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Immunoelectron Microscopic Study for the Influence of LH-RH by Interconnected Folliculo-Stellate Cells

Hong Jian Wang

The First Department of Anatomy Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi, 467-8601, Japan

Running Title: Immunoelectron Microscopic Study for LH-RH Influence
Key Words: Rat, Anterior pituitary, Pars tuberalis, Folliculo-stellate cell, LH-RH, Castration
Adress for correspondence: Hong Jian Wang, The First Department of Anatomy, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, 467-8601, Japan
Tel (+81) 52-853-8121; Fax (+81) 52-842-3210;
E-mail: hwang@med.nagoya-cu.ac.jp
Total numbers of pages: 20
Total numbers of figures: 4

SUMMARY
Intercellular gap junctions between folliculo-stellate cells of anterior pituitary glands indicate the existence of cell-to-cell communication within the organ. In the present study, we propose a possible role for regulation of hormone secretion in the anterior pituitary gland under castrated conditions.
Twenty Wistar-Imamichi strain male rats were separated into 2 groups. Whereas the first group was for not-castrated group, the second group was castrated on day 10, and each group was sub-divided into 2 subgroups as controls and LH-RH treated groups. The animals were killed on day 40, whose calvaria were opened and brains were removed. In case of LH-RH treatment groups, the animals were treated with LH-RH for 3 min, while in control groups this LH-RH treatment was omitted. Then, the pituitary glands were processed for immunohistochemical light microscopy and immunoelectron microscopy.
The reaction end-products were observed around the vessels especially in the peri-capillary area outside of the cell membrane only in castrated LH-RH treated rats at the light microscopic level. A few electron dense particles sized approximately 100-150 nm in diameter appeared in the peri-capillary space by using electron microscopy. The immunoelectron microscopic technique by using protein A gold showed that these electron dense particles contained LH immunoreactive substance.
I suppose that the stimulatory "message" of LH-RH can be transmitted via the folliculo-stellate cells to the granulated cells in the anterior lobe by the gap junctionally connected network to affect LH release from gonadotrophs in castrated rats.
INTRODUCTION
Endocrine glands such as adenohypophysis have been subjected to relate specific cell types to their hormonal species by many immunocytochemical investigators. The protein A labeled with colloidal gold can be used as a marker for the ultrastructural detection of intracellular antigens at electron microscopic levels (1). Because its characteristic ability to interact with Fc fragment of IgG molecules from several animal species, it presents a simple and reliable procedure for the good detection of intracellular antigens, and the protein A gold method has a theoretical advantage for the high quality of the fine labeling and the good preservation of the underlying cellular structures. On the other hand, there were many reports about the influence of LH-RH treatment over the gonadotrophs that had severely upregulated sensitibity in case of castrated animals for 1970's (2, 3, 4, 5, 6). In the present study, I tried to demonstrate that the stimulatory "message" of LH-RH could be transmitted via the gap junctionally connected network of the folliculo-stellate cells to the granulated cells in the anterior lobe to affect LH release from gonadotrophs by affecting castrated male rats gonadotrophs.
MATERIALS AND METHODS
Twenty Wistar-Imamichi strain male rats were separated into 2 groups. Whereas the first group was for not-castrated group, the second group was castrated at 10 day-old under anesthesia with Nembutal (phenobarbital) and each group was sub-divided into 2 subgroups as controls and LH-RH treated groups. The animals were killed on day 40, whose calvaria were opened and brains were removed and left ventricle of the heart opened for 3 min. under Nembutal anesthesia for controls. This procedure detached the pituitary gland from the hypothalamus and ceased the circulation to the hypophysis. In case of LH-RH treatment groups, the animals were treated with LH-RH for 3 min. by placing a small triangular piece of filter paper containing LH-RH [Tanabe Seiyaku Co., LTD, Tokyo; 0.8 mg/ml in distilled water] on the cut surface of the pituitary stalk immediately after the detachment. Exposure of the anterior lobe on the detached surface was confirmed microscopically.
Then the pituitary gland was removed from each animal and separated into five parts; one central area, two intermediate areas and two peripheral areas which were at the tips of both wings. The peripheral areas were then used in this study (Fig. 1).

Preparation for Immunohistochemical Light Microscopy:
For the immunohistochemical light microscopic observations, the pituitary glands were fixed overnight in Bouin's solution without acetic acid at 4C, dehydrated in an ethanol series, and embedded in Paraplast embedding media (Sigma Chem. Co,, St. Louis, MO, USA). Serial sagittal sections, 2 m in thickness, were mounted on poly-L-lysine-coated slide glasses, and used for immunohistochemistry. The sections were immunostained by the peroxidase-labeled antibody method of Nakane and Pierce (7) using a slight modification (8).
Preparation for Immunoelectron Microscopy:
The samples to be studied by electron microscopy were immersed for one hour in a fixative containing 4% paraformaldehyde and 2% sucrose buffered with 0.05M sodium cacodylate (pH. 7.4). After the fixation, postosmication was usually omitted, and the sections were rinsed in ice cold water for 5 min. and dehydrated in a graded series of ethanol. Following two rinses in 100% ethanol for 10 min. each, the specimens were then embedded in epoxy resin (9) and polymerized in lower temperature (45 degree centigrade) for the preservation of antigens. Thin sections were prepared, placed on uncoated nickel grids, and for labeling one side of the grid was incubated with LH antibody followed by incubation with 10 nm diameter protein A-gold particles solution (1). The sections were stained with uranyl acetate, and observed using a Hitachi H-7000 transmission electron microscope.
The antiserum against rat LH was a gift from Dr. A.P. Parlow (NIH, USA), and its specificity has been described in a previous publication (8).
RESULTS
Immunohistochemical Light Microscopy:
The intensity of immunoreactivity of intact groups was stronger than that of castrated rat pituitaries, however the cell size which could react to anti-LH was larger than that of intact groups (Figs. 2a and c). While a marginal zone of almost all cells in control rats showed more intensely reactivity to the anti-LH antibody, a perinuclear zone of those cells showed weak reactivity (Fig. 2a). An effect of castration was clearly observed on the cells.
In the anterior pituitaries of intact LH-RH treated groups and castrated LH-RH treated groups, the reaction end-products were conspicuous on vascular side in the cells (Fig. 2b and 2d, arrow heads). These reaction products were also observed around the vessels especially in the peri-capillary area outside of the cell membrane only in castrated LH-RH treated rats (Fig. 2d, arrows).
Immunoelectron Microscopy:
Peri-capillary spaces seemed to be slightly enlarged. After the LH-RH treatment, a few electron dense particles sized approximately 100-150 nm in diameter appeared in the peri-capillary space (Fig. 3 and 4). Concerned with immunohistochemical light microscopy of the matched condition of anterior pituitary, the area where these particles occupied were corresponded to the peri-capillary space which showed LH immunoreactivity in Fig. 2d. The immunoelectron microscopic technique by using protein A gold showed that these electron dense particles contained LH immunoreactive substance (Fig. 3 and 4), while the secretary granules within the granulated cell were also labeled similarly (Fig. 4).
DISCUSSION
The peri-capillary spaces of the anterior pituitary gland in castrated LH-RH treated 40 day-old age rats were intensely stained by LH antibody in this study. These stained substances were demonstrated to be the particles containing LH which accumulate around the peri-capillary space by using immunocytochemical electron microscopy with protein A gold technique.
Endocrine glands such as adenohypophysis have been subjected to relate specific cell types to their hormonal species by many immunocytochemical investigators. In this study, I found the particles which showed the presence of LH by mounting protein A gold complex in the pericapillary spaces. The LH containing granules could also be found in the granulated cells beside that pericapillary space. The particles were assumed secretary granules that were rest at the perivascular area because of the disturbance over the transport of blood flow hormone circulation.
Although the vascular circulation to the rat hypophysis had been ceased, administration of LH-RH to the proximal portion of stalk of the anterior pituitary gland was capable of influencing the adenohypophysis. Few nerve fibers were observed in the anterior lobe of the rat pituitary gland (10, 11, 12), and the intermediate lobe is richly innervated in some species (13), however the LH-RH given to the animals whose brains had been removed did not directly stimulate nerve fiber activity. In this study, the systemic circulation had clearly ceased after the procedure of brain removement, therefore the LH-RH treated on the cut surface of the gland could not get to the hypophysis via portal veins. In addition, the concept of diffusion from the surgical site to the pituitary gland has also been dismissed due to the speed by which LH-RH could diffuse within tissue fluids (14). Many investigators have examined gap junctions between endothelial cells of capillaries in rat embryos, rat kidney vessels and monolayer cultures of capillary endothelial cells (15, 16, 17). However, no report can be found demonstrating gap junctions between endothelial cells within the anterior pituitary gland, therefore information transport via junctionally coupled endothelial cells does not exist in adenohypophysis.
One further possibility by which the releasing hormone could have reached the gonadotrophs may have been through either the intermediate or posterior lobes; however, these avenues for LH-RH to reach the anterior lobe are remote (18) since there are only few direct anatomical connections including small sympathetic nerves between the three regions of the hypophysis (19, 20). Thus, a more rapid conduction system must be employed such as through the pars tuberalis located in the pituitary stalk. The pars tuberalis consists of two to four cell layers extending around the hypophysial stalk, and covers the anterior side of the median eminence. Early investigators thought that pars tuberalis had a simple function of mechanical support for the infundibular stem, however there is a trunk of interwoven portal capillaries where hypothalamo nerve ends are thought to release the hypophysiotrophic factors (21).
Further ultrasructural studies about the pars tuberalis of rat adenohypophysis revealed the presence of at least two cell types; one is a granulated cell, and the other is a non-granulated cell showing similar appearance to folliculo-stellate cells in anterior pituitary gland. Hormone containing cells could be directly identified for their locarization within the pituitary gland in the progress of immunocytochemical techniques (7). The presence of immunoreactive luteinizing hormone containing cells was demonstrated in the pars tuberalis of various species (22, 23). The following studies revealed that gonadotropes and thyrotropes can be generally found in pars tuberalis of mammalian species (24).
In another hand, the non-granulated cells in pars tuberalis have been thought to have junctional coupling to folliculo-stellate cells in the anterior pituitary gland. Agranular cells which showed star-like ultra structural characteristics within adenohypophysis of many mammalian species have been named folliculo-stellate cells. They were initially reported by Farquhar (25) as adrenocorticotrophs in the anterior pituitary gland. In spite of the great effort of many investigators, the precise physiological functions of these cells have not been fully elucidated. Intercellular junctions, including gap junctions, have been described between cells of the monkey, rat and teleost anterior pituitary glands, indicating the existence of cell-to-cell communication within the organ (26, 27, 28, 29, 30, 31, 32). The presence of gap junctions between folliculo-stellate cells may allow both metabolic and electrical coupling within anterior pituitary gland.
It has been suggested that some information conducting factors pass through gap junctions. While such factors still remain to be identified, several second messengers have been shown to pass through gap junctions, including cyclic AMP and Ca2+ ion. It has been reported that this cell-to-cell Ca2+ passage may lead the intercellular Ca2+ signaling observed in many cell types. Though the role of such intercellular signaling in keeping tissue homeostasis remains to be determined, this phenomenon may be important in normal functions in vivo.
Soji et al. (31) performed the quantitative electron microscopic study and described that the morphological formation of gap junctions within anterior pituitary gland could be firstly observed on day 20, and the frequency reached similar to that found in adults by day 40 in case of Wistar-Imamichi strain male rats. Soji and Herbert (32) demonstrated that the gap junctional formation between folliculo-stellate cells was delayed by castration and the slow rate in gap junctional formation in the castrated rats could be compensated by testosterone injection. The final populations of gap junction in the anterior pituitary gland was decided by a castrated age in their developmental stage (33). However, it must be stressed here that a few increment of population of gap junctions between folliculo-stellate cells can be observed in Wistar-Imamichi strain male rats under the castrated condition.
On the other hand, a castration can severely increase the sensitivity of gonadotrophs to environmental changes such as the change of LH-RH concentration. There were many reports about the influence of LH-RH treatment over the gonadotrophs in case of castrated animals for 1970's (2, 3, 4, 5, 6). According to Rennels et al. (2), castrated male rat gonadotrophs reacted to 100ng LH-RH injection and serum concentration of LH increased 13 times, while FSH increased 2 times within few minutes. Soji et al. (5) reported that ovariectomized female rat gonadotrophs responded 5 min. after LH-RH injection and rose the serum concentration of LH rose about 5 times higher than the initial state.
I suppose that the stimulatory "message" of LH-RH may be transmitted via the folliculo-stellate cells to the granulated cells in the anterior lobe by the gap junctionally connected network to affect LH release from gonadotrophs. Moreover, the gonadotrophs in castrated rats possessed hypersensitivity to the information of LH release. Therefore, the granules were secreted from the gonadotrophs in this study could be detected at the pericapillary space beside the vascular circulation ceased vessels.
ACKNOWLEDGEMENTS
The author thanks Professor Tsuyoshi Soji (The First Department of Anatomy, Nagoya City University Medical School) for his scientific advice on this study.

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Figure Legends
Fig. 1: The pituitary gland was separated into five parts; one central area, two intermediate areas and two peripheral areas. Only the peripheral areas were then used in this study.

Fig. 2: Immunohistochemical light microscopy;
a: Intact 40 day-old rat, b: Intact LH-RH treated 40 day-old rat, c: castrated 40 day-old rat and d: Castrated LH-RH treated 40 day-old rat;
In case of the castrated LH-RH treated rat (2d), the reaction products were observed in peri-vascular area (arrow heads) as well as vascular side in the cells (white arrows), the profile of reacted cells showed dull edge.
a, b, c, d X 400
Fig. 3 Electron micrograph of castrated LH-RH treated 40 day-old pituitary.
Small electron dense particles scattered in the enlarged peri-capillary space. A size of the particle was estimated approximately 100-150 nm in diameter. One granule in the pericapillary space was clearly labeled by the protein A gold complex. The area indicated by the arrow is also shown at higher magnification in the inset.
X 7,500
Inset X 12,000

Fig. 4 Electron micrograph of castrated LH-RH treated 40 day-old pituitary
The hypertrophied basophiles which contained enlarged endoplasmic reticulum were observed. The granule in the enlarged pericapillary space was labeled by the protein A gold complex (arrow), while the secretary granules within the granulated cell were also labeled similarly (arrow heads). The labeled granule in the pericapillary space is also shown at higher magnification (arrow in the inset).
X 7,500
Inset X 12,000


Nagoya City University
Medical School