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Regular Article |
a Department of Physiology and Biophysics and
b Department of Obstetrics and Gynecology, The University of Iowa, Iowa City, Iowa 52240
ABSTRACT
The presence of the LH receptor (LHR) in nongonadal tissues of the reproductive tract has been reported, but localization studies have not been performed. Our objectives were to demonstrate the presence of LHR in the reproductive tract and to localize receptor expression. Reproductive age rats and mice were obtained and 125I-hCG binding assays were performed on membrane preparations from the uterus, ovary, liver, and testis. In situ hybridizations were performed using 35S-labeled antisense and sense RNA probes prepared from nucleotides 1–591 of the mouse LHR cDNA. Specific hCG binding was detected in membrane preparations from the ovary, uterus, and testis but not in the liver in both the rat and mouse. In the ovary, LHR mRNA was localized in theca cells, large follicles, and corpora lutea as expected. In the uterus, LHR mRNA was expressed in stromal cells of the endometrium and in the uterine serosa. Uterine smooth muscle cells had low levels of expression, and the endometrial epithelium was negative. In the oviduct, high levels of LHR expression were noted on the serosa and in subepithelial cells. Oviductal smooth muscle had low expression, and the epithelium was negative. We conclude that functional, nongonadal LHR are expressed in the mouse reproductive tract. The presence and localization of LHR expression in the mouse reproductive tract lay the foundation for transgenic models to address the physiologic role of these receptors.
fallopian tubes, female reproductive tract, hormone action, LH, Müllerian ducts, oviduct, uterus
INTRODUCTION
The luteinizing hormone receptor (LHR) has been classically described as a receptor in the ovary and testis that controls reproductive processes. However, evidence is accumulating that the LHR may be present in extragonadal tissue, particularly in the female reproductive tract. Indeed, the LHR has been reported to be present in the bovine, porcine, rat, mouse, rabbit, and human uterus [1–11]. Although the presence of the LHR has been demonstrated in these species, there is little information regarding the localization of this receptor in the reproductive tract. The purposes of our study were to confirm the presence of the LHR in the rat and mouse uterus, to compare the relative levels of receptor binding in the uterus and gonads, and to localize the expression of this receptor using in situ hybridization. These experiments lay the foundation for future studies utilizing mouse cell-specific gene knock-out models to establish the physiologic importance of the extragonadal LH receptors.
MATERIALS AND METHODS
Hormones and Supplies
Highly purified hCG (CR-123) was kindly provided by the National Hormone and Pituitary Agency of the National Institute of Diabetes and Digestive and Kidney Diseases (Baltimore, MD). Human CG was iodinated as described previously [12]. Unlabeled hCG was obtained from Sigma (St. Louis, MO). The cDNA for the mouse LHR was kindly provided by Drs. Lutz Birnbaumer (UCLA School of Medicine) and Thomas Gudermann (Free University of Berlin, Germany).
Animals
Four-month-old Sprague-Dawley rats and 4-mo-old C57BL/6 mice were obtained from Harlan Sprague and sacrificed by cervical dislocation according to protocols approved by the Animal Care Committee of the University of Iowa.
Preparation of Tissue Membranes
The procedure for membrane preparation described by Bonnamy et al. [2] was adapted and modified in the present study. Female rats (four rats per membrane preparation) were sacrificed at the metestrous cycle stage after going through two consecutive estrous cycles as determined by daily vaginal smears. Female mice (24 mice per preparation) were sacrificed regardless of their estrous cycle stage. Male rats or mice were sacrificed on the day of membrane preparation. The uterus, ovary, or testis, and liver were removed from the animals and immediately washed in sterile, ice-cold 0.9% NaCl containing the following protease inhibitors: 200 mM PMSF at 5 µl/ml, 200 mM EDTA at 10 µl/ml, and 333 mM N-ethylmaleimide at 15 µl/ml. The tissues were trimmed, weighed, and cut into small pieces. They were then homogenized on ice with a polytron (Ultra-Turrax T25 by Janke & Kunkel IKA-Labortechnik) in 25 mM Tris-HCl, pH 7.4, containing 0.25 M sucrose and the above protease inhibitors using eight 5-sec bursts at the highest speed with 25-sec intervals between the bursts. The tubes containing tissues were kept on ice during the homogenization. The homogenate was centrifuged at 500 x g at 4°C for 30 min, the pellets were discarded, and the supernatant was centrifuged at 38 724 x g at 4°C for 20 min. The pellets were saved and resuspended by homogenization in 25 mM Tris-HCl, pH 7.2, containing 5 mM MgCl2 and the above protease inhibitors. An aliquot of the membrane sample was used for protein assay as previously described [13]. The rest of the sample was used immediately for 125I-hCG binding assays.
125I-Human Chorionic Gonadotropin Binding Assays
Three 12 x 75-mm plastic tubes were used for each variable, two for total binding activity and one for nonspecific binding. Membrane samples containing 300 µg protein in 500 µl 25 mM Tris-HCl, pH 7.2, containing 5 mM MgCl2, 1 mg/ml BSA, and the above protease inhibitors were added to each tube. Then, 50 IU unlabeled hCG in 50 µl 0.15 M NaCl, 20 mM Hepes, pH 7.4 containing 1 mg/ml BSA was added to the tubes for the nonspecific binding assay and 50 µl buffer only was added to the tubes for the total binding assays. The 125I-hCG in 50 µl 0.15 M NaCl, 20 mM Hepes, pH 7.4 containing 1 mg/ml BSA was added to all tubes to give a final hCG concentration of 100 ng/ml. The binding assay tubes were rotated end-over-end overnight at 4°C, and then the contents plus one subsequent wash were transferred to fresh plastic tubes on ice. The membranes were then centrifuged at 2135 x g at 4°C for 15 min. The membrane pellets were resuspended in 3 ml cold Hanks' balanced salt solution, pH 7.4 containing 1 mg/ml BSA and centrifuged as above. The resulting pellets were counted in a gamma counter.
In Situ Hybridization for Localization of the LHR in the Mouse Reproductive Tract
The ovary, uterus, oviduct, and cervical regions of the reproductive tract of randomly cycling female mice were rapidly excised and frozen in liquid nitrogen. Cryostat sections (20 µm) of each were mounted on Superfrost/Plus glass slides (Fisher Scientific, Pittsburgh, PA) and fixed for 20 min at room temperature in freshly prepared 4% paraformaldehyde. The slides were then treated with proteinase K (2 µg/ml) in Tris-HCl, pH 8.0 for 10 min at room temperature, followed by a 30-sec incubation in 0.2% glycine in PBS. The slides were washed with PBS and then with 0.1 M triethanolamine. They were then incubated in 0.1 M triethanolomine containing 0.25% acetic anhydride for 10 min at room temperature and dehydrated through a graded ethanol series (30–100%). Slides were then air dried and used immediately for prehybridization and hybridization. A 591-base pair cDNA fragment corresponding to nucleotides 1–591 of the mouse LHR was subcloned into Litmus28 (New England BioLabs, Beverly, MA). Sense and antisense RNAs were prepared using T7 RNA polymerase following digestion with SpeI or EcoR1, respectively. The slides were prehybridized for 2 h at 52°C in hybridization solution (1x Denhard's solution [0.02% BSA, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 3x SSC, where 1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.3 M NaCl, 5 mM EDTA, 10 mM Tris-HCl, pH 8.0, 500 µg/ml yeast tRNA, 100 µg/ml sonicated salmon sperm DNA, and 50% formamide). Sense and antisense probes were placed on the slide at a concentration of 2 x 107 cpm/ml in 100 µl hybridization buffer. The slides were overlaid with coverslips and incubated 20 h at 52°C. After hybridization, the coverslips were removed and the slides were incubated with 4x SSC for 10 min at room temperature and then with 20 µg/ml RNase A for 30 min at 37°C. After a 30-min incubation at room temperature with 10 mM Tris-HCl, pH 8.0, 0.5 M NaCl, the slides were washed with decreasing concentrations of SSC (2x to 0.1x). They were then dehydrated through a graded ethanol series (30–100%) and air dried. The slides were processed for liquid emulsion autoradiography using NTB-2 emulsion (Eastman Kodak, Rochester, NY). Slides were developed after 3–5 days and then counterstained with hematoxylin-eosin. All slides were then viewed under both darkfield and brightfield microscopy to look for the presence of specific binding of the cDNA probes. Photos were taken at either 100x or 200x total magnification.
RESULTS
Presence of LHR in the Rat Uterus
Our first objective was to repeat the experiments of Bonnamy et al. [2] and demonstrate the presence of LHR in the rat uterus at metestrous. Uterine membrane samples were prepared from rats that had gone through two consecutive estrous cycles and were at metestrous stages. The 125I-hCG binding assays were performed to determine the presence of hCG binding sites in uterine membrane samples. As shown in Figure 1, there were very few or no hCG binding sites detectable in rat liver, whereas significant and specific hCG binding was present in rat ovary, testis, and uterus. The levels of hCG binding in the uterus are comparable to those in the testis when expressed on a per microgram of membrane protein basis.
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Presence of LHR in the Mouse Uterus
Our next objective was to detect LHR in the mouse uterus using 125I-hCG binding assays. Membrane samples were prepared from the uteri of randomly cycling mice. Similar to the findings in the rat, membrane preparations from the mouse uterus, ovary, and testis all specifically bound hCG, indicating the presence of the LHR in all these tissues (Fig. 2). When expressed on a per microgram of protein basis, the mouse uterus and testes had an equal number of binding sites, although both of these tissues had substantially fewer binding sites than the ovary.
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Localization of LHR in the Mouse Reproductive Tract
In situ hybridizations were then performed to determine the cell-specific localization of the LHR mRNA in mouse reproductive tissues. An RNA probe corresponding to nucleotides 1–591 of the mouse LHR (mLHR) was utilized for these experiments. On Northern blots of mRNA isolated from transfected 293 cells, this probe recognized rat LHR mRNA and mouse LHR mRNA, but not rat FSH receptor mRNA (data not shown). In the ovary, LHR mRNA was localized in theca cells, large follicles, and corpora lutea as expected (Fig. 3). Figure 3B demonstrates expression of LHR mRNA in the corpus luteum. We also noted LHR mRNA in the surface epithelium of the ovary (Fig. 4). In the oviduct, LHR mRNA was localized in the serosa and in subepithelial cells (arrows and arrowheads of Fig. 4B). Oviductal smooth muscle had detectable LHR mRNA expression, but it was much reduced compared to amounts in the serosa and subepithelial cells (Fig. 4B). Oviductal epithelium had no detectable LHR mRNA expression. In the uterus, LHR mRNA was most highly expressed in stromal cells of the endometrium and in the uterine serosa. Figure 5B demonstrates LHR mRNA expression in the uterine stroma and in the uterine serosa. Figure 6 is a higher power view of the uterine stroma demonstrating increased LHR mRNA expression in cells just beneath the epithelium (subepithelial cells) as compared to the rest of the stroma. Figure 7 demonstrates LHR mRNA expression in the stroma of the lower uterine segment near the cervix. The LHR mRNA was detectable in the stroma of all uteri examined regardless of the phase of the estrus cycle. In addition, LHR was detected in the stroma at all levels of the uterus from the uterine horns to the cervix (Fig. 7). Uterine smooth muscle cells had low levels of expression of LHR mRNA and the endometrial epithelium did not express LHR mRNA. In all experiments, the control slides performed using a radiolabeled sense probe at the same concentration were negative, substantiating the specificity of our in situ hybridization results.
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DISCUSSION
Extragonadal LHR are present in both the mouse and rat reproductive tract. Specific binding of hCG was demonstrated in membrane preparations of the uterus of both species. Although levels of binding were much less than in the ovary, the levels of binding were equivalent to that found in the testes when the data are expressed as binding per milligram of membrane protein. In contrast, we found no significant binding of hCG in the liver or in the prostate (data not shown). We localized the receptor by in situ hybridization using a probe specific for the portion of the mLHR encoding the extracellular binding region. We demonstrated the presence of mRNA for LHR in the serosa of the fallopian tubes and uterus and in subepithelial cells in the fallopian tube. In addition, we demonstrated the presence of LHR mRNA in the uterine stroma, particularly in the cells just below the epithelial lining of the uterine lumen. The presence of LHR mRNA was demonstrated in multiple sections of the uterus, ranging from the fundus down to very near the cervix. The epithelium of the endometrium demonstrated no LHR mRNA and uterine musculature showed very weak binding of the probe for the LHR mRNA. Although we did not specifically perform studies looking at the different days of the estrous cycle, we did see LHR mRNA in multiple uterine sections of randomly cycling mice. Our data are the first to detect and localize the LHR in the mouse reproductive tract.
Extragonadal LHR have been described in the reproductive tract of several species. Ziecik et al. [1] first reported the presence of LHR in the porcine uterus by binding assays of membrane preparations both from the myometrium and endometrium. The presence of LHR in the porcine uterus was later confirmed [8]. Bonnamy et al. [2] detected the presence of LHR in the uterus of the rat using binding assays. They found the highest binding levels found in the metestrus stage of the reproductive cycle. In a separate study, these investigators described the presence of LHR in the uterus of pregnant rats with highest levels of binding detected on Days 2 and 3 of pregnancy [3]. The LHR have also been reported in the mouse [10], bovine [7], and rabbit uterus [4]. Although still controversial [14], the presence of LHR has also been reported in the human uterus [5, 9].
Although the presence of LHR has been described in the uterus, there had been little information available regarding the localization of these receptors. Reshef et al. [5] utilized immunocytochemistry to localize the LHR in the human uterus. This study described staining in nearly all portions of the uterus, although particularly strong staining was noted in the glandular and luminal epithelial cells of the endometrium. The antibody used in this study has not proven to be very sensitive or specific in our hands when used on Western blots (data not shown). We found no mRNA expression for the LHR in uterine endometrial epithelium of the mouse. The same investigators have reported the presence of LHR in the mucosa of the human fallopian tube [15]. We found no mRNA expression for the LHR in the oviductal mucosa of the mouse but rather found binding in the serosa and in subepithelial cells. Whether these differences reflect species differences in the localization of the LHR within the reproductive tract or experimental differences remains to be determined.
The functional role of extragonadal LHR in the reproductive tract is not known. Investigators have speculated that LHR may be functionally important in the process of decidualization of the endometrium and implantation of the embryo into the uterus [16, 17]. Others have speculated that LHR may lead to vascular dilation and thus mediate blood flow changes to the uterus during the reproductive cycle and during pregnancy [15]. Rao and coworkers [18, 19] have demonstrated that LH induces the cyclooxygenase 2 gene in both endometrial glandular epithelium and stroma, and thus some of the effects of LH on these tissues may be mediated by increased prostaglandin production. Finally, LH may have direct actions in the uterus to stimulate growth. The uteri of transgenic mice lacking LH gene expression are only 30% the weight of wild-type control mice and have an infantile appearance [20]. These effects occur despite equal estrogen concentrations in the blood of the respective animals. Furthermore, uterine weight can be restored to normal in the LH-deficient mice after the injection of gonadotropin hormones. It is not entirely clear if this increase in weight is secondary to a direct effect of gonadotropins on the uterus or via an increase in ovarian hormones resulting from the gonadotropin injections. Therefore, multiple suggestions for the physiologic role of LHR have been put forth including involvement in the processes of implantation, regulation of blood flow to the uterus, survival of the early pregnancy, decidualization, regulation of enzymes within the uterus, and growth of the uterus.
Most of the abovementioned physiologic roles of the LHR have been based on in vitro experimentation. However, the precise role for this important receptor in the reproductive tract awaits further elucidation. Only with tissue-specific transgenic knock-out studies will we fully understand the importance of nongonadal LHR in reproduction. Our experiments documenting the presence and localization of the LHR in nongonadal tissues in the mouse validates using this animal system for future studies using transgenic approaches to eliminate selectively LHR expression in a given tissue. These studies should lead to important observations about the physiologic role of this hormone receptor.
ACKNOWLEDGMENTS
We thank Jeanne Snyder, Ph.D. and Kelli Goss for help with the in situ hybridization studies. We also thank Cyndy Bohnenkamp for her excellent secretarial assistance.
FOOTNOTES
1 These studies were supported by National Institutes of Health grants HD33931 and HD22916 (D.L.S.) and a Carver Collaborative Project grant from the University of Iowa. The services and facilities of the University of Iowa Diabetes and Endocrinology Research Center, supported by DK-25295, are also acknowledged. 
2 Correspondence: Bradley J. Van Voorhis, Department of Obstetrics and Gynecology, The University of Iowa, 200 Hawkins Drive, Iowa City, IA 52242. FAX: 319 353 6659; brad-van-voorhis{at}uiowa.edu
Accepted: August 16, 2000.
Received: April 19, 2000.
REFERENCES
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