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Testis |
a Department of Biochemistry, University of Stellenbosch, Matieland, Stellenbosch 7602, Republic of South Africa
b Center for Molecular Medicine and Genetics,
c Department of Obstetrics and Gynecology, Institute for Scientific Computing, Wayne State University School of Medicine, Detroit, Michigan 48201
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gene regulation, gonadotropin-releasing hormone receptor, sperm, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The well-characterized pituitary GnRH receptor (or so-called type I receptor), a member of the seven transmembrane G-protein coupled family of receptors, has been cloned and its structure and function have been studied extensively in both mammalian and nonmammalian vertebrates [2–8]. Interestingly, most vertebrates, including man, have two or more forms of the GnRH peptide. In man, a second form of the GnRH hormone has been detected in the kidney, prostate, bone marrow, and brain [9]. In addition, GnRH receptors have been detected by ligand-binding studies in the pituitary, placenta, ovary, testis, adrenal glands, lymphocytes, and the central nervous system, as well as cancers of the breast and prostate [10–14]. Thus it is possible that GnRH(s) and its receptor(s) may, in addition to the established endocrine role, have paracrine and autocrine functions in several reproductive and nonreproductive tissues. In human testis, for example, it may act as a neuroendocrine hormone [15]. In amphibia, GnRH is present in the sympathetic ganglia where it acts as a neuromodulator [16]. In fish, GnRH-producing cells interact with neurons that control sperm duct and oviduct contractility [17]. Furthermore, several differentiated lymphocytes produce GnRH or a GnRH-like peptide [18]. Taken together, these observations suggest that more than one form of the GnRH receptor exists within a single species.
Indeed, a second form of the GnRH receptor has recently been cloned from several nonmammalian vertebrate organs such as goldfish brain and pituitary [2], frog midbrain and pituitary [3], and chicken pituitary [4], as well as from mammalian vertebrate organs such as marmoset pituitary [19], COS-1 (vervet monkey kidney) cells, and rhesus monkey pituitary [20]. The mRNAs of all the mammalian GnRH receptors cloned to date are encoded by three exons, and the gene structures are conserved. Database searches have revealed that apart from the known type I pituitary receptor gene there are at least two other GnRH receptor genes in the human genome, which are located on separate chromosomes. A gene that encodes exons 2 and 3 as well as intron 2 of a putative type II GnRH receptor is located on chromosome 14. A second human type II GnRH receptor gene containing all three putative exons (exons 1, 2, and 3) is located on chromosome 1 (accession AL160282). It has been reported that the chromosome 14 gene is abundantly expressed in a wide range of human tissues, but the transcripts are in the antisense orientation with respect to the type II receptor sequence [21]. Reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blotting analyses showed that the gene is transcribed in most human tissues examined. However, an intronic sequence equivalent to intron 2 was retained, reflecting the absence of donor and acceptor splice sites for these transcripts ([21] and our unpublished results). Interestingly, the chromosome 14 transcript encodes the 3' untranslated region of a novel human ribonucleoprotein mRNA, RBM8 (accession AF127761) [21, 22]. In comparison, little is known of the expression of the chromosome 1 gene. This includes whether a functional, full-length transcript for a human type II GnRH receptor, arising from transcription of the gene on chromosome 1, is expressed in human pituitary or any other tissue. The results of our studies that directly address this issue are presented.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For RT-PCR analysis, human semen samples pooled from a number of male donors were obtained from the Andrology Department, Groote Schuur hospital (Cape Town, South Africa). Adult human postmortem tissue was obtained from the Salt River Mortuary under the supervision of pathologists from the University of Cape Town (UCT) Medical School (Cape Town, South Africa), after approval from the Medical Ethics Committee at UCT. Postabortion human fetal tissue was obtained by Nicola Illing (Department of Molecular and Cell Biology, UCT, Cape Town, South Africa) who obtained permission from the Medical Ethics Committee at UCT. For in situ hybridizations, formalin-fixed, paraffin-embedded testicular samples were provided by Harper hospital (Detroit, MI). Human semen samples from a normal male donor were obtained from the in vitro fertilization (IVF) clinic, Hutzel hospital (Detroit, MI). Semen samples had an average concentration of 15 x 107 sperm/ml and contained less than 2% immature germ cells and/or somatic contaminants.
Human Dot Blot
Dot blot analysis was performed using radiolabeled, double-stranded exon 1-specific human type II GnRH receptor DNA to probe a commercially available human RNA Master blot (Clontech, Palo Alto, CA) containing poly(A)+ RNA from 50 different human tissues. The DNA probe was obtained by PCR amplification of a 402-base pair (bp) fragment from human genomic DNA using primer pair S1 (5' CCC ACC TTC TCG GCA GCA GCC 3') and AS2 (5' GAA ACT AAG TCC CCA GGC TGC 3'). A 25-ng aliquot of the probe was labeled for 1 h at 37°C with the use of the Megaprime DNA labeling system (Amersham, Buckinghamshire, England) and 50 µCi [
-32P]dCTP as described by the supplier. The labeled probe (109 dpm/µg) was purified on a G-25 spin column. Hybridization, using 25 ng of probe in 5 ml of solution, was performed as described in the Master blot manual, except for the replacement of sheared salmon testis DNA with sheared herring sperm DNA. Washes were as follows: five 20-min washes with solution 1 (2 x SSC, 1% SDS) at 65°C and two 20-min washes with solution 2 (0.1 x SSC, 0.5% SDS) at 55°C. The Master blot membrane was exposed to Hyperfilm for four overnights prior to autoradiographic detection.
Isolation of Total RNA
Total RNA was isolated from fresh human semen by cesium chloride-guanidinium isothiocyanate ultracentrifugation as described [23]. In brief, cells were pelleted for 10 min at 2000 x g and resuspended in 4.5 ml of guanidinium solution per 0.5-g pellet. Prior to ultracentrifugation, 1 g of cesium chloride was added per 2.5 ml of homogenate. Dithiothreitol (DTT) and RNasin ribonuclease inhibitor (Promega, Madison, WI) were added to the purified RNA to final concentrations of 1 mM and 1 U/µl, respectively. Human fetal RNA was obtained from Nicola Illing. Total RNA from other human tissues was isolated with the use of TRI-reagent (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer's protocol.
Reverse Transcriptase-Polymerase Chain Reaction
A 1-µg aliquot of total RNA was reverse-transcribed in a 20-µl reaction volume using 200 ng of a hexanucleotide mix (Roche, Randburg, South Africa) and 200 U of Superscript II RNaseH- reverse transcriptase (Gibco-BRL, Paisley, Scotland). Reverse transcriptase-reactions included 1 mM dNTPs, 1 x Gibco first strand cDNA buffer, 10 mM DTT, 20 U RNasin, and 0.1 mg/ml BSA and were performed at 42°C for 1 h. Polymerase chain reactions were performed with 10 µl of first-strand cDNA and 5 U Taq DNA polymerase in a 50-µl reaction volume using combinations of human type II GnRH receptor exon 1-, exon 2-, and exon 3-specific oligonucleotide primers (see Fig. 2). Twenty picomoles of each gene-specific primer were used. The sense primers were for exon 1, S2 (5'ACC TGG AAT ATC ACT GTT CAA TGG 3'); for exon 2, S3 (5' GCA AGA GAC CAC CTA TAA CCT 3'); and for mouse ß-actin, ß-S (5' CAC CAC ACC TTC TAC AAT GAG CTG 3'). Antisense primers were for exon 1, AS1 (5' CAT GCG ATG TCC ACA GCC AGC C 3') and AS2 (5' GAA ACT AAG TCC CCA GGC TGC 3'); for exon 2, AS3 (5' GGT TAT AGG TGG TCT CTT GC 3'); for exon 3, AS4 (5' GGT GTC CAG CAG AGG ATG AAG GTC AG 3') and AS5 (5' GGA GAG CAG GAG TAG AAG TGA G 3') for the 3' untranslated region; and for mouse ß-actin, ß-AS (5' GAT CTT CAT GAG GTA GTC TGT CAG G 3'). Cycling parameters were as follows: 2.5 min denaturing at 93°C, followed by 35 cycles of 1 min denaturing at 93°C, 1 min annealing at 55°C and 1 min extension at 72°C, and a final extension of 10 min at 72°C. The RT-PCR products were visualized on 1% agarose gels containing 0.5 µg/ml ethidium bromide.
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5' Rapid Amplification of cDNA Ends
Double-stranded Marathon-ready cDNA for 5' rapid amplification of cDNA ends (RACE) was prepared from 1 µg of total RNA from human semen. Hexanucleotide primers (5 µM) were used for first-strand cDNA synthesis using AMV reverse transcriptase and all other components of the Marathon cDNA amplification kit (Clontech). Adaptor-ligated double-stranded cDNA was diluted 1:25 in Tricine-EDTA buffer, which was supplied with the RACE kit. A 5-µl aliquot of RACE-ready cDNA and 10 picomoles of each primer were used per 50 µl reaction. Primary 5' RACE was performed with the adaptor-specific AP1 primer (5' CCA TCC TAA TAC GAC TCA CTA TAG GGC 3') in conjunction with exon 1-specific antisense oligo AS2. Nested 5' RACE was performed using 5 µl of the primary reaction, adaptor-specific AP2 primer (5' ACT CAC TAT AGG GCT CGA GCG GC 3') and antisense gene-specific primer AS1, which is internally nested to AS2, in a final volume of 50 µl. Touchdown PCR was performed according to the manufacturer's instructions using 36 cycles for the primary and 30 cycles for the secondary PCR reactions, respectively. Products of 5' RACE were purified, cloned, and sequenced as described below.
Southern Blot Confirmation of RT-PCR and 5' RACE Results
The PCR products were transferred to Hybond N+ nylon membranes by capillary blotting after electrophoresis. The DNA was cross-linked to the membranes with a UV crosslinker (Amersham). Membranes were subsequently probed with type II GnRH receptor gene-specific oligonucleotides that were labeled and detected using the ECL 3'-oligo labeling and detection system (Amersham). Hybridizations were performed at 42°C in 0.25 ml of hybridization solution per square centimeter of membrane. A 10-ng aliquot of fluorescein-11-dUTP-labeled oligonucleotide was added per milliliter of hybridization solution. Signals were detected by autoradiography.
Cloning and Sequencing of RT-PCR Amplification Products
The RT-PCR products were purified from low melting-point agarose using the Macherey-Nagel Nucleospin extract 2-in-1 system (Separations, Düren, Germany) according to the manufacturer's instructions. The purified DNA was ligated to the pMOSBlue vector and used for subsequent transformation of competent MOSBlue cells using the pMOSBlue blunt-ended cloning kit (Amersham). After plating, white colonies were isolated and screened by PCR for the presence of the expected size insert prior to plasmid DNA isolation and sequencing. Plasmid DNA was isolated with the use of the Wizard Plus SV miniprep DNA purification system (Promega) and sequenced at the Core Sequencing facility, University of Stellenbosch, South Africa. Several clones were sequenced in both directions to obtain a consensus sequence.
Synthesis of cRNA Probes for In Situ Hybridization Studies
A 402-bp subcloned fragment containing part of exon 1 of the human type II GnRH receptor gene (see above) was ligated into the multiple cloning site of the pGEM-T vector (Promega), possessing flanking T7 and SP6 bacterial phage promoters. The resulting construct was then sequenced to confirm orientation. The construct was linearized by digestion with either BamHI or NotI restriction endonucleases in order to generate the corresponding sense or antisense RNA probes, respectively. A 1-µg aliquot of purified linearized template and 80 µCi [-35S]UTP (>1000 Ci/mmole, Amersham) was subsequently used to synthesize cRNA probes using the Maxiscript in vitro transcription system (Ambion, Austin, TX) according to the manufacturer' instructions.
In Situ Hybridization
The hybridization protocol was similar to that described previously [24]. Both paraffin sections and sperm smears were prepared using Vectabond (Vector, Burlingame, CA) silane-coated slides. In brief, the slides were pretreated by digestion with 1 µg/ml proteinase K (Invitrogen, Carlsbad, CA) at 37°C for 30 min followed immediately by acetylation with 0.25% (v/v) acetic anhydride in 0.1 M triethanolamine-HCl, pH 8.0, at room temperature for 10 min. The pretreated slides were then hybridized at 42°C overnight in a 200-µl hybridization solution containing 106 cpm of the labeled cRNA probe, 1 mg/ml tRNA, 0.1 M DTT, 50% formamide, 300 mM NaCl, 50 mM Na2EDTA, 10% PEG-8000, and 1x Denhardt reagent. Following hybridization, the slides were washed through a series of progressively higher stringency washes at 50°C as described elsewhere [24]. Subsequent to washing, the slides were coated with a thin film of a 1:1 mixture of Kodak NBT-2 (Eastman Kodak, Newhaven, CT) emulsion in 0.3 M ammonium acetate prewarmed to 45°C. The slides were air-dried vertically at room temperature and then exposed at 4°C for 2–3 days. Following autoradiographic development, the tissue sections and sperm smears were counterstained with a hematoxylin and eosin histological stain.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our studies initially focused on determining whether exon 1-containing transcripts from the chromosome 1 gene were expressed in any human tissue(s). The rationale for this approach was that transcripts from the gene on chromosome 14, although abundant and widely distributed, would not contain exon 1. Expressed sequence tags containing exon 1 have been identified in the National Center for Biotechnology Information database from a prostate adenocarcinoma cell-line (accession BG036291) and from RNA pooled from testis, B-lymphocytes, and fetal lung cells (accession AA954764). The existence of these expressed sequence tags indicated that transcripts for the gene on chromosome 1 are expressed at least in these tissues, and possibly in others.
We examined the distribution of exon 1-containing human type II GnRH receptor transcripts in a variety of human tissues using dot blot analysis (Fig. 1). Hybridization with an exon 1-specific DNA probe under high stringency conditions yielded a convincing, albeit faint, positive signal for putamen (Fig. 1, B2). In addition, signals could also be detected for the following tissues (see Fig. 1): caudate nucleus (A3), cerebellum (A4), occipital lobe (B1), adult heart (C1), testis (D1), salivary gland (D7), peripheral leukocyte (E6), and lymph node (E7). Cross-hybridization with bacterial chromosomal DNA (Fig. 1, H4) is evident; however, the other negative controls including yeast total RNA, yeast tRNA, E. coli rRNA, Poly r(A), human Cot1 DNA, and human DNA (100 and 500 ng) did not react with the probe and confirm its specificity for the chromosome 1 type II GnRH receptor. The fidelity of the positive signals was assessed by RT-PCR on total RNA from human tissues that were available.
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RT-PCR was performed on total RNA from 22 different human tissues, cells, or cell lines that were available (see Fig. 2). The adult human tissues were cerebellum, cortex, hypothalamus, kidney, medulla, midbrain, pituitary, pons, and testis. The human fetal tissues were adrenals, cerebellum, frontal lobe, hypothalamus, medulla, midbrain, lumbar sympathetic chain, olfactory bulb, pituitary, pons, and retina. The human cells were total ejaculate and HepG2 hepatocyte carcinoma cells. Several sets of PCR primers were utilized. These included the exon 1-specific primers in combination with primers to either exon 2 (that would yield a 329-bp intronless amplicon) or exon 3 (that would yield a 559-bp intronless amplicon), as well as an exon 2–3 primer pair (that would yield a 660- or 250-bp amplicon, respectively, if an intron was present or absent). These results are summarized in Figure 3. As expected, the unprocessed 660-bp chromosome 14 product was amplified from almost all human RNA samples (Fig. 3 and results not shown). This product was also present in testis and mature sperm (Fig. 3A, lane 2, and Fig. 3B, lane 3, respectively). In contrast, the processed 250-bp exon 2–3 product, arising from transcription of the chromosome 1 gene, was detected only in testis and mature sperm (Fig. 3A, lane 2, and Fig. 3B, lane 3). Furthermore, exon 1-containing transcripts were detected only in mature sperm (Fig. 3B, lanes 1 and 2). Hybridization analysis of the RT-PCR products revealed the presence of both the exon 1–2 (329 bp) and exon 1–3 (559 bp) intronless chromosome 1 transcripts in sperm (Fig. 3C).
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To further examine the distribution of the human chromosome 1 type II GnRH receptor transcripts in testis and ejaculated sperm, in situ hybridization analyses were then performed using both sense and antisense exon 1-specific riboprobes. Bright-field photomicrographs summarizing these results are shown in Figures 4 and 5. In human testis the presence of chromosome 1 type II GnRH receptor sense transcripts is indicated by the marked deposition of silver grains within the adluminal region of the seminiferous epithelium (Fig. 4A). Although some luminal collapse resulting from the fixation process is evident, there is sufficient cellular detail to localize the signal. Silver grains were confined to the various types of differentiating haploid spermatids and not observed in association with spermatogonia, spermatocytes, Sertoli cells, or stromal cells (Fig. 4A). This is consistent with the view that the type II GnRH receptor gene on chromosome 1 is transcribed during the haploid phase of spermatogenesis. In contrast, human testis hybridized with the probe for the antisense transcript exhibited a sparse, nonspecific distribution of silver grains (Fig. 4B). In situ hybridization also revealed the persistence of human chromosome 1 type II GnRH receptor transcripts in mature sperm (Fig. 5). Sperm hybridized with the probe for the sense transcript displayed a concentrated, specific deposition of silver grains over the entire surface of the head, indicating the presence of human type II GnRH receptor sense transcripts (Fig. 5A). In contrast, human sperm hybridized with the corresponding control probe for the antisense transcript displayed a sparse and nonspecific background distribution of silver grains (Fig. 5B).
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Cloning and Sequencing of Human Type II GnRH Receptor Transcripts from Ejaculated Sperm
A processed human chromosome 1 type II GnRH receptor transcript of the predicted size and containing all three exons was amplified from human sperm total RNA by RT-PCR (see Fig. 3, B and C). The 5' end of the cDNA was determined by 5' RACE. A consensus sequence spanning the full putative coding region for the human type II GnRH receptor cDNA was compiled by cloning and sequencing of three overlapping RT-PCR products (accession AY077708) and is shown in Figure 6. Several clones from each individual PCR reaction were sequenced. Of note, a UGA translation stop or selenocysteine codon was detected within exon 2 of the chromosome 1 transcript. In addition, alignment of the human and vervet monkey (accession AF353988) protein sequences revealed that the human cDNA would require the insertion of a single G residue in the 5' end region to encode a protein homologous to the monkey type II GnRH receptor (Fig. 6). The consensus coding sequence was then compared to the corresponding chromosome 1 gene sequence (accession AL160282) and found to be identical except for two nucleotide positions within the coding region. This would result in the substitution of a valine for an alanine residue and a serine for an arginine residue at amino acid positions 220 and 232, respectively, if the cDNA is translated.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This is the first report of the cloning of a potentially full-length type II GnRH receptor transcript from the gene on chromosome 1 in any human tissue or cell type. Sequencing of the chromosome 1 sperm transcripts showed the presence of a UGA translation stop codon within exon 2, as well as a frame shift within exon 1 when compared to the recently cloned vervet monkey type II GnRH receptor (accession AF353988) [20] (see Fig. 6). Both the stop codon and frame shift are also found in the human type II GnRH receptor gene on chromosome 1 (accession AL160282). Overall there is a 99.7% identity between the cDNA and gene sequence, with only two nucleotide differences within the coding region, most likely due to variation between individuals. The site of initiation of translation was predicted from the cDNA sequence of the monkey type II GnRH receptor. Interestingly, translation of the human cDNA would be predicted to initiate at an ATG codon like most eukaryotic cDNAs, whereas translation of the monkey cDNA starts with an unusual ACG. Overall, there is a 96.5% identity between the coding regions of the human and monkey type II GnRH receptor cDNAs and a 92.9% identity at the amino acid level (see Fig. 6).
Many human tissues were screened by RT-PCR and/or RACE for the presence of intronless transcripts containing the three exons. Ejaculated sperm was the only source where a potential full-length intronless transcript, resulting from the gene on chromosome 1, was detected. The failure to detect exon 1-containing sperm transcripts in testis total RNA by RT-PCR may have been due to their degradation postmortem and/or during the RNA isolation procedure from the testis. However, in situ hybridization revealed the presence of exon 1-containing transcripts in both testis and mature sperm. The in situ localization of type II GnRH receptor transcripts to the adluminal region of the seminiferous epithelium is consistent with the distribution of other haploid-specific mRNAs [24] and suggests that the human chromosome 1 type II GnRH receptor gene is postmeiotically expressed in round and elongating spermatids. These transcripts are distributed throughout the entire sperm head in a similar manner as reported for other sperm transcripts [25]. A central query to be resolved is whether this transcript is functional in sperm.
Given that the mRNA contains a stop codon and would also require a nucleotide insertion to create the correct open reading frame, it seems likely that this gene is a transcribed pseudogene that, although functional in other primates, is nonfunctional in humans [20]. This may reflect the involvement of the type II GnRH receptor in the induction of mating behavior of other primates that are seasonal breeders [20], unlike humans. Pseudogenes are a consequence of gene duplication via either retrotransposition or duplication of genomic DNA [26]. The human genome contains a large number of pseudogenes, most of which are retrotransposons or processed pseudogenes, which lack introns and arise from single-stranded RNA. An intron-containing GnRH receptor pseudogene on chromosome 1 would thus have originated by DNA duplication. Pseudogenes derived from duplicated genomic DNA are most likely to be on the same chromosome as their paralogous functional partners, although they can also be inserted into a different chromosome by a duplication and translocation process [26]. Clearly, the latter process would have had to occur to result in a GnRH receptor pseudogene on chromosome 1 and a paralogous gene on chromosome 14. Most pseudogenes are promoterless and are therefore not transcribed. However, transcripts for some pseudogenes have been identified, arising most likely from DNA duplication of the promoter elements in parallel with the coding regions or from insertion of the pseudogene near the promoter of another gene [26]. Interestingly, spermatogenic cells have a high tendency to express processed retrotransposons. For example, 10 of the 14 retrotransposons that have been retained as functional genes in mammals are expressed in testis [27]. Taken together, although expression of pseudogenes does occur in mammalian spermatogenic cells, the expression in these cells of a GnRH receptor pseudogene on chromosome 1 would appear to be a rare event. Furthermore, the detection of immunoreactivity to the type II GnRH receptor protein in human pituitary and brain tissue by Millar et al. [19] would suggest that the gene on chromosome 1 is not a pseudogene.
Nevertheless, it is difficult to envisage how transcripts from a gene containing a stop codon and a frame shift within the coding region could result in a full-length, functional G-protein coupled receptor. It has, however, been shown that 5-transmembrane G-protein coupled receptors, lacking transmembrane helices 1 and 2, are functional [28]. Thus one possibility is that a functional, truncated, immunoreactive protein containing transmembrane helices 3–7 is expressed. This could occur if translation begins at the second AUG, situated at the end of transmembrane helix 2 (see Fig. 6), were it not for the stop codon within extracellular loop 2. RNA editing, with a single base transition within the UGA stop codon, could be involved in generating a functional truncated protein. Alternatively, a full-length functional protein could be generated by an additional RNA editing event involving the insertion of a G residue near the 5' end of the transcript. It has been shown that the monkey type II GnRH receptor cDNA contains a CGA arginine codon instead of a stop codon, creating an extended open reading frame [19, 20]. The presence of a UGA stop codon within the human sequence may represent a mechanism for the temporal translational regulation of the type II GnRH receptor during spermatogenesis. Many haploid expressed genes are under extensive translational control. For example, the mammalian protamines, which package the DNA within the sperm head, are initially transcribed at the round spermatid stage and subsequently stored as inactive ribonucleoprotein particles prior to their translation in elongating spermatids [29]. Differentiating spermatids might require a functional type II GnRH receptor at specific stages during spermatogenesis. RNA editing would therefore provide a similar means for the translational regulation of these transcripts.
Although there are no reports of the occurrence of RNA editing in human sperm, there are examples in the literature for other mammalian tissues and for other species. Substitution editing, including posttranscriptional U to C substitutions, has been previously described for mammalian and plant transcripts [30]. This has been shown to result in the removal of stop codons for some plants [30]. In addition, there are many examples in the literature of posttranscriptional insertion editing resulting in expression of alternative reading frames [30]. Although we are not aware of any cases reported to date of G insertions for mammalian transcripts, there are examples of A insertion in human mitochondrial transcripts [31], G insertions in viruses [30], and insertions of all four nucleotides in a slime mold [30]. Our inability to detect an edited type II GnRH receptor transcript may have been due to a short half-life or low abundance.
Evidence from others suggests another unusual mechanism whereby a functional type II GnRH receptor may be produced. This could involve the incorporation of a selenocysteine amino acid at the UGA position, rather than encoding a translation stop signal [32, 33]. A prominent role for selenium during spermatogenesis has been well established [34, 35]. Selenium, which is incorporated into selenocysteine, has been shown to be supplied to the testis with an apparent priority over other tissues [34], and the uptake thereof appears to be under gonadotropin control [35]. For example, the selenoenzyme phospholipid hydroperoxide glutathione peroxidase has been detected in mammalian spermatids, sperm, and testis [32, 33]. In addition, a number of mammalian proteins have been identified that contain selenocysteines encoded by in-frame UGA codons [36]. Therefore, the UGA codon in the type II GnRH receptor transcript may code for selenocysteine. Selenocysteine incorporation, however, requires a selenocysteine-insertion sequence (SECIS) motif of approximately 200 nucleotides that form a stem-loop structure in the 3' untranslated segment of the mRNA [37]. Because of the length and degeneracy of the SECIS sequence, it is difficult to assess whether a SECIS sequence occurs in the chromosome 1 human type II GnRH receptor transcript. The finding that selenocysteine is more efficiently incorporated when the UGA codon is positioned closer to the middle of the coding region, rather than close to one of the ends [38], as is the case for the human type II GnRH receptor mRNA, would be consistent with the selenocysteine hypothesis. Production of a selenocysteine-containing truncated protein would not require an RNA editing event. However, if the stop codon encodes selenocysteine, the production of a full-length protein would still require RNA insertion editing of the transcript to correct the frame shift in the amino terminus, an event that appears to be extremely rare.
Although the functionality of these transcripts remains to be confirmed, the presence of GnRH and GnRH receptors has been shown to play a role during spermatogenesis, sperm maturation, and fertilization [39]. GnRH hormone has been localized to the seminal plasma [39] and has been shown to increase the binding of sperm to the ovum, an effect that is inhibited by GnRH antagonists [40, 41]. In addition, GnRH has also been shown to function as a local regulator in human placenta, where GnRH or GnRH-like peptides are synthesized by cytotrophoblasts and syncytiotrophoblasts during embryogenesis [42]. The mammalian type II GnRH receptor has been demonstrated to specifically regulate FSH secretion, a peptide hormone involved in the development of meiotic spermatocytes and postmeiotic spermatids [43]. Sperm have also been shown to express receptors for other hormones or signaling molecules such as the estrogen receptor [44] and A1 adenosine receptor [45]. Furthermore, a number of neuroendocrine hormones and growth factors, including GnRH, are produced by the testis [15]. The presence of these receptors and hormones is consistent with the presence of a network of intratesticular hormonal regulators, where hormones can function in a paracrine or autocrine manner due to their isolation from the rest of the body by a blood-testis barrier [15]. The expression of functional type II GnRH receptor transcripts in human sperm could be part of the existing network of intratesticular or neuroendocrine hormonal regulation governing spermatogenesis. Although some of the above-mentioned functions could be mediated by the type I GnRH receptor, the expression of a functional type II GnRH receptor protein in the testis and sperm would be consistent with these reports.
In summary, we have cloned a transcript of the gene on chromosome 1 for the human type II GnRH receptor from human sperm. This transcript, although containing all the exons required for a full-length receptor protein, contains a stop codon and a frame shift, which are also present in the gene. Although this would suggest that the gene is a transcribed pseudogene, several lines of evidence from the literature suggest otherwise. There is evidence for a functional role for a type II GnRH receptor protein in human sperm and testis. Furthermore, immunoreactivity data strongly suggest that a protein is expressed from the human chromosome 1 gene [19]. Thus if the gene is not a pseudogene, the transcript could possibly be translated as a truncated, immunoreactive protein or edited to result in translation of a full-length protein, possibly containing selenocysteine. However, given that RNA editing and/or incorporation of selenocysteine are rare events, the latter possibility seems unlikely. Further experiments using specific antibodies directed against domains encoded by sequences both 5' and 3' to the stop codon would be necessary to clarify whether a full-length or truncated type II GnRH receptor protein is expressed in sperm or in the developing zygote.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: J. Hapgood, Department of Biochemistry, University of Stellenbosch, Private Bag X1, Matieland, Stellenbosch 7602, Republic of South Africa. FAX: 2721 808 5863; jhap{at}sun.ac.za
Received: 19 December 2001.
First decision: 4 January 2002.
Accepted: 25 June 2002.
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L. Ding, J. Xu, X. Luo, and N. Chegini Gonadotropin Releasing Hormone and Transforming Growth Factor {beta} Activate Mitogen-Activated Protein Kinase/Extracellularly Regulated Kinase and Differentially Regulate Fibronectin, Type I Collagen, and Plasminogen Activator Inhibitor-1 Expression in Leiomyoma and Myometrial Smooth Muscle Cells J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5549 - 5557. [Abstract] [Full Text] [PDF]
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 X. Luo, L. Ding, and N. Chegini Gonadotropin-releasing hormone and TGF-{beta} activate MAP kinase and differentially regulate fibronectin expression in endometrial epithelial and stromal cells Am J Physiol Endocrinol Metab, November 1, 2004; 287(5): E991 - E1001. [Abstract] [Full Text] [PDF]
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T. M. Siler-Khodr, F.-Q. Yu, P. Wei, S.-X. Tao, and Y.-X. Liu Contraceptive Action of a Gonadotropin-Releasing Hormone II Analog in the Rhesus Monkey J. Clin. Endocrinol. Metab., September 1, 2004; 89(9): 4513 - 4520. [Abstract] [Full Text] [PDF]
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 Y. Okada, A. Murota-Kawano, S. S. Kakar, and S. J. Winters Evidence that Gonadotropin-Releasing Hormone (GnRH) II Stimulates Luteinizing Hormone and Follicle-Stimulating Hormone Secretion from Monkey Pituitary Cultures by Activating the GnRH I Receptor Biol Reprod, October 1, 2003; 69(4): 1356 - 1361. [Abstract] [Full Text] [PDF]
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