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Mechanisms of Hormone Action |
Institute of Reproductive Medicine of the University, University of Münster, D-48129 Münster, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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human chorionic gonadotropin, luteinizing hormone, signal transducers, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The LHR belongs to the group of glycoprotein hormone receptors, comprising the closely related FSH receptor (FSHR) and TSH receptor (TSHR), which share the common feature of a large extracellular hormone-binding domain and a seven transmembrane domain, the hallmark of the G protein-coupled receptor family. At the genomic level, the three glycoprotein hormone receptors display striking similarities with respect to their genomic organization and exon size. However, while the extracellular domain of the FSHR and TSHR is encoded by nine exons, 10 exons are present in this region of the LHR gene [5–7]. The transmembrane domain is encoded in all three receptors by one large exon. The genomic organization is conserved throughout all different species analyzed, such as the human, mouse, ovine, and rat.
In the human, the LHR gene spans approximately 80 kilobase pairs (kbp) and is located on chromosome 2p21 (sequence AC073082). Exons 2 to 9 encode leucine-rich repeats believed to be involved in direct hormone binding, while parts of exon 9, the complete exon 10, and a 5'portion of exon 11 form the hinge region [7, 8]. Exon 10 consists of 81 bp encoding 27 amino acids. There are no clear-cut motifs present within this exon, but it contributes to the formation of a C-terminal cysteine cluster of the extracellular domain.
In several species, the LHR gene is alternatively spliced during transcription, either by exon skipping or by use of alternative splice sites, resulting in a number of potential LHR isoforms [9–14]. Nearly all the described LHR isoforms remain untranslated, or, if translated, they cannot be properly transported to the cell membrane and remain trapped inside the cytoplasm. Thus, according to current knowledge, such LHR isoforms do not play any significant physiological role in hormone binding and signal transduction of LH and CG.
Interestingly, no isoform lacking exon 10 has been reported in humans, rats, and mice [15], and there is only one report indicating that splicing of the LHR gene can result in an LHR isoform lacking exon 10 in the ewe [16]. The only species in which a constitutive skipping of exon 10 has been reported is the common marmoset monkey (Callithrix jacchus) [17]. In this species, the LHR mRNA lacking exon 10 represents the wild-type form. Functional studies on this LHR revealed that hormone binding and signal transduction are not impaired using hCG. However, so far such studies have not been conducted using LH.
In order to investigate whether the lack of exon 10 in the LHR mRNA of the common marmoset is an isolated, species-specific finding, we performed an RT-PCR-based phylogenetic screening for the presence LHR exon 10 using testicular mRNA from several primate species. Furthermore, considering that in the human LHR, exon 10 is necessary for LH but not for hCG bioactivity [18], we addressed the question whether the lack of exon 10 mRNA might be related to the pituitary expression of CG in the common marmoset.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oligonucleotides
The oligonucleotides used in the RT-PCR reactions were either designed as consensus sequences from nucleotide sequence stretches highly conserved within exon 9 and exon 11 of the human, mouse, rat, and common marmoset LHR, based on the marmoset or from human LHR cDNA sequences.
consensus exon 9 for1
5'-GG(G/C)T(G/A)GAGTCCATTCAGACGCT-3'
consensus exon 11 rev1
5'-GGG(G/A)TT(G/A)AAAGCATCTGGTTC(A/T)GGAG-3'
marmoset exon 9 for1
5'-GCCAATCTCCTGGATGCCACGC-3'
marmoset exon 11 rev1
5'-CAGGAGCACATCGGGGTGTCTTG-3'
human exon 9 for1
5'-CAGAGGCTAATTGCCACGTCATCC-3'
The following amplicons should be obtained when using these primer combinations: 1) consensus exon 9 for1/consensus exon 11 rev1: 335 bp with exon 10; 254 bp without exon 10; 2) consensus exon 9 for1/marmoset exon 11 rev1: 318 bp with exon 10; 237 bp without exon 10; 3) marmoset exon 9 for1/marmoset exon 11 rev1: 240 bp with exon 10; 159 bp without exon 10; and 4) human exon 9 for1/marmoset exon 11 rev1: 303 bp with exon 10; 222 bp without exon 10.
RT-PCR
RNA isolation from testicular tissues was performed using Ultraspec (AMS Biotechnology, Wiesbaden, Germany), followed by a DNAse digestion with the DNA free kit (Ambion, Austin, TX). Five micrograms total RNA were reverse transcribed using the appropriate reverse primer and Superscript transcriptase (Invitrogen, Karlsruhe, Germany). One to two micrograms of cDNA were used for each of the subsequent PCR reactions. PCR cycle conditions were as follows: 1 cycle at 94° 2 min; 30 cycles at 94°C 50 sec, 58°C 40 sec, and 72°C 1 min; a final extension step was performed at 72°C for 10 min. The reactions were electrophoresed on 2% agarose. Several primer combinations were used for each species; however, for some, only one primer combination worked. This is presumably due to sequence variations within this region preventing primer annealing.
RNA was isolated from common marmoset pituitaries and placenta using the Ultraspec method. RNA was reverse transcribed using the previously published reverse primer 5'- GCGGATTCAGAAGCCTTTATTG-3' [19]. Subsequent PCR was performed employing the forward primer 5'- GGGGACGCACCAAGGATG-3' and the reverse primer at the following cycling conditions: 1 cycle at 94°C for 1 min; 30 cycles at 94°C for 30 sec, 60°C for 30 sec, and 72°C for 60 sec; the PCR was terminated with a final extension step at 72°C for 56 min. The reactions were run on a 2% agarose gel. The corresponding bands were cloned into the pGEM-T easy vector (Clontech, Heidelberg, Germany) and the inserts sequenced.
DNA Sequencing
The amplicons obtained were purified by High Pure Purification kit (Roche, Mannheim, Germany) and cloned into the pGEM-T easy vector. A minimum of five different clones were sequenced from each amplicon. The DNA sequences given in Figure 3 represent the consensus sequence obtained from these clones using the sequencer software (Genecodes, Ann Arbor, MI). The CLUSTAL W align software (http://www.ebi.ac.uk/Tools) was used for the comparison of the LHR sequences from the different primates.
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Southern Blot
Ten micrograms of genomic DNA isolated from blood samples from the marmoset and cynomolgus macaques and humans were digested with EcoRI and run on a 0.8% agarose gel. The DNA was blotted onto nylon membranes (Pharmacia, Freiburg, Germany) and hybridized with a radioactive-labeled marmoset exon 10 probe using ExpressHyb (Clontech). The blot was exposed for 3 days and analyzed using a Phospho-Imager (Molecular Dynamics, Inc., Sunnyvale, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To cover all recent systematic groups among the primate order reflecting different levels of evolutionary progress, we chose Strepsirrhini and anthropoid species from the Platyrrhini (Cebidae and Callithrichidae) as well as Catarrhini (Cercopithecidae, cynomolgous macaque) and Hominoidea (Pongidae and humans [Hominidae]) (Fig. 1).
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RT-PCR of the Exon 9 to Exon 11 Region of the LHR
Testicular tissue was obtained from species shown in Figure 1 and used for RNA isolation. The region encompassing the 3' part of exon 9 to the 5' region of exon 11 of the LHR was amplified by RT-PCR using primer pairs directed to conserved nucleotide sequence stretches among the human, mouse, and rat LHR cDNA within these two exons or using human or marmoset-specific primer pairs. RT-PCR from testicular RNA should yield amplicons containing exon 10 (plus 81 bp) or amplicons lacking exon 10 (minus 81 bp) or both. Analysis of RT-PCRs from testes of different primate species revealed distinct patterns for two amplicons. The results showed only one amplicon including exon 10 in the Strepsirrhini gray mouse lemur (Microcebus murinus) and crowned lemur (Eulemur coronatus) (Lemuriidae), in the Old World cynomolgus macaque Macaca fascicularis, in the bonobo Pan paniscus (Pongidae), and in the human, while the cebid squirrel monkey (Saimiri sciureus), the brown capuchin (Cebus apella), the callithrichid common marmoset (Callithrix jacchus), and the cotton top tamarin (Saguinus oedipus) display an amplicon without exon 10 (Fig. 2). This indicates that Strepsirrhini, Catarrhini, Pongidae, and Hominidae correctly splice exon 10 of the LHR giving rise to an exon 9-exon 10-exon 11 amplicon, while in Platyrrhini exon 10 mRNA is skipped. DNA sequencing of the amplicons confirmed the findings. Interestingly, no alternative splicing variants lacking or including exon 10 were observed in all tissues investigated. This implies that exon 10 is not alternatively spliced in these species. Rather, splicing is very efficiently regulated in Strepsirrhini, Catarrhini, and Hominoidea.
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Sequence Analysis
A nucleotide sequence alignment of the amplified exon 9 to exon 11 regions revealed an 84% homology between all species (Fig. 3). The 94% homology between phylogenetically related taxonomic groups, such as the great apes and Old World monkeys Catarrhini, is higher (P. paniscus p./M. fascicularis) than in phylogenetically more distant primates, such as great apes and Platyrrhini (77%) (P. paniscus/S. sciureus). Within each systematic group, homology reaches 92%.
At the amino acid level, the overall homology drops to 70%, independent of which exon is analyzed (Fig. 4). Again, closely related species display a higher homology (P. paniscus/M. fascicularis: 92%) compared to more divergent primate species (P. paniscus/S. sciureus: 75%). Apparently similar homologies were observed at both the nucleotide and the amino acid level when exon 9, exon 10, and exon 11 were compared, thereby excluding that exon 10 might be a vestigial exon of the LHR. In all glycoprotein hormone receptors, splicing takes place at the second nucleotide of the last triplet of the corresponding exon, which then is joined to the first nucleotide of the next exon (phase 2 splicing). In Platyrrhini, the last two nucleotides of exon 9 are joined directly to the first nucleotide of exon 11, leading to a triplet encoding aspartic acid (D) instead of glutamic acid (E), which is the amino acid ensuing when exon 9 is joined to exon 10 in the other species.
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Southern Blot Hybridization of Exon 10
To investigate whether the absence of exon 10 of the LHR mRNA in Platyrrhini might be caused by a genomic deletion removing this part of the LHR gene, we performed a Southern blot using genomic DNA from Platyrrhini and Catarrhini and human (Fig. 5). Hybridizing female and male DNA with a probe corresponding to exon 10 of the LHR revealed distinct specific signals in female and male genomic DNA from all species tested, indicating both the presence of exon 10 sequences in Platyrrhini and the autosomal localization of the LHR gene.
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RT-PCR of CG ß mRNA from the Placenta and the Pituitary of the Common Marmoset Monkey (Callithrix jacchus)
In the human, LH is inactive on the LHR lacking exon 10, while hCG results in normal testicular response and testosterone production [18]. In order to analyze which gonadotropin sustains LHR function in the New World monkeys lacking exon 10, we took advantage of the published marmoset CG cDNA sequence [19] and performed RT-PCR from placenta and pituitary tissues of the common marmoset (Callithrix jacchus). Using primers previously reported to be capable of amplifying the ß subunit of marmoset CG, we consistently obtained distinct amplicons of approximately 520 bp in size in both tissues (Fig. 6). DNA sequencing of the amplicons (n = 8) revealed 100% nucleotide sequence identity when comparing the placenta CG ß cDNA to the cDNA sequence obtained from the pituitary. No amplicons encoding for a putative LH ß subunit, which is expected to have an additional A nucleotide in the 3' region of the mRNA, were found using this strategy. Attempts were made to amplify the expressed LH ß subunit by using primers designed as consensus from the LH ß sequences known from other species, but again no LH ß could be obtained. Thus, in the marmoset monkey, the CG ß subunit is highly expressed in the pituitary. Pituitary glands from other species were not available.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There is no indication that exon 10 skipping/splicing is gender or testis specific. In fact, exon 10 is present in the LHR mRNA obtained from ovaries of humans, rats, and mice [14, 15], while it is constitutively skipped in the ovary of the common marmoset (Fig. 2). The Southern blot experiment indicated that, similarly to the Catarrhini and the human, sequences encoding exon 10 must be present at the genomic level in the common marmoset. This is in agreement with previous findings showing cross hybridization of human exon 10 to marmoset genomic DNA [17]. Thus, the lack of exon 10 at the mRNA level is not due to the absence of genomic sequences encoding for it but, presumably, is caused by a transcriptional event in which a nucleotide change in exon 10 prevents splicing or a primate lineage-specific splicing failure caused by impaired action of splice factors.
Exon 10 is constitutively spliced not only in the human and some other primates but also in all lower species in which it has been cloned (e.g., rodents). This indicates the presence of strong splice recognition sites adjacent to exon 10, thereby preventing alternative splicing that instead has been shown for a number of other exons within the LHR gene [15]. This hypothesis is supported by the fact that, up to now, no such LHR isoform lacking exon 10 has been identified in the human, mouse, and rat. Exon 10 mRNA skipping is not an isolated species-specific finding but instead takes place in at least four different monkey species from Platyrrhini and occurs presumably in the whole taxonomic lineage. We, therefore, propose that the LHR exon 10 is the wild-type form for the complete Platyrrhini lineage and forms a new subclass of LHRs in which exon 10 is a pseudoexon. According to the nomenclature of other G protein-coupled receptors [20], this receptor could be defined as LHR type II, where LHR type I represents the complete receptor.
Although at present we do not know the molecular mechanism underlying the constitutive missplicing of exon 10, a chronological approximation of the origin of the observed defect during primate evolution can be deduced. Exon 10 mRNA is present in Strepsirrhini species, such as Microcebus and Eulemur, indicating that exon 10 splicing is normal up to this level of evolution. Therefore, the molecular event leading to the observed missplicing of exon 10 must have occurred after splitting of the Platyrrhini but before splitting of the three suborders Callithrichidae, Calliconidae, and Cebidae within these New World monkeys. In fact, a scenario where such a defect has occurred independently in all subgroups of the Platyrrhini is very unlikely. This leads to the assumption that this change must have taken place during a time interval between 40 and 35 million years ago.
Does exon 10 skipping have any consequences for receptor function, hormone binding, and primate reproductive physiology? Obviously, the common marmoset has normal sexual differentiation and normal reproductive function even without LHR exon 10. Previously, it has been shown that the marmoset LHR binds hCG with normal affinity, indicating normal hormone binding and signal transduction [21]. Recently, we reported a male hypogonadal patient with very high serum LH levels in whom a genomic deletion caused the complete loss of exon 10 of the LHR [18]. Complete inactivation of the LHR in the human leads to pseudohermaphroditism with female phenotype in genotypically male subjects [4]. The normal sexual differentiation of the patient indicated that during embryonic development, hCG was capable of interacting with this truncated receptor, while the high LH levels were unable to sustain pubertal development. Subsequent treatment of the patient with hCG induced androgenization and spermatogenesis onset, strongly supporting our hypothesis that the absence of exon 10 affects LH but not hCG action [18]. Furthermore, functional studies revealed that the human LHR lacking exon 10 is normally expressed at the cell surface and that hCG could evoke normal signal transduction, in terms of cAMP production, while LH signal transduction was severely impaired in the presence of normal hormone binding (data not shown). Therefore, in the human, the absence of exon 10 is not compatible with normal receptor function in the presence of LH. This raises the question of which gonadotropin is involved in LHR activation in the marmoset and, more generally, in the Platyrrhini.
In order to assess whether the endogenous LH is active on the common marmoset LHR lacking exon 10, it will be necessary to clone the marmoset LH ß subunit and express recombinant marmoset LH in vitro. In fact, we found that human LH is active on the marmoset LHR (data not shown), but the well-known species specificity of the LH/LHR interaction [22] suggests caution, and the results obtained by using human LH and common marmoset LHR are not necessarily indicative of the physiological situation in the marmoset. In any case, the pituitary CG is an obvious candidate for the luteotropic action in this species, especially considering its high level of expression and the evolution of the LH beta;/CG ß gene cluster. The CG ß gene derived from the duplication and a single base deletion of the LH ß gene that incorporated its 3'-untranslated region into the coding region, giving rise to a 24 amino acids carboxy-terminal extension [23]. Recently, it has been shown that the duplication of the LH ß gene, which resulted in the formation of the first CG ß subunit gene, should be approximately dated to 30–40 million years ago, after splitting of the Strepsirrhini from the anthropoid line but before splitting of Platyrrhini and Catarrhini [24]. Therefore, two separate LH ß and CG ß genes and a dual hormone system consisting of LH and CG exist in the entire Platyrrhini lineage. We speculated that, in the common ancestor of this lineage, a mutational event occurred in the LHR gene resulting in the loss of exon 10, but this event remained without functional consequences for reproduction because receptor function could be driven, at least in part, by CG. This hypothesis requires that CG ß is expressed at the pituitary level. Indeed, we found the CG ß mRNA is highly expressed (by RT-PCR) in the pituitary of male and female Callithrix jacchus, supporting this hypothesis and suggesting that CG has some function beyond pregnancy. The pituitary expression of CG ß is also compatible with the evolutionary events of LH ß gene duplication. In fact, it should be assumed that, in the species in which it occurred, the duplication also involved the regulatory sequences, implying pituitary expression of the new subunit [25]. This expression pattern might have been maintained in the common marmoset and, presumably, in other Platyrrhini, owing to the LHR exon 10 deletion, independently on the expression at the placenta level, which might be an earlier event in evolution [25]. In this context, it is interesting to observe that with our RT-PCR strategies, we were unable to amplify LH-specific sequences from pituitary RNA. Further investigation of the LH ß/CG ß gene cluster of the marmoset monkey at the genomic level is necessary to understand better the genomic organization, function, and control of gene expression of LH and CG in Platyrrhini.
In summary, these data suggest that a very ancient evolutionary step in the development of the dual LH/CG hormone system is present in the common marmoset. In this species, pituitary expression of CG is maintained, unlike in other primate species in which the expression of CG is restricted to the placenta. Together with the presence of the LHR type II, this finding suggests that, in Platyrrhini, CG plays a more crucial role in reproductive physiology compared to higher primates and the human, where the physiological CG action is restricted to pregnancy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Jörg Gromoll, Institute of Reproductive Medicine, University of Münster, Domagkstrasse 11, 48129 Münster, Germany. FAX: 49 2518356093; gromolj{at}uni-muenster.de
Received: 20 December 2002.
First decision: 13 January 2003.
Accepted: 6 February 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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