even distribution of immunoreactive cells both in the anterior and posterior portions of the expanding pars distalis. Note the LH-immunoreactive cells in the pars intermedia (arrows). X 100. Fig. 18. A section of developing pituitary (I week postnatal) incubated with anti-human FSH shows several immunoreactive cells in the pars distalis (arrows). The infundibulum (I), pars intermedia (PI), and lumen of the pituitary cleft also are shown. X 250. Fig. 19. A region of developing pituitary from a 2 week old opossum incubated with anti-human FSH demonstrates several immunoreactive cells (large arrows) in the narrow region between the larger anterior and posterior portions of the pars distalis. Note the 3 FSH-immunoreactive cells in the pars intermedia (small arrows). X 250. Fig. 20. A portion of developing pituitary from a Il~ d opossum embryo incubated with anti-human prolactin demonstrates scattered, occasional immunoreactive cells in the presumptive pars distalis (arrows). The infundibulum (I) is shown at the upper right. X 250. Fig. 21. A section of developing pars distalis taken from a I week old opossum represents a similar area but with further development to that shown in Fig. 20. Note the scattered prolactin-immunoreactive cells (arrows). The infundibulum (I), pars intermedia (PI), and a portion of the pituitary cleft lumen (L) are shown at the right of the photomicrograph. X 250.

Development of opossum pituitary

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Acta histochem., Bd. 89, 1

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D. M. SHERMAN and W. J. KRAUSE

intermedia, numerous granular cells similar to those comprising the pars distalis are present in the external layer and obscure the contiguous layer of stellate cells. The large, electron-lucent ciliated cells of the pars intermedia are not observed in the external layer. All 3 cell types are clearly defined in the majority of the pars intermedia by beginning of weaning (lIth postnatal week) and are similar to those of the mature opossum. Cilia extending from the apices of the light staining, bulbous cells are present but seen less frequently when compared to cells examined in fully mature animals. Secretory granules within granular cells at this stage of development are numerous and cisternae of the rough endoplasmic reticulum often appear expanded with a light staining amorphous material. 3.3. Immunohistochemistry 3.3.1. Adult Immunoreactive cells from the 5 categories examined (somatotropes, corticotropes, thyrotropes, gonadotropes, lactotropes) are present scattered in variable numbers throughout all regions of the pars distalis. Cells immunoreactive for corticotropin (ACTH) and thyrotropin (TSH) were concentrated primarily in the anterior region of the pars distalis, whereas cells immunoreactive for somatotropin (GH) appeared to be the more common cell type in the posterior region. Cells immunoreactive for prolactin (PRL) also showed a tendency to be concentrated in the posterior region of the pars distalis. Although specific cells show immunoreactivity utilizing the anti-rat prolactin antiserum to demonstrate lactotropes in the opossum pituitary, the staining intensity is considerably weaker when compared to staining using the anti-human prolactin antiserum. For this reason the latter was the antiserum of choice. The reason for the marked difference in staining intensity is unknown. Gonadotropes containing either LH or FSH are found fairly evenly scattered throughout the pars distalis but in some sections appear more concentrated anteriorly. All 6 types of immunoreactive cells are observed scattered within the epithelium forming either the internal wall (pars intermedia) or the external wall of the pituitary cleft. 3.3.2. Development Cells immunoreactive to anti-human corticotropin, anti-human somatotropin, and anti-human prolactin are observed in the adenohypophysis of the first embryonic stage examined immunohistochemically, the II Y2 d opossum embryo (Figs. 10, 13, 20, Table 2). Corticotropinimmunoreactive cells are numerous at this stage of development and found in nearly equal numbers throughout the presumptive pars distalis (Fig. 10). ACTH-immunoreactive cells are for the most part large, oval or round in shape, and show intense immunoreactive staining. In contrast, cells immunoreactive for both somatotropin (Fig. 13) and prolactin (Fig. 20), are very few in number, usually spindle in shape and for the most part show only light immunoreactive Table 2. Appearance of immunoreactive cells in opossum pituitary 11 Vz d embryo ACTH GH PRL LH FSH TSH

*** * *

birth

****

** * * * *

Relative frequency of immunoreactive cells present:

****

1 week

****

****

** ** ** ** *

numerous;

2 weeks

*** ** *** *** ** ***

moderate;

** few; * rare;

- not observed

Development of opossum pituitary

51

staining. Within 24 h, cells immunoreactive for the latter 2 probes, stain more intensely and appear more round in shape (Figs. 12. 41). At the time of birth light immunoreactive staining also is observed in scattered occasional cells incubated with anti-human luteotropin, anti-human folliculotropin, and anti-human thyrotropin antisera. Although seen on rare occasion in the newborn opossum pituitary, both gonadotropes did not appear consistently in each section of pituitary until at least 1 week into the postnatal period (Figs. 16, 17, 18, 19). Likewise, thyrotropin-immunoreactive cells, although seen earlier, were not observed consistently in each section examined until 2 weeks into the postnatal period (Fig. 15). ACTH-immunoreactive cells show a definite tendency to be concentrated anteriorly in the pars distalis of the newborn opossum (Fig. 11) and the adult distribution of this cell type is well established by the end of the 2nd postnatal week (Fig. 12). Immunoreactive cells are initially observed in the pars intermedia at birth. The first of these to be seen with consistency are corticotropin-immunoreactive cells. The other immunoreactive cell types, although observed, do not appear with consistency within the pars intermedia until after the 2nd postnatal week.

4. Discussion The development of the pituitary in Didelphis follows the general pattern described for most eutherian (NELSON 1933; SANO and SASAKI 1969; SVALANDER 1974; KRAUSE and CUTTS 1986) and metatherian mammals (PARKER 1917; HALL and HUGHES 1985). In marsupial species examined thus far (Didelphis and lsoodon) the dorsal portion of the multichambered pituitary vesicle that results from Rathke's pouch comes to lie in close association with the presumptive infundibulum of the diencephalon by the 10th d of gestation. At this stage of development, the pituitary vesicle is of uniform thickness in both species. In Didelphis the epithelial wall of the vesicle consists of spindleshapes cells and is pseudostratified in character. The dorsal wall of the pituitary vesicle remains uniformly thin and as development continues forms the pars intermedia; the ventral wall thickens due a subsequent proliferation of cells from within and gives rise to the pars distalis. At the time of birth (l2!!z d gestation in Didelphis), the lumen of the multichambered pituitary vesicle has been transformed into a flattened, curved cleft that continues to clearly delineate the forming pars intermedia from the pars distalis. By the end of the 2nd postnatal week the relationships between the pars nervosa, pars intermedia, pituitary cleft, and pars distalis are well established and show little change other than size during development in Didelphis. Similar observations have been reported on the early development of the pituitary in the bandicoot [(lsoodon macrourus); (HALL and HUGHES 1985)]. Utilizing immunohistochemical probes the 5 cell types (cortioctropes, somatotropes, lactotropes, gonadotropes, thyrotropes) examined in this study could be demonstrated at birth in Didelphis and ACTH-, GH-, and PRLimmunoreactive cells also are observed in the 11 Yz d opossum embryo. Corticotropes are numerous in the presumptive pars distalis of the 11!!z d opossum embryo, whereas immunoreactivities to both human somatotropin and human prolactin are seen only in occasional, scattered cells. The latter are elongate and spindle in shape suggesting the initial differentiation of these cell types in the epithelial wall of the pituitary vesicle. In contrast, cells immunoreactive for corticotropin were round or oval in shape with considerable cytoplasm surrounding a central nucleus. The morphology together with the intense immunoreactive staining of this cell type suggests that they had differentiated somewhat earlier. At birth, immunoreactivities for both somatotropin and prolactin are confined primarily to similar appearing round or oval shaped cells. An extensive proliferation of epithelial cells occurs in the ventral region of the pituitary vesicle during the fIrst 2 postnatal weeks. As these differentiating cells continue to divide, there is a net accumulation of cells away from the original ventral wall of the pituitary vesicle that results in the formation of the anterior and posterior regions of the pars distalis. By the end of the 2nd postnatal week the relative proportions and the distribution of all 5 cell types associated with the pars distalis appear to have been established. Thus, the pars intermedia (internal wall of the pituitary cleft) represents the dorsal wall of the original pituitary vesicle and although appearing to be 2 to 3 cells thick remains a simple epithelium. It subsequently thins with development. Although obscured by the various epithelial cell types it has in common with the pars distalis, the ventral wall of the pituitary vesicle also retains its identity as the external wall of the pituitary cleft even in the adult. A distinct basal lamina separates the internal wall (pars intermedia) of the pituitary cleft from the pars nervosa and ultimately is continuous with the limiting basal lamina separating the external wall of the pituitary cleft from the pars distalis. Because of this. perhaps both walls bounding the pituitary cleft would more appropriately be considered the pars intermedia in Didelphis rather than just the internal one. Both walls represent an epithelial continuum around the pituitary cleft that is limited by a continuous basal lamina. Thus, the stellate (marginal) cells lining both sides of the pituitary cleft represent cells that were derived for the original cells of the pituitary vesicle. It also is from this 4*

52

D. M. SHERMAN and W. J. KRAUSE

population of cells that various types of pituitary hormone-producing cells arise. Similar observations have been made by Yoshimura et al. (l977a, b) in the rat. In Didelphis it is the corticotrope that is the first to appear in the presumptive pituitary and in this regard is similar to eutherian species (CONKLIN 1968; WATANEBE and DAIKOKU 1979). In contrast, thyrotropes are reported to occur first in the bandicoot (HALL and HUGHES 1985). Like Didelphis, all 5 pituitary cell types could be demonstrated immunohistochemically I d after birth in the Tammer wallaby with somatotropes being the predominate cell type at this time in this species (LEATHERLAND and RENFREE 1983b). Developing pituitaries of other Australian marsupials (Dasyurus hallucatus, Trichosurus vulpecula, Isoodon macrourus), which have shorter gestation times than the Tammar, also exhibit ultrastructural features suggestive that these developing endocrine organs are functional at or shortly after birth (HALL and HUGHES 1985; GEMMELL and NELSON 1988). The adrenal glands of both Dasyurus hallucatus and Trichosurus vulpecula also exhibit ultrastructural features suggesting these endocrine glands are functional at birth (GEMMELL and NELSON 1988). In addition to ultrastructural observation suggesting function, the adrenal of Isoodon macrourus and Macropus rufogriseus have been shown capable of producing 0.094 ng and 0.5 ng of cortisol per adrenal at birth, respectively (GEMMELL et al. 1982; WALKER and GEMMELL 1983). In comparison, the development of chromaffin cells in the adrenal medulla of Didelphis does not occur until the postnatal period following the entrance of pheochromoblasts into the cortical anlage (CARMICHAEL et al. 1987). Although cortical cells have not been examined ultrastructurally in this species nor the amount of cortisol production measured, the large number of ACTH-immunoreactive cells present in the pituitary I d before birth and their abundance thereafter would suggest that cells of the adrenal cortex are functional at birth in Didelphis. In contrast to the adrenal, the thyroid of Didelphis is very rudimentary at birth and it is not until the postnatal 11th d that the first primary follicles begin to form (CUTTS and KRAUSE 1983). Similarly, thyrotropes could not be demonstrated in the pituitary unti I after birth and did not constitute a significant population even at 2 weeks into the postnatal period. The thyroid of the bandicoot (Isodoon macrourus) also is very immature in appearance at birth and not fully active until about 7th week of postnatal life (JOHNSTON and GEMMELL 1987). Likewise, gonadotropes were not apparent in Didelphis until after birth and sexual differentiation occurs postnatally in this species rather than in utero as in the case for most eutherian mammals. Differentiated endocrine function of the testes and ovaries occurs as soon as the development of the scrotum in males and the pouch in females becomes apparent (about 7 d postnatal in Didelphis). The testes synthesize testosterone and the ovaries are capable of aromatizing androgens to estrogens at this time (GEORGE et al. 1985). The endocrine differentiation of both gonads appears to occur prior to the differentiation of male and female genital tracts and corresponds to the increase in number of gonadotropes observed during the first weeks of postnatal life. Generally, the distribution of the various cell types in the opossum pituitary is similar to that of the Tammar wallaby (LEATHERLAND and RENFREE 1982, 1983b) as well as the majority of eutherian mammals examined utilizing immunohistochemical probes. Throughout development and in the adult, the pars distalis remains separated from the pars nervosa and its overlying pars intermedia by a well defined pituitary cleft. This space was never observed to be crossed by vessels or other structures but remained a narrow space throughout the life-history of Didelphis. Nerve terminals constituting the lobules of the pars nervosa have been shown to be filled with electron dense secretory granules (ROTH and LUSE 1964). As direct vascular channels do not cross the pituitary cleft, releasing factors and other substances that enter the vasculature from the pars nervosa must first pass dorsally into the region of pituitary stalk prior to entering the vasculature that supplies the pars distalis. In addition, stellate (marginal) cells which are the most abundant cell type within the lining epithelium of the pituitary cleft, also may be involved in the transport of materials released from the pars nervosa. It has been proposed that stellate cells in the pars distalis of eutherian species transport materials from the vasculature to pituitary cell types comprising the epithelial cords (LEATHERLAND 1970; LEATHERLAND et al. 1975; SHIOTANI 1980; DE BOLD et al. 1980; LEATHERLAND and BAKER 1982). The non-granulated (stellate) cells of the wallaby pars distalis have been proposed to form a link between the center of the epithelial cords and the surrounding vasculature (LEATHERLAND and RENFREE 1982, 1983a). Similar cells are seen in the pars distalis of Didelphis. As in the wallaby, the folliculostellate cells which are indistinguishable from the stellate (marginal) cells lining the pituitary cleft in Didelphis may be involved in transport and/or intercellular communication. Soji and HERBERT (1989) have suggested that the folliculostellate cells of the rat pars distal is form a functional syncytium that acts as a system for intercellular communication between cells through gap junctions. Thus, it may very well be that the nongranular stellate or marginal cells forming the walls of the original pituitary vesicle not only give rise to the various epithelial cell types associated with hormone production in the pars distalis but continue to remain intimately linked with them physiologically. The precise function of stellate cells in Didelphis is unknown and will require further study using additional techniques. Cells immunoreactive for human-corticotropin were first apparent and a consistent observation in the pars intermedia of the 11 y, d opossum embryo. The remaining 4 cell types (somatotropes, gonadotropes, thyrotropes,

Development of opossum pituitary

53

lactotropes) although observed scattered within the epithelium of the pars intermedia during the Ist postnatal week were not a consistent observation until after the 2nd week of postnatal life. The 5 types of immunoreactive ceJls demonstrated in the pars intermedia and pars distalis correspond to the granulated cells observed at the ultrastructural level. The various immunoreactive ceJl types within the pars intermedia and external wall of the pituitary cleft may only represent residual ceJls that have differentiated in this region but failed to migrate with the majority to form the pars distalis rather than having special physiological significance. The examination of ceJls in adjacent serial sections of the developmental series of opossum pituitaries also suggests a possible co-localization of luteotropin and folliculotropin within some but not all gonadotropes. Similar co-localization of LH- and FSH-immunoreactivity in some gonadotropes has been suggested previously for some eutherian species (PHIFER et al. 1973; MORIARTY 1976) including man (NEWMAN et al. 1989). On occasion, prolactin-immunoreactivity also appeared to be co-localized within opossum pituitary cells that showed LHimmunoreactivity. Whether or not this is indeed the case needs to be confirmed utilizing the double labelling technique on the same ceJl preferably at the ultrastructural level. Prolactin-immunoreactive granules have been demonstrated in LH-immunoreactive cells of the human pituitary (NEWMAN et al. 1989).

Acknowledgements We would like to thank Dr. SALVATORE RAITI, Director, National Hormone and Pituitary Program, Baltimore, Maryland, for providing several antigens and antisera used in this study. We would also like to acknowledge MIDGE M. LIND for the preparation of the manuscript.

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CUTTS, H. J., KRAUSE, W. J. and LEESON, C. R., General observations on the growth and development of the pouch young opossum, Didelphis virginiana. Bio\. Neonate 33, 264-272 (1978). DAIKOKU, S., KINUTANI, M., and WATANABE, Y. G., Role of hypothalamus on the development ofadenohypophysis. An electron microscopic study. Neuroendocrinology 11, 284-305 (1973). DAWSON, A. B., The epithelial components of the pituitary gland of the opossum. Anat. Rec. 72,181-193 (1973). DE BOLD, A. J., DE BOLD, M. L., and KRAICER, J., Structural relationships between parenchymal and stromal elements in the pars intermedia of the rat adenohypophysis as demonstrated extracellular space markers. Cell Tissue Res. 207, 347-359 (1980). DIHL, F., BEGEOT, M., LOEVENHRUCK, C., DUBOIS, M. P., and DUBOIS, P. M., Ontogeny of gonadotropic and thyrotropic cells in fetal mouse anterior pituitary. Comparison between two species C57 Bib and Balble. Anat. Embryo!. 178, 21-27 (1988). DUBOIS, P. M., BEGEOT, M., DUBOIS, M. P., and HERBERT, D. C., Immunocytologicallocalization of LH, FSH, TSH and their subunits in the pituitary of normal or anencephalic fetuses. Cell Tissue Res. 191, 249-265 (1978). DuPOuy, J. P., and DUBOIS, M. P., Ontogenesis of the