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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 10 5161-5167
Copyright © 2004 by The Endocrine Society

Leydig Cell Hypoplasia: Absent Luteinizing Hormone Receptor Cell Surface Expression Caused by a Novel Homozygous Mutation in the Extracellular Domain

A. Richter-Unruh, M. Verhoef-Post, S. Malak, J. Homoki, B. P. Hauffa and A. P. N. Themmen

Department of Pediatric Hematology, Oncology and Endocrinology (A.R.-U., S.M., B.P.H.), University Children’s Hospital, University of Essen, 45122 Essen, Germany; Department of Internal Medicine (M.V.-P., A.P.N.T.), Erasmus Medical Center, 3000 DR Rotterdam, The Netherlands; and Department of Pediatrics (J.H.), University Children’s Hospital, 89075 Ulm, Germany

Address all correspondence and requests for reprints to: Annette Richter-Unruh, Department of Hematology, Oncology and Endocrinology, University Children’s Hospital, Hufelandstrasse 55, 45122 Essen, Germany. E-mail: annette.richter-unruh{at}uni-essen.de.


   Abstract
Top
 Abstract
Introduction
 Patient and Methods
 Results
 Discussion
 References
 
Leydig cell hypoplasia is a rare autosomal recessive condition that interferes with normal development of male external genitalia in 46,XY individuals. We have studied a family with a 46,XY girl due to a new homozygous mutation (V144F) in the extracellular ligand-binding domain. HEK 293 cells transfected with the mutant LH receptor exhibited a marked impairment of human chorionic gonadotropin binding. Using Western blotting of the expressed V144F mutant LH receptor protein showed the absence of the glycosylated cell surface form. Treatment of the mutant LH receptor with N-glycosidase F or endoglycosidase-H demonstrated that the mutant receptor is retained in the endoplasmic reticulum. Expression and study of enhanced green fluorescent protein-tagged receptors confirmed that the mutant LHR-V144F receptors do not migrate to the cell surface, and the fluorescence remains intracellular and colocalizes with an endoplasmic reticulum marker, ER-tracker Blue-white DPX. Comparison of the theoretical molecular models of the extracellular domain of the wild-type and the mutant receptor suggests that the mutation LHR-V144F, located in the outer circumference in a {alpha}-helix of the leucine-rich repeat 4, may induce a conformational strain on the molecule. F144 of the mutant LH receptor has overlapping interactions with F119, which V144 in the wild-type receptor has not.


   Introduction
Top
 Abstract
 Introduction
Patient and Methods
 Results
 Discussion
 References
 
NORMAL MALE SEX differentiation is dependent of the testicular secretion of testosterone. If testosterone biosynthesis is reduced or absent, virilization will be diminished or missing, and individuals with a 46,XY karyotype will have a female or ambiguous phenotype. Testosterone biosynthesis is dependent on the stimulation of testicular Leydig cells through activation of the LH receptor (LHR) by LH and the placental LH homolog human chorionic gonadotropin (hCG). The resistance of Leydig cells to the action of hCG and LH, due to inactivating mutations of the LHR gene, causes Leydig cell hypoplasia, characterized by a phenotype that ranges from complete 46,XY male pseudohermaphroditism to normal male sex differentiation with hypospadias or a micropenis (1). Because a number of inactivating mutations have been found in the LHR gene, a correlation can be made between the phenotype of patients with Leydig cell hypoplasia and the activity of their LHR alleles. The presence of the A593P mutation on both alleles (2) or compound heterozygosity of the C343S and C543R (3) mutations, all of which completely inactivate the LHR, is associated with complete pseudohermaphroditism. Truncation of the LHR protein by a homozygous nonsense mutation causes a similar phenotype (4, 5). Patients homozygous for mutations that cause even less complete impairment of LHR function, such as S616Y (4, 6) and I625L (7), show a mild phenotype with micropenis. A carrier of a homozygous mutant LHR that is severely but not completely affected in its activity, such as the LHR-C131R mutant (8), has a micropenis with hypospadias. This mutation is located in exon 5, which encodes a part of the extracellular domain of the LHR.

In the present study, we report a new missense mutation, V144F, also located in exon 5 of the LHR gene, 11 amino acids C terminal of C131R. In contrast, this patient presented as a 46,XY girl.


   Patient and Methods
Top
 Abstract
 Introduction
 Patient and Methods
Results
 Discussion
 References
 
Patient

The girl was born as the first of three children from healthy, related parents from Turkey. At birth, she presented with clitoral enlargement and labial synechia. At the age of 4 months, a right-sided inguinal hernia was discovered. Surgery showed an incarceration of the right gonad. The organ was displayed back to the abdominal cavity without taking a biopsy. At the left side, a testis was found in the inguinal region by sonography. Müllerian structures were not seen. The karyotype was 46,XY; serum testosterone levels were very low and hardly could be stimulated by hCG treatment (basal serum testosterone 0.0 nmol/liter, after 5000 IU/m2 body surface increase of serum testosterone to 0.322 nmol/liter). At the age of 10.5 yr, she was referred to the Department of Pediatric Endocrinology in Ulm, Germany. She had a female phenotype with slight clitoral hypertrophy. Testosterone and dihydrotestosterone were not detectable in serum. EDTA-blood was taken for mutation analysis. The patient’s parents were informed about all diagnostic steps and gave their written consent for the surgery and analysis of the LHR gene. Additionally, the studies in this patient were part of a more comprehensive study that was approved by our Institutional Review Board.

DNA analysis

Genomic DNA was extracted from peripheral blood using QIAamp DNA mini kit (Qiagen, Hilden, Germany). Exons 1–10 and two overlapping fragments of exon 11 of the LHR gene as well as the adjacent exon-intron boundaries partly up to 70 intron nucleotides (intron 1) were amplified by PCR using primers and conditions described previously (9, 10). The PCR fragments were checked on agarose gels, purified with the High Pure PCR Purification kit (Roche Molecular Biochemicals, Mannheim, Germany), and sequenced using the Thermo Sequenase Dye Terminator Cycle sequencing kit for PCR fragments (Amersham Pharmacia Biotech, Little Chalfont, UK).

Construction of mutant human LHR (hLHR) expression vector

The expression plasmid pSG5 containing the wild-type hLHR cDNA extended by an immunotag [hemagglutinin 1 (HA1)], followed by the green fluorescent protein (GFP) at the C terminus (pSG5-hLHR), has been described previously (3). Mutations were introduced into this construct using standard PCR mutagenesis (11, 12) with primers (MWG-Biotech AG, Ebersberg, Germany) as described below. The nucleotides that differ from the wild-type hLHR cDNA are indicated in bold and underlined: LHR181FOR, 5'-CTCCCTCTCAAAGTGATCC-3'; LHR11. 1R EV, 5'-GGAGCAAAAGCGTACACGTTAG-3'; LHR(V144F)FOR, 5'-GTTACGAAGTTCTTCTTCTCTG-3'; LHR(V144F)REV, 5'-CAGAGAAGAAGAACTTCGTAAC-3'.

To construct pSG5-hLHR-V144F, primer sets LHR181FOR/LHR(V144F)REV and LHR(V144F)FOR/LHR11.1REV were used separately to perform the first PCR amplification. After mixing of the fragments, the final mutant fragment was obtained by using the primer set LHR181FOR and LHR11.1REV. To construct the mutant human LHR expression vector, a HindIII-Bsu36 I fragment of the reamplified fragment was used to replace the wild-type sequence in pSG5-hLHR, resulting in pSG5-hLHR-V144F. The correct nucleotide sequence of the exchanged fragment was verified by DNA sequencing.

cAMP-responsive luciferase reporter activity measurements

HEK 293 cells, maintained as described previously (7), were seeded at 15% confluence in 75-cm2 flasks (Nunc, Roskilde, Denmark) and transfected the next day with 1 ml calcium phosphate precipitate containing 20 µg DNA [10 µg expression construct, 1 µg pRSVlacZ (13), 2 µg pCRE6Lux (14), and 7 µg of carrier DNA per milliliter of precipitate]. Three days after transfection, the hCG-dependent luciferase response was determined in 24-well tissue culture plates (Costar, Cambridge, MA) by incubating the cells for 4 h in culture medium containing 0.1% BSA and increasing concentrations of hCG (0.001–1000 ng/ml; urinary hCG; Organon International, Oss, The Netherlands). Subsequently, the cells were lysed, and luciferase activity was measured (15). ß-Galactosidase activity of the lysates was determined to correct for transfection efficiency (13).

Scatchard analysis

To determine the binding affinity (Kd) and total receptor number (Bmax) of the cell surface-expressed LHR protein, HEK 293 cells were transfected with pSG5, pSG5-hLHR, and pSG5-hLHR-V144F. Three days after transfection, Scatchard analysis using chloramine T [125I]-labeled hCG was performed on intact cells as described previously (7, 16)

Construction of stable cell lines with inducible hLHR expression

We used the TRex system (Invitrogen, Carlsbad, CA) to construct stable cell lines with indocile expression of the hLHR. The entire LHR coding sequence, including the HA1 tag and GFP sequence, was isolated from the pSG5-base vectors by digestion with EcoRI and HpaI and placed in the EcoRI/EcoRV-digested pcDNA4/TO vector, resulting in pcDNA4/TO-hLHR and pcDNA4/TO-hLHR-V144F. These expression plasmids were transfected into TRex-293 cells (Invitrogen), and after 3 d, the cells were split 1:4 in DMEM/F12 medium plus 10% fetal calf serum (FCS) and selected in the response of 400 µg/ml Zeocine (Invitrogen) and 5 µg/ml Blasticidine (Invitrogen). After 2 wk, surviving clones were pooled and kept under selection in DMEM/F12 with 10% FCS containing 400 µg/ml Zeocine and 5 µg/ml Blasticidine. These manipulations resulted in the cell lines TRex-hLHR and TRex-hLHR-V144F, respectively.

Western blotting

TRex-hLHR and TRex-hLHR-V144F cells were induced overnight in Costar six-well plates (Corning Inc., Corning, NY) with 15 ng/ml tetracycline (Sigma, St. Louis, MO). Cells were rinsed with PBS, detached in PBS, and centrifuged, and the cell pellets were frozen at –20 C. Before treatment, cell pellets were dissolved in the supplied buffer at 1 µg/µl protein concentration. Cells were dissolved completely by passing through a 25-gauge needle and left at room temperature for 30 min, followed by freezing at –20 C. For N-glycosidase F (PNGase F; New England Biolabs, Beverly, MA) treatment (removal of N-linked glycosylation side chains), 500 U PNGase F were added to 10 µl dissolved cell pellet and incubated for 4 h at 37 C. Protein was separated using PAGE after the addition of 2.5 µl of 5x Laemmli sample buffer. For Endoglycosidase H (EndoH; New England Biolabs) treatment, the same procedure was used with the supplied EndoH buffer. The Western blotting procedure and subsequent LHR detection was performed as described previously (3) using a conjugated HA1-horseradish peroxidase monoclonal antibody (Roche Diagnostics, Mannheim, Germany) in combination with the Renaissance chemiluminescence detection kit (NEN Life Science Products, Du Pont den Nemours, Dreieich, Germany).

Live cell imaging of TRex-hLHR and TRex-hLHR-V144F cells

For determination of cellular localization of LHR-GFP fluorescence, cells were grown on coverslips coated with polylysine and induced overnight with 15 ng/ml tetracycline. Coverslips were mounted and studied in DMEM/F12 and 10% FCS at 37 C at 5% CO2. For endoplasmic reticulum colocalization studies, cells were incubated with ER-tracker Blue-white DPX (Molecular Probes, Leiden, The Netherlands) at a concentration of 1 µM. Cells were analyzed at 37 C on a Zeiss LSM510NLO confocal laser-scanning microscope (Zeiss, Oberkochen, Germany). The optical slice (z-dimension) was set to 0.5 µm. Other settings that were used (such as laser intensity and gain value) were adapted to obtain optimal signal to noise ratios. One image was taken from the middle of the stack and prepared for use in Figs. 5Go and 6Go using the Zeiss LSM510 software.



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  		FIG. 5. LHR-V144F is mainly colocalized with an endoplasmic reticulum marker. HEK 293 cells were transfected with GFP-tagged wild-type (wt; top) or V144F mutant LHR (bottom) expression plasmids, incubated with ER-tracker Blue-white DPX. GFP (green) and ER-Tracker (red) fluorescence in living cells was determined using the confocal laser microscope. In the photomicrographs on the right, a merge of the two images is presented. LHR-V144F colocalizes with the ER-Tracker fluorescence, whereas wild-type LHR is expressed at the plasma membrane and shows almost no endoplasmic reticulum localization.

 


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  		FIG. 6. A, Top view of a model of the extracellular domain of the hLHR (http://www.expasy.org/spdbv/). The model is presented with a view parallel to the ß-strands that are formed by the leucine-rich repeat structures. The wild-type predicted structure is shown on the left, and the F144 mutant receptor is on the right. V144, F144, and the cysteine bridge between C131 and C156 are indicated. Secondary structures are indicated by colors: red, helix; turquoise, strand; green, coil. B, Detailed view of the area surrounding V/F144. V144 is at the top, and F144 is at the bottom, the third to fifth {alpha}-helix of the extracellular domain. The mutant receptor F144 has overlapping interactions with F119 that V144 has not. F119 is located at the interface between the third {alpha}-helix and the fourth ß-sheet of the extracellular domain.

 

   Results
Top
 Abstract
 Introduction
 Patient and Methods
 Results
Discussion
 References
 
Sequence analysis of the LHR gene

Sequencing of the complete coding sequence of the LHR gene derived from genomic DNA revealed a homozygous missense mutation. In exon 5 at codon 144 (nucleotide 430), we identified a GTC to TTC change resulting in a missense amino acid change from valine to phenylalanine (V144F). The father, mother, and one of the brothers were all heterozygous at codon 144 for the same mutation.

Biochemical characterization and functional analysis of the mutant receptors

To determine the effects of the missense mutation on the LHR protein, wild-type LH and mutant receptors were expressed in TRex-293 cells and visualized using Western blotting (Fig. 1Go). The wild-type receptor migrates mostly as a 130-kDa band, representing the mature plasma membrane form of the receptor, although a 100-kDa band, which represents an immature, incompletely processed form of the receptor, is found also (3). The LHR-V144F migrates as the smaller 100-kDa band, with no mature 130-kDa band present. The 100-kDa form represents the immature LHR precursor glycoprotein that is localized in the endoplasmic reticulum, whereas the mature receptor with an apparent mass of 130 kDa arises from the maturation during trafficking of the100-kDa form to the cell surface (Ref. 3 and see below). The sizes of the LHR protein are larger than those reported previously (3) because the proteins were extended with the 27-kDa GFP protein. Treatment of the wild-type receptor and the mutant LHR-V144F protein with PNGase F removed the oligosaccharide chains from both the 100- and 130-kDa forms of the receptor to yield a 90-kDa band, demonstrating that both receptors contain N-linked oligosaccharide chains. In contrast, the 130-kDa form of the wild-type receptor is not susceptible to EndoH, a glycosidase that removes the high-mannose type of carbohydrate side chains associated with immature glycoproteins.



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  		FIG. 1. Expression of wild-type (wt) and mutant hLHR-V144F receptors in TRex cells, visualized using Western blotting. Shown are untreated receptors (left), after digestion with PNGase F (middle), and after treatment with EndoH (right). The wild-type receptor migrates mostly as a 130-kDa band, representing the mature plasma membrane form of the receptor, although also as a 100-kDa band, representing an immature, incompletely processed form of the receptor. The smaller-sized 100-kDa mutant protein represents the immature LHR precursor glycoprotein, which is localized in the endoplasmic reticulum. The sizes of the LHR protein are larger than those reported previously (3 ) because the proteins are extended with the 27-kDa GFP protein. Treatment of the receptors with PNGase F removed the oligosaccharide chains from both the 100- and 130-kDa forms of the receptors to yield a 90-kDa form, demonstrating that the mutant receptor, like the wild-type receptor, contains N-linked oligosaccharide chains. The lower band in the wild-type receptor lane is probably a breakdown product. The 100-kDa bands are sensitive to EndoH treatment, suggesting that these are immature forms localized in the endoplasmic reticulum. In contrast, the mature form at 130 kDa of the wild-type receptor cannot be cleaved by EndoH.

 
The 100-kDa bands found in both the wild-type and LHR-V144F-transfected cells show sensitivity to EndoH treatment, suggesting that these are immature glycoproteins localized in the endoplasmic reticulum. Although the shift of the bands after EndoH treatment is not large, the colocalization of the LHR-V144F with an endoplasmic reticulum marker confirms this suggestion (see below and Fig. 5Go)

The Western blot results indicate that LHR-V144F is processed differently from the wild-type LHR. Therefore, we examined whether the mutant V144F receptors were transported properly to the cell surface after synthesis and were able to display proper ligand binding. Cells transfected with pSG5-hLHR-GFP showed high-affinity binding (Kd = 0.57 ± 0.08 nM), whereas only very weak binding was detectable in intact cells transfected with pSG5-hLHR-V144F-GFP (Fig. 2Go).



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  		FIG. 2. Scatchard analysis of [125I]hCG binding to HEK 293 cells expressing wild-type and pSG5-hLHR-V144F receptors. The mutant receptor ({blacksquare}; enlarged in inset) shows only very week hormone binding compared with the wild-type receptor (•). B/F, Bound/free.

 
HEK 293 cells were transfected with the pSG5-LHR or pSG5-LHR-V144F expression vectors and incubated with increasing concentrations (1–1000 ng) of hCG (Fig. 3Go). The cells transfected with pSG5-LHR showed a vigorous response to increasing concentrations of hCG. As expected, based on the deficient hormone binding, the response of cells transfected with pSG5-LHR-V144F was markedly impaired, although at high concentrations of hCG a limited response was observed.



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  		FIG. 3. hCG-dependent cAMP-responsive luciferase activity measured in wild-type LHR (•), LHR-V144F mutant receptor ({blacksquare}), and empty expression plasmid pSG5 ({diamondsuit}) expressing HEK 293 cells. The biological activity of the mutant receptor is markedly impaired. At high concentrations of hCG, a limited response is observed. ß gal, ß-Galactosidase; RLU, relative luciferase units.

 
Intracellular localization of the wild-type and mutant receptors was studied in TRex-293 cells, which were stably transected with pcDNA4/TO-hLHR and pcDNA4/TO-hLHR-V144F (i.e. tetracycline-inducible, GFP-tagged LHR expression constructs). After induction with tetracycline, the wild-type LHR showed clear expression at the cell surface, with some intracellular fluorescence. The pattern for the V144F mutant LHRs was clearly different. They did not traffic to the cell surface, and fluorescence remained intracellular, with a widespread and heterogenic distribution. Because this pattern was reminiscent of localization in the endoplasmic reticulum, cells were incubated with an endoplasmic reticulum marker, ER-tracker Blue-white DPX (17), and fluorescent images of the marker and GFP fluorescence were taken (see Fig. 5Go). As also shown in Fig. 4Go, wild-type GFP-tagged receptors localize mainly at the cell surface, whereas the ER-tracker fluorescence is mostly found perinuclear and in the cytoplasm, consistent with endoplasmic reticulum localization. Only limited colocalization of the wild-type receptor and ER-tracker is found, as indicated by the yellow color in the merged ER-tracker (red) and GFP (green) images (Fig. 5Go). In contrast, the majority of the GFP signal in the cells expressing LHR-V144F is found colocalized with the red ER-tracker fluorescence. Thus, LHR-V144F appears to be trapped in the endoplasmic reticulum without subsequent processing and trafficking to the cell surface, consistent with the absence of the large 130-kDa band on the Western blot (Fig. 1Go).



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  		FIG. 4. Subcellular localization of enhanced GFP-tagged wild-type and LHR-V144F receptors. A, Cell surface localization of wild-type receptors (indicated by an arrow). B, Mutant receptors did not migrate to the cell surface and remained intracellular (indicated by an arrow).

 

   Discussion
Top
 Abstract
 Introduction
 Patient and Methods
 Results
 Discussion
References
 
Abnormalities of sex differentiation should not only be suspected in infants with ambiguous genitalia but also in apparent females with clitoromegaly (>0.9 cm in length), or presence of posterior labioscrotal fusion, inguinal masses, or an inguinal hernia(e), especially those containing a palpable mass (18). Our 46,XY girl, born of consanguineous parents, presented after birth with clitoral hypertrophy, and at the age of 4 months, a right-sided inguinal hernia was discovered. The phenotype was due to a new homozygous missense mutation in exon 5. Both parents and a brother were heterozygous for the V144F LHR allele. Our study underscores the necessity to investigate apparent girls with inguinal hernia(e).

The mutation is located in the extracellular domain, 13 amino acids C terminal from the LHR-C131R mutation that was described previously (8). In contrast to our phenotypical girl, the patient reported in the study by Misrahi et al. (8) presented at birth as a male infant with a hypoplastic phallus associated with hypospadias. Cells transfected with mutant receptors C131R and V144F showed some stimulation of adenylyl cyclase at high concentrations of hCG. The experiments are in accordance with the hCG stimulation test performed in the patient (C131R: after 500 IU hCG/d for 7 d, serum testosterone increased insufficiently from 0.13–0.347 nmol/liter; V144F: after 5000 IU hCG/m2 body surface, there was an insufficient increase of serum testosterone from undetectable to 0.135 nmol/liter). In cells transfected with the LHR-C131R mutant, very weak binding was found, approximately 5% of the wild-type receptor. These results are similar to our results with LHR-V144F.

Trafficking to surface was not explored in the patient carrying the C131R mutation, whereas we found, using GFP-tagged receptors, that the LHR-V144F did not process to the cell surface and that fluorescence remained intracellular, colocalizing with a fluorescent endoplasmic reticulum marker. The lack of processing was confirmed by the absence on Western blots of the mature LHR with an apparent mass of 130 kDa. Only a 100-kDa mutant protein was found on Western blots, representing the immature LHR precursor glycoprotein localized in the endoplasmic reticulum.

The extracellular domain of the LHR is responsible for the high-affinity ligand binding of the LHR. Bhowmick et al. (19) developed a model incorporating residues 27–235 of the extracellular domain of the rat LHR. Sequence homology and alignment revealed nine leucine-rich regions, with N-terminal and C-terminal flanking cysteine clusters as found in a number of other leucine-rich region proteins (20, 21, 22).

To better understand the difference between the C131R and V144F LHR mutations, we used the theoretical model of Jiang et al. (http://www.expasy.org/spdbv/ and Ref. 23 ; Fig. 6Go). C131 is located close to the fourth ß-sheet in the inner concave face of the horseshoe-shaped extracellular binding domain, and its change to R might directly interfere in ligand binding or signal transduction. In addition, the absence of the Cys131 for formation of the cysteine bridge with Cys156 might result in aberrant conformation of the extracellular domain of the C131R LHR mutant. In contrast, V144 is positioned in the fourth {alpha}-helix at the concave outer face of the binding domain and is directed toward the third {alpha}-helix. In the model of Jiang and co-workers (http://www.expasy.org/spdbv/ and Ref. 23), the F144 mutant amino acid at that position has overlapping interactions with F119, whereas V144 in the wild-type receptor has not (Fig. 6BGo). F119 is located at the interface of the third {alpha}-helix and the fourth ß-sheet of the extracellular domain. Steric hindrance may result in a conformational change that causes aberrant processing and trafficking of the protein, although it must be stressed that these observations are based on theoretical model and await structure determination of the extracellular domain of the LHR (http://www.expasy.org/spdbv/).

An important observation is the quite different phenotype of the patients carrying the C131R and V144F LHR mutations. The C131R patient presented at birth as a male with a hypoplastic phallus associated with hypospadias, whereas the V144F patient was a girl with clitoral hypertrophy, although both mutant LHRs revealed a very similar pattern of stimulation of adenylyl cyclase and binding. An explanation might be that notwithstanding the low binding and activation activity of the C131R mutant LHR protein, enough of the protein is transported to the plasma membrane to allow for some activation by hCG and LH, whereas the V144F mutation hinders trafficking to the cell surface almost completely, causing a more severe phenotype.

Alternatively, differences in genetic background of the patients could play a crucial role. The relationship between genotype and phenotype can be quite variable as exemplified by similarity in syndromes of the androgen insensitivity syndrome, particularly in its partial forms (24). Also, in the case of the activating LHR mutation M398T, the genotype does not always correlate with phenotype (25). It will be interesting to have a second patient with same LHR mutation comparing phenotypes.

In conclusion, 46,XY male pseudohermaphroditism in the present patient is due to a new missense mutation, V144F, in the extracellular domain of the LHR. The V144F LHR protein does traffic very inefficiently to the plasma membrane, indicating that the mutation does not cause deficient signaling or binding per se, but rather is recognized by the folding quality system of the cell and is refused for additional transportation to the membrane.


   Acknowledgments
 
We thank Dr. W. A. van Cappellen (Department of Reproduction and Development, Erasmus Medical Center, Rotterdam, The Netherlands) for help with the use of the confocal laser scanning microscope.


   Footnotes
 
This work was supported by a grant (Lise-Meitner-Habilitationsstipendium) from the Ministerium für Wissenschaft und Forschung des Landes Nordrhein-Westfalen, Germany (to A.R.-U.).

Abbreviations: EndoH, Endoglycosidase H; FCS, fetal calf serum; GFP, green fluorescent protein; HA1, hemagglutinin 1; hCG, human chorionic gonadotropin; hLHR, human LHR; LHR, LH receptor; PNGase F, N-glycosidase F.

Received February 17, 2004.

Accepted June 23, 2004.


   References
Top
 Abstract
 Introduction
 Patient and Methods
 Results
 Discussion
 References
 

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