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Immunohistochemical and Ultrastructural Study of the Rat Anterior Pituitary Gland
Conduction of Information of LH-RH by the Folliculo-Stellate Cells

Eiji Yamada, Morihiro Yoshida, Hiroyuki Kimura, Jun Kurita, and Chieko Kurono

Department of First Anatomy, Nagoya City University Medical School,
1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya City, Aichi, 467-8601, Japan
Running Title: Possible system of conduction of information

KEY WORDS: Rat, Anterior pituitary, Pars tuberalis, LH-RH

Correspondence: Eiji Yamada
Address: Department of First Anatomy, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya City, Aichi, 467-8601, Japan

ABSTRACT
The folliculo-stellate cell of the anterior pituitary gland was initially reported by Farquhar (1). Intercellular junctions, including gap junctions, have been described between parenchyma cells of the monkey, rat and teleost anterior pituitary glands, indicating the existence of cell-to-cell communication within the organ. We have previously examined their possible role in the rapid dissemination of information through a complex system of interconnecting follicles and reported that gap junction formation in the male rat pituitary gland appears to be under hormonal control. In this study 20 and 60 day-old male rats were each divided into 3 groups. The first group consisted of 20 and 60 day-old animals sacrificed as the intact controls; in the second group, the brain of the rats was removed3 min before sacrifice; and the third group animals were treated in a similar manner as the second except that they were given LH-RH for 3 min. after removal of the brain. The pituitary glands were then analyzed using immunohistochemistry and histomorphometry or processed for ultrastructural examination. The morphological changes were rather slight, with the number of secretory granules in some LH-type cells decreasing following LH-RH treatment. Gap junctions were not present between folliculo-stellate cells in the hypophysis of the 20 day-old rats nor were significant changes in LHb immunoreactive cells noted. The density of the LH cells increased slightly after brain detachment but declined significantly in the presence of LH-RH on day 60. These changes were confirmed by histomorphometry. The present study shows that cell-to-cell conduction of the folliculo-stellate cells through gap junctions acts to aid in the regulation of hormone secretion, presumably LH, from the hypophysis. Moreover, these findings also indicate the possibility that the signal provided by the releasing hormone was amplified through a network of folliculo-stellate cells, perhaps located in the pars tuberalis.

INTRODUCTION
Farquhar (1) initially reported the folliculo-stellate cell of the anterior pituitary gland as an adrenocorticotroph. Intercellular Junctional specializations, including gap junctions, have been described between cells of the mammals and teleost anterior pituitary glands, indicating the existence of cell-to-cell communication within the organ (2-8). We had demonstrated the presence of gap junctions in the adult male rat hypophysis, noting that they were only associated with folliculo-stellate cells and not with any of the granular cell types (6-8). Moreover, we have pointed to their possible role in the rapid dissemination of information through a complex system of interconnecting follicles and that gap junction formation in the male rat pituitary gland appears to be under hormonal control (6-8). In the present report, we document the presence of a possible role for another regulatory mechanism of hormone secretion in the anterior pituitary gland.

MATERIALS AND METHODS
Fifteen of 20 day-old and 15 of intact 60 day-old male Wistar-Imamichi rats were used. Two intact groups of animals divided into 3 sub-groups. Each sub- group consisted of 5 animals. As the first group, each 5 of 20 and intact 60 day-old animals were sacrificed under Nembutal (Phenobarbital) anesthesia and served as the intact controls. In the second and third groups, the animals were anesthetized with Nembutal, their calvaria opened and brains removed. Then the intermediate and posterior lobes were removed from the grand to avoid the influence of both lobes. The all animals were treated with The Guidelines for Animal Experimentation of the Experimental Animal Science Center of the University. This procedure detached the pituitary gland from the hypothalamus and circulation to the hypophysis ceased. The animals in the second group were treated saline on the cut surface of the pars tuberalis for 3 min. under low magnification binocular, then sacrificed. Those in the third groups were treated with LH-RH for 3 min. by placing on the cut surface of the pars tuberalis a small triangular piece of filter paper containing LH-RH [Tanabe Seiyaku Co., LTD, Tokyo; 0.8 mg/ml in saline (Fig. 1)]. Following this, they were sacrificed. Exposure of the anterior lobe (pars tuberalis) on the detach surface was confirmed microscopically (Fig. 2).The pituitary glands were 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 (Fig. 3). The peripheral areas were then used in this study. The samples to be studied by electron microscopy were immersed for one hour in a fixative containing 2.5% glutaraldehyde and 2% sucrose buffered with 0.05M sodium cacodylate (pH 7.4). The specimens were washed for half an hour with the same buffer solution as used for fixation and postfixed for two hours with a 1% osmium tetroxide fixative containing 2% sucrose, buffered with 0.05M sodium cacodylate (pH 7.4). After postfixation, the tissues were subsequently dehydrated in a graded series of ethanol for 10 min. each, immersed twice in propylene oxide for 15 min., and embedded in epoxy resin (9). Thin sections were obtained and then stained with uranyl acetate and lead citrate before being observed using a Hitachi H-7000 transmission electron microscope.
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. Twenty specimens were obtained from each animals for immuno-histomorphometry. The sections were immunostained by the peroxidase-labeled antibody method of Nakane and Pierce (10) using a slight modification (11). 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 (11). On each obtaiend specimen, 20 fields were rundomly sellected for histomorphometry. The intensity of the pituitary sections immunostained with the antibody to LH was measured using a light microscope equipped with a CCD-TV camera (Nikon-Hitachi, Tokyo, Japan), and analyzed with a computer by image-analyzer software (NIH Image 1.5). The treatment means were tested for homogeneity using one-factor analysis of variance (ANOVA), and the difference between specific means was tested for significance using Scheffe's multiple range test. A difference between two means was considered statistically significant when p was <0.05. The all results were expressed into meanSE.

RESULTS
Changes in the 20 day-old rat pituitary
No significant morphological changes were observed between the normal controls, the animals in which the brain had been removed and detached from the pituitary stalk for 3 min, and those given LH-RH following removal of the brain (Fig. 4a,b,c). Gap junctions between the non-granulated folliculo-stellate cells were not found. Results of the histomorphometry studies of the LH-containing cells showed no significant changes in cell area between the normal controls (403255034 sq.pixel), animals in which the brain had been removed and given saline (406243239 sq.pixel) and in those given LH-RH (450544080 sq.pixel) (Fig. 5). These findings were confirmed by immunohistochemistry (Fig. 6).
Changes in intact 60 day-old rat pituitary
The morphological changes were rather slight between the normal controls (Fig. 7a), the animals in which the brain had been removed and detached from the pituitary stalk for 3 min., and those given LH-RH following removal of the brain (Fig. 7b); secretory granule number appeared to have decreased after LH-RH treatment in some of the LH-type cells (Figs. 7).
The results of histomorphometry studies in which the area of LH-secreting cell was quantified (Fig. 8). Cell areas which were reactive to the anti-LH serum changed from 162932148 sq.pixel (Figs. 8 control, 9a) to 205102216 sq.pixel (Figs. 8 cut after 3 min., 9b) in the controls vs. animals in which the saline treated, then decreased to 119252939 sq.pixel (Figs. 8 LH-RH, 9c) in those given LH-RH. The reduction as a result of LH-RH treatment was significant when compared to either of the other animal groups (Fig. 9). The immunocytochemical studies corroborated these observations (Fig. 9).

DISCUSSION
This study demonstrates that although the vascular circulation to the rat hypophysis had been removed, administration of LH-RH to the proximal portion of stalk of the anterior pituitary gland was capable of influencing the hypophysis in the 60 day-old rat pituitary gland, while having no significant effect on the LH containing cells in the 20 day-old animals.
The peroxidase-labeled antibody method (10), the peroxidase-anti-peroxidase method (12), and the avidin-biotin-peroxidase complex method (13) have been widely used to detect particular proteins in histological sections. An peroxidase-labeled antibody method was developed to quantify the procedure using a model system (11,14, 15). These investigators showed that the magnitude of the reaction was parallel to the amount of antigen present in a solid-phased matrix, which was made up of gelatin, polyacrylamide, or bovine serum albumin. Therefore, the reduction in immunostained area should reflect a reduced amount of an antigen in cells. Thus, the immunohistochemical method which was used in this study provided an estimate of the hormonal content of the hypopyseal cells.
There was a significant decrease in LH cell area between day 60 (162932148 sq.pixel in control) and day 20 (403255034 sq.pixel in control). These differences may be due in part to changes in the weight of the hypophysis with age and differential changes in the cells. The weight of the organ increased about 3 times between 20 and 50 days of age [2.30.2 vs. 7.10.4 mg in Sprague-Dawley male rats (16)]. It is possible that the number and volume of pituitary cells which were not immunoreactive to the anti-rat-LH serum increased more than the incremental change in anti-rat-LH reacted cells. Therefore, although the total number of LH cells increased with age, their density decreased (Fig. 5a and 9a).
While the anterior lobe of the rat pituitary gland contains few nerve fibers (17-19), the LH-RH given to the animals whose brains had been removed did not directly stimulate nerve fiber activity. In these same animals, the systemic circulation had clearly ceased suggesting that the LH-RH placed on the cut surface of the gland could not have been distributed to the hypophysis by the hypophyseal portal veins. Additionally, concerned with the molecular weight, LH-RH is too large to diffuse to 3 mm within 3 min. The concept of diffusion from the central site, which is surgical site, to peripheral area of the pituitary gland has also been dismissed due to the speed by which LH-RH could diffuse within tissue fluids (20). Marshall (20) described when the certain molecule was dropped in a water, the diffusion speed of 3mm/3min. was expected about 100 MW or less. The molecule weight of LH-RH which was used in this study was a deca-peptide. Even in water, such large molecule needed 3 or more hours to diffuse 3mm. Therefore the discharge of the LH reactive substance from certain cells in this study was difficult to concern direct effect of administrated LH-RH. In some species, the intermediate lobe is richly innervated (21). 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 (22) since there are few direct anatomical connections including small sympathetic nerves between the three regions of the hypophysis (23, 24). Thus, a more rapid conduction system must been employed such as through the pars tuberalis located in the pituitary stalk, a region which is considered as the most highly vascularized subdivision of the hypophysis (25). This region consists of small agranulated cells, cells which contain LH and thyrotrophs and cells which form follicle-like structures in humans (26) and monkeys (27).
Our previous studies showed that the agranular folliculo-stellate cells in the sexually mature adult were connected by gap junctions (6). No gap junctions were observed before day 20 but once they appeared, the density of gap junction in the gland increased with development until the time the animals (Wistar-Imamichi rats) had undergone puberty, at approximately 45 days of age (7). Thereafter, gap junction numbers were influenced by the sexual state of the animal, such as the development of the reproductive axis, estrous cycle, pregnancy and ovariectomy/castration (28, 29, 30). Recently, Yanagisono et al. (31) found that the LH-RH positive neurons terminated on the S-100 positive cells in the pars tuberalis. The findings from our previous reports indicate that the agranular folliculo-stellate cells play an important role in the physiology of the pituitary gland from information of the releasing hormon, perhaps by participation in the regulation of hormone secretion.
The present study indicates that no influence of LH-RH was observed on day 20 when no gap junctional connections were present between adjacent folliculo-stellate cells. However, a measurable influence of LH-RH was apparent in the 60 day-old animals when gap junctional connections were well established. Therefore, these findings lead us to postulate that the stimulatory "message" from LH-RH may have been transmitted via the folliculo-stellate cells in the pars tuberalis to the granulated cells in the anterior lobe through the network of gap junctions to ultimately affect LH release.

ACKNOWLEDGMENT
The author gives great thanks to Professor Damon C. Herbert (The Health Science Center at San Antonio, University of Texas), Dr. Nobuyuki Shirasawa (Department of Anatomy. Wakayama Medical Collage) and Professor Tsuyoshi Soji (Nagoya City University, Medical School) for their support and advice during the course of this study.

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FIGURE LEGENDS
Figure 1: LH-RH was administrated through the tip of triangular piece of filter paper on the cut surface of the gland (arrow). A: Anterior lobe. P: Posterior lobe
Figure 2: Light micrograph of the cut surface of the hypophyseal stalk in 60 day-old rat. No intermediate lobe is observed. A: Anterior lobe. P: Posterior lobe
Truisin blue X 20
Figure 3: A schematic representation of the morphology of the anterior pituitary gland. Only the peripheral areas were used in the present study.
Figure 4: Electron micrographs of gonadoptrophs from the anterior lobe of a 20 day-old a) intact control, b) animal whose brain had been removed for 3 min prior to sacrifice, and c) animal treated with LH-RH for 3 min. following removal of the brain. Morphologically no marked changes were observed between the three animal groups.
a X3,500
b X4,800
c X3,500
Figure 5: The areas of LH cells in 20 day-old rats as identified by immunocytochemistry; no significant differences were noted amongst the three animal groups.
Figure 6: Immunohistochemical verification of the changes in the LH cells in the 20 days-old rat following removal of the brain in the absence (b) or presence (c) of LH-RH; a) is from an intact control.
X 200
Figure 7: Electron micrographs of gonadoptrophs from the anterior lobe of a 60 day-old a) intact control, and b) animal treated with LH-RH for 3 min. following removal of the brain. In the animals given LH-RH, some gonadotrophs displayed fewer secretary granules.
a and b X4,800
Figure 8: The areas of LH cells in the 60 day-old animals as identified by immunocytochemistry revealed marked differences amongst the three animal groups in the study.
* p<0.05, ** p<0.01
Figure 9: Immunohistochemical verification of the changes in the LH cells in the 60 day-old rat following removal of the brain in the absence (b) or presence (c) of LH-RH; a) is from an intact control. Compared with the 20 day-old animals, the LH cells were less numerous.
X 400

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