0013.7227/92/1312-0947$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine
Vol. 131, No. 2 Printed in U.S.A.
Society
Expression of Epidermal Growth Factor and its Ligands, EGF and Transforming a!, in Human Fallopian Tubes* Z. M. LEI
AND
Department 40292
of
(EGF) Receptor Growth Factor-
Ch. V. RAO Obstetrics
and Gynecology,
University
of
Louisville,
School of Medicine,
Louisville,
Kentucky
tory molecules varied. For all of them, however, ampullary segments contained more than isthmus; proliferative phase and/or postpartum specimens contained more than secretory phase; and postmenopausal specimens contained the lowest amounts. The cell periphery and nuclear/perinuclear area of the cells contained EGF, TGF-(u, and their receptors. Immunogold electron microscopy showed the receptors to be present in cell membranes, cilia, basal bodies which control ciliary activity, endoplasmic reticulum, nuclear membranes, and chromatin. In summary, human fallopian tubes contain EGF, EGF/TGF-(Y receptor mRNA and protein, and TGF-cu protein. The expression of all these regulatory molecules was dependent on anatomical region, cell type, and reproductive state of the fallopian tubes. These findings suggest that EGF and TGF-ol may regulate numerous tubal functions, thus potentially influencing fertility in women. (Endocrinology 131:
ABSTRACT
Although human uterus is known to contain epidermal growth factor (EGF) and its receptors, it is virtually unknown whether human fallopian tubes, which are an anatomical continuation of the uterus, also contain them. Therefore, the present studies investigated whether EGF and its structural and functional homolog, i.e. transforming growth factor-a (TGF-ol), and their common receptor are expressed in human fallopian tubes. Human fallopian tubes contain major 10.5-kilobase (kb) and minor 6.0-kb receptor messenger RNA (mRNA) transcripts, a single 5.0-kb EGF mRNA transcript, and a single 170-kilodalton receptor protein. The transcripts, along with their corresponding proteins and TGF-ol protein, are present in ciliated and nonciliated epithelial cells, tubal smooth muscle, vascular smooth muscle, and endothelium. The cellular distribution and reproductive state dependency of these three regula-
947-957,1992)
E
whether fallopian tubes from humans or from any animal speciescontain EGF or its structural and functional homolog, TGF-LU,and their common receptor. As a first step toward a better understanding of possible regulation of human fallopian tubes by EGF/TGF- (Y, we investigated whether this organ might expressthese regulatory molecules and whether cellular, anatomical, and reproductive state differences exist in their expression.
PIDERMAL growth factor (EGF) and transforming growth factor-0 (TGF-a) are single-chain polypeptides of similar size [-6 kilodaltons (kDa), structure, and function, and are encoded by different genes(1,2). EGF was originally discovered in submaxillary glands of mice and TGF-a in transformed cells (3, 4). Now they have been found in many normal and cancer tissues(5). EGF and TGF-(Uare functionally similar, and they bind to a common membrane receptor (5). The receptor is a transmembrane glycoprotein of 170 kDa consisting of extracellular ligand binding domain connected to cytoplasmic kinase domain by a short transmembrane region (6, 7). EGF and TGF-a binding results in a stimulation of phosphorylation of their receptor as well as numerous other target proteins in their tyrosyl residues (6,
Materials
and Methods
Materials Vectastain ABC kits were purchased from Vector Labs (Burlingame, CA); [cy-32P]deoxycytodine triphosphate and Genescreen plus hybridization transfer membrane from New England Nuclear-DuPont Company Biotechnology Systems (Boston, MA); monoclonal antibody to TGF-ol and purified TGF-(U from Oncogene Science (Manhasset, NY); polyclonal antibody to human EGF (hEGF) from Biomedical Technologies Inc. (Stoughton, MA); monoclonal antibody to EGF receptors (clone 29.1), polyclonal antibody to mouse EGF, proteinase 2K, sonicated and denatured salmon sperm DNA, poly(A)+ RNA, transfer RNA, and ribosomal RNA from Sigma Chemical Co. (St. Louis, MO); oligo(dT) cellulose and 0.24-9.5 kilobase (kb) RNA ladder from Gibco BRL (Gaithersburg, MD); XbaI, HindIII, EcoRI, and a random-primed DNA labeling kit from United States Biochemicals (Cleveland, OH); chemicals and supplies for light microscope autoradiography from Polysciences (Warrington, PA); colloidal gold (15 nm)-labeled goat antimouse immunoglobulin G (IgG) from Janssen Life Science Products (Piscataway, NJ); and other immunochemicals, reagents, molecular biology grade chemicals, and solvents from regular commercial sources. Monoclonal antibody to EGF receptors (IgG 528) was obtained from Dr. Hideo Masui (Sloan-Kettering Cancer Center, New York, NY). The recombinant hEGF was provided by Dr. Pablo Valenzuela (Chiron Corporation, Emeryville, CA). Highly purified mouse EGF was obtained from Dr. Gregory Schultz
7).
The fallopian tube, an anatomical continuation of the uterus, is a dynamic and cyclically changing structure which responds to ovarian steroid hormones and prostaglandins (8-14). Fallopian tubes contain multiple cell types, and this cellular diversity helps in the maturation and transport of gametesand embryo, fertilization, and early development of the embryo (8-14). Thus, fallopian tubes play a pivotal role in normal fertility in women. Although human and animal uteri are known to contain EGF and its receptors and are subject to their regulation (15-27), it is virtually unknown Received March 9, 1992. Address all correspondence and reprint requests to: Dr. Ch. V. Rao, Department of Obstetrics and Gynecology, 438 MDR Building, University of Louisville, Louisville, Kentucky 40292. * This work was presented at the 23rd Annual Meeting of the Society for the Study of Reproduction, Knoxville, TN, 1990 (Abstract 99). 947
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VIP-IR
968
CELLS
IN RAT PITUITARY
C
25
” 2: 20
0
male
diesuous
ovx+oil
ovx;+B7pg
ovxgopg
female FIG. 3. Pituitary VIP content (top), PRL content (middle), and serum PRL levels (bottom) in intact male and female rats and OVX rats injected SCwith 7 or 70 rg EB dissolved in sesame oil or oil alone and decapitated 3 days later (AM). Each column represents the mean + SEM of individual values obtained from six to nine rats. a, P < 0.05 us. diestrous female and OVX plus oil group. b, P < 0.05 us. male and diestrous female group. c, P c 0.05 us. all other groups.
in the OVX rats in which EBtreatment resulted in an increase in the pituitary VIP content. As we have demonstrated before, this was the caseonly in the group treated with the highest dose of EB. This effect of 70 pg EB on the pituitary VIP content and serum PRL levels is probably causedby the significantly greater levels of serum estradiol present in the 70 pg group compared to the 7 pg group (9). However, both dosesof EB resulted in serum estradiol levels considerably
Endo. Voll31.
1992 No 2
greater than those found throughout the estrous cycle (9). Thesefindings support the suggestionthat pituitary VIP may be related to the development of estrogen-induced hyperprolactinemia (26-28). The inverse relationship found between serum and pituitary PRL levels and pituitary VIP content in the untreated male ZIS.female rat suggests,however, that VIP is not related to basal PRL secretions. Therefore, although these and other data show that VIP is produced and releasedby rat adenohypophyseal cells, the functional significance of this VIP remains to be defined. In vitro data clearly show that VIP releasedfrom anterior pituitary cells has a stimulatory role in PRL release (3-5). The most compelling evidence that VIP has a role in the regulation of PRL releasecomes from studies in which circulating PRL levels were reduced after the biological action of VIP was neutralized by treatment of rats with a VIP antiserum or antagonist (23, 24, 28-34). Whereas those VIP neutralization studies clearly demonstrate a role for VIP in the regulation of PRL release, they provide no information on the origin of the active VIP in vim. This is critical, because VIP may be released from the median eminence (18, 35) or posterior pituitary (35, 36) in addition to the adenohypophysis and affect pituitary function. Several laboratories have attempted to study the role of anterior pituitary VIP in the regulation of PRL in vim by following the changesin the contents of these two hormones after various endocrine manipulations known to alter PRL content and secretion (2,6-10). The results have been equivocal because, for example, pituitary VIP and PRL content were increased equally by the high dosesof EB (70 pg/rat), while pituitary PRL content, but not VIP, was increasedafter lower dosesof EB (7 pg/rat) (9). This was confirmed in the present study. In hypothyroid rats, the pituitary VIP content was markedly increased, while pituitary PRL was reduced (2, 10). In the same manner, comparing VIP and PRL in anterior pituitaries of untreated males and females suggests that VIP has little to do with the regulation of PRL. However, statements about hormone function based on tissue content must be interpreted with caution, becausecontent may be a poor reflection of hormone secretory dynamics. In summary, these data demonstrate that VIP-IR cells are not lactotrophs and are numerous throughout the anterior pituitary gland of untreated male and female rats, Furthermore, changesin the pituitary content of VIP are probably a reflection of parallel changes in the cellular content of VIP, rather than in the number of VIP-producing cells. Acknowledgments This publication is dedicated to the memory of Dr. Mary Notter, who taught Dr. Phelps to “quantify” rather than “quantitate;” her accidental death on November 2, 1991, was both a personal and professional loss to the author. The technical assistance of Ms. Myra Vaccarella and the photographic expertise of Ms. Dorothy Herrera and Ms. Nancy Dimmick are gratefully acknowledged. We thank Dr. A. Arimura for his generous gift of PACAP38. Materials for PRL RIA and ICC were gifts from the NIDDK and the National Hormone and Pituitary Program at the University of Maryland.
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EGF, TGF-a,
AND
THEIR
RECEPTOR
Results EGFITGF-a distribution
receptor and EGF mRNAs and their cellular
Northern blot analysis shows that human fallopian tubes contain major 10.5-kb and minor 6.0-kb receptor mRNA transcripts (Fig. lA, lane 2) and a single 5.0-kb EGF mRNA transcript (Fig. 1C). The HN5 cells, which served as a positive control, also showed the same major and minor receptor transcripts (Fig. lA, lane l), but their relative abundance is much higher than in the human fallopian tubes. In situ hybridization analysis shows that tubal epithelium, smooth muscle, and blood vessels contain receptor mRNA (Fig. 2, A-C) and EGF mRNA (Fig. 2, E-G) transcripts. These transcripts disappeared in the control sections which were pretreated with RNase before hybridization (Fig. 2, D and H). Ciliated epithelial cells contain more receptor transcripts than the nonciliated cells (Fig. 2B), whereas ciliated and nonciliated cells show a similar distribution of EGF transcripts (Fig. 2F). Immunoreactive EGF/TGF-CY receptor protein Western immunoblot analysis shows that human fallopian tubes (Fig. 1, lane 2) contain, like A431 cells (Fig. 1, lane l), a single 170-kDa protein. This protein became nondetectable when the receptor antibody preabsorbed with term human placental microsomes was used (Fig. 1, lane 3) or when unabsorbed receptor antibody was substituted with nonspecific mouse IgG (Fig. 1, lane 4). Immurwstaining
for EGF receptor protein
Light microscope immunocytochemistry shows that tubal epithelium, smooth muscle (Fig. 3, A-C), vascular smooth muscle, and endothelium (Fig. 4A) are immunostained for the receptors. This immunostaining was absent when the receptor antibody was preabsorbed with term human placen-
IN HUMAN
FALLOPIAN
TUBES
949
tal microsomes (Fig. 4D) or when unabsorbed receptor antibody was omitted or substituted with nonspecific mouse IgG during the immunostaining procedure (data not shown). Tubal epithelium contains more immunostaining than the other cells; ciliated epithelial cells contain more than nonciliated cells; and immunostaining in ciliated cells is present in cell periphery, cilia, cytoplasm, and nuclei (Fig. 3C). Ampullary segments contain more immunostaining than isthmus (Fig. 3, compare A with B). Proliferative phase and postpartum ampulla contain a similar amount of immunostaining which is higher than in the secretory phase (Fig. 5, compare A and B with C). Postmenopausal tubes contain the least amount of immunostaining (Fig. 5D). Isthmus segments also showed similar reproductive state differences as ampulla (data not shown). The immunostaining in ampullary and isthmic junctions and other tubal regions could not be evaluated. Another monoclonal receptor antibody (IgG 528) gave similar results as the receptor antibody (29.1) for which the data is presented in Figs,. 3-5, with the exception that the nuclei were not as extensively immunostained (data not shown). Immutwstaining
for EGF protein
The immunostaining for EGF is present in tubal epithelium, smooth muscle (Fig. 3, D-F), vascular smooth muscle, and ‘endothelium (Fig. 4B). This immunostaining is absent when the human EGF antibody was preabsorbed with excess purified human EGF (Fig. 4E) or when unabsorbed EGF antibody was omitted or substituted with nonspecific rabbit IgG (data not shown). Generally, vascular smooth muscle and epithelium contain a similar amount of EGF immunostaining except in the secretory phase, where heterogeneously immunostained epithelial cells contain more immunostaining than the vascular smooth muscle (compare Fig. 4B with Fig. 3, D and E, and Fig. 5G). Unlike receptors (Fig. 3C), there is no obvious difference between ciliated and nonciliated epithelial cells in
C 6.0, kb
FIG. 1. Northern blot analysis of EGF/TGF-(U receptors (A) and EGF (C) and Western blot analysis of EGF/TGF-(r receptors (B) in human fallopian tubes. HN5 cells in Northern blot analysis and A431 cells in Western blot analysis were used for positive controls. In A, lane 1 represents HN5 cells and lane 2 human fallopian tubes. In B, lane 1 represents A431 cells and lanes 2-4 human fallopian tubes. Lanes 3 and 4 are controls in which the receptor antibody was preabsorbed with human placental microsomes (lane 3) or unabsorbed primary antibody was substituted with nonspecific mouse IgG (lane 4). In Northern analysis, 3 pg poly(A)+ selected RNA was used per lane. In Western blot analysis, 10 pg protein in lane 1 and 20 pg protein in lanes 2-4 were used.
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EGF,
TGF-u,
AND
THEIR
RECEPTOR
IN HUMAN
FALLOPIAN
TUBES
Frc. 4. Light microscopic immunocytochemical localization of EGFITGF-a receptor (A), EGF (BI, and TGF-a (C) proteins in tubal blood vessels. In A-C, large arrowheads point out vascular smooth muscle, and small arrowheads paint out endothelium. D-F are controls in which the receptor antibody was preabsarbed with term human placental microsames (D), and EGF (E) and TGF-a (F) antibodies were preabsarbed with corresponding excess peptides before immunostaining. Magnifications, A and D, x550; B, x300; E, F, G, X140.
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EGF, TGF-a,
AND THEIR
RECEPTOR
IN HUMAN
FALLOPIAN
TUBES
953
. FIG. 5. Light microscopic immunocytochemical localization of EGFJTGF-a reeeptor (A-D), EGF (E-H), and TGF-a (J-M) proteins in human fallopian tubes from proliferative phase (A, E, and J), postpartum (B, F, and K), secretary phase (C, G, and L), and post menopause (D, H, and M). Magnifications, A-M, x140.
” ,.
is.
(Fig. 3F). In addition, cilia and irn&sr of nuclei are not immunostained. Ampullary segmentscontain more EGF immunostaining than isthmus (Fig. 3, compare D with E). Proliferative phase and postpartum ampulla contain a similar immunostaining which is higher than in postmenopausal specimens(Fig. 5, EC;F immunostaining
compare E and F with H). In the secretory phase, the immunostaining of epithelial cells is quite heterogenous and it is higher than those from the other reproductive states (Fig. 5G). Polyclonal antibody to mouse EGF gave similar results as the polyclonal antibody to human EGF for which the data is presented
in Figs. 3-5.
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EGF, TGF-a, AND THEIR RECEPTOR IN HUMAN
954 Immunostaining
for
TGF-a
protein
All the tubal cells are also immunostained for TGF-(r (Fig. 3, G-J; Fig. 4C). The immunostaining was absent when TGFQ antibody was preabsorbed with excessTGF-cupeptide (Fig. 4F) or when unabsorbed TGF-(Y antibody was omitted or substituted with nonspecific mouse IgG (data not shown). Vascular endothelial cells contain a similar immunostaining asepithelial cells which is higher than in other tubal cells (Fig. 3, G and H; Fig. 4C). Similar to EGF, TGF-(Y immunostaining is higher in ampulla than in isthmus (Fig. 3, compare G with H). Unlike EGF: 1) TGF-a immunostaining is higher in nonciliated epithelial cells than in ciliated cells (Fig. 3J); 2) the immunostaining is present primarily at the cell periphery (Fig. 3J); 3) proliferative phase specimenscontain more immunostaining than those from postpartum (Fig. 5, compare J with K); and 4) the immunostaining is relatively homogeneous in the secretory phase (Fig. 5L). Immunogold
electron
microscopy for EGF receptors
The immunogold particles reflecting the receptors are present over cell membranes, cilia, basal bodies, nucleus, and endoplasmic reticulum of ciliated epithelial cells (Fig. 6A). Compared to these cells, nonciliated cells contain a much lower number of gold particles, and they are primarily distributed over microvilli (Fig. 6B). Preabsorption of the receptor antibody with term human placental microsomes (Fig. 6C) or omissionor substitution of unabsorbed receptor antibody with nonspecific mouse IgG (data not shown), although keeping everything including colloidal gold-labeled antimouse IgG (secondary antibody), resulted in the absenceof gold particles. The distribution of gold particles with monoclonal receptor antibody, IgG 528, was similar to the receptor antibody, 29.1, for which the data is presented in Fig. 6. However, there was one exception, that is, nuclei contained relatively fewer gold particles with the receptor antibody, IgG 528 (data not shown). Discussion
The present studiesinvestigated the possibleexpression of EGF receptor and its ligands, EGF and TGF-(u, in human fallopian tubes by Northern blot analysis, in situ hybridization, Western immunoblot analysis, and light and/or electron microscopeimmunocytochemistry. TGF-a mRNA could not be measured because of the lack of cDNA probe in our laboratory. The resultsshow that fallopian tubes contain EGF receptor and EGF mRNA transcripts. The size of these transcripts are in agreement with those reported in other tissues and in HN5 cells, which were used as a positive control for the receptors (24, 25, 35-38). As expected, the relative abundance of the receptor transcripts in HN5 cells is higher than in fallopian tubes. The receptor antibody immunoreacted with a single 170kDa protein in fallopian tubes. This is identical to the molecular size of EGF receptors in A431 cells, which were used as a positive control, and what was previously reported for a variety of other tissues and cells (6, 7). The relative abundance of 170-kDa protein in A431 cells, which are known to
FALLOPIAN
TUBES
Endo. Vol131.
1992 No 2
contain l-3 million receptors per cell determined by ligand binding, is, however, similar to fallopian tubes even after taking into consideration that different amounts of protein were used. This is unexpected, and it may be related to A431 cells containing truncated receptors as well as some full length receptors with aberrant oligosaccharide moities (35, 39). Both these receptor forms may not be fully immunoreactive toward 29.1 receptor antibody, which is specific to carbohydrate structures on the extracellular receptor domain (40, 41). The carbohydrate structures of EGF receptors are related to those in blood group active antigens (40). Therefore, it is theoretically possible that the receptor antibody clone 29.1 can cross-react with these nonreceptor-related antigens. However, it is not known whether these antigens are present in fallopian tubes or other tissuesor bind to the receptor antibody. Nevertheless, the present studies used a second receptor antibody, which immunoreacts with the protein structure of the receptor (42), and it gave similar immunostaining results. Human fallopian tubes contain epithelial cells lining the tubal lumen, stroma, smooth muscle, and serosal layer on the outside (8-l 1). Blood vesselsare interspersed among the stroma and smooth muscle layers. Mature tubal epithelium consists of ciliated nonsecretory and nonciliated secretory epithelial cells (8-11). The relative proportion of these two cell types varies during the menstrual cycle (13). Ciliated cells through their ciliary beat can pick up ovum and transport gametes and embryo (8-11, 13). The nonciliated cells can synthesize and secrete macromolecules which provide milieu conducive for fertilization, nourishment, and early cleavage of embryos (8-11, 13). The contractions of tubal smooth muscle also help in the transport of gametes and embryo (8-14). All the above tubal cells contain EGF receptor and its ligands, EGF and TGF-(Y, which suggeststhat these two growth factors may potentially regulate ciliary beat, secretions, contractions, blood flow, cell division, and differentiation in the fallopian tubes. Recently, Takeuchi et al. (43) have shown that EGF can enhance proliferation of human fallopian tubal epithelial cells. There is also a precedencefor EGF regulating many of these events in other reproductive and nonreproductive tissues(23, 27, 44-46). The correspondence between the distribution of mRNA and the distribution of protein is excellent in tubal cells. For example, epithelial cells contain more receptor mRNA and receptor protein, and vascular smooth muscle contains more EGF mRNA and EGF protein than the other tubal cells. Ciliated cells contain more receptor mRNA and receptor protein than nonciliated cells, whereas ciliated and nonciliated cells contain similar amounts of EGF mRNA and EGF protein. Similarities as well as differences were found for EGF receptor and its ligands, EGF and TGF-(Y, distribution in tubal tissue/cells. Similarities include: ampulla, which is known to be more estrogen dependent, contain all three molecules more than the less estrogen-dependent isthmus; and proliferative (estrogen dominance) and/or postpartum specimenscontain all three molecules, more than secretory phase, whereas postmenopausal specimens contained the least amounts. Since there are only two specimensin the postmenopausalgroup, we cannot generalize these findings.
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EGF, TGF-a, AND THEIR
RECEPTOR
IN HUMAN
FALLOPIAN
TUBES
955
FIG. 6. Immunogold electron microscopic localization of EGF/TGF-(U receptors in ciliated (A) and nonciliated (B) epithelial cells. A, The immunogold particles reflecting the receptors are present in cell membranes, cilia, basal bodies, and nucleus. B, Fewer gold particles are present and they are mostly distributed over microvilli. Arrows point out basal bodies; large arrowheads point out plasma membranes; and small arrowheads point out endoplasmic reticulum. C is a control in which the receptor antibody was preabsorbed with human term placental microsomes. Magnification, A-C, X25,000.
The cellular and reproductive state differences include: 1) Epithelial cells contain more receptors than the other tubal cells; epithelial cells contain a similar amount of EGF as vascular smooth muscle except in secretory phase; and epithelial cells contain a similar amount of TGF-a as vascular endothelium. 2) Ciliated cells contain more receptors than nonciliated cells,
vice versufor TGF-(Y, and both cells contain a similar amount
of EGF. 3) Although proliferative and postpartum specimens contain similar amounts of receptors and EGF, proliferative phase specimens contain more TGF-a than postpartum specimens. 4) Although the distribution of receptors and TGF-(w is uniform in epithelial cells from the secretory phase, the EGF
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956
EGF, TGF-a,
AND
THEIR
RECEPTOR
distribution is quite heterogenous. Clearly, the cellular distribution and reproductive state dependency of these three regulatory molecules are not the same. This suggests that paracrine in addition to autocrine mechanisms are involved in the regulation of tubal functions by these growth factors. All the tubal cells contain both EGF and TGF-o(, and it appears that these cells contain more TGF01than EGF. However, we have not eliminated the possibilities that differences in the nature of EGF and TGF-a antibodies, IgG concentrations, and possible differential preservation of EGF and TGF-a proteins during tissue processing could account for the immunostaining differences between these two growth factors. If these two growth factors have identical functions because of structural similarity and binding to the same receptors, then there is no need for expression of both growth factors at the same time. On the other hand, if these two growth factors have some nonoverlapping functions, then the expression of both growth factors will be required to meet the functional needs in different cells and in different reproductive states. The present data is consistent with such a possibility. In fact, EGF and TGF-a have previously been shown to differ in some functional properties (46-50).
It is clear from the light microscope immunocytochemistry that the receptor immunostaining is present in cell periphery, cilia, cytoplasm, nuclear periphery, and interior of the nucleus. EGF immunostaining is present in cell periphery, cytoplasm, and nuclear periphery. TGF-a immunostaining is present in cell and nuclear periphery. Immunogold electron microscopy showed the receptors to be present in cell membranes, cilia, basal bodies which control ciliary beat frequency, endoplasmic reticulum, nuclear membranes, and chromatin. This distribution pattern is specific because the immunogold particles reflecting the receptors are absent in all three controls in which colloidal gold-labeled antimouse IgG is present. Moreover, Western immunoblot analysis showed that the receptor antibody recognized only EGF receptors. The nuclear distribution of receptors has alsobeen seen in human fallopian tubes with another receptor antibody (IgG 528), which is directed against the binding site on the extracellular receptor domain (42). In addition, using the sameor similar receptor antibodies, nuclei from other target tissuessuch as human placenta, bovine corpus luteum, and A431 cells have also been shown to contain EGF receptors (34, 41, 51, 52). We have not made a similar detailed analysis of subcellular distribution of EGF and TGF-CV.It is possible that immunostaining seenin cell periphery, cytoplasm (EGF), and the perinuclear region may represent receptor binding of thesegrowth factors. Thesefindings suggestthe possibility that EGF and TGF-ol may well have cell surface as well as intracellular sites of action. In fact, previous studies have shown that EGF can directly regulate target cell nuclear functions (53-55). In summary, human fallopian tubes contain EGF, EGF/ TGF-(r receptor mRNA and protein, and TGF-cuprotein. The expression of all these three regulatory molecules was dependent on anatomical region, cell type, and reproductive state of the fallopian tubes. These findings suggestthat EGF and TGF-a may regulate numerous tubal functions, thus potentially influencing fertility in women. The reproductive
IN HUMAN
FALLOPIAN
TUBES
Endo. Vol131.
1992 No 2
state dependency suggeststhat other reproductive hormones may modulate the actions of these growth factors. Acknowledgments We thank Dr. Dwight Pridham and numerous other obstetrics and gynecology physicians and pathologists at the University of Louisvilleaffiliated hospitals for their help in collecting the tissues, and Dr. Chandan Chakraborty for performing Western immunoblot analysis.
References 1. Scott J, Urdea M, Quiroga M, Sanchez-Pescador R, Fong N, Selby M, Rutter WJ, Bell GI 1983 Structure of a mouse submaxillary messenger RNA encoding epidermal growth factor and seven related proteins. Science 221:236-240 2. Derynck R, Roberts AB, Winkler ME, Chen EY, Goedded DV 1984 Human transforming growth factor-a: precursor, structure and expression in E. coli. Cell 38:287-297 3. Cohen S 1962 Isolation of a mouse submaxillary protein accelerating incisor eruption and eyelid opening in the newborn animal, J Biol Chem 237:1555-1562 4. Todaro GJ, Fryling C, DeLarco JE 1980 Transforming growth factors produced by certain human tumor cells: polypeptides that interact with epidermal growth factor receptors. Proc Nat1 Acad Sci USA 77:5258-5262 Burgess AW 1989 Epidermal growth factor and transforming growth factors. Br Med Bull 45:101-124 Carpenter G 1987 Receptors for epidermal growth factor and other polypeptide mitogens. Annu Rev Biochem 56:881-914 Carpenter G, Cohen S 1990 Epidermal growth factor. J Biol Chem 265:7709-7712 Novak ER, Woodruff JD 1967 Histology of fallopian tube. In: Novak ER, Woodruff JD (eds) Gynecologic and Obstetric Pathology. W.B. Saunders Co., Philadelphia 9. Pauerstein CJ, Woodruff JD, Zachary AS 1968 Factors influencing physiologic activities in the fallopian tube: the anatomy, physiology and pharmacology of tubal transport. Obstet Gynecol Survey 23:215-243 10. Woodruff JD, Pauerstein CJ 1969 The Fallopian tube: Structure, Function, Pathology and Management. Williams AZ Wilkins, Baltimore 11. Critoph FN, Dennis KJ 1977 The cellular composition of the human oviduct epithelium. Brit J Obstet Gynecol 84:219-221 12. Lindblom B, Hamberger L, Wiqvist N 1978 Differential contractile effects of prostaglandins E and F on the isolated circular and longitudinal smooth muscle of the human oviduct. Fertil Steril 30:553-559 13. Verhage HG, Bareither ML, Jaffe RC, Akbar M 1979 Cyclic changes in ciliation, secretion and cell height of the oviduct epithelium in women. Am J Anat 156:505-522 14. Lindblom B, Hamberger L, Ljung B 1980 Contractile patterns of isolated oviductal smooth muscle under different hormonal conditions. Fertil Steril 33:283-287 15. Hofmann GE, Rao ChV, Barrows GS, Schultz GS, Sanfilippo JS 1984 Binding sites for epidermal growth factor in human uterine tissues and leiomyomas. J Clin Endocrinol Metab 58:880-884 16. Chegini N, Rao ChV, Wakim N, Sanfilippo J 1986 Binding of ‘?e5;yermal growth factor to human uterus. Cell Tissue Res 246:54317. Sheets EE, Tsibris JCM, Cook NI, Virgin SD, DeMay RM, Spellacy WN 1985 In vitro binding of insulin and epidermal growth factor to human endometrium and endocervix. Am J Obstet Gynecol 153:60-65 18 Hofmann GE, Scott RT, Bergh PA, Deligdisch L 1991 Immunohistochemical localization of epidermal growth factor in human endometrium, decidua, and placenta. J Clin Endocrinol Metab 73:882-887 19 Kornyei J, Rao ChV Molecular analysis of epidermal growth factor action in human myometrial smooth muscle cells. Program of the 39th Annual Meeting of the Society for Gynecologic Investigation, San Antonio, TX, 1992, Abstract 581
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EGF,
TGF-a,
AND
THEIR
RECEPTOR
20. Rossi MJ, Chegini N, Masterson BJ 1992 Presence of epidermal growth factor, platelet-derived growth factor, and their receptors in human myometrial tissue and smooth muscle cells: their action in smooth muscle cells in vitro. Endocrinology 130:1716-1727 21. Mukku VR, Stance1 GM 1985 Receptors for epidermal growth factor in the rat uterus. Endocrinology 117:149-154 22. Tomooka Y, DiAugustine RP, McLachlan JA 1986 Proliferation of mouse uterine epithelial cells in vitro. Endocrinology 118:lOl l-1018 23. Gardner RM, Lingham RB, Stance1 GM 1987 Contractions of the isolated uterus stimulated by epidermal growth factor. FASEB J 1:224-228 24. Lingham RB, Stance1 GM, Loose-Mitchell DS 1988 Estrogen regulation of epidermal growth factor receptor messenger ribonucleic acid. Mol Endocrinol 2:230-235 25. DiAugustine RP, Petrusz P, Bell GI, Brown CF, Korach KS, McLachlan JA, Teng CT 1988 Influence of estrogens on mouse uterine epidermal growth factor precursor protein and messenger ribonucleic acid. Endocrinologv 122:2355-2363 26. Huet-Hudson YM, Chakrabvdrrty C, De SK, Suzuki Y, Andrews GK, Dey SK 1990 Estrogen regulates the synthesis of epidermal growth factor in mouse uterine epithelial cells. Mol Endocrinol 4:510-523 27. Nelson KC, Takahashi T, Bossert NL, Walmer DK, McLachlan JA 1991 Epidermal growth factor replaces estrogen in the stimulation of female genital-tract growth and differentiation. Proc Nat1 Acad Sci USA 88:21-25 28. Sambrook J, Fritsch EF, Maniatis T 1989 Molecular Cloning: A Laboratory Manual, ed 2. Cold Spring Harbor Laboratory, Cold Spring Harbor, book 2:7.12-7.52, 10.27-10.36 29. Angerer LM, Cox KH, Angerer RC 1987 Demonstration of tissue specific gene expression by in situ hybridization. Methods Enzymol 152:649-661 30. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193:265275 31. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage Tq. Nature 227:680-685 32. Dunn SD 1986 Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on western blots by monoclonal antibodies. Anal Biochem 157:144-153 33. Ramani N, Chegini N, Rao ChV, Woo& PG, Schultz GS 1986 The presence of epidermal growth factor binding sites in the intracellular organelles of term human placenta. J Cell Sci 84:19-40 34. Chegini N, Lei ZM, Rao ChV 1989 Light and electron microscope immunocytochemical localization of epidermal growth factor receptors in bovine corpora lutea of pregnancy. In: Hirshfield AN (ed) Growth Factors and the Ovary. Plenum Publishing Corp., New York, pp 213-220 35. Xu Y-H, Richert N, Ito S, Merlin0 GT, Pastan I 1984 Characterization of epidermal growth factor receptor gene expression in malignant and normal human cells. Proc Nat1 Acad Sci USA 81:7508-7312 36. Gubits RM, Shaw PA, Gresik EW, Onetti-Muda A, Barka T 1986 Epidermal growth factor gene expression is regulated differently in mouse kidney and submandibular gland. Endocrinology 119:13821387 37. Kasayama S, Ohba Y, Oka T 1989 Epidermal growth factor deficiency associated with diabetes mellitus. Proc Nat1 Acad Sci USA 86:7644-7648 38. Yoshida K, Kyo E, Tsuda T, Tsujino T, Ito M, Niimoto M, Tahara E 1990 EGF and TGF-ol, the ligands of hyperinduced EGFR in
IN HUMAN
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52. 53.
54.
55.
FALLOPIAN
TUBES
957
human esophageal carcinoma cells, act as autocrine growth factors. lnt J Cancer 45:131-135 Slieker LJ, Martensen TM, Lane MD 1986 Synthesis of epidermal growth factor in human A431 cells. Glycosylation-dependent acquisition of ligand binding activity occurs post-translationally in the endoplasmic reticulum. J Biol Chem 261:15233-15241 Childs RA, Gregoriou M, Scudder P, Thorpe SI, Rees AR, Feizi T 1984 Blood group-active carbohydrate chains on the receptor for epidermal growth factor of A431 cells. EMBO J 3:2227-2233 Carpentier J-L, Rees AR, Gregoriou M, Kris R, Schlessinger J, Orci L 1986 Subcellular distribution of the external and internal domains of the EGF receptor in A431 cells. Exp Cell Res 166:312326 Gill GN, Kawamoto T, Cachet C, Le A, Sato JD, Masui H, McLeod C, Mendelsohn J 1984 Monoclonal anti-epidermal growth factor receptor antibodies which are inhibitors of epidermal growth factor binding and antagonists of epidermal growth factor stimulated tyrosine protein kinase activity. J Biol Chem 259:7755-7760 Takeuchi K, Maruyama I, Yamamoto S, Oki T, Nagata Y 1991 Isolation and monolayer culture of human fallopian tube epithelial cells. In Vitro Cell Dev Biol 27A:720-724 Bhargava G, Rifas L, Makman MH 1979 Presence of epidermal growth factor receptors and influence of epidermal growth factor on proliferation and aging in cultured smooth muscle cells. J Cell Physiol 100:365-374 Fox CF, Linsley PS, Wrann M 1982 Receptor remodeling and regulation in the action of epidermal growth factor. Fed Proc 41:2988-2995 Gan BS, Hollenberg MD, MacCannell KL, Lederis K, Winkler ME, Derynck R 1987 Distinct vascular actions of epidermal growth factor-urogastrone and transforming growth factor (Y. J Pharmacol Exp Ther 242:331-337 Winkler ME, O’Connor LO, Winget M, Fendly B 1989 Epidermal growth factor and transforming growth factor a bind differently to epidermal growth factor receptor. Biochemistry 28:6373-6378 Stern PH, Krieger NS, Nissenson RA, Williams RD, Winkler ME, Derynck R, Strewler GJ 1985 Human transforming growth factoralpha stimulates bone resorption in vitro. J Clin Invest 76:20162019 Ibbotson KJ, Harrod J, Gowen M, D’Souza S, Winkler ME, Derynck R, Mundy GR 1986 Human recombinant transforming growth factor o( stimulates bone resorption and inhibits formation in vitro. Proc Nat1 Acad Sci USA 83:2228-2232 Schrieber AB, Winkler ME, Derynck R 1986 Transforming growth factor-a: a more potent angiogenic mediator than epidermal growth factor. Science 232:1250-1253 Rao ChV, Chegini N, Lei ZM 1988 Epidermal growth factor in human placenta. In: Mochizuki M, Hussa RO (eds) Placental Protein Hormones. Elsevier Science Publishers B.V. (Biomedical Division), Amsterdam, pp 171-182 Lei ZM, Chakraborty C, Rao ChV 1990 Nuclear epidermal growth factor receptors in human placentas. J Cell Biol 11 I:522 (Abstract) Schindler M, Jiang L-N 1987 Epidermal growth factor and insulin stimulate nuclear pore-mediated macromolecular transport in isolated rat liver nuclei. J Cell Biol 104:849-853 Buckley AR, Crowe PD, Russell DH 1988 Rapid activation of protein kinase c in isolated rat liver nuclei by prolactin, a known hepatic mitogen. Proc Nat1 Acad Sci USA 85:8649-8653 Jiang L-N, Schindler M 1990 Nucleocytoplasmic transport is enhanced concomitant with nuclear accumulation of epidennal growth factor (EGF) binding activity in both 3T3-1 and EGF reconstituted NR-6 fibroblasts. J Cell Biol 110:559-568
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Vol. 131, No. 2 Printed in U.S.A.
Society
Expression of Epidermal Growth Factor and its Ligands, EGF and Transforming a!, in Human Fallopian Tubes* Z. M. LEI
AND
Department 40292
of
(EGF) Receptor Growth Factor-
Ch. V. RAO Obstetrics
and Gynecology,
University
of
Louisville,
School of Medicine,
Louisville,
Kentucky
tory molecules varied. For all of them, however, ampullary segments contained more than isthmus; proliferative phase and/or postpartum specimens contained more than secretory phase; and postmenopausal specimens contained the lowest amounts. The cell periphery and nuclear/perinuclear area of the cells contained EGF, TGF-(u, and their receptors. Immunogold electron microscopy showed the receptors to be present in cell membranes, cilia, basal bodies which control ciliary activity, endoplasmic reticulum, nuclear membranes, and chromatin. In summary, human fallopian tubes contain EGF, EGF/TGF-(Y receptor mRNA and protein, and TGF-cu protein. The expression of all these regulatory molecules was dependent on anatomical region, cell type, and reproductive state of the fallopian tubes. These findings suggest that EGF and TGF-ol may regulate numerous tubal functions, thus potentially influencing fertility in women. (Endocrinology 131:
ABSTRACT
Although human uterus is known to contain epidermal growth factor (EGF) and its receptors, it is virtually unknown whether human fallopian tubes, which are an anatomical continuation of the uterus, also contain them. Therefore, the present studies investigated whether EGF and its structural and functional homolog, i.e. transforming growth factor-a (TGF-ol), and their common receptor are expressed in human fallopian tubes. Human fallopian tubes contain major 10.5-kilobase (kb) and minor 6.0-kb receptor messenger RNA (mRNA) transcripts, a single 5.0-kb EGF mRNA transcript, and a single 170-kilodalton receptor protein. The transcripts, along with their corresponding proteins and TGF-ol protein, are present in ciliated and nonciliated epithelial cells, tubal smooth muscle, vascular smooth muscle, and endothelium. The cellular distribution and reproductive state dependency of these three regula-
947-957,1992)
E
whether fallopian tubes from humans or from any animal speciescontain EGF or its structural and functional homolog, TGF-LU,and their common receptor. As a first step toward a better understanding of possible regulation of human fallopian tubes by EGF/TGF- (Y, we investigated whether this organ might expressthese regulatory molecules and whether cellular, anatomical, and reproductive state differences exist in their expression.
PIDERMAL growth factor (EGF) and transforming growth factor-0 (TGF-a) are single-chain polypeptides of similar size [-6 kilodaltons (kDa), structure, and function, and are encoded by different genes(1,2). EGF was originally discovered in submaxillary glands of mice and TGF-a in transformed cells (3, 4). Now they have been found in many normal and cancer tissues(5). EGF and TGF-(Uare functionally similar, and they bind to a common membrane receptor (5). The receptor is a transmembrane glycoprotein of 170 kDa consisting of extracellular ligand binding domain connected to cytoplasmic kinase domain by a short transmembrane region (6, 7). EGF and TGF-a binding results in a stimulation of phosphorylation of their receptor as well as numerous other target proteins in their tyrosyl residues (6,
Materials
and Methods
Materials Vectastain ABC kits were purchased from Vector Labs (Burlingame, CA); [cy-32P]deoxycytodine triphosphate and Genescreen plus hybridization transfer membrane from New England Nuclear-DuPont Company Biotechnology Systems (Boston, MA); monoclonal antibody to TGF-ol and purified TGF-(U from Oncogene Science (Manhasset, NY); polyclonal antibody to human EGF (hEGF) from Biomedical Technologies Inc. (Stoughton, MA); monoclonal antibody to EGF receptors (clone 29.1), polyclonal antibody to mouse EGF, proteinase 2K, sonicated and denatured salmon sperm DNA, poly(A)+ RNA, transfer RNA, and ribosomal RNA from Sigma Chemical Co. (St. Louis, MO); oligo(dT) cellulose and 0.24-9.5 kilobase (kb) RNA ladder from Gibco BRL (Gaithersburg, MD); XbaI, HindIII, EcoRI, and a random-primed DNA labeling kit from United States Biochemicals (Cleveland, OH); chemicals and supplies for light microscope autoradiography from Polysciences (Warrington, PA); colloidal gold (15 nm)-labeled goat antimouse immunoglobulin G (IgG) from Janssen Life Science Products (Piscataway, NJ); and other immunochemicals, reagents, molecular biology grade chemicals, and solvents from regular commercial sources. Monoclonal antibody to EGF receptors (IgG 528) was obtained from Dr. Hideo Masui (Sloan-Kettering Cancer Center, New York, NY). The recombinant hEGF was provided by Dr. Pablo Valenzuela (Chiron Corporation, Emeryville, CA). Highly purified mouse EGF was obtained from Dr. Gregory Schultz
7).
The fallopian tube, an anatomical continuation of the uterus, is a dynamic and cyclically changing structure which responds to ovarian steroid hormones and prostaglandins (8-14). Fallopian tubes contain multiple cell types, and this cellular diversity helps in the maturation and transport of gametesand embryo, fertilization, and early development of the embryo (8-14). Thus, fallopian tubes play a pivotal role in normal fertility in women. Although human and animal uteri are known to contain EGF and its receptors and are subject to their regulation (15-27), it is virtually unknown Received March 9, 1992. Address all correspondence and reprint requests to: Dr. Ch. V. Rao, Department of Obstetrics and Gynecology, 438 MDR Building, University of Louisville, Louisville, Kentucky 40292. * This work was presented at the 23rd Annual Meeting of the Society for the Study of Reproduction, Knoxville, TN, 1990 (Abstract 99). 947
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VIP-IR
968
CELLS
IN RAT PITUITARY
C
25
” 2: 20
0
male
diesuous
ovx+oil
ovx;+B7pg
ovxgopg
female FIG. 3. Pituitary VIP content (top), PRL content (middle), and serum PRL levels (bottom) in intact male and female rats and OVX rats injected SCwith 7 or 70 rg EB dissolved in sesame oil or oil alone and decapitated 3 days later (AM). Each column represents the mean + SEM of individual values obtained from six to nine rats. a, P < 0.05 us. diestrous female and OVX plus oil group. b, P < 0.05 us. male and diestrous female group. c, P c 0.05 us. all other groups.
in the OVX rats in which EBtreatment resulted in an increase in the pituitary VIP content. As we have demonstrated before, this was the caseonly in the group treated with the highest dose of EB. This effect of 70 pg EB on the pituitary VIP content and serum PRL levels is probably causedby the significantly greater levels of serum estradiol present in the 70 pg group compared to the 7 pg group (9). However, both dosesof EB resulted in serum estradiol levels considerably
Endo. Voll31.
1992 No 2
greater than those found throughout the estrous cycle (9). Thesefindings support the suggestionthat pituitary VIP may be related to the development of estrogen-induced hyperprolactinemia (26-28). The inverse relationship found between serum and pituitary PRL levels and pituitary VIP content in the untreated male ZIS.female rat suggests,however, that VIP is not related to basal PRL secretions. Therefore, although these and other data show that VIP is produced and releasedby rat adenohypophyseal cells, the functional significance of this VIP remains to be defined. In vitro data clearly show that VIP releasedfrom anterior pituitary cells has a stimulatory role in PRL release (3-5). The most compelling evidence that VIP has a role in the regulation of PRL releasecomes from studies in which circulating PRL levels were reduced after the biological action of VIP was neutralized by treatment of rats with a VIP antiserum or antagonist (23, 24, 28-34). Whereas those VIP neutralization studies clearly demonstrate a role for VIP in the regulation of PRL release, they provide no information on the origin of the active VIP in vim. This is critical, because VIP may be released from the median eminence (18, 35) or posterior pituitary (35, 36) in addition to the adenohypophysis and affect pituitary function. Several laboratories have attempted to study the role of anterior pituitary VIP in the regulation of PRL in vim by following the changesin the contents of these two hormones after various endocrine manipulations known to alter PRL content and secretion (2,6-10). The results have been equivocal because, for example, pituitary VIP and PRL content were increased equally by the high dosesof EB (70 pg/rat), while pituitary PRL content, but not VIP, was increasedafter lower dosesof EB (7 pg/rat) (9). This was confirmed in the present study. In hypothyroid rats, the pituitary VIP content was markedly increased, while pituitary PRL was reduced (2, 10). In the same manner, comparing VIP and PRL in anterior pituitaries of untreated males and females suggests that VIP has little to do with the regulation of PRL. However, statements about hormone function based on tissue content must be interpreted with caution, becausecontent may be a poor reflection of hormone secretory dynamics. In summary, these data demonstrate that VIP-IR cells are not lactotrophs and are numerous throughout the anterior pituitary gland of untreated male and female rats, Furthermore, changesin the pituitary content of VIP are probably a reflection of parallel changes in the cellular content of VIP, rather than in the number of VIP-producing cells. Acknowledgments This publication is dedicated to the memory of Dr. Mary Notter, who taught Dr. Phelps to “quantify” rather than “quantitate;” her accidental death on November 2, 1991, was both a personal and professional loss to the author. The technical assistance of Ms. Myra Vaccarella and the photographic expertise of Ms. Dorothy Herrera and Ms. Nancy Dimmick are gratefully acknowledged. We thank Dr. A. Arimura for his generous gift of PACAP38. Materials for PRL RIA and ICC were gifts from the NIDDK and the National Hormone and Pituitary Program at the University of Maryland.
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EGF, TGF-a,
AND
THEIR
RECEPTOR
Results EGFITGF-a distribution
receptor and EGF mRNAs and their cellular
Northern blot analysis shows that human fallopian tubes contain major 10.5-kb and minor 6.0-kb receptor mRNA transcripts (Fig. lA, lane 2) and a single 5.0-kb EGF mRNA transcript (Fig. 1C). The HN5 cells, which served as a positive control, also showed the same major and minor receptor transcripts (Fig. lA, lane l), but their relative abundance is much higher than in the human fallopian tubes. In situ hybridization analysis shows that tubal epithelium, smooth muscle, and blood vessels contain receptor mRNA (Fig. 2, A-C) and EGF mRNA (Fig. 2, E-G) transcripts. These transcripts disappeared in the control sections which were pretreated with RNase before hybridization (Fig. 2, D and H). Ciliated epithelial cells contain more receptor transcripts than the nonciliated cells (Fig. 2B), whereas ciliated and nonciliated cells show a similar distribution of EGF transcripts (Fig. 2F). Immunoreactive EGF/TGF-CY receptor protein Western immunoblot analysis shows that human fallopian tubes (Fig. 1, lane 2) contain, like A431 cells (Fig. 1, lane l), a single 170-kDa protein. This protein became nondetectable when the receptor antibody preabsorbed with term human placental microsomes was used (Fig. 1, lane 3) or when unabsorbed receptor antibody was substituted with nonspecific mouse IgG (Fig. 1, lane 4). Immurwstaining
for EGF receptor protein
Light microscope immunocytochemistry shows that tubal epithelium, smooth muscle (Fig. 3, A-C), vascular smooth muscle, and endothelium (Fig. 4A) are immunostained for the receptors. This immunostaining was absent when the receptor antibody was preabsorbed with term human placen-
IN HUMAN
FALLOPIAN
TUBES
949
tal microsomes (Fig. 4D) or when unabsorbed receptor antibody was omitted or substituted with nonspecific mouse IgG during the immunostaining procedure (data not shown). Tubal epithelium contains more immunostaining than the other cells; ciliated epithelial cells contain more than nonciliated cells; and immunostaining in ciliated cells is present in cell periphery, cilia, cytoplasm, and nuclei (Fig. 3C). Ampullary segments contain more immunostaining than isthmus (Fig. 3, compare A with B). Proliferative phase and postpartum ampulla contain a similar amount of immunostaining which is higher than in the secretory phase (Fig. 5, compare A and B with C). Postmenopausal tubes contain the least amount of immunostaining (Fig. 5D). Isthmus segments also showed similar reproductive state differences as ampulla (data not shown). The immunostaining in ampullary and isthmic junctions and other tubal regions could not be evaluated. Another monoclonal receptor antibody (IgG 528) gave similar results as the receptor antibody (29.1) for which the data is presented in Figs,. 3-5, with the exception that the nuclei were not as extensively immunostained (data not shown). Immutwstaining
for EGF protein
The immunostaining for EGF is present in tubal epithelium, smooth muscle (Fig. 3, D-F), vascular smooth muscle, and ‘endothelium (Fig. 4B). This immunostaining is absent when the human EGF antibody was preabsorbed with excess purified human EGF (Fig. 4E) or when unabsorbed EGF antibody was omitted or substituted with nonspecific rabbit IgG (data not shown). Generally, vascular smooth muscle and epithelium contain a similar amount of EGF immunostaining except in the secretory phase, where heterogeneously immunostained epithelial cells contain more immunostaining than the vascular smooth muscle (compare Fig. 4B with Fig. 3, D and E, and Fig. 5G). Unlike receptors (Fig. 3C), there is no obvious difference between ciliated and nonciliated epithelial cells in
C 6.0, kb
FIG. 1. Northern blot analysis of EGF/TGF-(U receptors (A) and EGF (C) and Western blot analysis of EGF/TGF-(r receptors (B) in human fallopian tubes. HN5 cells in Northern blot analysis and A431 cells in Western blot analysis were used for positive controls. In A, lane 1 represents HN5 cells and lane 2 human fallopian tubes. In B, lane 1 represents A431 cells and lanes 2-4 human fallopian tubes. Lanes 3 and 4 are controls in which the receptor antibody was preabsorbed with human placental microsomes (lane 3) or unabsorbed primary antibody was substituted with nonspecific mouse IgG (lane 4). In Northern analysis, 3 pg poly(A)+ selected RNA was used per lane. In Western blot analysis, 10 pg protein in lane 1 and 20 pg protein in lanes 2-4 were used.
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EGF,
TGF-u,
AND
THEIR
RECEPTOR
IN HUMAN
FALLOPIAN
TUBES
Frc. 4. Light microscopic immunocytochemical localization of EGFITGF-a receptor (A), EGF (BI, and TGF-a (C) proteins in tubal blood vessels. In A-C, large arrowheads point out vascular smooth muscle, and small arrowheads paint out endothelium. D-F are controls in which the receptor antibody was preabsarbed with term human placental microsames (D), and EGF (E) and TGF-a (F) antibodies were preabsarbed with corresponding excess peptides before immunostaining. Magnifications, A and D, x550; B, x300; E, F, G, X140.
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EGF, TGF-a,
AND THEIR
RECEPTOR
IN HUMAN
FALLOPIAN
TUBES
953
. FIG. 5. Light microscopic immunocytochemical localization of EGFJTGF-a reeeptor (A-D), EGF (E-H), and TGF-a (J-M) proteins in human fallopian tubes from proliferative phase (A, E, and J), postpartum (B, F, and K), secretary phase (C, G, and L), and post menopause (D, H, and M). Magnifications, A-M, x140.
” ,.
is.
(Fig. 3F). In addition, cilia and irn&sr of nuclei are not immunostained. Ampullary segmentscontain more EGF immunostaining than isthmus (Fig. 3, compare D with E). Proliferative phase and postpartum ampulla contain a similar immunostaining which is higher than in postmenopausal specimens(Fig. 5, EC;F immunostaining
compare E and F with H). In the secretory phase, the immunostaining of epithelial cells is quite heterogenous and it is higher than those from the other reproductive states (Fig. 5G). Polyclonal antibody to mouse EGF gave similar results as the polyclonal antibody to human EGF for which the data is presented
in Figs. 3-5.
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EGF, TGF-a, AND THEIR RECEPTOR IN HUMAN
954 Immunostaining
for
TGF-a
protein
All the tubal cells are also immunostained for TGF-(r (Fig. 3, G-J; Fig. 4C). The immunostaining was absent when TGFQ antibody was preabsorbed with excessTGF-cupeptide (Fig. 4F) or when unabsorbed TGF-(Y antibody was omitted or substituted with nonspecific mouse IgG (data not shown). Vascular endothelial cells contain a similar immunostaining asepithelial cells which is higher than in other tubal cells (Fig. 3, G and H; Fig. 4C). Similar to EGF, TGF-(Y immunostaining is higher in ampulla than in isthmus (Fig. 3, compare G with H). Unlike EGF: 1) TGF-a immunostaining is higher in nonciliated epithelial cells than in ciliated cells (Fig. 3J); 2) the immunostaining is present primarily at the cell periphery (Fig. 3J); 3) proliferative phase specimenscontain more immunostaining than those from postpartum (Fig. 5, compare J with K); and 4) the immunostaining is relatively homogeneous in the secretory phase (Fig. 5L). Immunogold
electron
microscopy for EGF receptors
The immunogold particles reflecting the receptors are present over cell membranes, cilia, basal bodies, nucleus, and endoplasmic reticulum of ciliated epithelial cells (Fig. 6A). Compared to these cells, nonciliated cells contain a much lower number of gold particles, and they are primarily distributed over microvilli (Fig. 6B). Preabsorption of the receptor antibody with term human placental microsomes (Fig. 6C) or omissionor substitution of unabsorbed receptor antibody with nonspecific mouse IgG (data not shown), although keeping everything including colloidal gold-labeled antimouse IgG (secondary antibody), resulted in the absenceof gold particles. The distribution of gold particles with monoclonal receptor antibody, IgG 528, was similar to the receptor antibody, 29.1, for which the data is presented in Fig. 6. However, there was one exception, that is, nuclei contained relatively fewer gold particles with the receptor antibody, IgG 528 (data not shown). Discussion
The present studiesinvestigated the possibleexpression of EGF receptor and its ligands, EGF and TGF-(u, in human fallopian tubes by Northern blot analysis, in situ hybridization, Western immunoblot analysis, and light and/or electron microscopeimmunocytochemistry. TGF-a mRNA could not be measured because of the lack of cDNA probe in our laboratory. The resultsshow that fallopian tubes contain EGF receptor and EGF mRNA transcripts. The size of these transcripts are in agreement with those reported in other tissues and in HN5 cells, which were used as a positive control for the receptors (24, 25, 35-38). As expected, the relative abundance of the receptor transcripts in HN5 cells is higher than in fallopian tubes. The receptor antibody immunoreacted with a single 170kDa protein in fallopian tubes. This is identical to the molecular size of EGF receptors in A431 cells, which were used as a positive control, and what was previously reported for a variety of other tissues and cells (6, 7). The relative abundance of 170-kDa protein in A431 cells, which are known to
FALLOPIAN
TUBES
Endo. Vol131.
1992 No 2
contain l-3 million receptors per cell determined by ligand binding, is, however, similar to fallopian tubes even after taking into consideration that different amounts of protein were used. This is unexpected, and it may be related to A431 cells containing truncated receptors as well as some full length receptors with aberrant oligosaccharide moities (35, 39). Both these receptor forms may not be fully immunoreactive toward 29.1 receptor antibody, which is specific to carbohydrate structures on the extracellular receptor domain (40, 41). The carbohydrate structures of EGF receptors are related to those in blood group active antigens (40). Therefore, it is theoretically possible that the receptor antibody clone 29.1 can cross-react with these nonreceptor-related antigens. However, it is not known whether these antigens are present in fallopian tubes or other tissuesor bind to the receptor antibody. Nevertheless, the present studies used a second receptor antibody, which immunoreacts with the protein structure of the receptor (42), and it gave similar immunostaining results. Human fallopian tubes contain epithelial cells lining the tubal lumen, stroma, smooth muscle, and serosal layer on the outside (8-l 1). Blood vesselsare interspersed among the stroma and smooth muscle layers. Mature tubal epithelium consists of ciliated nonsecretory and nonciliated secretory epithelial cells (8-11). The relative proportion of these two cell types varies during the menstrual cycle (13). Ciliated cells through their ciliary beat can pick up ovum and transport gametes and embryo (8-11, 13). The nonciliated cells can synthesize and secrete macromolecules which provide milieu conducive for fertilization, nourishment, and early cleavage of embryos (8-11, 13). The contractions of tubal smooth muscle also help in the transport of gametes and embryo (8-14). All the above tubal cells contain EGF receptor and its ligands, EGF and TGF-(Y, which suggeststhat these two growth factors may potentially regulate ciliary beat, secretions, contractions, blood flow, cell division, and differentiation in the fallopian tubes. Recently, Takeuchi et al. (43) have shown that EGF can enhance proliferation of human fallopian tubal epithelial cells. There is also a precedencefor EGF regulating many of these events in other reproductive and nonreproductive tissues(23, 27, 44-46). The correspondence between the distribution of mRNA and the distribution of protein is excellent in tubal cells. For example, epithelial cells contain more receptor mRNA and receptor protein, and vascular smooth muscle contains more EGF mRNA and EGF protein than the other tubal cells. Ciliated cells contain more receptor mRNA and receptor protein than nonciliated cells, whereas ciliated and nonciliated cells contain similar amounts of EGF mRNA and EGF protein. Similarities as well as differences were found for EGF receptor and its ligands, EGF and TGF-(Y, distribution in tubal tissue/cells. Similarities include: ampulla, which is known to be more estrogen dependent, contain all three molecules more than the less estrogen-dependent isthmus; and proliferative (estrogen dominance) and/or postpartum specimenscontain all three molecules, more than secretory phase, whereas postmenopausal specimens contained the least amounts. Since there are only two specimensin the postmenopausalgroup, we cannot generalize these findings.
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EGF, TGF-a, AND THEIR
RECEPTOR
IN HUMAN
FALLOPIAN
TUBES
955
FIG. 6. Immunogold electron microscopic localization of EGF/TGF-(U receptors in ciliated (A) and nonciliated (B) epithelial cells. A, The immunogold particles reflecting the receptors are present in cell membranes, cilia, basal bodies, and nucleus. B, Fewer gold particles are present and they are mostly distributed over microvilli. Arrows point out basal bodies; large arrowheads point out plasma membranes; and small arrowheads point out endoplasmic reticulum. C is a control in which the receptor antibody was preabsorbed with human term placental microsomes. Magnification, A-C, X25,000.
The cellular and reproductive state differences include: 1) Epithelial cells contain more receptors than the other tubal cells; epithelial cells contain a similar amount of EGF as vascular smooth muscle except in secretory phase; and epithelial cells contain a similar amount of TGF-a as vascular endothelium. 2) Ciliated cells contain more receptors than nonciliated cells,
vice versufor TGF-(Y, and both cells contain a similar amount
of EGF. 3) Although proliferative and postpartum specimens contain similar amounts of receptors and EGF, proliferative phase specimens contain more TGF-a than postpartum specimens. 4) Although the distribution of receptors and TGF-(w is uniform in epithelial cells from the secretory phase, the EGF
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956
EGF, TGF-a,
AND
THEIR
RECEPTOR
distribution is quite heterogenous. Clearly, the cellular distribution and reproductive state dependency of these three regulatory molecules are not the same. This suggests that paracrine in addition to autocrine mechanisms are involved in the regulation of tubal functions by these growth factors. All the tubal cells contain both EGF and TGF-o(, and it appears that these cells contain more TGF01than EGF. However, we have not eliminated the possibilities that differences in the nature of EGF and TGF-a antibodies, IgG concentrations, and possible differential preservation of EGF and TGF-a proteins during tissue processing could account for the immunostaining differences between these two growth factors. If these two growth factors have identical functions because of structural similarity and binding to the same receptors, then there is no need for expression of both growth factors at the same time. On the other hand, if these two growth factors have some nonoverlapping functions, then the expression of both growth factors will be required to meet the functional needs in different cells and in different reproductive states. The present data is consistent with such a possibility. In fact, EGF and TGF-a have previously been shown to differ in some functional properties (46-50).
It is clear from the light microscope immunocytochemistry that the receptor immunostaining is present in cell periphery, cilia, cytoplasm, nuclear periphery, and interior of the nucleus. EGF immunostaining is present in cell periphery, cytoplasm, and nuclear periphery. TGF-a immunostaining is present in cell and nuclear periphery. Immunogold electron microscopy showed the receptors to be present in cell membranes, cilia, basal bodies which control ciliary beat frequency, endoplasmic reticulum, nuclear membranes, and chromatin. This distribution pattern is specific because the immunogold particles reflecting the receptors are absent in all three controls in which colloidal gold-labeled antimouse IgG is present. Moreover, Western immunoblot analysis showed that the receptor antibody recognized only EGF receptors. The nuclear distribution of receptors has alsobeen seen in human fallopian tubes with another receptor antibody (IgG 528), which is directed against the binding site on the extracellular receptor domain (42). In addition, using the sameor similar receptor antibodies, nuclei from other target tissuessuch as human placenta, bovine corpus luteum, and A431 cells have also been shown to contain EGF receptors (34, 41, 51, 52). We have not made a similar detailed analysis of subcellular distribution of EGF and TGF-CV.It is possible that immunostaining seenin cell periphery, cytoplasm (EGF), and the perinuclear region may represent receptor binding of thesegrowth factors. Thesefindings suggestthe possibility that EGF and TGF-ol may well have cell surface as well as intracellular sites of action. In fact, previous studies have shown that EGF can directly regulate target cell nuclear functions (53-55). In summary, human fallopian tubes contain EGF, EGF/ TGF-(r receptor mRNA and protein, and TGF-cuprotein. The expression of all these three regulatory molecules was dependent on anatomical region, cell type, and reproductive state of the fallopian tubes. These findings suggestthat EGF and TGF-a may regulate numerous tubal functions, thus potentially influencing fertility in women. The reproductive
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state dependency suggeststhat other reproductive hormones may modulate the actions of these growth factors. Acknowledgments We thank Dr. Dwight Pridham and numerous other obstetrics and gynecology physicians and pathologists at the University of Louisvilleaffiliated hospitals for their help in collecting the tissues, and Dr. Chandan Chakraborty for performing Western immunoblot analysis.
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