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a Division of Animal Physiology, Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The differentially regulated expression of the tLH-R mRNA isoforms in nongonadal tissues and ovarian stromal tissues during various reproductive stages was quantified and analyzed by Northern blot and/or RT-PCR. Alternatively spliced tLH-R isoforms were differentially expressed in a tissue-specific manner in most of the tissues examined. The steady-state levels of tLH-R mRNA isoforms were relatively high in the hypothalamus and optic nerve and relatively low in the cortex, pituitary, and cerebellum when compared to levels in ovarian follicles. In nongonadal reproductive tissues, the steady-state levels of tLH-R mRNA isoforms were relatively high in the uterus and infundibulum and relatively low in the isthmus, oviduct, and magnum. In addition, in the nongonadal peripheral tissues, the steady-state levels of tLH-R isoforms were relatively high in the thyroid gland and relatively low in the spleen, adrenal gland, kidney, skin, bursa, and muscle.
The present study suggests that the alternative splicing of LH-R transcripts occurs in a tissue-specific manner and has been evolutionarily conserved (similar results were obtained in chicken and swine). These results raise fundamental questions as to the function of LH-R isoforms in nongonadal tissues.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit, while the heterologous ß subunit determines hormone specificity [1]. The ability of LH and FSH to modulate ovarian functions depends not only on the circulating levels of the gonadotropins but also on the expression of their receptors by potential target cells in the ovary [2,3]. With the recent cloning of LH receptor (LH-R) [4–8] and FSH receptor (FSH-R) [9–11] cDNAs, substantial progress has been made in elucidating the structures of the gonadotropin receptors. Both belong to the G protein-coupled receptor family that have seven membrane-spanning domains in common [2,3]. Multiple isoforms of LH-R mRNAs have also been detected in mammalian species [4–6], suggesting a common mechanism(s) that may be evolutionarily conserved (at least between avian and mammalian species).
While LH-R expression has been found to be strictly tissue specific, it is not known why various nongonadal and brain tissues also have been shown to express LH-R [12–21]. The isolation and functional characterization of LH-R from nongonadal tissues should help in determining their possible biological functions.
In the present study, three alternatively spliced turkey LH receptor (tLH-R) isoform cDNAs and tLH-R genomic sequences were isolated and partially characterized. The tLH-R isoforms are expressed in a tissue-specific manner in the nongonadal tissues of the turkey, and these expression patterns of LH-R isoforms for various nongonadal tissues are highly conserved in the chicken and swine.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Turkeys Nicholas Large White female turkeys that were laying regularly were used. Hens were killed at the same time of day to avoid possible diurnal changes in hormone levels.
Chickens Single-comb white Leghorn hens between the ages of 20 and 40 wk that had laid at least four eggs were used.
Pigs Five sexually immature 5-wk-old and two sexually mature male crossbred boars were killed for the collection of porcine tissues. Tissue samples were frozen immediately in liquid nitrogen.
Isolation and Characterization of a Partial tLH-R cDNA
Total cellular RNA (1–3 µg) from granulosa cells from F1 follicles was reverse transcribed into first-strand cDNA using oligo(dT) primer and reverse transcriptase in the presence of deoxynucleotides as recommended by the manufacturer (Perkin-Elmer, Norwalk, CT). Sequences from degenerate oligonucleotide primer pairs were based on the published human LH-R cDNA sequence [6] (the location of amino acids corresponding to the published human sequence are in parentheses): forward primer (amino acids 397–404), CCN MGN TTY CTG ATG TGY AAT CT; reverse primer (amino acids 590–584), AAG SWG ATK GGR GCC ATR CA; where M = A, C; Y = T, C; S = G, C; W = A, T; K = G, T; R = G, A; N = A, T, G, C.
First-strand cDNA was subjected to 35 cycles of PCR amplification using GeneAmp core reagents (Perkin-Elmer). Reaction times were 2-min denaturation at 95°C for the first cycle and 1 min per cycle thereafter; 1 min annealing at 55°C; and 1-min extension at 72°C for the first 34 cycles, 7-min extension on the final cycle at 72°C. The amplified 592-base pair (bp) PCR product was resolved on a 1.2% agarose gel, isolated, purified, and subcloned into the pBluescript KS(+) plasmid vector for large-scale plasmid preparation and nucleic acid sequence analysis.
Subsequently, the rapid amplification of cDNA ends (RACE) technique was used to further characterize the tLH-R cDNA sequence in the 5' and 3' directions, and was accomplished essentially as described by the manufacturer (Clontech Laboratories, Palo Alto, CA). Briefly, poly(A)+-enriched RNA from granulosa tissue of the second largest preovulatory F2 and prehierarchal (6–8 mm) follicles was isolated as described previously [22]. Double-stranded cDNA was synthesized and ligated to the Marathon cDNA amplification adaptor (Clontech). Nested gene-specific primers for the 5' direction (5RACE-1 and 5RACE-2) were designed from the transmembrane tLH-R PCR product described above. An initial amplification of the 5' end by PCR was performed using 5RACE-1 and Adaptor Primer 1 (Clontech) under the following conditions: 1 min at 95°C, 0.5 min at 95°C, and 2 min at 72°C (for five cycles); 0.5 min at 95°C and 2 min at 70°C (for five cycles); 0.5 min at 95°C and 2 min at 68°C (for 25 cycles). A second amplification was performed using one-tenth the volume of the first reaction as template, with the 5RACE-2 and Adaptor Primer 2 as internal primers and amplification conditions as described above. Three PCR products (ranging from 0.8 to 1.2 kilobases [kb] in length) were gel purified and ligated into the pBluescript KS(+); they were then further amplified and purified from plasmid preparations. Initial and secondary amplifications of the 3' end were conducted using 3RACE-1 and Adaptor Primer 1 for the initial and 3RACE-2 and Adaptor Primer 2 for the secondary, under amplification conditions similar to those described above with the exception that during the final 25 cycles, primer annealing was at 65°C for 2 min. A single PCR product from each primer combination (approximately 0.5 kb in length from 3RACE-1 and 0.9 kb from 3RACE-2) was gel purified and ligated into pBluescript KS(+) vector; it was then amplified and purified from plasmid preparations. Nucleotide sequence analysis of positive clones was performed on both strands using the dideoxy chain termination method with both the modified T7 polymerase (Sequenase; U.S. Biochemical Corp., Cleveland, OH) and Taq DNA polymerase, respectively. Gels were read manually or on an Applied Biosystems DNA Sequencer (Model 377; Foster City, CA; Advanced Genetic Analysis Center, University of Minnesota). The nucleotide and amino acid sequences of the tLH-R cDNAs were compiled, analyzed, and compared to chicken [8], quail [7], and mammalian LH-R cDNAs [4–6] using the homology search feature of Genetics Computer Group (Madison, WI).
Northern Blot Analysis
To evaluate tissue-specific expression of the tLH-R isoform transcripts, morphologically normal and regressed follicles were collected and processed for RNA without separating granulosa and theca layers. Follicles were identified as regressed based on the presence of follicle haemorrhagia, collapsed morphology, and an opaque appearance. Total cellular RNA (30–50 µg) was isolated from all tissues using Trizol reagent (Life Technologies, Gaithersburg, MD) and was separated by agarose gel electrophoresis in the presence of 6% formaldehyde, transferred to a nylon membrane (MSI, Westboro, MA), and then hybridized with a -32P-labeled tLH-R cDNA insert at 42°C for 16–18 h. After hybridization, the membrane was washed twice (30 min each) with double-strength SSC, 0.1% SDS (single-strength SSC is 150 mM sodium chloride, 15 mM sodium citrate) at room temperature; this was followed by 2–3 washes in 0.1-strength SSC, 0.1% SDS (30 min each wash) at 65°C. Membranes were exposed to autoradiographic film at -70°C for 1–5 days. After autoradiography, the membranes were boiled for 30 min in 0.1-strength SSC/0.1% SDS to remove the probe and rehybridized with chicken ß-actin insert [23].
The intensity of the hybridization band for steady-state mRNA levels of the tLH-R isoforms and ß-actin was quantified from autoradiographs using a scanning densitometer (Model 4000; Ambis, San Diego, CA). Results were normalized and compared with each other after dividing individual values by the intensity value of the ß-actin mRNA band plus the band intensity of the ethidium bromide-stained 28S and 18S rRNA. The rationale for this analysis is that ß-actin mRNA (a common housekeeping gene) was intended to be used as a quantity control for quantitative reverse transcription-polymerase chain reaction (RT-PCR) as described previously [24].
RT-PCR
Single-stranded cDNA was synthesized from 2 mg total cellular RNA using oligo(dT) primer and reverse transcriptase, as recommended by the manufacturer (Perkin-Elmer). Reaction mixtures (50 µl) were prepared as described previously [24] and amplified by 24–34 cycles of 30 sec at 95°C, 30 sec at 65°C, and 30 sec at 72°C. The amplified products of the reaction were separated by 2% agarose gel electrophoresis, visualized using Eagle Eye II (after staining with ethidium bromide) (Stratagene, La Jolla, CA), and then Southern transferred to a nylon membrane and hybridized to the 32P-labeled tLH-R insert probe. The steady-state levels of tLH-R and ß-actin mRNAs were measured by RT-PCR and were quantified and normalized as described previously [24].
Oligonucleotide Primer Sequences
The ß-actin-specific oligonucleotide sequences [23,24] used in the PCR reaction were Act-1, 5'-TCT GGT GGT ACC ACA ATG TAC CCT; Act-2, 5'-ACC AGT AAT TGG TAC CGG CTC CTC. Used for the amplification of tLH-R cDNA by means of 5'- and 3'-RACE were 5RACE-1 (1144–1170), 5'-CCA GCC ACC AAG CAT GAT GAG CAC GGC; 5RACE-2 (1054–1080), 5'-GGA GAT CAC CGT TAC CGT GTA CAC GGA; 3RACE-1 (482–508), 5'-AAT TCA GCA GTC TGC TGG AAG CTG TTC; and 3RACE-2 (1279–1306), 5'-ATA TTG CTG ATC TTA GTG CTC AAC GCT. Different combinations of oligonucleotide primers were used for the amplification of chicken and turkey total tLH-R transcripts (364 bp; S6F/S9R), coamplification of tLH-Rintact (416 bp) and tLH-Rinsert (502 bp) transcripts (splice-1/splice-2), and amplification of tLH-Rtrunc transcripts (325 bp) (trunc-1/trunc-2): S6F (106–130), 5'-CCA GAC TTG ACT CAG ATC TTC TTC T; S9R (446–470), 5'-GGC AGC CTC TTC AAT GAG TAC GAT G; splice-1 (446–470), 5'-GGC AGC CTC TTC AAT GAG TAC GAT G; splice-2 (838–862), 5'-AGT GGC TGG TTA TGA GGA CGA GGA G; trunc-1, 5'-CAT CGT ACT CAT TGA AGA GGC TGC C; trunc-2, 5'-CTG TGC AAC TTG TAA GGG TGA CTG A.
Oligonucleotide Primer Sequences for the Amplification of Porcine LH-R (pLH-R) Isoforms
Different combinations of oligonucleotide primers were used for the amplification of intact pLH-R transcript [5] (426 bp; pLH-2/pLH-3) and coamplification of truncated (307 bp) and intact (636 bp) pLH-R transcripts (pLH-1/pLH-6): pLH-1 (615–640), 5'-GGA GAA TGC ACA CCT GAA GAA GAT GC; pLH-6 (1228–1253), 5'-ACT GAG GCA ATG AGT AGC AGG TAC AG; pLH-2 (1399–1424), 5'-GTC CAG CTG AAT AGC ATA GGT GAT GG; pLH-3 (962–987), 5'-TTG CTG AGA GTG AAC TGA GTG ACT GG.
Statistical Analysis
All experiments were repeated two to five times. Data were examined by a one-way ANOVA using the General Linear Models procedure of the Statistical Analysis System (Cary, NC), and treatment means were compared using Duncan's Multiple Range test. Significance was reported at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An initial 592-bp tLH-R product was generated by RT-PCR using F1 granulosa RNA as template. Sequence analysis of the 592-bp product showed 98% and 85% amino acid similarity to chicken and rat LH-R, respectively. The structural organization of the partial tLH-R gene was compared to that of rat LH/CG receptor gene [25,26] (Fig. 1).
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The sequence information from the 592-bp RT-PCR fragment in combination with 5'- and 3'-RACE was used to generate a partial tLH-R cDNA (tLH-Rintact) clone that included a 1806-bp coding region and 101-bp 3'-untranslated region (3'-UTR) (Fig. 2A; also Fig. 1). One of two 5'-RACE products generated as described above contained an 86-bp insert tLH-R isoform (tLH-Rinsert) (Fig. 2E). This form was similar (98% identity) to that found in the chicken [7], although it has not been described previously in LH-R cDNAs from mammalian species. The remaining nucleotide sequence of tLH-Rinsert isoform was identical to that of tLH-Rintact form (Fig. 1). The 86-bp insert sequence was located within the extracellular (EC) domain 23 amino acids upstream from the predicted transmembrane (TM) domain and encoded 17 amino acids, followed by an in-frame TGA stop codon (Fig. 2, A and B; Fig. 1).
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The third tLH-R isoform was the product of 3'-RACE and contained a 171-bp sequence that was spliced out of the tLH-Rintact and tLH-Rinsert isoforms between the EC and TM domains (Fig. 2D). This truncated isoform (tLH-Rtrunc) completely lacked the last exon that encoded the TM and intracellular (IC) domains. Otherwise, the remaining 5' portion of the tLH-Rtrunc sequence was the same as for tLH-Rintact (Fig. 1).
To characterize the mechanism(s) generating the three different tLH-R cDNA isoforms, turkey genomic DNA was amplified by PCR using splice-1/splice-2 primers. An approximate 4-kb turkey genomic DNA was amplified by PCR and partially characterized by sequence analysis (Fig. 1 and Fig 2D). The 86 bp found in tLH-Rinsert isoform also was found in the genomic DNA (data not shown). The 171-bp sequence found in tLH-Rtrunc isoform was located at the beginning of the last intron (Fig. 2D; also Fig. 1). This 171-bp intronic fragment was spliced out of the tLH-Rintact and tLH-Rinsert isoforms.
Tissue-Specific Expression of Alternatively Spliced tLH-R mRNAs by Northern Blot Analysis
To test whether alternatively spliced tLH-R isoforms were expressed in a tissue-specific manner in nongonadal tissues, total RNA was subjected to Northern blot analysis. Figure 3A shows a representative Northern blot analysis of the tissue-specific expression of tLH-R mRNA using total RNA. Two distinct 3.0-kb and 1.5-kb tLH-R transcripts were detected in ovarian tissues (lanes 1 and 2) when the EC domain region was used as probe. However, the 1.5-kb transcript was not detected when the TM domain region was used as probe (Fig. 3C). For normalization of equal RNA loading, the intensity of ß-actin mRNA bands was used as a control (Fig. 3B). Turkey LH-R expression was not observed in either hypothalamic or thyroid gland tissues (Fig. 3, lanes 3 and 4). Overall, dramatic changes in tLH-R total mRNA levels were observed between morphologically normal F3 follicles (Fig. 3A, lane 1) and regressed follicles of the same size (Fig. 3A, lane 2) (17.6 ± 2.5 arbitrary densitometric units [ADU] in normal vs. 3.4 ± 5.2 ADU in regressed follicles).
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Tissue-Specific Expression of Three Different LH-R mRNA Isoforms by RT-PCR
Figure 4A illustrates the location of different primer combinations as well as the length of the expected PCR products. The PCR amplification rate of two different products (tLH-Rintact and tLH-Rinsert) was tested to assure that the number of PCR cycles was in the linear range. These values were analyzed and normalized as described above (data not shown). The amplification rate curves of these two different products remained constant from 28 to 36 cycles, with quantification of products every other cycle, in 6 independent experiments. A similar amplification efficiency was observed for the total tLH-R or tLH-Rtrunc isoform when coamplified with ß-actin-specific fragments. After initial characterization and optimization, total RNA from various tissues of laying turkeys were analyzed by RT-PCR. Amplified products of tLH-R mRNA from the three different primer combinations were found in several nongonadal tissues, including the hypothalamus (Fig. 4B, top, lane 4), thyroid gland (Fig. 4B, top, lane 9), uterus (Fig. 4B, bottom, lane 6), and optic nerve (Fig. 4B, bottom, lane 8), as well as in the ovary (regressed follicle; Fig. 4B, bottom, lane 10). The results of Figure 4B were normalized and are summarized in Table 1. To expand upon the results of nongonadal LH-R expression, total RNA from several tissues of laying chickens (including spleen, kidney, muscle, and regressed follicle) was isolated, and tLH-R oligonucleotide primers were used to amplify chicken LH-R (cLH-R) transcripts. Essentially the same pattern of expression was observed for the three different cLH-R transcripts as was shown for the tLH-R transcripts (Fig. 5).
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To determine whether the tissue-specific expression pattern observed in avian LH-R transcripts was the same as for non-avian species (suggesting an evolutionarily conserved splicing mechanism), the tissue-specific expression of pLH-R was examined. The locations of two different primer combinations are depicted and the expected sizes of pLH-R RT-PCR products [5] shown schematically in Figure 6A. Figure 6B shows expression of total pLH-R mRNA in nongonadal tissues as well as in testis. Of the different RNA analyzed (from brain, intestine, kidney, liver, muscle, tonsil, spleen, and testis), only brain (lane 1), kidney (lane 3), muscle (lane 5), spleen (lane 7), and testis (lane 8) showed an RT-PCR product. Figure 6C shows RT-PCR amplification of two different isoforms of pLH-R (the intact 636-bp and a truncated 307-bp fragment) not only in testis but also in nongonadal tissues. The resulting tissue-specific alternative splicing of pLH-R mRNA expression is summarized in Figure 6D.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Partial characterization of the tLH-R genomic sequence was used to determine the origin of the tLH-Rtrunc and tLH-Rinsert isoform transcripts. In contrast to mammalian gonadotropin receptor genes, which do not contain intronic sequences within the last TM encoding exon of the primary transcript, tLH-R (and cLH-R) genes have an 86-bp insertion in the upstream region of TM domain. This 86-bp insert was spliced out in the tLH-Rintact to produce intact tLH-R proteins that contained the TM domain. Otherwise, if this 86-bp fragment is inserted due to alternative splicing (the tLH-Rinsert isoform), it encodes 17 amino acids followed by an in-frame TGA stop codon that will produce a truncated tLH-R protein isoform lacking the TM region.
The most common feature of the mammalian LH-R gene is that the first 10 exons encode the majority of the EC domain, while exon 11 encodes less than 50 amino acid residues of the N-terminal EC domain, the seven TM-spanning domain, and the entire C-terminal cytoplasmic portion of the protein. It is interesting to note that common mechanisms must exist among avian and mammalian species for producing soluble or secreted binding proteins from a single LH-R gene due to alternative mRNA splicing. Multiple LH-R mRNA transcripts have been detected in mammalian species, and their size and relative abundance are both tissue- and species-specific [4–6,27,28]. In the rat ovary, the predominant mRNA is 6.7 kb in length with less abundant 1.2-, 2.6-, and 4.3-kb transcripts [29]. By comparison, both the ovary and testis of the pig express 1.4-, 2.6-, 4.0-, 4.7-, 5.8-, and 6.7-kb LH-R mRNAs, but in different relative proportions [5]. It has been demonstrated that the 1.2-kb LH-R mRNA from the rat encodes a truncated form of the receptor [27].
Northern blot analysis revealed a predominant 3.0-kb tLH-R transcript, in addition to a distinctive 1.5-kb tLH-R transcript that was also expressed in turkey ovarian tissue. This 1.5-kb tLH-R transcript (tLH-Rtrunc) was found to encode a truncated form of the tLH-R that lacked both TM and IC domains as confirmed by RNase protection assay, cloning, and sequence analysis. By comparison, there appears to be a single 3.0-kb LH-R transcript expressed in chicken ovarian tissues [7] and in quail testis [8]. However, in the present study, the identification of alternatively spliced LH-R transcripts in the chicken suggests that the steady-state levels of cLH-R mRNA could be the summation of two alternatively spliced cLH-R (LH-Rintact and LH-Rinsert) transcripts, since Northern blot analysis was unable to discriminate the 86-bp difference in size [7].
The physiological significance of the two different, alternatively spliced forms of tLH-R (tLH-Rinsert and tLH-Rtrunc) is not clear at this time, but they could be of particular importance as receptor variants that lack a TM region, since they would be expected to secrete proteins capable of binding LH. These soluble variants of the LH-R may be important in modulating extracellular LH levels by reducing the concentration of free LH available to bind to LH-R on the target cells. Recently, alternatively spliced avian FSH-R cDNA isoforms (FSH-Rintact and FSH-Rtrunc) from chicken and turkey ovarian tissues have been characterized [30]. Results showed multiple FSH-R transcripts (due to incompletely or alternatively spliced transcripts) similar to those in mammals [9,31–36]. Thus, the highly conserved alternative splicing appears to be an important mechanism for the synthesis of variant gonadotropin receptor proteins.
It has been determined that nongonadal tissues also express LH-R [13–21]. The novel expression of human LH-R gene in the brain [18] and in several nongonadal reproductive tissues has been characterized by immunocytochemistry, in situ hybridization, and traditional ligand binding [13–17,19–21]. This raises important fundamental questions as to why these nongonadal tissues express LH-R. RNase protection assay and sequence analysis of several LH-R cDNAs isolated from a human thyroid cDNA library have shown that the predominate human thyroid tissue LH-R transcript is an incompletely spliced isoform [12]. Also, LH-R mRNA was not efficiently spliced in thyroid and testis tissue (thus the amount of normal LH-R protein expressed in these tissues also might be regulated by splicing). The isolation and characterization of human LH-R from thyroid tissue raises the possibility of functional LH-R expression in the thyroid. This is further supported by indirect evidence that LH/hCG functions to regulate the thyroid during early pregnancy [35–41]. In addition, several studies have suggested the possible physiological action of LH/hCG in the brain via functional brain receptors. For example, stereotaxic implantation or microiontophoretic application of LH to the hypothalamus resulted in a decrease in pituitary or peripheral LH levels and caused changes associated with reproductive events [42–48]. Furthermore, it was shown that culturing GnRH-containing neurons with LH altered thesynthesis and release of GnRH [49]. Collectively, these studies suggest that LH can act on the hypothalamus and perhaps upon various regions of the central nervous system. Withuse of an RT-PCR assay, three different mRNA isoforms of tLH-R were detected in the hypothalamus and thyroid gland, with relatively lower levels in other nongonadal tissues.
The tissue-specific alternative splicing of LH-R has been documented in chickens and pigs, suggesting that the process is evolutionarily conserved. Differential expression of alternatively spliced avian FSH-R transcripts in nongonadal tissues has also been demonstrated [29]. The alternative splicing of gonadotropin receptor mRNA isoforms appears to be a general mechanism across a variety of species, although their physiological role is still unclear.
In the present study, we isolated and characterized alternatively spliced, partial, tLH-R cDNA isoforms and tLH-R genomic sequences. We also showed that alternatively spliced tLH-R mRNAs are expressed in a tissue-specific manner. Furthermore, it is possible that the differential regulation of alternatively spliced LH-R transcripts in nongonadal tissues may be a physiologically conserved mechanism. However, the molecular mechanisms that serve as important controlling elements in the expression of functional LH-R in these nongonadal tissues are yet to be determined.
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FOOTNOTES |
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1 GenBank Accession Number: U92082. 
2 Correspondence: Douglas N. Foster, Division of Animal Physiology, Department of Animal Science, University of Minnesota, 495 An Sci/Vet Med Building, 1988 Fitch Avenue, St. Paul, MN 55108. FAX: 612 625 2743; foste001{at}tc.umn.edu
Accepted: September 3, 1999.
Received: August 18, 1998.
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