0013-7227/90/1264-2214$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 4 Printed in U.S.A.
Cellular Localization of the Growth Hormone Receptor/ Binding Protein in the Male and Female Reproductive Systems* PETER E. LOBIEf, WINRICH BREIPOHL, JUANITA GARCIA ARAGON, AND MICHAEL J. WATERS Department of Physiology and Pharmacology (P.E.L., J.G.A., M.J.W.), Department of Anatomy (W.B.), University of Queensland, St. Lucia, Queensland 4067, Australia
ABSTRACT. We have used immunohistochemistry to localize GH receptor/binding protein (BP) in the male and female reproductive systems of adult rats. Testes and ovaries from neonatal animals were also examined to determine if GH receptor/ BP expression in these tissues is developmentally regulated. Two monoclonal antibodies (MAb 43 and 263) were immunoreactive in identical locations whereas no immunoreactivity was evident when control monoclonal antibodies 7 and 50.8 were used. Localization of the receptor/BP was observed in both the nucleus and cytoplasm of immunopositive cells confirming our recent report of a nuclear GH receptor. Intense GH receptor/BP immunoreactivity in the male reproductive system was evident in the epithelium of the vas deferens and coagulating gland, the prostatic epithelium during the secretory phase, and the ductular epithelium of the coagulating and bulbourethral glands, respectively. Strong immunoreactivity was detectable in the Leydig and Sertoli cells, the epithelium of the ductus epididymis and seminal vesicles and smooth muscle of the tunica muscularis of the vas deferens, septae of the seminal vesicles, and in prostatic fibromuscular stroma. Cells of the seminiferous tubules (spermatogonia, primary and secondary spermatocytes, and spermatids) were moderately immunoreactive. No immunoreactivity was detectable in spermatozoa in the ductus epididymis or vas deferens, in scattered epithelial cells of the ductus epididymis, the prostatic epithelium in the nonsecretory phase, and mucous secreting cells of the bulbourethral
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ONADAL development and differentiation is dependent upon GH. In cases of isolated GH deficiency puberty is delayed and can be readvanced by GH therapy (1). Similarly GH restores fertility in GH-deficient mice (2). In hypophysectomized male rats exogenous GH administration increases testicular steroidogenic responses and LH receptor content (3). SuppresReceived September 25,1989. Address requests for reprints to: Peter E. Lobie, Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Queensland 4067, Australia. * This work was supported by a National Health and Medical Research Council (Australia) grant to M.J.W. t Supported by the Australian Kidney Foundation, NHMRC, AMA JG Hunter Research Fellowship, and the William Nathaniel Robertson and Douglas H. K. Research Scholarships.
glands. Leydig cells of 10-day postnatal rat testis were intensely immunoreactive whereas seminiferous tubular cells displayed homogenous immunoreactivity from moderate to strong. Intense GH receptor/BP immunoreactivity in the female reproductive system was evident in the germinal epithelium, the vascular endothelium of the myometrium, the epithelial lining of the fimbriae and oviduct, the endometrial epithelium and scattered endometrial glands, the mesothelium of the perimetrium, and the vascular endothelium of the endometrium. Strong immunoreactivity was exhibited by scattered oocytes, lutein cells of the corpus luteum, scattered endometrial glands, and the vascular endothelium of the endometrium. Moderate immunoreactivity was evident in scattered oocytes, granulosa cells, theca interna and externa, smooth muscle of the oviduct and myometrium, scattered endometrial glands, and luminally placed endometrial stroma cells. Ovarian granulosa cells from 10-day postnatal rats displayed strong immunoreactivity in contrast to moderate immunoreactivity in adult granulosa cells. In conclusion, we report a widespread distribution of the GH receptor/BP in the reproductive system of the rat by which GH may exert a direct action on reproductive function. The distribution is concordant with a role for GH in epithelial function and/or maintenance and also with a possible role for GH in the integrity of the endometrial vasculature. (Endocrinology 126: 2214-2221, 1990)
sion of GH release in female rats reduces both ovarian LH receptor content and steroidogenic responses to human CG (hCG) (4, 5). Moreover, GH enhances FSHinduced differentiation of rat granulosa cells and cAMP production in vitro (5). The major postulated mechanism of GH action is via production of insulin like growth factor-1 (IGF-1). Both testicular (6) and ovarian (7) levels of IGF-1 are regulated by GH suggesting that GH may act locally rather than by hepatic IGF-1 production. Immunohistochemical studies have detailed gonadal cell types synthesizing IGF-1 (8, 9) and IGF-1 effects on reproductive function are well documented (10-17). However, the gonadal cell types which express the GH receptor and are therefore responsive to GH action remain unknown. In this study
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GH RECEPTOR/BP IN REPRODUCTIVE TISSUES
we report the immunohistochemical localization of the GH receptor in the male and female reproductive systems of the rat.
Materials and Methods Determination of estrous cycle Stages of estrus were determined by daily vaginal smears and subsequently confirmed by histological examination of the uterine epithelium. Production and characterization of GH receptor monoclonal antibodies (MAbs) MAbs (7, 43) to the rabbit GH receptor were produced by application of hybridoma technology to splenic lymphocytes from mice immunized with a human (hGH) affinity purified preparation of rabbit liver GH receptor (18). These antibodies recognize independent epitopes on the extracellular portion of the receptor, do not cross-react with insulin or PRL receptors in the appropriate receptor assays, and react specifically with the GH receptor in immunoblots (19). MAb 7 recognizes a species specific epitope on the rabbit GH receptor. MAb 263 which recognizes a cross-species determinant with high affinity (20) was similarly prepared by immunization of mice with purified rat GH receptor. MAb 263 is reactive against the GH receptor in a number of species and does not inhibit 125-I insulin m - i PRL binding to insulin or PRL receptors in rabbit or or rat liver. Under certain conditions MAb 263 precipitates rat and rabbit GH receptor, although it can also compete for hormone binding to subtypes of the GH receptor, as does MAb 7 (20). These antibodies have been previously used to detect GH receptors in cartilage (21), the central nervous system (22), and the gastrointestinal tract (23). Tissue preparation for immunohistochemistry Ten-day and 12-week Wistar rats were anesthetized by an ip injection of pentobarbitone (30 mg). Animals were perfused intracardially with PBS, pH 7.4, until blanching and subsequently with Bouin's solution [0.9% picric acid, 9% (vol/vol) formaldehyde, 5% acetic acid]. Reproductive tissues were dissected and post fixed in Bouin's solution for 4 h at 4 C. The tissues were embedded in paraffin by standard histological procedure. Semi-serial 5-/um sections were cut and collected onto gelatin/chromic potassium sulfate coated slides. Immunohistochemistry Sections were deparaffinized and subjected to immunohistochemical staining according to the following schedule; 1) elimination of endogenous peroxidase activity with 0.5% H2O2 in PBS for 15 min at 20 C; 2) elimination of nonspecific protein binding by incubation at 20 C with 10% normal goat or normal sheep serum for 1 h; 3) incubation overnight at 4 C with mouse anti-GH receptor [MAb 7 at 100 Mg/ml, MAb 43 at 8 Mg/ml, and MAb 263 at 25 Mg/ml or Brucella MAb at 25 Mg/ml in PBS- 1% BSA]; 4) incubation with goat anti-mouse or sheep anti-mouse biotinylated immunoglobulin G (IgG), (Amersham,
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Buckinghamshire, United Kingdom) for 2 h at 25 C (diluted 1:150 in PBS-1% BSA); 5) incubation with avidin (streptavidin)-biotin horse radish peroxidase complex (Amersham) for 1 h at 25 C (diluted 1:150 in PBS-1% BSA); and 6) treatment with 0.05 mg/ml diaminobenzidine in PBS containing 0.1% H2O2 for 3 min. Between each step sections were washed three times in PBS and once in PBS-1% BSA. All incubations were performed in a humidified chamber. Sections were left uncounterstained or counterstained in Mayer's haematoxylin, dehydrated, and mounted. Controls were performed by 1) omission of the primary antibody or 2) replacing the anti-GH receptor mouse IgG by unrelated primary antibodies [Brucella abortus (MAb 50.8) and MAb 7] of the same isotype (IgGKl) and at the same or greater concentration.
Results General The GH receptor binding protein (BP) displayed both nuclear and cytoplasmic immunoreactivity in some locations of both the male and female reproductive systems. Further histochemical, antigenic and physicochemical characterization of the nuclear GH receptor will be described elsewhere (24). The specificity of GH receptor localization in this study has been verified by abolition of immunoreaction when control MAb 7 and TABLE 1. Intensity of GH receptor/BP immunoreactivity in the male rat reproductive system Intense immunoreactivity Epithelium of vas deferens Epithelium of coagulating gland Ductular epithelium of coagulating gland Prostatic epithelium in secretory phase Ductular epithelium of bulbourethral gland Strong immunoreactivity Interstitial cells of Leydig Sustentacular cells of Sertoli Epithelium of ductus epididymis Tunica muscularis of vas deferens Seminal vesicular epithelium Smooth muscle septa of seminal vesicle Smooth muscle of prostatic fibromuscular stroma Moderate immunoreactivity Spermatogonia Primary spermatocytes Secondary spermatocytes Spermatids Mesothelial cells of vas deferens Smooth muscle coat of ductus epididymis Prostatic secretion Weak immunoreactivity Coagulation gland secretion Scattered mucous cells of bulbourethral gland No immunoreactivity Spermatozoa in ductus epididymis and vas deferens Scattered epithelial cells of the ductus epididymis Seminal vesicular secretion Prostatic epithelium in nonsecretory phase Mucous secreting cells of bulbourethral gland
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TABLE 2. Intensity of GH receptor/BP immunoreactivity in the female rat reproductive system Intense immunoreactivity Germinal epithelium of ovary Endothelial cells of ovarian vasculature Epithelial lining of fimbriae and oviduct Mesothelium of perimetrium Endometrial epithelium of uterus Scattered endometrial glands Vascular endothelium of myometrium Vascular endothelium of endometrium Strong immunoreactivity Germinal epithelium of ovary Scattered oocytes Granulosa lutein cells of corpus luteum Scattered endometrial glands Vascular endothelium of endometrium Moderate immunoreactivity Scattered oocytes Membrana granulosa Theca interna and externa Smooth muscle and oviduct Myometrium Luminal endometrial stromal cells Scattered endometrial glands Scattered endometrial vascular endothelia Weak immunoreactivity Ovarian stromal cells Scattered oocytes Tunica media of ovarian and endometrial vasculature Endometrial stromal cells No immunoreactivity Tunica media of ovarian and endometrial vasculature Cells of the lamina propria of the oviduct
MAb 50.8 were used as primary antibodies. Both MAb 43 and 263 which recognize independent epitopes were intensely immunoreactive in identical locations. Male reproductive system Testis. The sustentacular cells of Sertoli and Interstitial cells of Leydig were strongly immunoreactive. Spermatogonia, primary and secondary spermatocytes, and maturing spermatids were moderate to weakly immunoreactive (Fig. la). Some variability in the degree of immunoreaction was observed between seminiferous tubules. Leydig cells and cells of the seminiferous tubules of the 10-day postnatal rat testis were intensely and moderate strongly immunoreactive, respectively. Ductus epididymis. The epithelium of the ductus epididymis was strongly immunoreactive although a subpopulation of immunonegative cells was observed scattered throughout the epithelium (Fig. lb). The thin circular coat of smooth muscle displayed moderate immunoreactivity. GH receptor immunoreactivity was weak or absent on maturing spermatozoa present in the lumen of the duct.
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Vas deferens. The epithelium of the vas deferens exhibited intense immunoreactivity (Fig. lc) whereas cells in the connective tissue underlying the epithelium were negative as were spermatozoa present in the lumen of the duct. The tunica muscularis of the vas deferens displayed strong immunoreactivity. Mesothelial cells were moderately immunoreactive. Seminal vesicle. The seminal vesicular epithelium displayed moderate immunoreactivity as did the smooth muscle septa surrounding and/or between the lobules. Although immunoreaction was predominantly cytoplasmic, nuclear immunoreaction was also apparent. No immunoreactivity was detectable in the secretion present in the lumen of the vesicle. Coagulating gland. The epithelium of the coagulating gland displayed both intense nuclear and cytoplasmic localization of the GH receptor. Some heterogeneity was observed as low cuboidal cells generally tended to be immunonegative. Prostate. Intense nuclear and cytoplasmic localization of the GH receptor was evident in the epithelial lining of the prostate during the secretory phase. Cuboidal epithelia of the nonsecretory portions of the prostate were generally immunonegative although sometimes a prominent nuclear immunoreaction was evident in some cuboidal cells (Fig. le). Moderate immunostaining was also evident in the smooth muscle of the fibromuscular stroma. The prostatic secretion of lobules in the secretory phase were moderately immunoreactive whereas that of the nonsecretory lobules was negative. Bulbourethral gland. The mucous secreting cells of the bulbourethral gland were immunonegative although weak nuclear immunoreaction could be observed in scattered cells. The ductular epithelium of this gland was however intensely immunostained as was the urothelium into which the ducts merged. Female reproductive system Ovary. The germinal epithelium displayed intense to moderate immunoreactivity (Fig. lg). Although variable, immunoreactivity was generally greater in the germinal epithelium covering newly formed corpora lutea. Ovarian stromal cells were weakly immunostained or negative whereas the endothelial lining of the ovarian arteries and veins was intensely immunoreactive. Oocytes displayed heterogenous immunoreactivity (strong to weak) which was not correlated with stages of follicular development (Fig. If). Granulosa cells were moderately immunostained also regardless of the stage of follicular development (Fig. If). Lutein cells of the corpus luteum were strongly immunoreactive,' with immunoreactivity pre-
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FIG. 1. GH receptor/BP immunoreactivity in reproductive tissues of the rat. Magnification bar is 100 nm for a, c, d, i and 50 ^im for b and e-h. GH receptor/BP immunoreactivity is represented by the red-brown peroxidase reaction, a) MAb 43 immunoreactivity in the testis of an adult rat. Note general homogenous immunoreaction in the seminiferous tubule and strong immunoreactivity in the Interstitial cells of Leydig. Hematoxylin counterstain. b) GH receptor/BP localization in the ductus epididymis (MAb 263). Note the weak immunoreaction in spermatozoa present in the lumen and intermittent immunoreactivity in the epithelial cells lining the duct. Hematoxylin counterstain. c) MAb 263 immunoreactivity in the epithelial and smooth muscular components of the vas deferens. Again note the weak or absent immunoreactivity in the spermatozoa present in the lumen of the duct. Hematoxylin counterstain. d) Adjacent control section (MAb 7) to that of c. Note complete lack of immunoreaction in structures intensely immunostained in c. Hematoxylin counterstain. e) GH receptor/BP immunoreactivity in prostatic epithelial cells. Note the prominent immunoreaction in columnar cells of the secretory lobule but no or only nuclear immunoreaction in epithelial cells of the nonsecretory lobules. Hematoxylin counterstain. f) GH receptor/BP immunoreactivity (MAb 263) in a postantral follicle. Note immunoreactivity in both the cytoplasm and nucleus (also nucleolus) of the oocyte. Also note moderate GH receptor immunoreactivity in granulosa and theca cells of the follicle. No hematoxylin counterstain. g) MAb 263 immunoreactivity in lutein cells of the corpus luteum and germinal epithelium. Note the prominent cytoplasmic immunoreaction in lutein cells although some faint nuclear immunoreaction is evident. No hematoxylin counterstain. h) GH receptor/ BP immunoreactivity in the oviduct (MAb 43). Note the prominent immunoreaction in the epithelial component and moderate immunoreactivity in the smooth muscle coat. No hematoxylin counterstain. i) MAb 263 immunoreactivity in the endometrium (diestrus). Note the prominent immunoreaction in the uterine epithelium, heterogenous immunoreaction in the endometrial glands, intense immunoreaction in the endothelium of the endometrial vasculature, and weakly stained stromal cells. No hematoxylin counterstain.
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dominantly cytoplasmic (Fig. lg). Cells of the theca interna and externa were moderately immunoreactive (Fig. If). Oocytes, granulosa, thecal, and germinal epithelial cells of 10-day postnatal rat ovary were moderately to strongly immunoreactive. Oviduct. The columnar epithelial lining of the fimbriae (ostium) and oviduct (Fig. lh) were intensely GH receptor/BP positive. The immunoreactivity in these locations was predominantly cytoplasmic although some nuclei were immunostained. The cells of the lamina propria were immunonegative whereas the inner circular and outer longitudinal smooth muscle layers were moderately to strongly immunostained. The external mesothelial lining was also intensely GH receptor/BP positive. Uterus. Mesothelial cells of the perimetrium were intensely immunostained whereas the myometrium displayed moderate to weak immunoreactivity. The epithelium lining the endometrium displayed intense GH receptor/BP immunoreactivity during metestrus and diestrus (Fig. li). A marginal decrease in immunoreactivity (though possibly not significant) was noted for proestrus and estrus. Endometrial glands displayed heterogenous immunoreactivity ranging from intense to weak. Again immunoreactivity tended to be marginally greater in metestrus and diestrus rather than in proestrus and estrus. However, the distinction between these stages was difficult to ascertain due to heterogeneity of immunoreaction within a single uterus and also since such apparent small changes in immunoreactivity could be due to differential fixation. Cells of the endometrial stroma displayed moderate to weak immunoreactivity with the stronger reacting cells tending to be more luminally placed. During diestrus the endothelium of the antimesometrial endometrial vasculature was intensely immunoreactive whereas that of the mesometrial side was weakly immunoreactive. On both sides however the vascular endothelium of the myometrium was intensely immunoreactive in all four estrus stages although heterogeneity was observed from animal to animal. During proestrus and estrus heterogenous immunostaining (from strong to weak) of the endometrial vascular endothelial cells was observed whereas the immunoreactivity during metestrus varied from intense to moderate.
Discussion We have demonstrated a widespread distribution of the GH receptor in the male and female reproductive systems of the rat. The most prominent immunoreactivity was associated with epithelial/endothelial cell types suggesting that GH effects on reproductive function are at least partially mediated at this level. One of the major problems associated with defining GH action on the
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reproductive systems is the ability of primate GHs to bind to both somatogenic (GH) and lactogenic (PRL) receptors in nonprimates (25). The use of 125I-hGH in binding studies has therefore lead to some confusion between the roles of GH and PRL in the reproductive systems. This has been further compounded by the widespread distribution of PRL receptors in reproductive tissues, and their more clearly defined physiological roles (26-28). In support of our immunohistochemical localization of somatogenic receptors, GH receptor/BP mRNA is detectable in rat testis (29) and specific somatogenic binding of 125I-hGH has been reported in rat prostatic epithelial cells (30). Furthermore, GH receptors are detectable in neoplastic prostatic and mammary gland epithelium by preincubation of histological sections with ovine GH (oGH) and subsequent immunohistochemistry for GH (31). Hepatic or local production of IGF-1 is considered to mediate the effects of GH (32). Both testicular (6) and ovarian (7) levels of IGF-1 display GH dependence. In rat reproductive systems, IGF-1 is localized to spermatocytes, spermatids, epithelial cells of the epididymis, seminal vesicle and prostate, granulosa cells, theca interna, luteal bodies, germinal epithelium and the epithelium of the oviduct and uterus (8). All of these locations display GH receptor/BP immunoreactivity suggesting that GH may stimulate local production of IGF-1 for replicative and/or differentiative purposes. The localization of the GH receptor/BP to the testis is concordant with GH induction of testicular protein (33) and IGF-1 (6) synthesis. Even though Leydig cells possess PRL receptors (34, 35), GH and PRL exert differential effects on Leydig cell function. Thus, rat GH but not PRL up-regulates IGF-1 receptor in purified Leydig cells derived from hypophysectomized rats (36). Furthermore, in both immature and mature hypophysectomized rats PRL increases LH receptor content but has no significant effect on LH-stimulated steroidogenesis (3). GH, however, markedly enhances LH-induced testosterone formation (3, 37). IGF-1 immunoreactivity is not detectable on Leydig cells of the adult rat testis (8) which suggests that GH effects on Leydig cell function could be mediated independently of IGF-1. However, this study reported no IGF-1 immunoreactivity in Sertoli cells in contrast to the reported localization of IGF-1 to these cells in rat Sertoli-spermatogenic cell cocultures (9). It is therefore possible that the immunohistochemical techniques employed by Hansson et al. (8) were not sensitive enough to detect low levels of IGF-1 which may be present in Leydig cells. The localization of the GH receptor to the epithelial component of the seminal vesicles is interesting for several reasons. Shahani et al. (38) have reported a specific GH dependent increase in the seminal vesicular weight
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GH RECEPTOR/BP IN REPRODUCTIVE TISSUES of immature mice compared to that obtained for LH, FSH, and PRL. Furthermore, it has been reported that parenterally administered bGH produces a transient burst of secretory activity in the seminal vesicles resulting in the appearance of fructose and citric acid in electroejactulated semen of bulls (39). oGH also appears to potentiate testosterone-dependent seminal vesicular growth in castrated mice (40) and IGF-1 is localized to scattered epithelial cells of the rat seminal vesicle (8), suggestive of a specific growth promoting role for GH in this organ. Our localization of the GH receptor/BP to the epithelial component of the prostate is in agreement with the recent report of specific somatogenic binding to isolated rat prostatic epithelial cells (30). Human GH also stimulates leucine uptake by these cells in a dose-dependent manner (30). In support of our nuclear localization of the GH receptor we have characterized the rabbit hepatic nuclear GH receptor/BP and demonstrated its antigenic identity to the liver cytosolic GH receptor/BP (24). Approximately 500 receptors per nucleus remain closely associated with chromatin after solubilization with 2% TX-100 indicative of direct GH regulation of transcription. As such the prominent nuclear immunoreaction associated with some nonsecretory prostatic epithelial cells could precede a transition from the nonsecretory to the secretory phase and may be associated with transcriptional activation of tissue specific genes necessary for this transition. In support of our nuclear localization of the receptor, at least in the prostate and mammary gland, El Eltreby and Mahrous (31) have reported nuclear immunoreaction in histological sections preincubated with oGH before immunohistochemistry for GH. The mechanism by which GH acts on the male reproductive system appears to involve synergism with androgens. Bovine GH itself is ineffective in stimulating growth of accessory sex glands but enhances the testosterone-induced synthesis of RNA and protein and weight of these organs (41-43). Furthermore, GH selectively decreases the dose of methandrostenolone necessary to restore levator ani weight to 50% of normal in castrated, hypophysectomized rats (44). It is reported that GH participates preferentially in the anabolic rather than the androgenic action of steroids (45) concordant with a specific growth-promoting action for GH. It is unclear whether these effects are mediated by local production of IGF-1 although IGF-1 modulates Leydig cell function (14-17). Similarly in the female GH acts synergistically with FSH and estradiol to stimulate IGF-1 and progesterone production by granulosa cells although GH alone stimulates high levels of IGF-1 secretion (46). IGF-1 effects on thecal (10, 11) and granulosa (12, 13) cells are also well documented. It is perhaps surprising that only moderate GH recep-
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tor/BP immunoreactivity was detected in ovarian granulosa cells considering well characterized GH effects on these cells (5, 46). GH-stimulated porcine granulosa cell cultures secrete 7.8 times as much IGF-1 and 2.6 times as much progesterone per cell as control cultures (46). Cultures treated with estradiol and FSH secreted 7.4 times control value of progesterone whereas a synergistic interaction between GH, FSH, and estradiol was observed which resulted in progesterone values 33 times control levels (46). GH also augments LH receptor formation and progestin biosynthesis in granulosa cells associated with FSH stimulated cAMP production (5). The relatively low levels of GH receptor/BP detectable on granulosa cells may be sufficient to stimulate IGF-1 synthesis which would subsequently mediate GH effects. The predominant localization of the GH receptor to the epithelium of the endometrium and oviduct is concordant with a role for GH in epithelial maintenance. Porcine GH increases the wet weight and [ 14 C]aminoisobutyric acid uptake of immature mouse uteri (47) and hGH significantly increases uterine IGF-1 gene expression in ovariectomized hypophysectomized rats (48). Futhermore, oGH increases the concentration of the uterine cytosolic estrogen receptor (49). An additional function of GH receptor/BP synthesis by oviductal and endometrial epithelia (further to stimulation of replication/growth) may be to provide free circulating GH B P to uterine fluid. Recombinant GH binding protein inhibits the biological response to GH in vitro (50). The uterine IGF binding protein has been proposed to be important in the paracrine regulation of trophoblast growth and penetration (51). The intense immunoreactivity observed in the endothelial cells of the endometrial vasculature does not correspond to any known role for GH in the maintenance of the uterine vasculature. The polarization of GH receptor endothelial cell immunoreactivity predominantly to the antimesometrial border is interesting as this location is the site of blood vessel proliferation during diestrus in the rat (52). Although there appears to be a slight decrease in endothelial cell GH receptor immunoreactivity during proestrus and estrus we cannot be definitive as immunohistochemistry does not allow such precise quantitation. Also we are unable to explain why the medial smooth muscle cells are immunonegative in the endometrium but not in other examined locations (unpublished observations). In conclusion, we have shown the presence of GH receptor/BP immunoreactivity in a range of cell types within the male and female reproductive systems. A variety of biological actions of GH on many of these reproductive tissues argues strongly for a functional significance of these receptors. However, until the relationship between the GH receptor and the GH BP is fully
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clarified, it is not possible to be unequivocal about the functional role of the GH receptor/BP immunoreactivity we have described. It is currently unresolved whether the BP in the rat is derived by cleavage from the full-length receptor as proposed by Spencer et al. (53) or whether it is synthesized from a shorter length mRNA transcript as recently reported (54-56). However, since our affinity cross-linking studies on the chromatin GH receptor indicate that the BP associates directly with chromatin (24) it may be possible that the BP itself has functional significance in GH-regulated transcriptional events.
16.
17. 18. 19.
Acknowledgments
20.
We wish to thank Walter Thomas for staging of the estrus cycle in female rats. We also thank Agen Ltd. (Acacia Ridge, Queensland) for generous assistance in the preparation of the MAbs used in this study.
21.
References 1. Sheikholislam BM, Stempfel RS 1972 Hereditary isolated somatotropin defiency: effects of human GH administration. Pediatrics 49:362 2. Bartke A 1964 Histology of the anterior hypophysis, thyroid and gonads of two types of dwarf mice. Anat Rec 149:225 3. Zipf WB, Payne AH, Kelch RP 1978 Prolactin, growth hormone, and luteinizing hormone in the maintenance of testicular luteinizing hormone receptors. Endocrinology 103:595 4. Advis JP, White SS, Ojeda SR 1981 Activation of growth hormone short loop negative feedback delays puberty in the female rat. Endocrinology 108:1343 5. Jia XC, Kalmijn J, Hseuh AJW 1986 Growth hormone enhances follicle stimulating hormone induced differentiation of cultured rat granulosa cells. Endocrinology 118:1401 6. D'Ercole AJ, Stiles AD, Underwood LE 1984 Tissue concentrations of somatomedin C: further evidence for multiple sites of synthesis and paracrine or autocrine mechanisms of action. Proc Natl Acad Sci USA 81:935 7. Davoren JB, Hseuh AJW 1986 Growth hormone increases ovarian levels of immunoreactive levels of somatomedin C/insulin like growth factor 1 in vivo. Endocrinology 118:888 8. Hansson HA, Nilsson A, Isgaard J, Billig H, Isaksson OGP, Skottner A, Andersson IK, Rozell B 1988 Immunohistochemical localization of insulin like growth factor-1 in the adult rat. Histochemistry 89:403 9. Tres LL, Smith EP, Van Wyk JJ, Kierszenbaum AL 1986 Immunoreactive sites and accumulation of somatomedin C in rat sertoli spermatogenic cell co-cultures. Exp Cell Res 162:33 10. Cara JF, Rosenfield RL 1988 Insulin like growth factor 1 and insulin potentiate luteinizing hormone induced androgen synthesis by rat ovarian thecal-interstitial cells. Endocrinology 123:733 11. Hernandez ER, Resnick CE, Svoboda ME, Van Wyk JJ, Payne DW, Adashi EY 1988 Somatomedin C/insulin like growth factor 1 as an enhancer of androgen biosynthesis by cultured rat ovarian cells. Endocrinology 122:1603 12. Adashi EY, Resnick CE, D'Ercole AJ, Svoboda ME, Van Wyk JJ 1985 Insulin like growth factors as intraovarian regulators of granulosa cell growth and function. Endocr Rev 6:400 13. Veldhuis JD, Rodgers RJ, Dee A, Simpson ER 1986 The insulin like growth factor, somatomedin C, induces the synthesis of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin in ovarian cells. J Biol Chem 261:2499 14. Lin T, Haskell J, Vinson N, Terracio L 1986 Direct stimulating effects of insulin like growth factor-1 on leydig cell steroidogenesis in primary culture. Biochem Biophys Res Commun 137:950 15. Lin T, Haskell J, Vinson N, Terracio L 1986 Characterization of insulin and insulin like growth factor 1 receptors of purified leydig
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38. 39.
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cells and their role in steroidogenesis in primary culture: a comparative study. Endocrinology 119:1641 Bernier M, Chatelain P, Mather JP, Saez JM 1986 Regulation of gonadotropin receptors, gonadotropin responsiveness and cell multiplication by somatomedin C and insulin in cultured pig leydig cells. J Cell Physiol 129:257 Kasson BG, Hseuh AJW 1987 Insulin like growth factor augments gonadotropin stimulated androgen biosynthesis by cultured rat testicular cells. Mol Cell Endocrinol 52:27 Barnard R, Bundesen PG, Rylatt DB, Waters MJ 1984 Monoclonal antibodies to the growth hormone receptor: production and characterization. Endocrinology 115:1805 Leung DW, Spencer SA, Cachianes G, Hammonds RG, Collins C, Henzel WJ, Barnard R, Waters MJ, Wood WI 1987 Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature 330:537 Barnard R, Bundesen PG, Rylatt DB, Waters MJ 1985 Evidence from the use of monoclonal antibody probes for structural heterogeneity of the GH receptor. Biochem J 231:459 Barnard R, Haynes KM, Werther GA, Waters MJ 1988 The ontogeny of growth hormone receptors in the rabbit tibia. Endocrinology 122:2562 Lobie PE, Lincoln DT, Breipohl W, Waters MJ, Growth hormone receptor localization on the central nervous system. Program of the 71st Annual Meeting of The Endocrine Society, Seattle, WA, 1989, p 771 (Abstract) Lobie PE, Breipohl W, Waters MJ 1990 Growth hormone receptor expression in the rat gastrointestinal tract. Endocrinology, 126:299 Lobie PE, Barnard R, Waters MJ, The growth hormone nuclear receptor/binding protein: antigenic and physicochemical characterization. Proceedings of the Endocrine Society of Australia 32:21 Posner BI, Kelly PA, Shiu RPC, Friesen HG 1974 Studies of insulin, growth hormone and prolactin binding: tissue distribution, species variation and characterization. Endocrinology 95:521 Williams GH, Hammond JM, Weisz J, Mortel R 1978 Binding sites for lactogenic hormone in the rat uterus. Biol Reprod 18:697 Amit T, Barkey RJ, Youdim MBH 1983 Effect of prolactin, testosterone and estrogen on prolactin binding in the rat testis, prostate, seminal vesicle and liver. Mol Cell Endocrinol 30:179 Orgebin-Crist MC, Djiane J 1979 Properties of a prolactin receptor from the rat epididymis. Biol Reprod 21:135 Mathews LS, Enberg B, Norstedt G 1989 Regulation of rat growth hormone receptor gene expression. J Biol Chem 264:9905 Prieto JC, Carmena MJ 1987 Growth hormone binding and stimulation of amino acid uptake in epithelial cells of rat ventral prostate. Cell Biochem Funct 5:63 El Eltreby MF, Mahrous AT 1979 Immunocytochemical technique for detection of prolactin (Prl) and growth hormone (GH) in hyperplastic and neoplastic lesions of dog prostate and mammary gland. Histochemistry 64:279 Daughaday WH 1989 A personal history of the origin of the somatomedin hypothesis and recent challenges to its validity. Persp Biol Med 32(2):194 Lawrence NR, Davies AG 1977 Stimulation of testicular protein synthesis in vivo by gonadotropins and growth hormone in hypophysectomized adult mice. J Reprod Fertil 49:41 Charreau EH, Attramadal A, Torjesen PA, Purvisk K, Calandra R, Hansson V 1977 Prolactin binding in rat testis: specific receptors in interstitial cells. Mol Cell Endocrinol 6:303 Bonifacino JS, Dufau ML 1985 Lactogen receptors in rat leydig cells: analysis of their structure with bifunctional cross linking reagents. Endocrinology 116:1610 Lin T, Blasdell J, Haskell JF 1988 Hormonal regulation of type 1 insulin like growth factor receptors of leydig cells in hypophysectomized rats. Endocrinology 123:134 Swerdloff RS, Odell WD 1977 Modulating influences of FSH, GH and prolactin on LH stimulated testosterone secretion. In: Troen P, Nankin HR (eds) The Testis in Normal and Infertile Men. Raven Press, New York, p 395 Shahani SK, Raman G, Rao SS 1977 Mouse seminal vesicle weight as an index of growth hormone activity. Indiana J Exp Biol 15:134 Mann T, Rowson LEA, Baronos S, Karagiannidis A 1971 The
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GH RECEPTOR/BP IN REPRODUCTIVE TISSUES
40. 41. 42.
43. 44. 45. 46. 47.
48.
androgenic potency of dihydrotestosterone and testosterone in the bull, and the effect of growth hormone on the bovine seminal vesicle. J Endocrinol 51:707 Keenan EJ, Thomas JA 1975 Effects of testosterone and prolactin or growth hormone on the accessory sex organs of castrated mice. J Endocrinol 64:111 Lostroh AJ 1962 Effect of testosterone and growth hormone on nucleic acid and protein in the sex accessory glands of Long Evans and Sprague Dawley rats. Endocrinology 70:747 Ebling FJ, Ebling E, Randall V, Skinner J 1975 The effects of hypophysectomy and of bovine growth hormone on the responses to testosterone of prostate, preputial, harderian and lachrymal glands and of brown adipose tissue in the rat. J Endocrinol 66:401 Steelman SL, Morgan ER 1972 The effect of sparganum growth factor on the action of androgen. Endocrinology 90:315 Desaulles PA 1960 Les hormones anabolisantes du point de vue experimental. Helv Med Acta 27:479 Steinetz BG, Giannina T, Butler M, Popick F 1972 The role of growth hormone in the anabolic action of methandrostenolone. Endocrinology 99:1396 Hsu CJ, Hammond JM 1987 Concomitant effects of growth hormone on secretion of insulin like growth factor 1 and progesterone by cultured porcine granulosa cells. Endocrinology 121:1343 Biswas S, Hindocha P 1973 Effects of human chorionic somatomammotrophin and porcine growth hormone on the weight of immature mouse uterus and ovary and uptake of a a-aminoisobutyric acid by these organs. J Endocrinol 58:343 Murphy LJ, Friesen HG 1988 Differential effects of estrogen and
49. 50. 51.
52. 53.
54. 55. 56.
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growth hormone on uterine and hepatic insulin like growth factor 1 gene expression in the ovariectomized hypophysectomized rat. Endocrinology 122:325 Chilton BS, Daniel JC 1987 Differences in the rabbit uterine response to progesterone as influenced by growth hormone or prolactin. J Reprod Fertil 79:581 Lim L, Waters MJ 1989 Recombinant growth hormone binding protein inhibits the biological effect of growth hormone. Proceedings of the Endocrine Society of Australia 32:204 Fazleabas AT, Jaffe RC, Verhage HG, Waites G, Bell SC 1989 An insulin like growth factor binding protein in the baboon (Papio anubis) endometrium: synthesis, immunocytochemical localization and hormonal regulation. Endocrinology 124:2321 Williams MF 1948 The vascular architecture of the rat uterus as influenced by estrogen and progesterone. Am J Anat 83:247 Spencer SA, Hammonds RG, Henzel WJ, Rodriguez H, Waters MJ, Wood WI 1988 Rabbit liver growth hormone receptor and serum binding protein: purification, characterization and sequence. J Biol Chem 263:7862 Smith WC, Linzer DIH, Talamantes F 1988 Detection of two growth hormone receptor mRNAs and primary translation products in the mouse. Proc Natl Acad Sci USA 85:9576 Smith WC, Kuniyoshi J, Talamantes F 1989 Mouse serum growth hormone (GH) binding protein has GH receptor extracellular and substituted transmembrane domains. Mol Endocrinol 3:984 Baumbach WR, Homer DL, Logan JS 1989 The growth hormone binding protein in rat serum is an alternatively spliced form of the rat growth hormone receptor. Genes Dev 3:1199
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Vol. 126, No. 4 Printed in U.S.A.
Cellular Localization of the Growth Hormone Receptor/ Binding Protein in the Male and Female Reproductive Systems* PETER E. LOBIEf, WINRICH BREIPOHL, JUANITA GARCIA ARAGON, AND MICHAEL J. WATERS Department of Physiology and Pharmacology (P.E.L., J.G.A., M.J.W.), Department of Anatomy (W.B.), University of Queensland, St. Lucia, Queensland 4067, Australia
ABSTRACT. We have used immunohistochemistry to localize GH receptor/binding protein (BP) in the male and female reproductive systems of adult rats. Testes and ovaries from neonatal animals were also examined to determine if GH receptor/ BP expression in these tissues is developmentally regulated. Two monoclonal antibodies (MAb 43 and 263) were immunoreactive in identical locations whereas no immunoreactivity was evident when control monoclonal antibodies 7 and 50.8 were used. Localization of the receptor/BP was observed in both the nucleus and cytoplasm of immunopositive cells confirming our recent report of a nuclear GH receptor. Intense GH receptor/BP immunoreactivity in the male reproductive system was evident in the epithelium of the vas deferens and coagulating gland, the prostatic epithelium during the secretory phase, and the ductular epithelium of the coagulating and bulbourethral glands, respectively. Strong immunoreactivity was detectable in the Leydig and Sertoli cells, the epithelium of the ductus epididymis and seminal vesicles and smooth muscle of the tunica muscularis of the vas deferens, septae of the seminal vesicles, and in prostatic fibromuscular stroma. Cells of the seminiferous tubules (spermatogonia, primary and secondary spermatocytes, and spermatids) were moderately immunoreactive. No immunoreactivity was detectable in spermatozoa in the ductus epididymis or vas deferens, in scattered epithelial cells of the ductus epididymis, the prostatic epithelium in the nonsecretory phase, and mucous secreting cells of the bulbourethral
G
ONADAL development and differentiation is dependent upon GH. In cases of isolated GH deficiency puberty is delayed and can be readvanced by GH therapy (1). Similarly GH restores fertility in GH-deficient mice (2). In hypophysectomized male rats exogenous GH administration increases testicular steroidogenic responses and LH receptor content (3). SuppresReceived September 25,1989. Address requests for reprints to: Peter E. Lobie, Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Queensland 4067, Australia. * This work was supported by a National Health and Medical Research Council (Australia) grant to M.J.W. t Supported by the Australian Kidney Foundation, NHMRC, AMA JG Hunter Research Fellowship, and the William Nathaniel Robertson and Douglas H. K. Research Scholarships.
glands. Leydig cells of 10-day postnatal rat testis were intensely immunoreactive whereas seminiferous tubular cells displayed homogenous immunoreactivity from moderate to strong. Intense GH receptor/BP immunoreactivity in the female reproductive system was evident in the germinal epithelium, the vascular endothelium of the myometrium, the epithelial lining of the fimbriae and oviduct, the endometrial epithelium and scattered endometrial glands, the mesothelium of the perimetrium, and the vascular endothelium of the endometrium. Strong immunoreactivity was exhibited by scattered oocytes, lutein cells of the corpus luteum, scattered endometrial glands, and the vascular endothelium of the endometrium. Moderate immunoreactivity was evident in scattered oocytes, granulosa cells, theca interna and externa, smooth muscle of the oviduct and myometrium, scattered endometrial glands, and luminally placed endometrial stroma cells. Ovarian granulosa cells from 10-day postnatal rats displayed strong immunoreactivity in contrast to moderate immunoreactivity in adult granulosa cells. In conclusion, we report a widespread distribution of the GH receptor/BP in the reproductive system of the rat by which GH may exert a direct action on reproductive function. The distribution is concordant with a role for GH in epithelial function and/or maintenance and also with a possible role for GH in the integrity of the endometrial vasculature. (Endocrinology 126: 2214-2221, 1990)
sion of GH release in female rats reduces both ovarian LH receptor content and steroidogenic responses to human CG (hCG) (4, 5). Moreover, GH enhances FSHinduced differentiation of rat granulosa cells and cAMP production in vitro (5). The major postulated mechanism of GH action is via production of insulin like growth factor-1 (IGF-1). Both testicular (6) and ovarian (7) levels of IGF-1 are regulated by GH suggesting that GH may act locally rather than by hepatic IGF-1 production. Immunohistochemical studies have detailed gonadal cell types synthesizing IGF-1 (8, 9) and IGF-1 effects on reproductive function are well documented (10-17). However, the gonadal cell types which express the GH receptor and are therefore responsive to GH action remain unknown. In this study
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GH RECEPTOR/BP IN REPRODUCTIVE TISSUES
we report the immunohistochemical localization of the GH receptor in the male and female reproductive systems of the rat.
Materials and Methods Determination of estrous cycle Stages of estrus were determined by daily vaginal smears and subsequently confirmed by histological examination of the uterine epithelium. Production and characterization of GH receptor monoclonal antibodies (MAbs) MAbs (7, 43) to the rabbit GH receptor were produced by application of hybridoma technology to splenic lymphocytes from mice immunized with a human (hGH) affinity purified preparation of rabbit liver GH receptor (18). These antibodies recognize independent epitopes on the extracellular portion of the receptor, do not cross-react with insulin or PRL receptors in the appropriate receptor assays, and react specifically with the GH receptor in immunoblots (19). MAb 7 recognizes a species specific epitope on the rabbit GH receptor. MAb 263 which recognizes a cross-species determinant with high affinity (20) was similarly prepared by immunization of mice with purified rat GH receptor. MAb 263 is reactive against the GH receptor in a number of species and does not inhibit 125-I insulin m - i PRL binding to insulin or PRL receptors in rabbit or or rat liver. Under certain conditions MAb 263 precipitates rat and rabbit GH receptor, although it can also compete for hormone binding to subtypes of the GH receptor, as does MAb 7 (20). These antibodies have been previously used to detect GH receptors in cartilage (21), the central nervous system (22), and the gastrointestinal tract (23). Tissue preparation for immunohistochemistry Ten-day and 12-week Wistar rats were anesthetized by an ip injection of pentobarbitone (30 mg). Animals were perfused intracardially with PBS, pH 7.4, until blanching and subsequently with Bouin's solution [0.9% picric acid, 9% (vol/vol) formaldehyde, 5% acetic acid]. Reproductive tissues were dissected and post fixed in Bouin's solution for 4 h at 4 C. The tissues were embedded in paraffin by standard histological procedure. Semi-serial 5-/um sections were cut and collected onto gelatin/chromic potassium sulfate coated slides. Immunohistochemistry Sections were deparaffinized and subjected to immunohistochemical staining according to the following schedule; 1) elimination of endogenous peroxidase activity with 0.5% H2O2 in PBS for 15 min at 20 C; 2) elimination of nonspecific protein binding by incubation at 20 C with 10% normal goat or normal sheep serum for 1 h; 3) incubation overnight at 4 C with mouse anti-GH receptor [MAb 7 at 100 Mg/ml, MAb 43 at 8 Mg/ml, and MAb 263 at 25 Mg/ml or Brucella MAb at 25 Mg/ml in PBS- 1% BSA]; 4) incubation with goat anti-mouse or sheep anti-mouse biotinylated immunoglobulin G (IgG), (Amersham,
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Buckinghamshire, United Kingdom) for 2 h at 25 C (diluted 1:150 in PBS-1% BSA); 5) incubation with avidin (streptavidin)-biotin horse radish peroxidase complex (Amersham) for 1 h at 25 C (diluted 1:150 in PBS-1% BSA); and 6) treatment with 0.05 mg/ml diaminobenzidine in PBS containing 0.1% H2O2 for 3 min. Between each step sections were washed three times in PBS and once in PBS-1% BSA. All incubations were performed in a humidified chamber. Sections were left uncounterstained or counterstained in Mayer's haematoxylin, dehydrated, and mounted. Controls were performed by 1) omission of the primary antibody or 2) replacing the anti-GH receptor mouse IgG by unrelated primary antibodies [Brucella abortus (MAb 50.8) and MAb 7] of the same isotype (IgGKl) and at the same or greater concentration.
Results General The GH receptor binding protein (BP) displayed both nuclear and cytoplasmic immunoreactivity in some locations of both the male and female reproductive systems. Further histochemical, antigenic and physicochemical characterization of the nuclear GH receptor will be described elsewhere (24). The specificity of GH receptor localization in this study has been verified by abolition of immunoreaction when control MAb 7 and TABLE 1. Intensity of GH receptor/BP immunoreactivity in the male rat reproductive system Intense immunoreactivity Epithelium of vas deferens Epithelium of coagulating gland Ductular epithelium of coagulating gland Prostatic epithelium in secretory phase Ductular epithelium of bulbourethral gland Strong immunoreactivity Interstitial cells of Leydig Sustentacular cells of Sertoli Epithelium of ductus epididymis Tunica muscularis of vas deferens Seminal vesicular epithelium Smooth muscle septa of seminal vesicle Smooth muscle of prostatic fibromuscular stroma Moderate immunoreactivity Spermatogonia Primary spermatocytes Secondary spermatocytes Spermatids Mesothelial cells of vas deferens Smooth muscle coat of ductus epididymis Prostatic secretion Weak immunoreactivity Coagulation gland secretion Scattered mucous cells of bulbourethral gland No immunoreactivity Spermatozoa in ductus epididymis and vas deferens Scattered epithelial cells of the ductus epididymis Seminal vesicular secretion Prostatic epithelium in nonsecretory phase Mucous secreting cells of bulbourethral gland
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TABLE 2. Intensity of GH receptor/BP immunoreactivity in the female rat reproductive system Intense immunoreactivity Germinal epithelium of ovary Endothelial cells of ovarian vasculature Epithelial lining of fimbriae and oviduct Mesothelium of perimetrium Endometrial epithelium of uterus Scattered endometrial glands Vascular endothelium of myometrium Vascular endothelium of endometrium Strong immunoreactivity Germinal epithelium of ovary Scattered oocytes Granulosa lutein cells of corpus luteum Scattered endometrial glands Vascular endothelium of endometrium Moderate immunoreactivity Scattered oocytes Membrana granulosa Theca interna and externa Smooth muscle and oviduct Myometrium Luminal endometrial stromal cells Scattered endometrial glands Scattered endometrial vascular endothelia Weak immunoreactivity Ovarian stromal cells Scattered oocytes Tunica media of ovarian and endometrial vasculature Endometrial stromal cells No immunoreactivity Tunica media of ovarian and endometrial vasculature Cells of the lamina propria of the oviduct
MAb 50.8 were used as primary antibodies. Both MAb 43 and 263 which recognize independent epitopes were intensely immunoreactive in identical locations. Male reproductive system Testis. The sustentacular cells of Sertoli and Interstitial cells of Leydig were strongly immunoreactive. Spermatogonia, primary and secondary spermatocytes, and maturing spermatids were moderate to weakly immunoreactive (Fig. la). Some variability in the degree of immunoreaction was observed between seminiferous tubules. Leydig cells and cells of the seminiferous tubules of the 10-day postnatal rat testis were intensely and moderate strongly immunoreactive, respectively. Ductus epididymis. The epithelium of the ductus epididymis was strongly immunoreactive although a subpopulation of immunonegative cells was observed scattered throughout the epithelium (Fig. lb). The thin circular coat of smooth muscle displayed moderate immunoreactivity. GH receptor immunoreactivity was weak or absent on maturing spermatozoa present in the lumen of the duct.
Endo • 1990 Vol 126 »No 4
Vas deferens. The epithelium of the vas deferens exhibited intense immunoreactivity (Fig. lc) whereas cells in the connective tissue underlying the epithelium were negative as were spermatozoa present in the lumen of the duct. The tunica muscularis of the vas deferens displayed strong immunoreactivity. Mesothelial cells were moderately immunoreactive. Seminal vesicle. The seminal vesicular epithelium displayed moderate immunoreactivity as did the smooth muscle septa surrounding and/or between the lobules. Although immunoreaction was predominantly cytoplasmic, nuclear immunoreaction was also apparent. No immunoreactivity was detectable in the secretion present in the lumen of the vesicle. Coagulating gland. The epithelium of the coagulating gland displayed both intense nuclear and cytoplasmic localization of the GH receptor. Some heterogeneity was observed as low cuboidal cells generally tended to be immunonegative. Prostate. Intense nuclear and cytoplasmic localization of the GH receptor was evident in the epithelial lining of the prostate during the secretory phase. Cuboidal epithelia of the nonsecretory portions of the prostate were generally immunonegative although sometimes a prominent nuclear immunoreaction was evident in some cuboidal cells (Fig. le). Moderate immunostaining was also evident in the smooth muscle of the fibromuscular stroma. The prostatic secretion of lobules in the secretory phase were moderately immunoreactive whereas that of the nonsecretory lobules was negative. Bulbourethral gland. The mucous secreting cells of the bulbourethral gland were immunonegative although weak nuclear immunoreaction could be observed in scattered cells. The ductular epithelium of this gland was however intensely immunostained as was the urothelium into which the ducts merged. Female reproductive system Ovary. The germinal epithelium displayed intense to moderate immunoreactivity (Fig. lg). Although variable, immunoreactivity was generally greater in the germinal epithelium covering newly formed corpora lutea. Ovarian stromal cells were weakly immunostained or negative whereas the endothelial lining of the ovarian arteries and veins was intensely immunoreactive. Oocytes displayed heterogenous immunoreactivity (strong to weak) which was not correlated with stages of follicular development (Fig. If). Granulosa cells were moderately immunostained also regardless of the stage of follicular development (Fig. If). Lutein cells of the corpus luteum were strongly immunoreactive,' with immunoreactivity pre-
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FIG. 1. GH receptor/BP immunoreactivity in reproductive tissues of the rat. Magnification bar is 100 nm for a, c, d, i and 50 ^im for b and e-h. GH receptor/BP immunoreactivity is represented by the red-brown peroxidase reaction, a) MAb 43 immunoreactivity in the testis of an adult rat. Note general homogenous immunoreaction in the seminiferous tubule and strong immunoreactivity in the Interstitial cells of Leydig. Hematoxylin counterstain. b) GH receptor/BP localization in the ductus epididymis (MAb 263). Note the weak immunoreaction in spermatozoa present in the lumen and intermittent immunoreactivity in the epithelial cells lining the duct. Hematoxylin counterstain. c) MAb 263 immunoreactivity in the epithelial and smooth muscular components of the vas deferens. Again note the weak or absent immunoreactivity in the spermatozoa present in the lumen of the duct. Hematoxylin counterstain. d) Adjacent control section (MAb 7) to that of c. Note complete lack of immunoreaction in structures intensely immunostained in c. Hematoxylin counterstain. e) GH receptor/BP immunoreactivity in prostatic epithelial cells. Note the prominent immunoreaction in columnar cells of the secretory lobule but no or only nuclear immunoreaction in epithelial cells of the nonsecretory lobules. Hematoxylin counterstain. f) GH receptor/BP immunoreactivity (MAb 263) in a postantral follicle. Note immunoreactivity in both the cytoplasm and nucleus (also nucleolus) of the oocyte. Also note moderate GH receptor immunoreactivity in granulosa and theca cells of the follicle. No hematoxylin counterstain. g) MAb 263 immunoreactivity in lutein cells of the corpus luteum and germinal epithelium. Note the prominent cytoplasmic immunoreaction in lutein cells although some faint nuclear immunoreaction is evident. No hematoxylin counterstain. h) GH receptor/ BP immunoreactivity in the oviduct (MAb 43). Note the prominent immunoreaction in the epithelial component and moderate immunoreactivity in the smooth muscle coat. No hematoxylin counterstain. i) MAb 263 immunoreactivity in the endometrium (diestrus). Note the prominent immunoreaction in the uterine epithelium, heterogenous immunoreaction in the endometrial glands, intense immunoreaction in the endothelium of the endometrial vasculature, and weakly stained stromal cells. No hematoxylin counterstain.
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GH RECEPTOR/BP IN REPRODUCTIVE TISSUES
dominantly cytoplasmic (Fig. lg). Cells of the theca interna and externa were moderately immunoreactive (Fig. If). Oocytes, granulosa, thecal, and germinal epithelial cells of 10-day postnatal rat ovary were moderately to strongly immunoreactive. Oviduct. The columnar epithelial lining of the fimbriae (ostium) and oviduct (Fig. lh) were intensely GH receptor/BP positive. The immunoreactivity in these locations was predominantly cytoplasmic although some nuclei were immunostained. The cells of the lamina propria were immunonegative whereas the inner circular and outer longitudinal smooth muscle layers were moderately to strongly immunostained. The external mesothelial lining was also intensely GH receptor/BP positive. Uterus. Mesothelial cells of the perimetrium were intensely immunostained whereas the myometrium displayed moderate to weak immunoreactivity. The epithelium lining the endometrium displayed intense GH receptor/BP immunoreactivity during metestrus and diestrus (Fig. li). A marginal decrease in immunoreactivity (though possibly not significant) was noted for proestrus and estrus. Endometrial glands displayed heterogenous immunoreactivity ranging from intense to weak. Again immunoreactivity tended to be marginally greater in metestrus and diestrus rather than in proestrus and estrus. However, the distinction between these stages was difficult to ascertain due to heterogeneity of immunoreaction within a single uterus and also since such apparent small changes in immunoreactivity could be due to differential fixation. Cells of the endometrial stroma displayed moderate to weak immunoreactivity with the stronger reacting cells tending to be more luminally placed. During diestrus the endothelium of the antimesometrial endometrial vasculature was intensely immunoreactive whereas that of the mesometrial side was weakly immunoreactive. On both sides however the vascular endothelium of the myometrium was intensely immunoreactive in all four estrus stages although heterogeneity was observed from animal to animal. During proestrus and estrus heterogenous immunostaining (from strong to weak) of the endometrial vascular endothelial cells was observed whereas the immunoreactivity during metestrus varied from intense to moderate.
Discussion We have demonstrated a widespread distribution of the GH receptor in the male and female reproductive systems of the rat. The most prominent immunoreactivity was associated with epithelial/endothelial cell types suggesting that GH effects on reproductive function are at least partially mediated at this level. One of the major problems associated with defining GH action on the
Knclo- 1990 Vol l'2(i«No4
reproductive systems is the ability of primate GHs to bind to both somatogenic (GH) and lactogenic (PRL) receptors in nonprimates (25). The use of 125I-hGH in binding studies has therefore lead to some confusion between the roles of GH and PRL in the reproductive systems. This has been further compounded by the widespread distribution of PRL receptors in reproductive tissues, and their more clearly defined physiological roles (26-28). In support of our immunohistochemical localization of somatogenic receptors, GH receptor/BP mRNA is detectable in rat testis (29) and specific somatogenic binding of 125I-hGH has been reported in rat prostatic epithelial cells (30). Furthermore, GH receptors are detectable in neoplastic prostatic and mammary gland epithelium by preincubation of histological sections with ovine GH (oGH) and subsequent immunohistochemistry for GH (31). Hepatic or local production of IGF-1 is considered to mediate the effects of GH (32). Both testicular (6) and ovarian (7) levels of IGF-1 display GH dependence. In rat reproductive systems, IGF-1 is localized to spermatocytes, spermatids, epithelial cells of the epididymis, seminal vesicle and prostate, granulosa cells, theca interna, luteal bodies, germinal epithelium and the epithelium of the oviduct and uterus (8). All of these locations display GH receptor/BP immunoreactivity suggesting that GH may stimulate local production of IGF-1 for replicative and/or differentiative purposes. The localization of the GH receptor/BP to the testis is concordant with GH induction of testicular protein (33) and IGF-1 (6) synthesis. Even though Leydig cells possess PRL receptors (34, 35), GH and PRL exert differential effects on Leydig cell function. Thus, rat GH but not PRL up-regulates IGF-1 receptor in purified Leydig cells derived from hypophysectomized rats (36). Furthermore, in both immature and mature hypophysectomized rats PRL increases LH receptor content but has no significant effect on LH-stimulated steroidogenesis (3). GH, however, markedly enhances LH-induced testosterone formation (3, 37). IGF-1 immunoreactivity is not detectable on Leydig cells of the adult rat testis (8) which suggests that GH effects on Leydig cell function could be mediated independently of IGF-1. However, this study reported no IGF-1 immunoreactivity in Sertoli cells in contrast to the reported localization of IGF-1 to these cells in rat Sertoli-spermatogenic cell cocultures (9). It is therefore possible that the immunohistochemical techniques employed by Hansson et al. (8) were not sensitive enough to detect low levels of IGF-1 which may be present in Leydig cells. The localization of the GH receptor to the epithelial component of the seminal vesicles is interesting for several reasons. Shahani et al. (38) have reported a specific GH dependent increase in the seminal vesicular weight
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GH RECEPTOR/BP IN REPRODUCTIVE TISSUES of immature mice compared to that obtained for LH, FSH, and PRL. Furthermore, it has been reported that parenterally administered bGH produces a transient burst of secretory activity in the seminal vesicles resulting in the appearance of fructose and citric acid in electroejactulated semen of bulls (39). oGH also appears to potentiate testosterone-dependent seminal vesicular growth in castrated mice (40) and IGF-1 is localized to scattered epithelial cells of the rat seminal vesicle (8), suggestive of a specific growth promoting role for GH in this organ. Our localization of the GH receptor/BP to the epithelial component of the prostate is in agreement with the recent report of specific somatogenic binding to isolated rat prostatic epithelial cells (30). Human GH also stimulates leucine uptake by these cells in a dose-dependent manner (30). In support of our nuclear localization of the GH receptor we have characterized the rabbit hepatic nuclear GH receptor/BP and demonstrated its antigenic identity to the liver cytosolic GH receptor/BP (24). Approximately 500 receptors per nucleus remain closely associated with chromatin after solubilization with 2% TX-100 indicative of direct GH regulation of transcription. As such the prominent nuclear immunoreaction associated with some nonsecretory prostatic epithelial cells could precede a transition from the nonsecretory to the secretory phase and may be associated with transcriptional activation of tissue specific genes necessary for this transition. In support of our nuclear localization of the receptor, at least in the prostate and mammary gland, El Eltreby and Mahrous (31) have reported nuclear immunoreaction in histological sections preincubated with oGH before immunohistochemistry for GH. The mechanism by which GH acts on the male reproductive system appears to involve synergism with androgens. Bovine GH itself is ineffective in stimulating growth of accessory sex glands but enhances the testosterone-induced synthesis of RNA and protein and weight of these organs (41-43). Furthermore, GH selectively decreases the dose of methandrostenolone necessary to restore levator ani weight to 50% of normal in castrated, hypophysectomized rats (44). It is reported that GH participates preferentially in the anabolic rather than the androgenic action of steroids (45) concordant with a specific growth-promoting action for GH. It is unclear whether these effects are mediated by local production of IGF-1 although IGF-1 modulates Leydig cell function (14-17). Similarly in the female GH acts synergistically with FSH and estradiol to stimulate IGF-1 and progesterone production by granulosa cells although GH alone stimulates high levels of IGF-1 secretion (46). IGF-1 effects on thecal (10, 11) and granulosa (12, 13) cells are also well documented. It is perhaps surprising that only moderate GH recep-
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tor/BP immunoreactivity was detected in ovarian granulosa cells considering well characterized GH effects on these cells (5, 46). GH-stimulated porcine granulosa cell cultures secrete 7.8 times as much IGF-1 and 2.6 times as much progesterone per cell as control cultures (46). Cultures treated with estradiol and FSH secreted 7.4 times control value of progesterone whereas a synergistic interaction between GH, FSH, and estradiol was observed which resulted in progesterone values 33 times control levels (46). GH also augments LH receptor formation and progestin biosynthesis in granulosa cells associated with FSH stimulated cAMP production (5). The relatively low levels of GH receptor/BP detectable on granulosa cells may be sufficient to stimulate IGF-1 synthesis which would subsequently mediate GH effects. The predominant localization of the GH receptor to the epithelium of the endometrium and oviduct is concordant with a role for GH in epithelial maintenance. Porcine GH increases the wet weight and [ 14 C]aminoisobutyric acid uptake of immature mouse uteri (47) and hGH significantly increases uterine IGF-1 gene expression in ovariectomized hypophysectomized rats (48). Futhermore, oGH increases the concentration of the uterine cytosolic estrogen receptor (49). An additional function of GH receptor/BP synthesis by oviductal and endometrial epithelia (further to stimulation of replication/growth) may be to provide free circulating GH B P to uterine fluid. Recombinant GH binding protein inhibits the biological response to GH in vitro (50). The uterine IGF binding protein has been proposed to be important in the paracrine regulation of trophoblast growth and penetration (51). The intense immunoreactivity observed in the endothelial cells of the endometrial vasculature does not correspond to any known role for GH in the maintenance of the uterine vasculature. The polarization of GH receptor endothelial cell immunoreactivity predominantly to the antimesometrial border is interesting as this location is the site of blood vessel proliferation during diestrus in the rat (52). Although there appears to be a slight decrease in endothelial cell GH receptor immunoreactivity during proestrus and estrus we cannot be definitive as immunohistochemistry does not allow such precise quantitation. Also we are unable to explain why the medial smooth muscle cells are immunonegative in the endometrium but not in other examined locations (unpublished observations). In conclusion, we have shown the presence of GH receptor/BP immunoreactivity in a range of cell types within the male and female reproductive systems. A variety of biological actions of GH on many of these reproductive tissues argues strongly for a functional significance of these receptors. However, until the relationship between the GH receptor and the GH BP is fully
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GH RECEPTOR/BP IN REPRODUCTIVE TISSUES
clarified, it is not possible to be unequivocal about the functional role of the GH receptor/BP immunoreactivity we have described. It is currently unresolved whether the BP in the rat is derived by cleavage from the full-length receptor as proposed by Spencer et al. (53) or whether it is synthesized from a shorter length mRNA transcript as recently reported (54-56). However, since our affinity cross-linking studies on the chromatin GH receptor indicate that the BP associates directly with chromatin (24) it may be possible that the BP itself has functional significance in GH-regulated transcriptional events.
16.
17. 18. 19.
Acknowledgments
20.
We wish to thank Walter Thomas for staging of the estrus cycle in female rats. We also thank Agen Ltd. (Acacia Ridge, Queensland) for generous assistance in the preparation of the MAbs used in this study.
21.
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