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Immunohistochemical Evidence That Follicle-Stimulating Hormone and Luteinizing Hormone Reside in Separate Cells in the Chicken Pituitary¹

^a Germplasm and Gamete Physiology Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20705 ^b Laboratory of Neuroendocrinology and Immunological Biotechnology, Zoological Institute, Catholic University of Leuven, B-3000 Leuven, Belgium

	ABSTRACT

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As is the case in other tetrapod species, the chicken gonadotropins LH and FSH consist of a common subunit and a hormone-specific ß subunit. Gonadotrophs containing LH were shown earlier to be distributed throughout both the caudal and cephalic lobes of the chicken anterior pituitary, but the cellular distribution of FSH in avian species is still uncertain. The purpose of this study was to determine the cellular distribution of FSH-containing chicken gonadotrophs by use of FSH-specific monoclonal antibodies (mAbs). Three new mAbs toward chicken FSH were proven hormone specific by immunodetection of purified hormones on dot blots and by dual-label immunohistochemistry (IHC) on sagittal sections of chicken pituitaries. A rabbit antibody was used to detect chicken LH.

Results showed that LH-containing gonadotrophs were densely distributed throughout the anterior pituitary, whereas gonadotrophs containing FSH were much less numerous; in addition, while also present in both lobes, FSH-positive cells were largely absent from the outer margin of the gland. Dual-label IHC revealed that LH and FSH reside almost exclusively in separate gonadotrophs. The identity of FSH-containing cells was further confirmed through use of an antibody to the chicken subunit, which showed that FSH immunoreactivity was always colocalized with the subunit.

Our results suggest the possibility that production and secretion of LH and FSH may be regulated differently in chickens than in most other species studied to date.

	INTRODUCTION

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The cellular localization of gonadotropins has been determined by immunohistochemical (IHC) techniques in a variety of species [1–15]; except in cattle [3], the majority of gonadotrophs in each species contain both LH and FSH. However, the cellular localization of LH and FSH in birds is enigmatic. A series of histochemical studies have provided conflicting evidence regarding whether there are one or two types of gonadotroph cells in the bird and whether they are found in both lobes or in just the caudal lobe of the anterior pituitary (see [16, 17] for reviews). IHC studies have variously shown avian LH to be present only in the caudal lobe [18] or in both lobes of the anterior pituitary [16, 17, 19]. Mikami [16] reported that Japanese quail pituitary cells that bound anti-chicken FSH serum also bound anti-chicken LH serum. However, cells that bound either antiserum also reacted with an antiserum to rat thyroid-stimulating hormone (TSH), suggesting that problems of antiserum specificity may contribute to the confusion concerning the hormonal content of avian gonadotrophs.

Production of antisera that are highly specific for gonadotropins is difficult because of structural similarities of FSH, LH, and TSH. These glycoprotein hormones consist of a common subunit and a hormone-specific ß subunit. Few antibodies to chicken FSH are available [20, 21], and since they are polyclonal antisera to the intact hormone, the interpretation of IHC results may be difficult. Although antibodies cross-reacting with LH were removed from one of these antisera [21] by immunoaffinity chromatography, this antibody is in very limited supply and is used only for RIA. Therefore, the present project was undertaken to produce monoclonal antibodies (mAbs) to chicken FSH that would be devoid of cross-reaction with LH, and to employ these antibodies to resolve the question of the cellular localization of FSH in the chicken.

Our results have demonstrated for the first time that FSH and LH reside in separate populations of gonadotrophs in the chicken pituitary, and that LH- and FSH-immunoreactive (ir) cells are present in both lobes of the anterior pituitary but are distributed differently within the gland.

	MATERIALS AND METHODS

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mAb Production

Three 6-wk-old female mice were immunized s.c. three or four times with 25 µg of purified chicken FSH (USDA-cFSH-I-1) at 3-wk intervals. The first and second immunizations employed a 50:50 (v:v) emulsion with Freund's complete adjuvant, while subsequent immunizations used incomplete adjuvant. One mouse was killed 4 wk after the third immunization, and two mice were killed 4 wk after the fourth immunization. In each case, mice were boosted by i.p. injection of 50 µg of chicken FSH in PBS 4 and 2 days prior to fusion. A suspension of activated splenocytes was prepared, and a portion was fused with SP2/0 myeloma cells by electrofusion [22]. The fused cells were plated into a total of thirty 96-well plates at a density of 20 000 cells per well in the presence of 100 U recombinant human interleukin-6 (Boehringer-Mannheim, Indianapolis, IN) to support single-cell growth. Plates were screened 2 wk after fusion using an IHC screening protocol [23] on 5-µm sagittal paraffin sections of nonperfused Bouin-Hollande Sublimate-fixed pituitaries from juvenile broiler chickens. (For Bouin-Hollande Sublimate, Bouin-Hollande solution is prepared first, by dissolving 4 g anhydrous picric acid [or 6 g of wet picric acid] and 2.5 g of anhydrous copper acetate in 90 ml distilled water, then adding 10 ml of 40% formaldehyde and 1 ml of glacial acetic acid. The Sublimate is prepared just before use by mixing 90 ml of the solution with 10 ml of saturated mercuric chloride. Saturated mercuric chloride solution is prepared by adding 8 g mercuric chloride to 100 ml water, boiling, and cooling.) Positive cultures producing relevant IHC staining patterns were propagated and cloned by limiting dilution.

IHC Characterization of mAbs

The mAbs were initially characterized during the IHC screening protocol using a peroxidase-conjugated goat anti-mouse IgG as described previously [24]. Final characterization of chicken gonadotrophs employed 4-µm sagittal sections of nonperfused chicken pituitaries both from 4-wk-old broiler chickens and from laying Leghorn hens at the end of the reproductive season (> 70 wk of age). Gonadotrophs expressing chicken LH were detected using a rabbit anti-chicken LH polyclonal antiserum (USDA-AcLH-5) provided by the USDA-ARS Animal Hormone Program (Beltsville, MD). All steps were conducted at room temperature. Tissue sections were incubated overnight with primary antibody at a dilution of 1:10 000 in Tris-saline buffer containing 0.1% Triton X-100, pH 7.4 (TBS). Detection employed a 1-h incubation with peroxidase-conjugated goat anti-rabbit IgG (1:2000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and color development with diaminobenzidine (DAB) [24]. In experiments using dual labeling with chromogenic detection, the appropriate rabbit polyclonal primary antibody was mixed with mouse anti-chicken FSH (ascites diluted 1:3000 to 1:10 000) for the initial incubation. After the first staining sequence with DAB was terminated, sections were incubated with biotin-conjugated goat anti-mouse IgG (1:5000; Jackson) for 1 h, followed by incubation for 30 min with alkaline phosphatase-conjugated streptavidin (1 µg/ml; Jackson). Color development with naphtol AS-MX phosphate and Fast Blue BB salt (Sigma Chemical Co., St. Louis, MO) [23] yielded blue cells that contrasted well with DAB-labeled cells (brown).

For dual-label fluorescent IHC for LH and FSH, tissue sections were first incubated for 1 h in 2.5% normal goat serum and then incubated overnight with primary antibodies as described above. Sections were then incubated with biotinylated goat anti-rabbit IgG (1:5000; Jackson) for 1 h. After washing, sections were incubated with a mixture of lissamine-conjugated anti-mouse IgG (1:100; Jackson) and avidin-fluorescein isothiocyanate (25 µg/ml; Vector Laboratories, Burlingame, CA) for 2 h in the dark. After washing with TBS, coverslips were applied using Vectashield (Vector), and FSH-ir cells (red) and LH-ir cells (green) were studied using fluorescence microscopy (Axioplan microscope; Zeiss, New York, NY).

Dual-label fluorescent IHC for FSH and subunit was performed as described above utilizing a rabbit polyclonal antiserum to chicken subunit [25] at a dilution of 1:5000. Cells that contained only subunit yielded green fluorescence, while cells that contained both FSH and subunit were yellow/orange.

Antibody Specificity

The potential cross-reactivity of anti-chicken FSH mAbs for chicken LH was assessed by incubating each antibody overnight with a nitrocellulose disk (7-mm diameter) that had been spotted with either 5 or 10 µg of purified chicken FSH (USDA-cFSH-K-1) or 10 µg of purified chicken LH (USDA-cLH-K-3). Similarly, the specificity of USDA-AcLH-5 was confirmed by incubation with spot blots containing 5 or 10 µg of chicken LH or 5 µg of chicken FSH. The membranes were blocked by incubation with 1% BSA prior to exposure to primary antibody. Detection of bound antibody was accomplished by incubating disks with either goat anti-rabbit IgG (for AcLH) or goat anti-mouse IgG (for mAbs) diluted 1:50 000 for 1 h at room temperature. After washing in TBS, the disks were incubated with streptavidin-alkaline phosphatase as described for IHC. Color was developed using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate (Sigma) as substrate [24] to produce a purple precipitate.

	RESULTS

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IHC screening of fusion plates was used to identify several wells containing mAbs that bound to cells present in both lobes of the anterior pituitary. Since only gonadotrophs are known to populate the entire avian anterior pituitary, the hybridoma cells in these wells were propagated, a portion was frozen for storage in liquid nitrogen, and several monoclonal cell lines were produced that secreted mAbs providing consistent immunostaining after repeated single-cell cloning. Three independently derived monoclonal cell lines (5B8G11, 16E4B6, and 20A3B5) produced a staining pattern in both lobes of the anterior pituitary (Fig. 1A) that differed from the pattern observed with the antiserum to chicken LH (Fig. 1B). The cells stained with anti-chicken FSH mAbs were obviously less numerous than those stained with anti-chicken LH, and FSH-ir cells were largely absent from the margin of each pituitary section (arrows, Fig. 1, A and B). The appearance of FSH-ir gonadotrophs under high magnification (Fig. 1C) was similar to that of LH-ir gonadotrophs (Fig. 1D) and of the gonadotrophs described by Mikami [16].

View larger version (147K): [in this window] [in a new window]   FIG. 1. IHC localization of FSH (A) and LH (B) in sagittal sections of chicken anterior pituitary (x200), demonstrating the differences in number and distribution of immunostained cells. Note the relative absence of cells immunostaining for FSH at the margin of the section, while LH-ir cells are found throughout this area (arrows). C and D show FSH-ir and LH-ir gonadotrophs at higher magnification (x1000). Reproduced at 60%.

The specificity of the mAbs for FSH was shown by detection of purified FSH on spot blots. Incubation of antibodies with spot blots containing purified LH or FSH showed that the mAbs bound to FSH but not to LH (Fig. 2). The specificity of the polyclonal LH antiserum has previously been shown by RIA [19, 26] and was confirmed for use in IHC by spot blots (Fig. 2).

View larger version (63K): [in this window] [in a new window]   FIG. 2. Specificity of three independently derived mouse mAbs to chicken FSH and a rabbit polyclonal antibody to chicken LH as demonstrated by immunostaining of nitrocellulose membranes spotted with either purified FSH (USDA-cFSH-K-1) or LH (USDA-cLH-K-3). Only background staining was observed on spots that received no hormone (0) or on spots that received the potentially cross-reacting gonadotropin, while the gonadotropin used as immunogen was strongly recognized.

Dual-label IHC for LH and FSH using chromogenic stains (Fig. 3A) clearly showed that most gonadotrophs contained either LH (brown) or FSH (blue). The FSH mAb seen in this figure was 5B8G11, but similar results were obtained with 16E4B6 and 20A3B5 (not shown). Dual-label IHC using FSH mAbs and rabbit polyclonal antisera to chicken GH or chicken prolactin (PRL) confirmed that GH-ir cells were localized in the caudal lobe and that PRL-ir cells were localized in the cephalic lobe, whereas FSH-ir cells were present in both lobes and distinct from somatotrophs and lactotrophs (data not shown). Because it is difficult to unambiguously identify dual-labeled cells with chromogenic staining, we used dual-label fluorescent IHC to confirm that LH and FSH were present in distinct cell populations that were distributed in different patterns throughout the anterior pituitary (Fig. 3, B and C). As observed with DAB staining, LH-ir cells were evenly distributed throughout the gland, while FSH-ir cells were largely absent from the perimeter of sagittal sections (Fig. 3B). Gonadotrophs that were dual labeled for both LH and FSH were occasionally observed (arrow, Fig. 3D), but were very rare.

View larger version (85K): [in this window] [in a new window]   FIG. 3. Dual-label IHC for chicken FSH and LH. A Demonstrates the localization of FSH (blue) and LH (brown) in separate gonadotrophs using chromogenic staining (x400). Use of immunofluorescent stains demonstrates the clear differences in organization of FSH-ir cells (red) and LH-ir cells (green) at low magnification (B; x200). High magnification (x1000) confirms the virtually complete separation of LH and FSH immunoreactivity into separate populations of gonadotrophs (C), although cells that appeared to contain both hormones were observed infrequently (arrow, D). To independently confirm that the cells identified as FSH-ir cells by the mAbs did, indeed, contain gonadotropin, E demonstrates dual labeling with a rabbit polyclonal antibody to the subunit (green) and a mAb to FSH (red). All FSH-ir cells also contained the subunit, yielding an orange/yellow stain. Reproduced at 64%.

Dual-label IHC for FSH and the subunit revealed that all pituicytes containing FSH-ir also contained the subunit (Fig. 3E). Many cells contained subunit without FSH-ir, presumably identifying LH-containing gonadotrophs and thyrotrophs.

Since the possibility exists that populations of gonadotrophs may vary with age or reproductive state, we included pituitary sections from both juvenile broilers and aged Leghorn laying hens (n = 3 individuals of each type) in both single- and dual-label fluorescent LH/FSH IHC procedures. In all cases, FSH and LH were detected in separate cell populations, with only rare appearance of dual-labeled cells. No conspicuous differences were observed in either the distribution or the relative abundance of LH-ir and FSH-ir cells between birds differing in both age and reproductive state (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Immunization of mice with intact chicken FSH produced at least three different mAbs that specifically recognize epitopes on chicken FSH not present on chicken LH. Although purified chicken TSH is not available for testing, our IHC results suggest that these antibodies do not recognize TSH, since thyrotrophs are located exclusively in the cephalic lobe of the chicken anterior pituitary [19] and we observed no concentration of immunostained cells in either lobe. Chicken FSH-ir was always colocalized with the subunit, as expected. Interestingly, our IHC screening procedure detected no antibodies producing a pituitary staining pattern that would be consistent with binding to the subunit. The choice of an IHC procedure for screening mAbs was highly advantageous because it detects mAbs of moderate or low affinity that might be overlooked in more conventional screening procedures. Although most IHC studies of LH and FSH localization in other species have employed antibodies raised to the ß subunit of one or both hormones [2, 4, 6, 8–14], this approach is not always suitable for localizing the native hormone [3]. Such an approach was not possible with chicken FSH because of the scarcity of purified hormone [27].

The IHC evidence presented here clearly demonstrates that LH and FSH reside in separate populations of gonadotrophs in the chicken pituitary. Quantization of the proportions of each cell type in the chicken pituitary was not attempted because FSH-ir gonadotrophs were not randomly distributed; but FSH-ir cells were clearly less numerous than LH-ir cells. Dual labeling with fluorescent detection showed that pituicytes containing both gonadotropins were very rare in both immature broiler (meat-type) chickens and in aged laying (egg-type) chickens. Thus, it seems likely that production of FSH and LH in separate cells is a characteristic of the species and not a function of age, strain, or reproductive state. Such a characteristic is highly unusual among the wide range of species studied to date and does not appear to have a phylogenetic basis. In pigs [10] and lizards [8], all gonadotrophs reportedly contain both LH and FSH. In other species ranging from the human [15] to the frog [2], at least two thirds of gonadotrophs contain both hormones. However, the bovine pituitary, like that of the chicken, produces LH and FSH in separate cells. Bastings et al. [3] suggested that technical problems related to specificity of antisera may contribute to "ambiguous" IHC results in other species (i.e., existence of LH-only and FSH-only gonadotrophs among a preponderance of gonadotrophs containing both hormones). However, a recent study of frog [2] gonadotrophs, utilizing dual immunofluorescent labeling with highly characterized mAbs to the ß subunits of bullfrog LH and FSH, has confirmed a high frequency of colocalization within gonadotrophs. The authors suggest that all gonadotrophs synthesize both hormones and that the presence of a small number of cells immunoreactive to only one gonadotropin results from differential secretion. However, the regulatory mechanism(s) by which one gonadotropin could be selectively depleted from a subpopulation of bihormonal gonadotrophs remains unclear.

The peculiar cellular distribution of FSH-ir gonadotrophs in the chicken pituitary was unexpected, and we know of no reports of a comparable anatomical organization of pituitary cells. It is possible that blood supply to the outer "cortex" of the gland may differ from that which supplies cells in the interior of the gland and leads to preferential regulation or depletion of FSH in one area. However, the similarity of this pattern in very young, reproductively inactive birds as well as old laying hens suggests that other explanations should be sought.

Our findings that LH and FSH reside in different cells suggest that the chicken may be an excellent model for studying the differential regulation of gonadotropin secretion. Although two forms of GnRH have been isolated in the chicken, little is known of their effects on FSH secretion. Krishnan et al. [21] demonstrated a significant stimulation of LH secretion by both mammalian GnRH and chicken GnRH-I in immature hens and young cockerels with no concomitant increase in FSH secretion. The effect of chicken GnRH-II on chicken FSH secretion has not been studied.

	ACKNOWLEDGMENTS

 
The authors thank the USDA-ARS Animal Hormone Program for the gift of chicken gonadotropins and LH antiserum. The assistance of Dr. Ramesh Ramachandran in immunofluorescent techniques is gratefully acknowledged.

	FOOTNOTES

 
¹ L.R.B. was granted as a Senior Research Assistant by the Flanders Fund for Scientific Research (F.W.O.-Vlaanderen). J.A.P. and L.R.B. are collaborators via a fellowship under the OECD Co-operative Research Programme: Biological Resource Management for Sustainable Agricultural Systems.

² Correspondence: J.A. Proudman, USDA-ARS, Germplasm and Gamete Physiology Laboratory, Bldg. 262, BARC-East, Beltsville, MD 20705. FAX: 301 504 8546; johnp{at}lpsi.barc.usda.gov

³ Current address: Poultry Science Department, Texas A&M University, College Station, TX 77843.

Accepted: January 11, 1999.

Received: November 18, 1998.

	REFERENCES

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal My Folders Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Proudman, J.A. Articles by Berghman, L.R. Search for Related Content PubMed PubMed Citation Articles by Proudman, J.A. Articles by Berghman, L.R. Agricola Articles by Proudman, J.A. Articles by Berghman, L.R.