REPRODUCTIVE BIOLOGY. Analysis of the Cys82Arg mutation in follicle-stimulating hormone beta (FSH ) using a novel FSH expression vector

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1 FERTILITY AND STERILITY VOL. 79, NO. 2, FEBRUARY 2003 Copyright 2003 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. REPRODUCTIVE BIOLOGY Analysis of the Cys82Arg mutation in follicle-stimulating hormone beta (FSH ) using a novel FSH expression vector Andrew D. Clark Ph.D., a,b and Lawrence C. Layman, M.D. a,b The Medical College of Georgia, Augusta, Georgia Received May 24, 2002; revised and accepted August 14, Supported by National Institutes of Health (PHS- NICHD HD33004), Society of Gynecologic Investigation. Reprint requests: Lawrence C. Layman, M.D., Section of Reproductive Endocrinology, Infertility and Genetics, Department of Obstetrics and Gynecology, 1120 Fifteenth Street, BB-7514, Augusta, Georgia (FAX: ; llayman@mail.mcg.edu). a Section of Reproductive Endocrinology, Infertility and Genetics, Department of Obstetrics and Gynecology. b Program in Neurobiology, Institute of Molecular Medicine and Genetics /03/$30.00 PII S (02) Objective: To determine the effect of the Cys82Arg FSH mutation from a patient with isolated FSH deficiency upon follicle-stimulating hormone (FSH) levels in vitro. Design: In vitro analysis of the Cys82Arg mutation and comparison with the phenotype. Setting: Tertiary medical center setting. Patient(s): DNA sequence of the FSH gene and clinical description from a patient with isolated FSH deficiency. Intervention(s): Construction of a new vector containing the cdnas for the -subunit and -subunit of FSH (p FSH ) followed by mutagenesis and transfection into Chinese hamster ovary cells. Main Outcome Measure(s): Immunoreactive and bioactive FSH levels from the CHO cellular media. Result(s): Although expression of both subunits was present, both immunoreactive and bioactive FSH levels were unmeasurable from cellular media containing the mutation versus wild type. Conclusion(s): The Cys82Arg mutation in a male with normal puberty and azoospermia results in profound deficiency of FSH in vitro, thereby confirming the molecular basis of hypogonadism in this patient and documenting the importance of the Cys residue at position 82 of the FSH subunit. (Fertil Steril 2003;79: by American Society for Reproductive Medicine.) Key Words: Isolated FSH deficiency, follicle-stimulating hormone, p FSH, FSH gene mutation Follicle stimulating hormone (FSH) is a dimeric protein consisting of an -subunit common to thyroid-stimulating hormone (TSH), luteinizing hormone (LH), and chorionic gonadotropin (CG), as well as a unique subunit that confers biological specificity (1). Both subunits are glycosylated, and the noncovalent association of the two subunits is required for biological activity. The cellular action of FSH is mediated through a heptihelical domain receptor that is coupled with a G protein, which upon activation increases intracellular camp. Follicle-stimulating hormone is required for the maturation of the ovarian follicle beyond the antral stage and it induces the aromatization of testosterone to estradiol, which is essential for normal puberty and fertility in females (2). It also is necessary for puberty and fertility in males, as it activates prenatal Sertoli cell mitogenesis, as well as maintains spermatogenesis in adulthood (2). Several human FSH mutations in males and females with isolated FSH deficiency have been described, as has the creation of the homozygous FSH knockout mice. Although uncommon, these FSH mutations have contributed to the understanding of FSH function in reproduction. Only three human males with FSH mutations (Val61X, Tyr76X, and Cys82Arg) have been described. All affected individuals had small testes, azoospermia, low immunoreactive FSH, and normal to elevated LH levels, while two had normal serum testosterone concentrations and one had a low serum testosterone level (3, 4). In vitro analysis of the mutant FSH gene has been performed for both the Val61X and Tyr76X mutations; both of these result in unmeasurable levels of immunoreactive and bioactive FSH in cellular media. Although the Cys82Arg FSH mutation in a male with isolated FSH deficiency is predicted 379

2 TABLE 1 Nucleotide sequences of used for amplification of FSH and subunit cdnas, sequencing of plasmid inserts, Cys82Arg mutagenesis, and RT-PCR. Sense Primer Antisense Primer NotI-alpha subunit- ACG TT GCGG CCGC TC TTC GGA TCC ACA GTC AAC ACT CCAGATCTG AAG CGT GTC AAA GTG GTA TG BglII Alpha subunit insert sequencing GCC TCA GAC AGT GGT TCA AA CGA GGA GAG GGT TAG GGA TAG HindIII-FSH - AAA CAC TAG AAGCTT CGA CCA CAG CCA GGA TGA A CTG TCA GTC GAC GAG TTC CCT CTG CCA GTA GAC CA SalI FSH insert sequencing TCG AAA TTA ATA CGA CTC ACT A TTT GTT CGG ATC CTC TAG AAG T C82R mutagenesis CCA CCC AGC GTC ACT GTG G CCA CAG TGA CGC TGG GTG FSH RT-PCR GGC AAG TGT GAC AGC GAC AG TGC CAG TAG ACC AGG GAT CAG T subunit RT-PCR TCT TTG TGG TCA CAT TGT CGG T CCT TAG TGG AGT GGG ATA TGC TC Note: Primers to amplify both subunits of FSH contained restriction sites (double underlined) to facilitate cloning into the pbud4.1 expression vector with 5 to 6 base pairs of random sequence added to the 5 end of the primer to facilitate enzyme digestion. To confirm insert orientation and sequence, that flank the two multiple cloning sites of the pbud4.1 expression vector were designed. Generation of the Cys82Arg mutant used internal that spanned the targeted base pair with the base pair change incorporated into the primer (underlined). Primers specific for the cdna for the and subunits of FSH were used to confirm expression of both subunits by RT-PCR. Clark. Cys82 Arg mutation in FSH. Fertil Steril to affect dimer formation, its in vitro analysis has not been reported. The purpose of the present study was to determine whether the Cys82Arg FSH mutation impairs FSH in vitro, thereby further supporting that the clinical phenotype in this patient is caused by this mutation. Because the previously described plasmid (pm2 FSH ) to study FSH mutations is quite large and requires multiple cloning steps (5), a new, smaller plasmid (p FSH ) was constructed to facilitate the in vitro analysis. MATERIALS AND METHODS All protocols were reviewed and approved by the Medical College of Georgia Human Assurance Committee. Patient Description The patient is a 28-year-old Serbian man of normal height and weight who sought medical attention for infertility in each of two marriages. He reported normal puberty, libido, and potency, yet had small testes and azoospermia when evaluated for infertility (3). The baseline level of FSH was low and LH was normal; following GnRH stimulation, the FSH level remained low and LH increased markedly. Upon DNA sequencing, he was found to be homozygous for a mutation in codon 82 in the FSH gene, in which a TGT (Cys) was changed to an CGT (Arg) (3). Generation of the p FSH Plasmid To facilitate the in vitro analysis, a smaller expression vector was first constructed. Instead of using the gene sequences of FSH and the -subunit, which make the vector large (more than 20 kb) (5) and require multiple cloning steps, a smaller vector containing the cdnas for the FSH and the -subunit genes was designed. Insertion of the Alpha Subunit ( S) We reverse transcribed 2 g of total RNA from CHO pm 2 /FSH (gift from J. Larry Jameson); 2 L was used in a PCR reaction to generate the S cdna. The PCR reactions contained 0.75 U Taq Polymerase, 50 mm KCl, 10 mm Tris-HCl (ph 9.0 at 25 C), 0.1% Triton X-100, 0.2 mm of each dntp, 2.0 mm MgCl 2, 25 pmol of each primer, and 200 ng of the DNA template. Negative controls (1 tube without RNA and 1 tube without reverse transcriptase enzyme) were included in each reaction. To facilitate ligation into NotI and BglII sites of the plasmid, cut sites were added to the 5 region of the along with 6 to 9 base pairs to protect the cut site (Fig. 1, Table 1). Cycling parameters for the PCR reaction consisted of denaturation for 3 minutes at 94 C, followed by 30 cycles at 94 C (1 minute), 55 C (45 seconds), 72 C (45 seconds), and a final extension using 72 C for 7 minutes. The samples were electrophoresed on a 1.2% agarose gel containing ethidium bromide with 123 bp ladder (Promega Corp., Madison, WI) and viewed with UV transillumination. To ensure a lack of DNA contamination, a negative control was also included that contained all the necessary reagents but lacked the DNA template. 380 Clark and Layman Cys82Arg mutation in FSH Vol. 79, No. 2, February 2003

3 FIGURE 1 Schematic diagram of the p FSH expression plasmid. The p FSH is a dual expression vector that constitutively expresses the and subunits of FSH. The subunit is inserted using BglII and NotI restriction sites present in the cloning site of the vector and is under the control of the human elongation factor 1 (EF-1 ) promoter. The subunit is flanked by a HindIII and SalI site and is under the control of a human cytomegalovirus promoter (CMV). In addition, the p FSH expression construct includes a Zeocin resistance cassette for cell selection and a puc origin to allow transfection and replication in bacterial cells. Clark. Cys82 Arg mutation in FSH. Fertil Steril The remaining PCR product was purified with PCR Prep spin columns (Qiagen, Valencia, CA) and double digested with 5 units of NotI and BglII overnight at 37 C. A total of 2 g of pbud 4.1 dual expression vector (Invitrogen, Carlsbad, CA) was subjected to the same digestion conditions, and both were extracted using the PCR prep kit. Each sample was quantified and combined in a 17 C overnight ligation reaction containing 300 mm Tris-HCl, 100 mm MgCl 2, 100 mm DTT, and 10 mm ATP, 4 units T4 DNA ligase, 80 ng of the NotI- S-BglII PCR product, and 100 ng of the vector. We transfected 2 L of the ligation product into TOP10F cells (Invitrogen) and cultured it overnight on LB agar plates in the presence of 30 g/ml of Zeocin for selection. Colonies were further amplified by overnight growth in LB broth in the presence of 30 g/ml of Zeocin. To select cells with insert, 13 colonies on the LB plate were labeled, and half of the colony was picked with a pipette tip and placed directly in a 25- L PCR reaction with that flanked the insert site (see Table 1). For those samples that contained inserts, as evidenced by a band of 600 bp, the remainder of the colony was grown overnight, DNA was extracted using a Quantum prep kit (BioRad, Hercules, CA), and sequenced using the identical used to test for the presence of insert to confirm the absence of exogenous mutations. Insertion of the FSH Subunit The insertion of the subunit of FSH was generated similarly to the S using the HindIII and SalI sites of the pbud plasmid (see Fig. 1, Table 1). Human pituitary cdna (Life Technology, Carlsbad, CA) was used as a template in a PCR using conditions as above and that contained HindIII and SalI cut sites on either ends. The resulting PCR product was purified, and both the PCR product and 2 g/ L of the pbud- subunit plasmid were digested with 5 units of HindIII at 37 C. Samples were extracted and placed in new reactions to be digested overnight at 37 C in the presence of SalI. Each sample was extracted and ligated at 17 C for 6 hours in a reaction containing 300 mm Tris-HCl, 100 mm MgCl 2, 100 mm DTT, and 10 mm ATP, 4 units of T4 DNA ligase, 60 ng of the purified HindIII-FSH-SalI PCR product, and 100 ng of the vector. Two L was transfected into TOP10F cells, grown overnight, and confirmed by PCR amplification of the flanking the insert site (see Table 1) and DNA sequencing as described below. This plasmid contains the S driven by an EF-1 promoter, the FSH subunit driven by a CMV promoter, and a Zeocin resistance gene for selection (see Fig. 1). DNA Sequencing The inserts were sequenced per the protocol provided by Perkin-Elmer for the ABI BigDye Terminator kit using 20 ng of DNA for PCR products and 700 ng for plasmid sequencing. Each product was sequenced three times in both directions in a total reaction volume of 20 L followed by purification with Centri-Sep spin columns (Princeton Separations, Trenton, NJ). Samples were dried for 20 minutes under vacuum; 12 L of template suppression reagent (Perkin Elmer Corp., Foster City, CA) was added to each sample, vortexed briefly, and heat denatured for 2 minutes at 95 C. After denaturation, sequencing products were placed on ice for 2 minutes and vortexed again to mix. Samples were kept on ice until being loaded on an ABI Prism 310 Genetic analyzer for analysis. PCR-Based Mutagenesis of FSH An overlap extension site-directed mutagenesis protocol (6) was used to generate the mutant FSH cdna subunit. Internal were designed that span the targeted mutation site, including the TGT (Cys) to CGT (Arg) mutation incorporated in the middle of each internal primer. These were combined with the used for the amplification of wild-type FSH subunit cdna ( HindIII-FSH -SalI ), which contain restriction sites for HindIII and SalI onthe sense and antisense, respectively (see Table 1). Stable CHO Cell Transfection The p FSH construct was transfected into a Chinese hamster ovary (CHO) cell line using the Lipofectamine Plus Reagent transfection kit (Life Technologies). A total of 2 g FERTILITY & STERILITY 381

4 of purified plasmid DNA with either the wild-type FSH or mutant Cys82Arg construct was transfected into 70% confluent CHO cells in media lacking antibiotic to improve transfection efficiency. Cells were allowed to grow at 37 C in 5% CO 2 for 48 hours before being trypsanized and plated in 100-mm cell culture plates with fresh media. The media contained 10% FCS, 292 ng/ml of glutamine, 100 U/mL of penicillin G, 250 of ng/ml amphotericin B, 100 U/mL of streptomycin, and 500/ g/ml of Zeocin. Cells were selected with Zeocin for 2 weeks before being trypsanized and diluted into 60-mm plates to isolate single colonies. A kill curve for Zeocin using untransfected CHO cells showed that 500 g/ml of Zeocin was effective in killing cells after 2 weeks of exposure. After 4 days of cell growth, cells from an individual colony were again trypsanized and harvested using a sterile pipette tip and plated in a 60-mm plate with Zeocin. Once a cell colony reached 90% confluence, the colony was split into three separate 60-mm cell culture dishes and Zeocin selection was discontinued. RNA Expression of the WT and Cys82Arg FSH Subunits To document the expression of the and FSH subunits, RT-PCR was performed using RNA extracted from the transfected CHO cells. We used 2 g of total RNA in duplicate reactions where one tube contained reverse transcriptase ( RT) and the other lacked reverse transcriptase ( RT). We amplified 3 L of cdna by PCR with (Table 1). To rule out DNA contamination in the PCR reaction, an additional negative control tube was included that contained the same concentrations of reagents as other tubes, with deionized water added in place of DNA template. Immunoreactivity and Bioassays After 2 to 4 days in media identical to what we have described, but without Zeocin, the media from the CHO cells were removed and spun for 3 minutes at 2500 rpm to remove residual cells. We removed 2 ml of the supernatant and transferred it to a sterile tube for storage at 80 C. The remaining cells in the culture dish were removed, and total protein, RNA, and DNA were extracted using Tri-reagent (Molecular Research Center Inc., Cincinnati, OH). Frozen media from cells transfected with wild-type FSH, media from Cys82Arg FSH transfected CHO cells, media from untransfected CHO cells, and fresh cell culture media were sent to Massachusetts General Hospital for FSH immunoreactivity and bioactivity studies. FSH immunoreactivity were assayed using a two-site sandwich assay (Abbott Laboratories, Abbott Park, IL) where FSH is captured by a monoclonal antibody to the subunit attached to latex particles. The particles are washed and then probed for dimeric FSH hormone using a polyclonal antibody to the subunit (lower limit of detection 1.6 miu/ml, interassay coefficient of variation 3% to 6%, intra-assay coefficient of variation 4% to 7%). Relative protein concentrations were ascertained by comparison of samples to a protein concentration curve using the WHO 71/223 FSH protein as the standard. Free alpha subunit (FAS) was measured in the cell media by radioimmunoassay (RIA) using a polyclonal antibody that is specific for the unbound conformation of the subunit and 125 I-alpha subunit as the competitor for the antibody (lower limit of detection 1.6 miu/ml, interassay coefficient of variation 4% to 12%, intra-assay coefficient of variation 3% to 9%). Samples were run in triplicate and normalized to total protein and total RNA extracted from trypsanized cells. A homologous in vitro bioassay for FSH developed by Christin-Maitre et al. (7) was used (lower limit of detection 1 miu/ml, interassay coefficient of variation 17%, intra-assay coefficient of variation 8%) to assess bioactivity of the various samples. Between 5 and 50 L of cell media was diluted into 100 L of assay medium and placed in a 96-well culture plate seeded with CHO/FSH-R cells. These cells were stably transfected with both the FSH receptor and a luciferase reporter gene under the control of a camp responsive promoter. As a reference standard, identical assays are run using increasing known concentrations of hfsh (WHO 71/223). Activation of the FSH-R present on the cell surface of the CHO cell results in an increase in intracellular camp, thus activating the camp responsive promoter of the luciferase gene. The cell media containing the FSH reacted with the CHO/FSH-R cells for 4.5 hours before being lysed, resuspended in a luciferase assay buffer, and combined with luciferin (Sigma Chemical Co., St. Louis, MO) for light detection. The relative light units were assessed for 10 minutes in a luminometer and the unknown FSH samples were determined by comparing the light units to a calibration curve determined by the samples with known FSH concentrations. Statistics To compare the bioactivity and immunoreactivity of FSH, and the RIA of FAS for each of the individual samples, the relative value was expressed as activity/ g total protein extracted from the cell. The relative values were also expressed as activity/ g total RNA; however, because similar results were obtained as activity/ g protein, they are not included. The relative amounts of FSH and FAS were not normally distributed and were compared using a nonparametric Mann Whitney test, with P.05 being considered statistically significant. The data were graphed as the mean of the raw data (without normalization) because the total protein extracted showed no statistically significant difference between samples. RESULTS To investigate if insertion of cdna for the and subunits was sufficient for transcription of bioactive FSH, a 382 Clark and Layman Cys82Arg mutation in FSH Vol. 79, No. 2, February 2003

5 construct was made containing two separate cloning sites under the activation of two separate promoters and a Zeocin resistance cassette for cell selection. The S was under the control of an EF-1 promoter while the transcription of the FSH subunit cdna was regulated by a CMV promoter. The cdna for the S was obtained from reverse transcription of RNA extracted from a CHO pm 2 /FSH cell line that has been previously described, which contains the entire FSH coding region and a minigene for the S (5). To facilitate cloning later, the S cdna was amplified with containing restriction sites for NotI on the 5 end of the upstream primer and BglII on the 5 region of the downstream primer with 6 to 9 bp of random sequence added to allow cleavage by a restriction enzyme. These cut sites were also present in the multiple cloning site of the pbud4.1 dual expression vector (see Fig. 1) that is under the direction of the EF-1 promoter. We were unable to achieve successful RT-PCR step for amplification of the FSH subunit from the CHO pm 2 / FSH cell line, so a human pituitary cdna library was used as the template to amplify the FSH cdna. The upstream primer included a HindIII site added to the 5 region, and the downstream primer included an SalI restriction site added to the 5 end of the primer, both of which were also present in the multiple cloning site regulated by the CMV promoter. Each of the cdnas was inserted separately into the pbud4.1 expression vector and confirmed with amplification using targeted to the vector adjacent to the insert region. Those colonies with no insert yielded a product size of 50 to 100 bp and those with insert present amplified a product of about 600 bp. The colonies with inserts were sequenced to ensure no exogenous mutations had been introduced during PCR amplification. We found a very large percentage of colonies tested (85% to 90%) contained the insert and were the proper sequence. The FSH secreted from the CHO cells stably transfected with the wild-type p FSH construct was detectable by immunoassay that only recognized the dimerized FSH molecule (Fig. 2). Wild-type FSH was also found to be capable of binding and activating the FSH receptor in the bioassay (see Fig. 2). To determine whether this expression vector would be useful in testing the immunoreactivity and bioactivity of human FSH mutations, a previously described (but not studied in vitro) Cys82Arg missense mutation identified by Lindstedt et al. (3) was created in the FSH subunit of the expression vector. This mutation was identified in a 28-yearold Serbian man who had sought medical attention for infertility in each of two marriages. Subsequent DNA sequencing of FSH exon 3 showed a homozygous substitution of a T to C which changed the primary amino acid sequence from a cysteine to an arginine at amino acid 82 (3). The investigators hypothesized that such a mutation would alter the FIGURE 2 Bioassay and immunoassay of the Cys82Arg mutant. Biological activity of the Cys82Arg construct was compared to wild-type FSH transfected CHO cells (Wild type), CHO cells not transfected with any expression construct (Untrans.), as well as fresh media (Media). In addition, the ability of the wild-type and the mutant C82R to be detected by immunoassay was also determined. Values are expressed as the raw data of immunoactivity or bioactivity when compared to identical tests run with known concentrations of WHO 71/223 hfsh protein standard. Normalization of the raw data to total protein extracted gave similar results. Each sample bar represents the mean of three separate cell colonies with 95% confidence intervals error bars. Values for Cys82Arg, Untrans, and media only gave undetectable levels, so no confidence intervals could be calculated. Clark. Cys82 Arg mutation in FSH. Fertil Steril tertiary structure, dimerization, and glycosylation of the mature peptide, although no in vitro analysis was performed. Using the PCR-based mutagenesis protocol described above, this mutation was introduced into the FSH cdna, ligated into the p FSH plasmid, and stably transfected into CHO cells for in vitro analysis. Our studies demonstrate that both immunoactive and bioactive FSH levels of Cys82Arg were not detectable in the media and were statistically different from CHO cells transfected with the wild-type FSH construct (see Fig. 2). These data serve to confirm the hypothesis that the Cys82Arg mutation alters the ability of the mature peptide to activate the signaling cascade. As compared to the wild-type construct, Cys82Arg showed no statistically significant difference in the levels of FAS measured in the wild-type transfected cells (Fig. 3). RT-PCR demonstrated that both the and FSH subunits of the wild-type and Cys82Arg constructs were expressed in vitro (data not shown). DISCUSSION Human FSH mutations have been identified in four women (8 11) and three men (3, 4, 11) with isolated FSH deficiency. Three women had absent breast development and primary amenorrhea, while one had evidence of partial FERTILITY & STERILITY 383

6 FIGURE 3 Radioimmunoassay for FAS of the Cys82Arg mutant. Media from CHO cells transfected with the Cys82Arg mutant (Cys82Arg), from wild-type FSH transfected CHO cells (Wild Type), untransfected CHO cells (Untrans.), and fresh cell media (Media) were assayed for FAS. FAS was detected using a polyclonal antibody specific for the conformation of FAS, which is slightly altered in its unbound state. Data are expressed as the mean of the raw data from three separate cell colonies. Cell colonies for Cys82Arg and wild type include error bars with 95% C.I. Untransfected cells and fresh culture media had no detectable FAS, so no confidence intervals could be calculated. (Statistical analysis to compare samples was done with raw values normalized to total protein extracted; only the raw data are presented in this graph.) Clark. Cys82 Arg mutation in FSH. Fertil Steril breast development and primary amenorrhea (8 11). All affected men had small testes and azoospermia, but two had normal testosterone levels; surprisingly, one man had absent puberty and a low testosterone level (3, 4). All humans with FSH deficiency had low serum FSH and high LH levels, except our present case, who had a normal LH level. The Val61X and Tyr76X mutations in two of the reported FSH-deficient men have been studied in vitro using a previously published expression vector pm 2 FSH, with stable transfection into Chinese hamster ovary (CHO) cells (10, 11). Both mutants were shown to impair the production of immunoreactive and bioactive FSH when compared with wild-type FSH genes, even though the Val61X mutation was observed in a man with absent puberty and the Tyr76X mutation was present in a man with pubertal development and a normal testosterone level. However, there has not been any in vitro analysis of the Cys82Arg described in the male patient as previously published by Lindstedt et al. (3). The purpose of our present study was to determine if the Cys82Arg mutation in a male with isolated FSH deficiency would affect FSH production in vitro. This man had presented for infertility and had normal puberty, small testes, and normal testosterone level, with low FSH and normal LH levels. As expected, with GnRH stimulation, serum LH rose further and deficient FSH was unable to do so. Upon semen analysis, he was found to be azoospermic. We hypothesized that, because the phenotype was not as severe as found in the male patient with absent puberty described by Phillip et al. (4), there would be some measurable, but lower, levels of immunoreactive and bioactive FSH produced in vitro. However, our in vitro analysis revealed that both immunoreactive and bioactive FSH were absent compared with wild-type and appropriate negative controls. The unmeasurable FSH levels were not because of deficient transcription; both the -subunit and FSH expression were present, as demonstrated by RT- PCR. Furthermore, our immunoactivity and bioactivity studies confirm that the Cys82Arg indeed causes the translation of a nonfunctional protein that is undetectable by immunoassay and unable to activate the FSH receptor in vitro (3). It is highly likely that the Cys82Arg mutation causes a posttranslation effect, disrupting FSH stability with a loss of a conserved Cys residue, known to be important in disulfide bond stabilization of the subunit. In addition, the Cys82 residue has been shown to be important in FSH dimer formation and may contribute to the cysteine knot of the FSH formation (12), as well as being critical for the stability of the subunit (13). In vitro analysis of the human FSH gene has been quite complicated because of the large insert size of the pm 2 FSH vector, and the requirement of multiple subcloning steps. For these reasons, we designed a smaller vector that we designated p FSH, composed of the cdnas for both the -subunit and the FSH subunit genes, driven by separate promoters. Both promoters have similar relative levels of expression of the products, and this is supported by our finding that immunoreactive and bioactive FSH was successfully produced following transfection into CHO cells. The wild-type FSH produced by this plasmid was found to react with an FSH antibody that requires the dimerization of the and subunits for detection; furthermore, it was able to activate an FSH receptor mediated signaling cascade in the FSH bioassay. These data suggest that insertion of the cdnas for the and subunits of FSH into this dual expression system is sufficient for the translation of a mature, biologically functional peptide. Assays for the FAS levels in the media revealed a large amount of FAS in the cell transfected both for wild-type and for Cys82Arg in similar concentrations. This is in contrast to the serum levels FAS in the patient, which were within the normal range (3). Free alpha subunit levels in humans range from pg/ml; in our system, the FAS concentration was about 10 5 times higher. This could be in part due to constitutive expression of the -subunit in vitro, as well as the lack of 384 Clark and Layman Cys82Arg mutation in FSH Vol. 79, No. 2, February 2003

7 degradation, clearance mechanisms, and feedback regulation in our system. Despite this drawback, our FSH expression vector is capable of producing biologically active FSH when stably transfected into CHO cells. Furthermore, this construct is useful for analyzing the immunoreactivity and bioactivity of human FSH mutations. The process of introducing a mutation in our system uses two separate PCR steps, a simultaneous restriction enzyme digestion of the plasmid and PCR product followed by a ligation step, which can be completed in less than 12 hours. Although we have used this expression vector solely for analysis of the subunit of FSH, it should also prove useful for the introduction of cdnas for the other pituitary glycoprotein subunits or for analysis of mutations compromising the function of the common S. We have demonstrated that the Cys82Arg FSH mutation in this patient with isolated FSH deficiency affects assayable immunoreactive and bioactive FSH in vitro. When taken in the context of the patient s phenotype, these findings suggest that FSH is necessary for normal testicular size and normal spermatogenesis, as has been suggested by the phenotypes of the FSH and FSHR knockout mice (14 17). It is interesting that this Cys82Arg mutation was associated with the phenotype of partial pubertal development and a normal testosterone level. A male patient described by Phillip et al. (4) with a Val61X mutation had absent puberty, a low serum testosterone, and azoospermia. This mutation was also shown to result in absent immunoactive and bioactive FSH in vitro, suggesting that these in vitro assays cannot distinguish between milder (Cys82Arg) and more severe (Val61X) mutations. Acknowledgments: The authors thank Dr. Jeffery Weiss, Northwestern University School of Medicine, Chicago, IL, for his helpful advise in completing this project, and Dr. Patrick M. 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