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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 2 952-956
Copyright © 2004 by The Endocrine Society

Human Fetal Nongonadal Tissues Contain Human Chorionic Gonadotropin/Luteinizing Hormone Receptors

M. A. Abdallah, Z. M. Lei, X. Li, N. Greenwold, S. T. Nakajima, E. Jauniaux and Ch. V. Rao

Department of Obstetrics, Gynecology, and Women’s Health (M.A.A., Z.M.L., X.L., S.T.N., Ch.V.R.), University of Louisville Health Sciences Center, Louisville, Kentucky 40292; and Academic Departments of Obstetrics and Gynecology (N.G., E.J.), Royal Free and University College Medical School, London WC1 E6BT, United Kingdom

Address all correspondence and requests for reprints to: Ch. V. Rao, Ph.D., Division of Research, Department of Obstetrics, Gynecology, and Women’s Health, 438 MDR Building, 511 South Floyd Street, University of Louisville Health Sciences Center, Louisville, Kentucky 40292. E-mail: crvrao001{at}gwise.louisville.edu; http://www.louisville.edu/medschool/obgyn/molrepro/.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human chorionic gonadotropin (hCG), a heterodimeric glycoprotein hormone produced in abundance by placental syncytiotrophoblasts, is preferentially secreted into maternal circulation. Fetal circulation also contains low levels of hCG that are probably derived from fetal kidney, liver, anterior pituitary gland, etc. In addition, the fetus has access to hCG present in exocoelomic and amniotic fluids. hCG has been found in a number of fetal tissues known to stimulate fetal adrenal and testicular steroidogenesis and is also thought to play a role in growth and differentiation of fetal tissues. This led us to test the hypothesis that fetal nongonadal tissues, as in the adult, may also contain hCG/LH receptors. This hypothesis was tested by immunocytochemistry, Western blotting, in situ hybridization, and RT-PCR. The results demonstrate that kidney, liver, pancreas, lung, small and large intestines, and adrenals contained hCG/LH receptors. Although the role of fetal nongonadal hCG/LH receptors is not known, they may mediate the pleiotropic actions of hCG in the growing human fetus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HUMAN CHORIONIC GONADOTROPIN (hCG) is a heterodimeric hormone primarily produced by placenta (1). LH, a structural and functional homolog of hCG, is produced by the anterior pituitary gland (1). These two hormones belong to the glycoprotein hormone as well as to the cystine knot growth factor families, which include FSH, TSH, nerve growth factor, platelet-derived growth factor-ß, TGF-ß, and vascular endothelial growth factor (1, 2). Both hormones, especially hCG, are also produced by many other normal and cancer tissues in small amounts (3). Although hCG and LH bind to the same transmembrane glycoprotein receptor that belongs to the G protein-coupled receptor superfamily (4, 5), hCG binds with higher affinity than LH (6). The receptors have about equal size exo and endo domains (4, 5). The exodomain contains the hormone-binding sites, and the endodomain contains seven transmembrane-spanning regions and a short cytoplasmic tail coupled to signal transduction pathways (4, 5). The work published from around the world in the last 20 yr has demonstrated that many nongonadal tissues in adult also contain low levels of functional hCG/LH receptors (7).

Maternal serum hCG levels exponentially increase and reach a peak by about the end of the first trimester, followed by a rapid decline to low steady-state levels of approximately one tenth of the peak values (3). hCG is also present in fetal circulation, amniotic fluid, extraembryonic coelome (exocoelomic or chorionic fluid), and yolk sac fluid (8, 9, 10, 11, 12, 13, 14, 15, 16). Although the maternal serum hCG is derived from placental syncytiotrophoblasts (3), the hCG in fetal circulation probably comes from fetal kidney, liver, anterior pituitary gland, and from the exocoelomic fluid via the secondary yolk sac and amniotic fluids (14, 15, 16, 17, 18, 19, 20). hCG is found in fetal ovary, testes, kidney, lung, liver, adrenal, thymus, spleen, and muscle (21), which implies that this hormone may have been sequestered with the help of its receptors, at least in some of these tissues. The sequestration suggests that hCG may play a role in growth and differentiation of fetal tissues. The available data suggest that hCG serves as an adrenocorticotropic hormone by stimulating dehydroepiandrosterone production (22, 23, 24). In addition, fetal hCG may also stimulate testosterone production by fetal testes (25, 26). Because hCG has a broader tissue distribution in fetus (21) and it targets nongonadal tissues in adult (7), we hypothesized that hCG may also target nongonadal tissues in fetus. In such a case, fetal nongonadal tissues should contain hCG/LH receptors. This hypothesis was tested by using immunocytochemistry, Western blotting, in situ hybridization, and RT-PCR on several fetal nongonadal tissues. However, functional studies could not be performed due to technical and logistical reasons.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissues

Forty human fetal specimens from 7 to 16 wk gestation (four each for wk 7 and 9; six each for wk 8, 10, 12, and 16; seven for wk 14; and one for wk 15) were obtained at the time of elective surgical terminations performed under general anesthesia for psychosocial reasons. Only uncomplicated pregnancies were recruited, and, in all cases, gestational age was calculated using maternal last menstrual period and confirmed by ultrasound measurements of the crown-to-rump length of the fetus. The tissues, which were initially identified by gross examination and later confirmed by histology, were fixed overnight in Bouin’s solution, processed, and embedded in paraffin on the same day. A minimum of three sections of each tissue was used in immunocytochemistry and in situ hybridization analyses. Fresh fetal intestines and placentas were locally obtained from three pregnancies at the 14th week of gestation. The use of tissues was approved by the University College London/University College Hospitals Committees on the Ethics of Human Research (study 2538) and by the Human Studies Committee at the University of Louisville.

Immunocytochemistry

This procedure was performed as previously described (27). Five-micrometer-thick sections were cut from paraffin-embedded tissue blocks. The sections were treated with 0.3% hydrogen peroxide in methanol to block endogenous peroxide activity. After three washes with PBS, nonspecific binding sites were blocked and then incubated overnight at 4 C with a 1:350 dilution of the polyclonal hCG/LH receptor antibody raised against the synthetic 15–38 N-terminus amino acid sequence (provided by Dr. Patrick Roche, who is now at Ventana Medical Systems, Tucson, AZ). The samples were then washed and incubated with a secondary antibody, an avidin-biotin-immunoperoxidase complex (Vector Laboratories, Burlingame, CA), and exposed to diaminobenzidine and hydrogen peroxide. The receptor antibody preabsorbed with excess receptor peptide was used in the procedural control.

Western blotting

The fresh tissues were homogenized for 1 min at 4 C with a Tissue Tearor (Research Products International Corp., Mt. Prospect, IL) in 50 mM Tris HCl (pH 8.0) containing 150 mM NaCl, 5 mM MgCl2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate, and 10 µg/ml each of leupeptin, aprotinin, and pepstatin. The homogenates were centrifuged for 15 min at 12,000 x g, the protein content in supernatants was determined, and 20-µg protein aliquots were separated by continuous SDS-PAGE under reducing conditions (28). The separated proteins were electroblotted onto immobilon P membranes (29). After blocking the nonspecific binding sites with 5% nonfat dry milk in 10 mM Tris-HCl (pH 7.5) containing 150 mM NaCl and 0.1% Tris-buffered saline with Tween 20 (TBS-T), the blots were washed for 5 min and incubated overnight at 4 C with 1:1500 dilution of the hCG/LH receptor antibody and then washed three times with TBS-T buffer. The washed blots were again incubated for 1 h at 22 C with a 1:1500 dilution of horseradish peroxidase-labeled secondary antibody and then washed four times for 15 min each time with TBS-T. The binding of receptor antibody was detected by an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ). The molecular size of hCG/LH receptor protein was determined by running molecular size marker proteins in an adjacent lane. The receptor antibody preabsorbed with excess receptor peptide was used in the procedural controls.

In situ hybridization

This procedure was performed as previously described (30, 31). Briefly, the sections were treated with 50 mM Tris-HCl (pH 7.5) containing 5 µg/ml proteinase K and 5 mM EDTA for 30 min at 37 C. Prehybridization mixture containing 50% formamide, 5x sodium chloride/sodium citrate (SSC), 1x Denhardt’s solution, 1 mg/ml yeast tRNA, 100 µg/ml heparin, and 5 mM EDTA was applied to the sections and left for 3 h at 55 C in moist chambers. The hybridization was carried out overnight in the same mixture containing antisense or sense riboprobes transcribed from human hCG/LH receptor cDNA. After hybridization, the sections were washed twice at 65 C for 30 min with 1x SSC and 0.2x SSC. Hybridization signals were detected by using an antifluorescein alkaline phosphatase conjugate and 4-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolylphosphate, which gave a blue color. The sense riboprobe was used for a procedural control.

RT-PCR

Total RNA was isolated using a single-step acidic guanidinium thiocynate extraction method (32). RNA aliquots (4 µg) were reverse-transcribed using 5'-primer (5'-CCCTTGGTTGGGAATCAAC-3') from Operon Technologies (Alameda, CA) and 1 µl avian myeloblastosis virus reverse transcriptase (Promega Corp., Madison, WI) in 5x buffer containing 250 mM Tris-HCl (pH 8.3), 250 mM KCl, 50 mM MgCl2, 50 mM dithiothreitol, 25 mM spermidine, and 0.5 µl deoxynucleotide triphosphate mix [20 µM of deoxy (d)ATP, dCTP, dGTP, and dTTp] from Promega Corp. Two- to 5-µl of reverse-transcribed cDNA were initially denatured for 3 min at 94 C and then amplified for 35 cycles with each cycle consisting of denaturation (55 sec at 94 C), annealing (55 sec at 59 C), and extension (55 sec at 72 C) with 0.5 µl Taq DNA polymerase (Promega), 0.5 µl (20 µM) of the primer pairs (forward, 5'-TGAACTGAGTGGCTGCCACTAT-3'; and reverse, 5'-CCCTTGGTTGGGAATCAAC-3') in 2.5 µl 10x buffer containing 2.5 mM MgCl2. Final extension was performed for 7 min at 72 C. The amplified fragments were resolved by Nusieve agarose gel electrophoresis. A 123-bp DNA ladder was run for determining the molecular size of amplified products. The omission of template cDNA served as a procedural control. The amplified expected size hCG/LH receptor fragment (300 bp) was sequenced.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunocytochemistry was first used to determine the distribution of hCG/LH receptors in human fetal nongonadal tissues. The results indicated that kidney, liver, lung, pancreas, small and large intestines, and adrenals contained the receptor immunostaining that dramatically decreased after the preabsorption of the receptor antibody with receptor peptide (Fig. 1Go). The receptor immunostaining was variable between and within the tissues. For example, adrenals contained the lowest amount of immunostaining compared with the other fetal organs. Proximal and distal collecting ducts contain more staining than glomerular capsule in kidney. Hepatocytes in liver, alveoli in developing lung, pancreatic acini, and mucosa of small and large intestines contained considerable amounts of receptor immunostaining compared with the other cellular elements in these tissues. The higher magnification picture of small intestine revealed that the receptor immunostaining is present not only on the cell surface, but also in the intracellular locations. Determination of immunostaining intensities by visual examination of sections under microscope revealed that there was no consistent change in tissues other than liver in which it increased from wk 7 through wk 14 gestation, followed by a modest decline (data not shown). In agreement with previous studies (3, 7), placenta contained receptor immunostaining (data not shown). Fetal skeletal and cardiac smooth muscle, adrenal medulla, and connective tissue elements in all the receptor-positive tissues tested negative for receptor immunostaining.



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  		FIG. 1. Immunostaining for hCG/LH receptors in human fetal kidney (A and B), liver (C and D), lung (E), pancreas (F), small intestine (G and H), large intestine (I and J), and adrenal (K and L). Kidney, liver, pancreas, small and large intestines, and adrenals were from the 14th gestational week, and the lung was from the 8th gestational week. Unabsorbed receptor antibody was used in panels A, C, E, F, G, H, I, and K, and preabsorbed receptor antibody was used in panels B, D, J, and L. Blue arrows in panel A indicate glomerular capsule, and red arrows indicate distal and proximal tubules. Blue arrows in panel E indicate alveoli, blue arrows in panel F indicate acini, and blue arrows in panels G, H, and I indicate mucosal folds. Black arrow in panel K indicates medulla. The higher magnification of small intestinal mucosal fold in panel H shows that the receptor immunostaining was not only present in cell membranes, but was also located in intracellular locations. Magnification, A–G and I–L, x150; H, x600.

 
Western blot analysis demonstrated that fetal intestines, as human placental tissue used as positive control, contained an 80-kDa receptor protein (Fig. 2Go). This protein was not detected when the receptor antibody preabsorbed with receptor peptide was used (Fig. 2Go).



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  		FIG. 2. Western blotting for hCG/LH receptors in term human placenta used as a positive tissue control (lanes 1 and 3) and in fetal intestine from 14th gestational week (lanes 2 and 4). Preabsorbed receptor antibody was used in lanes 3 and 4.

 
In situ hybridization analysis revealed that intestinal mucosa, renal glomerular capsule, proximal and distal collecting ducts, and hepatocytes in liver contained hybridization signals when receptor antisense riboprobe was used (Fig. 3Go). These signals were absent when sense riboprobe was used for controls (Fig. 3Go).



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  		FIG. 3. In situ hybridization for hCG/LH receptors in fetal intestine (A and B), kidney (C and D), and liver (E and F) from the 14th gestational week. Antisense probe was used in panels A, C, and E, and sense probe was used in panels B, D, and F. A, The red arrows indicate mucosal folds. C, The blue arrow indicates glomerular capsule, and red arrows indicate distal and proximal tubules. E, Red arrow indicates central vein. Magnification, A, C, E, and F, x300; B and D, x150.

 
The presence of hCG/LH receptor mRNA was further confirmed by RT-PCR, which demonstrated amplification of an expected size fragment from fetal intestines, as in the human placental tissue used as a positive control (Fig. 4Go). The sequencing of the amplified product from fetal intestines revealed a perfect match with a published human receptor sequence.



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  		FIG. 4. RT-PCR for hCG/LH receptors in term human placenta used as a positive tissue control (lanes 1 and 3) and fetal intestine from the 14th gestational week (lanes 2 and 4). cDNA template was omitted in lanes 3 and 4.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cord blood contains hCG (8, 9, 33), and there is no evidence that it comes from fetal blood cells. It is likely to come from fetal kidney, liver, and anterior pituitary gland, which synthesize hCG (17, 18, 19, 20), and also from uptake of hCG in exocoelomic fluid via the secondary yolk sac, which is connected to the fetal digestive system, and amniotic fluid, which the fetus swallows (8, 10, 11, 12, 13, 14, 15, 16). The hCG found in the yolk sac fluid is not secreted by the yolk sac cells (14, 15). Instead, it comes exclusively from absorption from exocoelomic fluid, which contains higher hCG levels than even maternal blood (14, 15). The amniotic fluid hCG levels, which are much lower than in coelomic and yolk sac fluids, are probably derived from fetal urine and some diffusion from the maternal compartment (8, 10, 11, 12, 13, 14, 15).

Although the role of hCG in the fetus is largely unknown, it is generally considered to serve as an adrenocorticotropic hormone by stimulating the production of dehydroepiandrosterone, which is later converted into estriol in the placenta (22, 23, 24). This conversion, as measured by maternal serum or urinary estriol levels, reflects fetal health and well being. In addition, hCG can stimulate testosterone production by Leydig cells, which is essential for masculinization of fetal tissues (25, 26). Considering that hCG was found in several nongonadal tissues of fetus (21), probably reflecting a sequestration with the help of its receptors, and that the sequestered hormone could play a role in their growth and differentiation, hCG is likely to have other functions. Thus, hCG could act on fetal nongonadal tissues, as in the adult. In such a case, fetal nongonadal tissues, which are likely to respond, should contain hCG receptors.

The search for hCG/LH receptors was made by using four complementary receptor detection techniques. Two of them (immunocytochemistry and in situ hybridization) were used in parallel on several nongonadal tissues, whereas the Western blotting and RT-PCR were only used on fetal intestines. The results demonstrated the presence of hCG/LH receptors in fetal kidney, liver, lung, pancreas, small and large intestines, and adrenals. The presence of the receptor suggests a role for hCG, which is known for fetal adrenals (22, 23, 24). However, it is not known what hCG does in liver, kidney, lung, pancreas, small and large intestines, which contain even more receptors than adrenals. Because hCG has properties of growth factors and cytokines, it is possible that it has growth and developmental roles, in addition to serving as a potential immunosuppressive agent and survival factor in fetal tissues (3, 7, 34, 35). Some of these roles may depend on the stage of fetal development. Consistent with this possibility, liver hCG/LH receptors changed with the gestational age.

Human fetal nongonadal tissues such as kidney, liver, lung, adrenal, thymus, spleen, and muscle contain hCG (21). Because some of these tissues are now found to contain hCG receptors, it is possible that hCG could be an autocrine factor for some and endocrine factor for other fetal nongonadal tissues.

In summary, this is the first report to demonstrate the presence of hCG/LH receptors in several human fetal nongonadal tissues. The hCG, which activates these receptors, is present in fetal circulation as well as in fluid compartments that are accessible to the fetus. Thus, it is possible that hCG may contribute to fetal growth and development by activating its receptors in nongonadal tissues.


   Footnotes
 
Abbreviations: d, Deoxy; hCG, human chorionic gonadotropin; SSC, sodium chloride/sodium citrate; TBS-T, Tris-buffered saline with Tween 20.

Received May 27, 2003.

Accepted October 20, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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