Ultrastructural and immunocytochemical studies of prolactin-secreting cells (PRL cells) in the anterior pituitary gland of the female musk shrew (Suncus murinus L.)код для вставкиСкачать
THE ANATOMICAL RECORD 220:415-423 (1988) Ultrastructural and Immunocytochemical Studies of Prolactin-Secreting Cells (PRL Cells) in the Anterior Pituitary Gland of the Female Musk Shrew (Suncus murinus L.) TOSHIKO ISHIBASHI A N D MASATAKA SHIINO Department of Anatomy, Wakayama Medical College, SKyubancho, Wakayama-shi, 640, Japan ABSTRACT Prolactin-secreting cells (PRL cells) in the anterior pituitary gland of the female musk shrew were identified by the protein A-gold procedure combined with electron microscopy. Their secretory granules were spherical, and showed various sizes ranging in diameter from 100 nm to 800 nm according to the difference in physiological conditions of the animals. In pregnant and lactating animals, the PRL cells exhibited morphologically active features, i.e., a large prominent Golgi apparatus and a well-developed rough endoplasmic reticulum (RER) consisting of densely packed parallel lamellae. In pregnant and estrogen-treated animals, the secretory granules of PRL cells significantly increased in size as compared with those of virgin animals. The most remarkable ultrastructural change observed in PRL cells of pregnant, lactating, and estrogen-treated female musk shrews was the occurrence of “intracisternal” granules. They were small and spherical, and were located without limiting membranes inside the dilated cisternae of the RER. Intracisternal granules, as well as ordinary secretory granules, were found to be immunoreactive for PRL by the protein A-gold procedure. These results suggest that PRL cells of the female musk shrew may possibly utilize a “bypass” route to form hormone-containing granules under highly activated cellular conditions. Recently, the musk shrew became available as a new laboratory animal. There is little information, however, concerning the morphology of the anterior pituitary cells of the musk shrew, except for a few light microscope reports (Naik and Dominic, 1972, 1978). We have been interested in studies on the anterior pituitary cells of the musk shrew, since it appears to be a unique animal that shows different cytological profiles than those of other laboratory animals. We have recently demonstrated in somatotrophs of the musk shrew’s pituitary gland the presence of rod-shaped secretory granules (Ishibashi and Shiino, 1988). In the present study, we have concentrated on some specific ultrastructural features of the secretory granules in PRL cells of the female musk shrew as seen in different physiological states, such as during pregnancy and lactation. MATERIALS AND METHODS Ten virgin female (1 month of age), three late-pregnancy, and four early-lactating musk shrews were employed in the present study. They were kept in airconditioned and light-controlled (0600-2000 hr) quarters, and fed Purina laboratory chow and tapwater ad libitum. For estrogen treatment, the 10 virgin females were divided into two groups: One group consisted of seven animals, which received subcutaneous injections 0 1988 ALAN R. LISS, INC. of 0-estradiol (E2, 20 pg dissolved in 0.05 ml of sesame oil) once a day for 2 weeks and the other group consisted of three animals, which served a s vehicle injected controls. The animals were killed by decapitation after light anesthesia with ether. The anterior pituitary glands were removed from the sella turcica and cut in half. Small pieces of the pituitary glands were fixed in a solution consisting of 3% paraformaldehyde and 3%glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 hr a t room temperature, and then postfixed in 1% osmium tetroxide dissolved in 0.1 M cacodylate buffer for 1hr a t 0°C. After a routine procedure of dehydration, the tissues were embedded in Spurr’s plastic mixture. For general electron microscope observation, thin sections were prepared and stained with uranyl acetate and lead citrate. For immunocytochemical observations with the electron microscope, thin sections were reacted by the protein A-gold method described by Bendayan and Zollinger (1983). Control immunocytochemical tests were carried out by substituting normal rabbit serum or phosphate-buffered saline for the specific antiserum. Electron microscope observations were made with a Hitachi H-300 electron microscope. Antiserum rat prolactin was Received July 20, 1987; accepted October 9, 1987. 416 T. ISHIBASHI AND M. SHIINO TABLE 1. The size of secretory granules in PRL cells of the female musk shrew Group Virgin Pregnant Laciating E2-injecteda No. of animals Diameter of granules, nm (means f SE) 3 3 313.0 467.2 341.0 382.0 4 7 & 22.7 & 30.4* f 23.1 If: 21.6* =E2:0-estradiol. * P i0.01 as compared with values of the virgin animals. a gift from Dr. A.F. Parlow a t the Pituitary Hormone and Antisera Center, Los Angeles, and also from Dr. Katsumi Wakabayashi a t the Gunma Institute of Endocrinology, Gunma, Japan. Protein A-gold complex was obtained from Janssen Products, Belgium. Experimental animals used in the present study were kindly provided by Dr. M. Miyajima of the Experimental Animal Center in our institute. For measuring the size of secretory granules, 80-120 PRL cells were selected at random from tissue sections of each group (virgin, late-pregnancy, early-lactating, and Ez-injected), and photographs taken at a magnification of 5,000 were enlarged to ~10,000.The diameters (the long axis) of 25 secretory granules from each cell were measured on the photographs. Significant differences in granule diameters were determined by one-way analysis of variance, followed by the least-significance test. RESULTS Prolactin-secreting cells of the anterior pituitary gland of the female musk shrew were identified by the protein ‘1 virgin A-gold procedure combined with electron microscopy. Their secretory granules were spherical, and showed various sizes ranging in diameter from 100 nm to 800 nm, owing to the difference in physiological conditions of the animals as summarized in Table 1. The distribution of granule size of the PRL cells in each physiological state is shown in Figure 1. In pregnant animals, PRL cells contained secretory granules with the largest average diameter, as shown in Table 1. Some of the PRL cells contained extremely large, mature granules (maximum 800 nm), which were fully distributed throughout the cytoplasm. Most of them revealed a large, prominent Golgi region associated with numerous immature granules and a well-developed rough endoplasmic reticulum (RER) consisting of densely packed parallel lamellae (Fig. 2). Extruded granular material was frequently observed in the intercellular spaces and, like the peripherally located secretory granules, they were found to be immunoreactive by the protein A-gold procedure (Fig. 3). Numerous cytoplasmic processes forming complicated interdigitating structures filled the spaces between adjacent PRL cells (Fig. 4). Occasionally the cytoplasmic processes of several PRL cells protruded into a large follicle in which irregularly shaped dense materials were observed. These materials were also seen to be immunoreactive by the protein Agold procedure. In lactating animals, PRL cells exhibited a well-developed Golgi zone consisting of stacked cisternae, numerous vesicles, condensing vacuoles, and prominent whorls of the RER (called Nebenkern) in the cytoplasm. Several multivesicular bodies were often seen in the Golgi area. Secretory granules of lactating animals were much smaller than those of pregnant animals ( P < 0.01). In general, morphologically enhanced PRL cells of lactat- 30 “I/ 30 Pregnant Lac t a t i n g 30 I 1 t 10 20 30 I0 50 80 10 10 20 30 Diameter (10 nm) Fig. 1. Frequency distribution histogram of the diameters of prolactin secretory granules in different physiological conditions of the female musk shrew. Fig. 2. Typical PRL cells observed in a pregnant animal. Note the Fig. 3. Immunoreactive PRL secretory granules in a pregnant animal revealed by the protein A-gold procedure and immunoreactive extruded prominent Gulgi complex and well-developed RER. x 6,300. secretory granules seen in the intercellular spaces (arrows). x 30,000. 418 T. ISHIBASHI AND M. SHIINO Fig. 4. PRL cells of a pregnant animal having numerous cytoplasmic processes (F‘RC). Note irregularly shaped dense materials between cytoplasmic processes (arrows). x 30,000. ing animals revealed rather smaller mature secretory granules. In E2-injectedanimals, many PRL cells contained numerous larger secretory granules, which were fully distributed in the cytoplasm. The Golgi apparatus and the RER were well developed but the Nebenkern formations were not observed. The most remarkable ultrastructural change in PRL cells of pregnant, lactating, and also the E2-injected female musk shrews was the occurrence of “intracisternal” granules. Intracisternal granules were small and spherical, and were present, without limiting membranes, inside the dilated cisternae of the RER (Fig. 5, and inset). The size of these granules appeared variable, ranging from 100 nm to 400 nm in diameter. Intracisternal granules were demonstrated to be immunoreactive by the protein A-gold procedure, as were peripheral secretory granules, which were distributed in the cytoplasm (Figs. 6 , 7). The formation of intracisternal granules was not observed in the virgin animals. Two types of PRL cells containing intracisternal granules were distinguished: One type had a prominent Golgi complex in which immature secretory granules were actively formed; the other was fully granulated with larger mature secretory granules, although the cytoplasmic organelles were not prominent. Furthermore, the formation of intracisternal granules could be divided into two cases: One case showed intracisternal granules of a uniform size (about 150-200 nm in diameter) that were closely aligned in rows within the dilated RER cisternae, which formed anastomosing tubules; in the other case smaller and more heterogeneous dense granules occupied the lumen of the irregularly expanded cisternae (Fig. 8).Lamellae of the RER containing intracisternal granules in their cisternae were frequently distributed in close proximity to the cell membrane. However, we observed neither an image of the RER lamellae fused with the cell membrane to open to extracellular spaces nor intracisternal granules that had been extruded from the RER lamellae into the cytoplasm or extracellular spaces. In early-lactacting animals, welldeveloped PRL cells contained intracisternal granules inside of the innermost cisternae of the Nebenkern (Fig. 9). These granules were also shown to be immunoreactive by the protein A-gold procedure. Another ultrastructural characteristic observed in PRL cells of the female musk shrew was the occurrence of low-density granules among the highly dense secretory granules. (Fig. 10). This phenomenon was often observed in late-pregnancy and E2-injected animals. These granules were limited by rough-contoured membranes and their markedly lower density contents were often spotted with much denser materials (Fig. 11).These Figs. 5-7. PRL cells containing intracisternal granules in the anteFig. 6. Intracisternal granules (arrowheads) are demonstrated to be rior pituitary gland of pregnant animals. immunoreactive by the protein A-gold procedure as are the ordinary secretory granules (SG). ~ 2 0 , 0 0 0 . Fig. 5. Small, spherical and dense granules (arrows) are seen in the dilated cisternae of the RER even while ordinary secretory granules Fig. 7. Higher magnification of the area enclosed in the rectangle in are actively forming within the Golgi area (GI. x 12,000. Inset: Higher Figure 6. Arrows indicate lamellae of RER. ~ 6 0 , 0 0 0 . magnification of the area enclosed in the rectangle. Note ordinary secretory granules (SG) and intracisternal granules (*) located inside the dilated cisternae of RER. ~ 6 0 , 0 0 0 . Figs, 8,9. Various types of intracisternal granules are observed in pregnant (Fig. 8) and lactating animals (Fig. 9). large mature secretory granules and small intracisternal granules (arrows), which are located in irregularly dilated RER lamellae. x 20,000. Fig. 8. T w o types of PRL cells in which intracisternal granules are contained. The PRL cell on the left contains numerous middle-sized prominent intracisternal granules that exist in rows within the cisternae of RER-like anastomosing tubules; the cell on the right contains Fig. 9. Note various sizes and densities of intracisternal granules present inside the innermost cisternae of Nebenkern. X 30,000. PRL-CELLS OF THE FEMALE MUSK SHREW Figs. 10-12. Examples of fully granulated PRL cells in the anterior pituitary glands of Ez-injected animals. Fig. 10. Low-density granules (arrows) are accumulated together with ordinary-density secretory granules in the whole cytoplasm. G: Golgi area. ~20,000. Fig. 11. Higher magnification of the area enclosed in the rectangle in Figure 10.Low-density granules (*) often show heterogeneous contents that are spotted with dense materials (arrows). ~40,000. 42 I Fig. 12. The immunoreactivity of low-density granules (arrows) visualized by the protein A-gold procedure is weak compared with that of ordinary secretory granules. ~40,000. 422 T. ISHIBASHI AND M. SHIINO low-density granules were revealed to be weakly immu- firmed by the protein A-gold procedure that these intranoreactive by the protein A-gold procedure (Fig. 12) and cisternal granules, as well as the mature secretory ordinarily seemed to be forming from the Golgi vesicles. granules, were immunoreactive. Farquhar et al. (1978) stated, in regard to the intracelDISCUSSION lular transport and packaging of prolactin, that the horWe first identified the PRL cells of the anterior pitu- mone is synthesized on attached ribosomes, segregated itary gland in the musk shrew by the protein A-gold inside the RER, and rapidly transported into the Golgi procedure combined with electron microscopy and dem- cisternae to be concentrated, where first the earliestonstrated their ultrastructural features. The PRL cells forming granules are recognized. The results obtained definitely showed morphologically active features in in the present study indicate that morphologically actipregnant and lactating animals. They were character- vated PRL cells are capable of forming visible and imized by a well-developed Golgi apparatus in which im- munoreactive granules inside RER cisternae without mature secretory granules were present and proliferated the process of transforming a prehormone to the Golgi RER consisting of densely packed parallel lamellae. Nu- apparatus and condensing it there. It is worth noting merous cytoplasmic processes were formed between ad- that some PRL cells observed in a pregnant animal jacent PRL cells. We often observed that the cytoplasmic contained numerous dense, clear-cut intracisternal granprocesses of PRL cells protruded and surrounded a large ules in slightly dilated cisternae of RER, while displayfollicle in which extruded-granules were stored as dense, ing a prominent Golgi zone that was actively forming immunoreactive material. It is a very interesting fact secretory granules. That is, the formation of granules in that secretory products from PRL cells are “pooled” in the Golgi apparatus and in the cisternae of RER was in the extracellular spaces during pregnancy. Secretory progress at the same time within a PRL cell. Chang and granules were spherical and they exhibited various sizes Nikitovitch-Winer (1976) stated that PRL cells stimuladepending upon the cellular status of secretion. We care- ted by suckling and lactating rats could possibly release fully observed changes in granule size according to the a soluble form of hormone stored within the RER cisterphysiological conditions and noted that PRL cells under nae directly onto the cell surface. Hausler et al. (1978) enhanced cellular activity induced by pregnancy or es- also suggested that a dual mechanism of hormone retrogen treatment generally contained larger secretory lease, i.e., a granular and a nongranular form of release, granules. PRL cells of lactating animals, which showed might exist in PRL cells of lactating rats. Recently, the preembedding techniques of the peroximorphologically the most hypertrophied cellular features, had a tendency to contain smaller secretory gran- dase-antiperoxidase method have enabled investigators ules rather than remarkably large granules as seen in to detect ultrastructural localizations of PRL not only in pregnant animals. It appears that the secretory gran- secretory granules but in both of the Golgi saccules and ules of PRL cells in the musk shrew generally increase the cisternae of RER (Tougard et al., 1980; Osamura et in size under enhanced states of hormone synthesis but al., 1982). Judging from the immunocytochemical and decrease in size under highly stimulated conditions of morphological findings by means of the technique mensecretion during lactation. tioned above, Osamura et al. (1982) stated that PRL It is well known that estrogen stimulates the secretory might diffuse directly from the RER into extraceIlular activity of PRL cells in the female rat (Zambrano and spaces. We observed, in the present study, that immuDeis, 1970; Shiino and Rennels, 1976; Aumuller et al., noreactive granules existed inside the cisternae of RER 1978). Similarly, in the present study, PRL cells of fe- in PRL cells and that these granule-containing cisternae male musk shrews were activated by Ez-injection and were often located close to the cell membrane. However, showed prominent Golgi complexes and a significant we have no morphological evidence that RER lamellae increase in the size of secretory granules. Although we containing immunoreactive granules can open toward have not observed the formation of Nebenkern in prolif- cytoplasmic or extracellular space to extrude their granerated RER, which was frequently seen in Ez-injected ules. rats, we did observe the presence of unusual low-density The finding of intracisternal granules in PRL cells in secretory granules characterized by weak immunoreac- the present study suggests that PRL cells of the female tivity. It seems likely that these low-density granules musk shrew may possibly utilize a “bypass” route to were the result of an incomplete condensation of secre- form hormone-containing granules. It will be very intertory granules within the Golgi complexes due to accel- esting to investigate how the intracisternal granules erated secretion. The lack of more dramatic ultra- inside the cisternae are finally altered. Further studies structural changes of PRL cells in the musk shrew, like will be needed to solve the problem. those seen in the rat, might be due to a n inappropriate ACKNOWLEDGMENTS dose or treatment period of estrogen. The occurrrence of intracisternal granules in PRL cells The authors wish to express their thanks to Dr. Edof pregnant, lactating, and E2-injected animals was one ward G. Rennels for his kind advice on preparing the of the most characteristic ultrastructural features in the manuscript. The authors also wish to thank Dr. A.F. musk shrew. The formation of intracisternal granules Parlow, of the Pituitary Hormones and Antisera Center, in the anterior pituitary cells is well known in thyroid- Harbor-UCLA Medical Center, Los Angeles, and the ectomy cells of the rat and other species, and a few cases National Hormone and Pituitary Program, University of intracisternaI granules in PRL cells of lactating rab- of Maryland School of Medicine, Baltimore, for their bits have been illustrated by Young et al. (1967). How- generous gifts of antigens and antisera. This study was supported by a Grant for Scientific ever, the intracisternal granules observed in PRL cells of the musk shrew are more numerous and prominent Research from the Ministrv of Education. Science and than those of Young et al. (1967). Additionally, we con- Culture, Japan, No. 62570016. PRL-CELLS OF THE FEMALE MUSK SHREW LITERATURE CITED Aumuller, G., R. Wagner, and K.J. 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