Mol. Hum. Reprod. Advance Access originally published online on May 28, 2004
Molecular Human Reproduction 2004 10(8):599-603; doi:10.1093/molehr/gah076
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Alternative splicing of the human luteal LH receptor during luteolysis and maternal recognition of pregnancy
1Obstetrics and Gynaecology, Department of Reproductive and Developmental Sciences, University of Edinburgh and 2MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, Chancellor's Building, Royal Infirmary of Edinburgh, Little France, Edinburgh
3 To whom correspondence should be addressed at: Obstetrics and Gynaecology, Department of Reproductive and Developmental Sciences, University of Edinburgh, Royal Infirmary of Edinburgh – Little France, 49 Little France Crescent, Old Dalkeith Road, Edinburgh EH16 4SB, UK. Email: w.c.duncan{at}ed.ac.uk
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Abstract |
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Deletion of exon 10 of the human LH receptor impairs LH but not hCG action. Other splice variants of the LH receptor impair both LH and hCG action in other species. We hypothesized that alternatively spliced LH receptors are involved in luteolysis and luteal rescue with hCG in women. mRNA was extracted from human luteinized granulosa cells and from corpora lutea from across the luteal phase and after luteal rescue in vivo with exogenous hCG. Splice variants were detected by RT–PCR using carefully designed primer pairs. Products were visualized on agarose gels, extracted, purified and sequenced. Three splice variants of the human LH receptor were detected and characterized. These demonstrate a region of multiple splicing between exons 8 and 11 of the receptor. A naturally occurring splice variant with exon 10 alone removed was not identified. There was no obvious change in the pattern of splice variants across the luteal phase in the presence or absence of hCG. These data do not support the hypothesis that qualitative changes in LH receptor splicing have a role in luteolysis or that a naturally occurring LH receptor lacking exon 10 has a role in maternal recognition of pregnancy.
Key words: corpus luteum/hCG/LH receptor/splice variants
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Introduction |
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Luteal progesterone synthesis is dependent on LH from the pituitary gland (Hutchinson and Zeleznik, 1984
; Fraser et al., 1986). However, during luteolysis, progesterone secretion falls in the presence of maintained concentrations of LH (Hutchison et al., 1986), LH receptors (Duncan et al., 1996) and other key factors in the steroidogenic pathway (Duncan et al., 1999). In a non-conception cycle, the corpus luteum becomes less sensitive to gonadotrophic support and its LH receptors become increasingly unable to stimulate the steroidogenic pathway. However, in a conception cycle, the increasing block to progesterone synthesis is overcome by logarithmically increasing concentrations of hCG acting through the LH receptor.
Previous investigations into the expression of the LH/hCG receptor in the pig (Loosfelt et al., 1989), rat (Aatsinki et al., 1992) and sheep (Bacich et al., 1994) have described multiple splice variants generated by alternative splicing of the primary transcript. It appears that the human LH/hCG receptor may also be alternatively spliced. Minegishi et al. (1997) detected a splice variant of the human luteal LH receptor that bound both LH and hCG but did not activate adenylate cyclase. We therefore hypothesized that as the corpus luteum ages, alternative splicing of the LH receptor increases and more non-functioning receptors are produced. We felt that this may explain the reduction in progesterone synthesis in the presence of maintained LH receptor expression.
This hypothesis could not explain how hCG was able to ‘rescue’ the corpus luteum and maintain progesterone synthesis. Recently, however, it has been shown that a variant of the LH receptor, where exon 10 is deleted, responds differently to LH and hCG (Müller et al., 2003). This variant binds both LH and hCG but the second messenger cascade is activated by hCG and not LH (Müller et al., 2003). This provides a mechanism by which the corpus luteum could remain receptive to hCG, but become desensitized to LH. This variant receptor was originally discovered in a male patient with a homozygous deletion of the exon 10 region of the LH/hCG gene (Gromoll et al., 2000). Although it has never been examined in the human ovary, it has been reported in sheep corpora lutea (Bacich et al., 1994). We hypothesized that a naturally occurring LH receptor variant with exon 10 removed functioned as an hCG receptor during maternal recognition of pregnancy.
We therefore aimed to characterize the nature of any LH receptor splice variants involving the hinge region around exon 10, in human luteinized granulosa cells and corpora lutea of different stages of the luteal phase and after simulated early pregnancy.
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Materials and methods |
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Tissue collection
The collection of human corpora lutea and luteinized granulosa cells were separately approved by the Reproductive Medicine Subcommittee of the Lothian Research Ethics Committee. Human corpora lutea (n=12) were collected and dated on the basis or the urinary LH surge as described previously (Duncan et al., 1996
). In this study, three corpora lutea were classified as early luteal (LH+1 to LH+5), three as mid-luteal (LH+6 to LH+10) and three as late luteal (LH+11 to LH+14). In addition, three corpora were rescued in vivo with daily injections of exogenous hCG to mimic the hormonal changes of early pregnancy (Duncan, 2000). Follicular fluid was collected from women undergoing transvaginal oocyte retrieval for IVF following ovarian stimulation using a standard procedure (Stamouli et al., 1996). Luteinized granulosa cells were obtained from the pooled follicular aspirates after the removal of the oocytes after Percoll separation (Stamouli et al., 1996). After washing, 75 000 viable cells were plated onto each well of 24-well plates pre-coated with Matrigel (40 µl/well; Beckton Dickinson UK Ltd, UK) and cultured in serum-free supplemented medium as described previously (Stamouli et al., 1996) for 48 h.
Extraction of mRNA and preparation of cDNA
At operation, the whole corpus luteum was enucleated from the ovary by blunt dissection and the ovary oversewn. A piece of corpus luteum was snap-frozen in liquid nitrogen and stored at –70°C until mRNA was batch extracted from using TRI-reagent (Sigma Chemical, Poole, UK). The luteinized granulosa cell culture experiments were carried out on three separate occasions using luteinized granulosa cells from different women. Each experiment was carried out with two replications and cells from identical wells were combined prior to RNA extraction. After removal of culture medium, cells were rinsed in phosphate-buffered saline and TRI-reagent was added. The resulting solution was stored at –70°C until batch extraction of RNA. Extracted RNA was treated with DNAse I and reverse-transcribed using a random hexamer priming (PE Applied Biosystems, UK). Two controls were used: one omitted the multiscribe enzyme and the other the template RNA.
PCR
Primers were designed using the Primer3 program (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) and the cDNA sequence of the LH/hCG receptor obtained from Nucleotide (Accession No: NM_000233). Primers (Table I) were synthesized by MWG-Biotech UK Ltd (UK). One microlitre of cDNA was used as the template in subsequent optimized PCR reactions, which were carried out using Taq DNA Polymerase (Promega, UK). Reaction conditions were 0.25 IU/µl Taq, 0.5 µmol/l each primer, 0.2 mmol/l dNTP, 0.1% Triton X-100, 50 mmol/l KCl, 10 mmol/l Tris–HCl, 1.5 mmol/l MgCl2. Each PCR reaction was overlayed with mineral oil and incubated at 95°C for 5 min, followed by 30 cycles of 95°C for 30 s, 62°C for 60 s, 72°C for 90 s, and a final elongation step of 72°C for 10 min. PCR products were separated by applying 100 V for 75 min to 1% aragose gels with ethidium bromide, and were visualized under UV transillumination.
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Real-time quantitative PCR was carried out using the LightCycler (Roche Diagnostics Ltd, UK). The Faststart S Y B R Green Master Mix (Roche Diagnostics Ltd) was used for all reactions in the LightCycler. Rescued corpus luteum cDNA was used to generate standards for quantification and conditions were optimized as recommended by the manufacturer. Primers F1 and R1 (Table I) amplified a 185 bp region at the 5'' end of the gene (Figure 1). The final optimized conditions were used as appropriate in duplicate, and all results for LH receptor expression are relative to glucose-6-phosphate dehydrogenase (G6PDH) expression (Table I). LightCycler assay analysis was carried out using the LightCycler software (Version 3.01). Amplified DNA fragments were recovered from agarose gels by MinElute Gel Purification Columns (QiaGen Ltd, UK). One hundred nanograms of each of the purified products were sequenced by cycle sequencing using ABI Prism Big Dye Terminator Sequencing Kits (Applied Biosystems). The fragments were separated on an Applied Biosystems 377XL sequencer by the MRC Human Genetics Unit, Edinburgh, UK.
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Results |
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Expression of LH receptor in the human corpus luteum
In order to validate the human corpora lutea and RT–PCR system used in these experiments, we chose to investigate total LH receptor expression across the cycle. Our previous studies using northern blotting, mRNA in situ hybridization and in situ binding showed a non-significant trend to reduction in the late luteal phase with maintenance of expression in the presence of hCG (Duncan et al., 1996
). The primer pair (F1/R1) detected a single 185 bp fragment at the 5'region of the LH receptor cDNA (Figure 1a, d). As this is outside the region of alternative splicing in other species (Aatsinki et al., 1992; Bacich et al., 1994), it is likely to include all variants of the LH receptor. Quantitative RT–PCR across the luteal phase and after luteal rescue with hCG demonstrated the expected pattern of LH receptor expression (Figure 2a). We therefore concluded that these samples were valid to study the pattern of splice variants across the cycle.
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Expression of splice variants of the luteal LH receptor
Multiple primer pairs were designed to span the region around exon 10 and the regions of alternative splicing seen in other species. We report here the set of primers that included all of the splice variants that we identified (F2/R2) (Table I). This primer pair should detect a 921 bp fragment that includes part of the extracellular domain, the hinge region and part of the transmembrane region (Figure 1d). When cDNA from human corpora lutea were analysed by RT–PCR using this primer pair, four bands were detected suggesting the presence of splice variants (Figure 1c). The full-length receptor and these three splice variants were detected in luteinized granulosa cells (Figure 1b) and corpora lutea from across the luteal phase and after luteal rescue with hCG in all samples analysed (Figure 2b). Because of the nature of the splice variants, we were unable to design appropriate primers to quantify the expression of each individual cDNA species across the cycle. We therefore have been unable to confirm our suspicion that the shorter forms of the LH receptor are more abundant than the full-length receptor and that the full-length receptor is differentially reduced in the late luteal phase (Figure 2b). Notably, this trend was detected in all late luteal samples analysed and the shorter forms of the LH receptor seem less abundant in the luteinized granulosa cells that will go on to form the early corpus luteum (Figure 1b).
The nature of LH receptor splice variants in the human corpus luteum
Band ‘a’ (921 bp) was determined by sequencing to be the full-length receptor species (Figure 3). Band ‘b’ (735 bp) was the truncated LH receptor as described by Minegishi et al. (1997) in which exon 9 was removed by an in-frame splice. Band ‘c’ (621 bp) was a novel splice variant in which the sequence encoding the first 89 amino acids of exon 11 was spliced out, leading to a frame-shift and a premature stop signal, 18 codons after the 3'splice site. Band ‘d’ (432 bp) was another novel splice variant which had both exon 9 spliced out, in addition to the 89 amino acids of exon 11, again leading to a stop codon shortly after the 3'splice site (Figure 3). We were unable to detect, in any experiment, an LH receptor variant with just exon 10 missing that would correspond to the putative hCG receptor.
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Discussion |
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This paper describes the presence of alternative splicing around the hinge region of the LH receptor in human corpora lutea and luteinized granulosa cells. We have detected the full-length LH receptor transcript and three additional splice variants. One previous study described a splice variant of the human LH receptor that had exon 9 removed. That variant bound LH and hCG but ligand binding did not stimulate adenylate cyclase (Minegishi et al., 1997
). We have confirmed the presence of this splice variant. Studies in the rat have characterzed LH receptor splice variants including a full-length receptor with exon 9 removed in-frame (Segaloff et al., 1990; Aatsinki et al., 1992). Studies of the ovine LH receptor described three splice variants in the hinge region but, as the primer was situated on exon 9, did not report any with exon 9 spliced out (Bacich et al., 1994).
We have also characterized two additional splice variants of the human LH receptor that contain premature stop codons. Splice variants containing premature stop codons have been described in the pig (Loosfelt et al., 1989), rat (Aatsinki et al., 1992) and sheep (Bacich et al., 1994) that are almost identical to Figure 3c. The same splicing pattern as Figure 3d has been reported in the rat ovary (Bernard et al., 1990). Similar splice variants were not detected when the bovine LH receptor was investigated (Robert et al., 2003), possibly as the primer was situated in the 5'region of exon 11 that is removed. These data show that there is some conservation of alternative splicing patterns between the species investigated to date. This is of interest as, unlike other species, the LH receptor is fundamental to primate luteal function during luteolysis and maternal recognition of pregnancy (Auletta and Flint, 1988; Stouffer, 1988).
We hypothesized that a naturally occurring LH receptor lacking exon 10 could function as an hCG receptor and have a role in maternal recognition of pregnancy. We have been unable to detect a splice variant lacking only exon 10, despite repeated attempts to do so and carefully designed primers. Variants of the LH receptor, lacking exon 10, have been described in the sheep (Bacich et al., 1994), a species that does not use CG to rescue the corpus luteum. Interestingly, it is the default LH receptor of the marmoset monkey (Zhang et al., 1997) that does use CG. However, if such a splice variant is present in the human ovary, it is present at extremely low abundance relative to other isoforms. These data do not support the concept that luteal rescue by hCG is through a novel splice variant lacking exon 10. Indeed, as exon 10 is present in these and in rat splice variants it may have a crucial role in human and rat receptor structure or function.
We have investigated the pattern of splice variants in luteinized granulosa cells and in corpora lutea from throughout the luteal phase and after luteal rescue in simulated early pregnancy. We have detected full-length receptor and the three variants in all tissues examined. As the qualitative pattern of splice variants does not change across the functional life-span of the corpus luteum, our hypothesis, that the expression of different variants as the corpus luteum matures reduces the sensitivity to LH, is not supported. What is not clear, however, is whether the relative abundance of the splice variants changes across the luteal phase. Quantitative assessment of the abundance of individual splice variants is not possible using these techniques. Whether the impression that the intensity of the band representing full-length receptors in the late luteal phase is reduced, while other bands are not, means that there is higher proportion of non-functioning LH receptors as the corpus luteum ages is unclear. However, one observation that is consistent with there being more truncated receptors as the corpus luteum ages is that there appears to be fewer truncated splice variants in luteinized granulosa cells. The role of splice variants and non-functioning LH receptors in luteolysis remains to be determined.
It is not known whether these splice variants are translated into proteins. Previous studies have used models where, as LH receptor is down-regulated by its ligand, the receptor dynamics are different to the human (Ascoli et al., 2002). However, in other species, and in expression studies, some transcripts are translated into protein and truncated LH receptors are synthesized (Minegishi et al., 1997). Whether, in the human, the apparently abundant truncated receptors lacking the transmembrane and intracellular domains, are synthesized, trapped inside the cell or secreted is not known. Certainly the extracellular domain of the LH receptor binds hCG with high affinity (Xie et al., 1990), and the soluble extracellular domain of the human LH receptor can inhibit the binding of hCG to the full-length receptor (Osuga et al., 1997). Studies have suggested the presence of a soluble LH receptor subspecies in the rat, which has binding activity (Tsai-Morris et al., 1990). The presence of an LH binding inhibitor has been described in the primate corpus luteum and its potential role in luteolysis has been highlighted (Auletta et al., 1990). Whether secreted, truncated LH receptors are involved remains to be determined.
In summary, we have confirmed that the human luteal LH receptor has multiple splice variants and have identified two variants not previously reported in the human. The role of these in luteal function is not clear. It is not known whether quantitative differences in the expression of LH receptor splice variants across the luteal phase occurs but it is clear that the pattern of splice variants does not change across the luteal phase and after luteal rescue with hCG. In addition the putative hCG receptor, which is a variant of the LH receptor without exon 10, does not occur naturally in the human corpus luteum at any stage of the cycle.
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Acknowledgements |
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The authors would like to acknowledge the skilled technical help of Julie Bell. We are grateful to Dr Peter Illingworth, Dr Richard Anderson and Dr Joo Thong for their support in collecting the human corpora lutea and luteinized granulosa cells. We also thank the research nurses involved in patient recruitment and the women themselves who consented to their tissues being used in this research. Dr Colin Duncan gratefully acknowledges the Wellcome Trust for providing project grant funding.
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Submitted on March 30, 2004; accepted on May 13, 2004.
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