Alternatively spliced variants of the follicle-stimulating hormone receptor gene in the testis of infertile men

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1 FERTILITY AND STERILITY VOL. 77, NO. 3, MARCH 2002 Copyright 2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Alternatively spliced variants of the follicle-stimulating hormone receptor gene in the testis of infertile men Gyun Jee Song, Ph.D., a,b Yong-Seog Park, Ph.D., a You Sik Lee, M.D., c Chung Choo Lee, Ph.D., b and Inn Soo Kang, M.D. d Laboratory of Reproductive Biology and Infertility, Samsung Cheil Hospital and Women s Healthcare Center, Sungkyunkwan University School of Medicine, Seoul, Korea Received May 11, 2001; revised and accepted September 20, Presented at the 56th Annual Meeting of the American Society for Reproductive Medicine, San Diego, California, October 21 26, Reprint requests: Gyun Jee Song, Ph.D., Samsung Cheil Hospital and Women s Healthcare Center, 1-19 Mookjung- Dong, Chung-Ku, Seoul, Korea (FAX: ; phdsong@samsung.co.kr). a Research Laboratory of Reproductive Biology and Infertility. b Department of Biology, Seoul National University, Seoul, Korea. c Department of Urology. d Department of Obstetrics and Gynecology /02/$22.00 PII S (01) Objective: To investigate whether or not alternatively spliced variants of the FSH receptor gene occur in human testis and whether the presence of the splicing variants is associated with spermatogenic defects and serum FSH concentration in infertile men. Design: A prospective case control study. Setting: An IVF clinic and infertility laboratory at a university hospital. Patient(s): Forty-three infertile patients undergoing testicular biopsy. Intervention(s): Total RNA was extracted from the testicular tissues and used for reverse transcriptasepolymerase chain reaction (RT-PCR). Main Outcome Measure(s): Expression pattern was analyzed by nested RT-PCR using primers designed to amplify a fragment of FSH receptor gene. PCR products of splicing variants were cloned and sequenced. Result(s): The PCR products showed three kinds of additional bands corresponding to alternatively spliced isoforms of the FSH receptor gene. Exon 9 deleted variant was detected in all patients and inclusion variant of small extra exon was detected in 64% (9/14) of the patients with normal spermatogenesis and 34% (10/29) of the patients with spermatogenic defects. The presence of inclusion variant was not significantly associated with spermatogenic defects but was associated with a low level of serum FSH. On the other hand, exon 6 deleted variant was detected in only one patient having a high level of FSH concentration (30 IU/L) and Sertoli cell only syndrome. Conclusion(s): We identified three different types of alternatively spliced variants of the human FSH receptor. However, it is not clear whether or not there is an association between three variants and spermatogenic defects. (Fertil Steril 2002;77: by American Society for Reproductive Medicine.) Key Words: Infertility, FSH receptor, splicing variant, spermatogenic defects Approximately 15% of couples attempting pregnancy meet with failure. Mostly, these patients are defined as infertile if they have been unable to achieve a pregnancy after 1 year of unprotected intercourse (1). In general, the male factor is at least partly responsible for about 50% of infertile couples (2). Testicular function is controlled by two gonadotropins, FSH and LH. The decrease of testicular FSH receptor binding ability was observed in infertile men (3). Therefore, we speculated that abnormality of the FSH receptor gene could be a cause of spermatogenic defects. The FSH plays a key role in the regulation of ovarian and testicular functions through binding with the FSH receptor (4). The FSH receptor can be found in granulosa cells and Sertoli cells (5). The FSH receptor belongs to the large family of GTP-binding protein (G protein)-coupled receptors and are located at the plasma membrane of target cells (6). In contrast to almost all other G protein-coupled receptors, the gonadotropin receptor is the socalled subgroup of glycoprotein hormone receptor that has a large glycosylated extracellular domain of approximately the same size as the transmembrane domain. The extracellular domain is involved in hormone binding and is encoded by the first nine exons of the FSH receptor gene, whereas the last exon encodes 499

2 both the transmembrane and the intracellular domains (7). The FSH receptor gene is located on chromosome 2p21, spans 54 kb of genomic DNA, and encodes a 695-aminoacid predicted protein (8, 9). Shortly after the gonadotropin receptor cdnas were cloned, it became apparent that alternatively spliced transcripts were coexpressed with the full-length receptor mrna transcript in the gonads of various species, including sheep, rat, mouse, and human (10 12). It has been proposed that these alternatively spliced forms may have some regulatory roles modulating cellular effects of gonadotropins. A carboxy-terminally truncated ovine FSH receptor has been described in sheep. This alternatively spliced receptor appeared on the cell surface, bound the hormone FSH with high specificity, but was incapable of activating adenylate cyclase (13). On the other hand, it has also been reported that there are no functional consequences of coexpression of several splicing variants of the mouse FSH receptor (14). To date, no clear evidence exists that shows the involvement of splicing variants of the FSH receptor gene in male infertility. This study attempted to investigate whether the presence of the alternatively spliced variants of human FSH receptor is correlated with testicular histopathology and FSH concentration in infertile men. MATERIALS AND METHODS This study was approved by our institutional review board. Patients Testicular biopsies were taken from 43 infertile patients, of whom 14 had obstructive azoospermia and 29 had nonobstructive azoospermia. Plasma concentrations of FSH and LH were measured in each subject by radioimmunoassay using 125 I-labeled FSH and LH (BioChem Immnuno System Inc., Biolona, Italy). Normal values of FSH and LH are 1 14 miu/ml and miu/ml, respectively. Plasma concentrations of testosterone were measured by RIA according to manufacturer s instructions (DPC, Los Angeles, CA). Patients with a documented cause for infertility such as congenital abnormality or chromosomal aberration were excluded from the analysis. RNA Extraction and Reverse Transcriptase- Polymerase Chain Reaction A piece of testicular tissue obtained by multisite testicular biopsy was used for reverse transcriptase-polymerase chain reaction (RT-PCR) from azoospermic patients. A small tissue specimen ( 5 7 mg) was obtained for histological study. Total RNA was extracted with Trizol reagents according to the manufacturer s instruction (GIBCO BRL, Grand Island, NY). The concentration of extracted RNA was determined by spectrophotometer at 260 nm absorbance. One microgram of total RNA was transcribed into cdna by FIGURE 1 Genomic structure and location of primers in the FSH receptor gene. First RT-PCR was performed using L1F and L10R primers. Its expected size was 2.4 kb. E1F and E4R primers for 402 bp, E3F and E9R primers for 618 bp, and E6F and E10R primers for 564 bp were used to produce nested RT- PCR products. incubation at 42 C for 60 min in 10 mmol/l Tris-HCl (ph 8.3), 50 mmol/l KCl, 5 mmol/l MgCl 2, 1 mmol/l dntps, 5 g oligo(dt) 12, 20 U RNAsin, and 200 IU AMV reverse transcriptase (Boehringer Mannheim, Germany) in a total volume of 20 L. The reaction was then heated to 95 C for 10 minutes and cooled down to 4 C. Two microliters of the reverse transcription product was amplified in a 20- L PCR mixture. The PCR mixtures were overlaid with mineral oil and amplified using a Perkins & Elmer cycler; 2 minutes of denaturation at 95 C, followed by 40 cycles, 94 C for 40 seconds, 60 C for 1 minute, 72 C for 1 minute. The cdna was also amplified by nested PCR using outer primers designed to amplify a 2.4-kb fragment (spanning exons 1 10) and inner primers for 402-bp, 618-bp, and 564-bp fragments spanning exon 1 to exon 4, exon 3 to exon 9, and exon 6 to exon 10, respectively. Two microliters of reverse transcription product was subjected to cycles of first PCR, and the first PCR product was used for nested PCR with Taq polymerase (BM) and three sets of specific primers (Fig. 1). The amplification products were electrophoresed through 2.0% agarose gel and visualized by ethidium bromide staining. Each PCR product was identified by digestion with specific enzyme having restriction site in the PCR product. 500 Song et al. Splicing variants of the FSH receptor gene Vol. 77, No. 3, March 2002

3 Cloning and DNA Sequencing The PCR products of alternatively spliced variants were cloned into the pt7 blue T-vector (Novagen, ). The DNA sequencing was performed using an automatic DNA sequencer (model ABI 3700). Sequence analyses were done by BLAST homology and GenBank database search. FIGURE 2 Agarose gel electrophoresis of RT-PCR products. (A), PCR products containing exon 1 to exon 4 (402 bp). (B), PCR products containing exon 6 to exon 10 show three kinds of bands, one expected band of 564 bp and two unexpected bands of 666 bp and 376 bp. (C), PCR products containing exon 3 to exon 9 show three kinds of bands, one expected band of 618 bp and two unexpected bands of 540 bp and 720 bp. M 100 bp ladder. P1 P6 testicular tissues of six infertile patients. RESULTS Several Splicing Variants of Human FSH Receptor Gene The levels of FSH receptor transcripts could be estimated by semiquantitative RT-PCR using GAPDH as a control in the testicular tissues from patients with spermatogenic defects and normal spermatogenesis. The association between the levels of FSH receptor transcripts and spermatogenic defects was not observed in this study (data not shown). Because of low amounts of transcripts, nested RT-PCR was used to amplify additional bands showing lower intensity as well as a main band. The first PCR was performed using L1F and L10R primers; the expected size was 2.4 kb (Fig. 1). To detect alternatively spliced variants of the FSH transcripts, three sets of nested primers spanning exon 1 to exon 10 were used. As expected, PCR product containing exon 1 to exon 4 showed a single band of 402 bp in size, which was detected in all patients (Fig. 2A). On the other hand, PCR products spanning from exon 6 to 5 end of exon 10 showed three bands corresponding to discrete, alternatively spliced forms of the FSH receptor gene (Fig. 2B). A 376-bp band of the exon 9 deleted variant (E9Del) and a 666-bp band of the inclusion variant of a small extra exon (E8 Inc) as well as a 564-bp band of normal size were detected in human testis. Exon 9 deleted variant may have produced a small polypeptide. This variant was expressed in all patients at low levels. From the sequence analysis using the GenBank database, it turned out that the E8 Inc variant had an extra exon originating from sequence in intron 8 far from 100 bp of exon 9 (Fig. 3). Splicing donor/acceptor nucleotides, gt/ag were conserved in the extra exon. This inclusion variant (E8 Inc) contained an additional 102 bp of nucleotides, 34 amino acids without frame shift. The PCR product containing exon 3 to exon 9 showed another additional band in only one patient (Fig. 2C). Sequence analysis showed it was exon 6 deleted variant. This splice variant did not lead to a frame shift in the open reading frame and premature termination of the polypeptide but resulted in a deletion of 26 amino acids. Expression Pattern of the Alternatively Spliced Variants in Infertile Men A total of 43 patients were analyzed for the expression pattern of the FSH receptor gene transcripts. Thirty-four percent of the patients with spermatogenic defects had E8 Inc variant of the FSH receptor gene in their testis tissues, whereas 64% of the patients with normal spermatogenesis had E8 Inc variant. However, the presence of this splice variant was not significantly associated with spermatogenic defects based on 2 test (Table 1). On the other hand, serum FSH and LH levels were significantly higher in the patients without expression of the E8 Inc variant (Table 2). Exon 6 deleted variant (E6Del) was detected in one patient who had a high level of FSH concentration (30 IU/L) and Sertoli cell only syndrome. In summary, three different types of alternatively spliced variants of the human FSH receptor were identified (Fig. 4). However, it is not clear whether there is an association FERTILITY & STERILITY 501

4 FIGURE 3 Inclusion variant (E8 Inc) of the FSH receptor gene. (A) Genomic structure of inclusion variant. Extra exon is located in intron 8. Splicing consensus sequences of acceptor/donor sequence, gt/ag were conserved in the boundary region of the extra exon. (B) DNA sequences of the extra exon. between common splicing variants (E9del and E8 Inc) and spermatogenic defects. DISCUSSION The FSH is known to be important for normal reproductive function. At present, several reports on the structure, genomic organization, and expression of FSH and FSH receptor have been presented (6, 15). In women, FSH is required for ovarian development and follicle maturation, whereas in men FSH determines Sertoli cell number and quantity and quality of spermatogenesis (5). To date, little is known about mutations and defects of regulation of the FSH receptor gene in infertile men. Tapanainen et al. (16) reported the first characterization of males homozygous for the Ala189Val FSH receptor mutation in Finnish families. Finnish males with this mutation have variable degrees of spermatogenic failure, but, surprisingly, do not show azoospermia or absolute infertility. Simoni et al. (17) investigated the occurrence of FSH receptor mutations in 48 men with idiopathic male infertility. One nonpolymorphic mutation (Val341Ala) was identified but showed no functional defects in vitro. Tuerlings et al. (18) also performed mutation screening in 28 selected infertile patients. No pathogenic FSH receptor mutation was detected in the TABLE 1 The relationship between the expression of inclusion variant (E8 Inc) and spermatogenic defects. Tesicular histology Present E8 Inc variant Absent Total Normal spermatogenesis 9 (64%) 5 (36%) 14 (100%) Spermatogenic defects 10 (34%) 19 (66%) 29 (100%) Total patients 19 (41%) 24 (59%) 43 (100%) Note: Values are the number of patients. Fisher s exact test, P Song et al. Splicing variants of the FSH receptor gene Vol. 77, No. 3, March 2002

5 TABLE 2 Comparison of clinical profiles between two patient groups with or without E8 Inc variant. Clinical profiles Present E8 Inc variant Absent P value a FSH LH Testosterone Testicle size Note: Values are mean SD. a Student s t-test. Netherlands population. Therefore, there is no clear evidence that mutation in the FSH receptor is a cause of spermatogenic defects. In this study, expression pattern of the FSH receptor gene was analyzed in the testicular tissues of infertile men with normal spermatogenesis and spermatogenic defects. We did not observe any difference in the level of FSH receptor gene transcripts between two groups as the level of transcripts is very low. Therefore, we performed nested RT-PCR. Interestingly, three different types of alternatively spliced variants of the FSH receptor gene could be observed: E9Del, E6Del, and E8 Inc variants. E9Del variant was previously reported by Gromoll (19) from Northern blot analysis using extracted RNA from the testes of three patients undergoing orchidectomy. The nucleotide deletion did not lead to a shift in the open reading frame and premature termination of the polypeptide but resulted in a deletion of 62 amino acids. Deletion of exon 9 would lead to removal of two highly conserved vicinal residues at positions 275 and 276. It is suggested that conformational change elicited by deletion of the exon 9 leads to normal regulatory function of the receptor (19). In this study, we identified the E8 Inc and E6Del variants of the FSH receptor gene in human testis for the first time. The expression of the E8 Inc variant tends to occur in patients with normal spermatogenesis, but it is not statistically significant. It is clear that expression of the E8 Inc variant is associated with normal serum FSH level. These results could be explained by a two-step hypothesis: [1] the expression of common splicing variants (E9Del and E8 Inc) may represent a general mechanism to modulate target cell responsiveness to gonadotropins; and [2] spermatogenic defects resulting from other causes may induce the defects of several translational regulators. Therefore, alternatively spliced variants of the FSH receptor gene tend not to occur in patients with spermatogenic defects. In fact, the exact physiologic role of this alternatively spliced variant has not yet been determined. From previous reports on functional studies of splicing variants in an animal model, however, we could expect the role of human testicular splicing variants observed in this study. In the mouse FSH receptor, four different splicing variants, selectively lacking exon 2, exon 2 and exon 5, exon 5 and exon 6, and 2, 5, and 6 of the coding region, were cloned (14). A receptor transcript that lacked exons 5 and 6 produced a mutant receptor that could not bind to FSH and failed to induce any detectable camp and P responses in transfected KK-1 cells (14). Therefore, the E6Del variant detected in infertile men may have a deleterious effect on FSH receptor functions. In the future, it would be of interest to determine whether levels of alternatively spliced transcripts of the human FSH receptor gene are related to testicular histopathology and FSH concentration. It would also be interesting to determine the binding affinity and signal transduction of these splicing variants. In conclusion, three types of alternatively spliced variants of the human FSH receptor were observed in human testicular tissues. It was observed that there is no association between common splicing variants and spermatogenic defects. The presence of these variants does not appear to be a cause of spermatogenic defects, but a FIGURE 4 Schematic diagram of three kinds of alternatively spliced variants of the FSH receptor gene. E9Del, E6Del, and E8 Inc variants were identified in human testicular tissues. FERTILITY & STERILITY 503

6 possibility still remains that they may play a regulatory role in normal spermatogenesis. Acknowledgments: The authors thank the IVF team and Joo Tea Seo, M.D. in Samsung Cheil Hospital and Women s Healthcare Center for assistance in the collection of materials. We also thank Hyung-Song Lee, M.S., in Samsung Cheil Hospital and Women s Healthcare Center, Seoul, Korea, for comments on the experiment. The authors especially thank Yong S. Moon, M.D., at the University of British Columbia, Vancouver, Canada, for kind review of the manuscript. References 1. Nieschlag E. Scope and goals of andrology. In: Nieschlag E, Behre MB, eds. Andrology: male reproductive health and dysfunction. Springer Verlag: Berlin, 1997: Moscher WE. Reproductive impairments in the United States, Demography 1985;22: Namiki M, Koide T, Okuyama A, Sonoda T, Itatani H, Miyake A, et al. Abnormality of testicular FSH receptors in infertile men. Acta Endocrinol 1984;106: Conway GS. Clinical manifestations of genetic defects gonadotrophins and their receptors. Clin Endocrinol 1996;45: Heckert LL, Griswold MD. Expression of follicle-stimulating hormone receptor mrna in rat testes and Sertoli cells. Mol Endocrinol 1991;5: Sprengel R, Braun T, Nikolics K, Segaloff DL, Seeburg PH. The testicular receptor for follicle stimulating hormone: structure and functional expression of cloned cdna. Mol Endocinol 1990;4: Gross B, Misrahi M, Sar S, Milgrom E. Composite structure of the human thyrotropin receptor gene. Biochem Biophys Res Commun 1991;177: Gromoll J, Pekel D, Nieschlag E. The structure and organization of the human FSH receptor gene. Genomics 1996;35: Gromoll J, Ried T, Holtgreve-Grez H, Nieschlag E, Gudermann T. Localization of the human FSH receptor to chromosome 2p21 using a genomic probe comprising exon 10. J Mol Endocrinol 1994;12: Themmen AP, Kraaij R, Grootegoed JA. Regulation of gonadotropin receptor gene expression. Mol Cell Endocrinol 1994;100: Kraaij R, Verhoef-Post, M, Grootegoed JA, Themmen APN. Alternative splicing of follicle-stimulating hormone receptor pre-mrna: cloning and characterization of two alternatively spliced mrna transcripts. J Endocrinol 1998;158: Yaron Y, Schwartz D, Evans MI, Lessing JB, Rotter V. Alternatively spliced mrna transcripts encoding the extracellular domain of the FSH receptor gene. J Reprod Med 1998;42: Sairam MR, Jiang LG, Khan H. Follitropin signal transduction: alternative splicing of the FSH receptor gene produces a dominant negative form of receptor which inhibits hormone action. Biochem Biophys Res Com 1996;226: Tena-Sempere M, Manna PR, Huhtaniemi I. Molecular cloning of the mouse follicle-stimulating hormone receptor complementary deoxyribonucleic acid: functional expression of altenatively exon 7 of the coding sequence. Bio Reprod 1999;60: O Shaughnessy PJ, Dudley K, Rajapaksha WS. Expression of follicle stimulating hormone-receptor mrna during gonadal. Mol Cell Endocrinol 1996;125: Tapanainen JS, Aottomake K, Kin J, Vaskiwuo T, Huhtaniemi IT. Men homozygous for an inactivating mutation of the FSH receptor gene present variable suppression of spermatogenesis and fertility. Nature Genet 1997;15: Simoni M, Gromoll J, Hoppner W, Kamischke A, Ktafft T, Stahle D, et al. Mutational analysis of the follicle-stimulating hormone (FSH) receptor in normal and infertile men: identification and characterization of two discrete FSH receptor isoforms. J Clin Endocrinol Metab 1999; 84: Tuerlings JHAM, Ligtenberg MJL, Kremer JAM, Sieres M, Meuleman EJH, Braat DDHM, et al. Screening male intracytoplasmic sperm injection candidates for mutations of the follicle stimulating hormone receptor gene. Human Reprod 1998;13: Gromoll J, Gudermann T, Nieschlag E. Molecular cloning of a truncated isoform of the human follicle stimulating hormone receptor. Biochem Biophys Res Commun 1992;199: Song et al. Splicing variants of the FSH receptor gene Vol. 77, No. 3, March 2002